Centro Congressi d’Ateneo Federico II - Via Partenope, Napoli
May 24-27, 2008
ELEVENTH
NAPLES WORKSHOP ON BIOACTIVE PEPTIDES
New Frontiers in the Search of Bioactive
Molecules: from Peptides to Drugs
Organized by
Università di Napoli “Federico II”
Dipartimento delle Scienze Biologiche
Centro Interuniversitario di Ricerca sui Peptidi Bioattivi (C.I.R.Pe.B.)
Istituto di Biostrutture e Bioimmagini del Consiglio Nazionale delle Ricerche
Centro Regionale di Competenza Diagnostica e Farmaceutica Molecolari
Under the auspicies of the
European Peptide Society
Consiglio Nazionale delle Ricerche
ELEVENTH NAPLES WORKSHOP ON BIOACTIVE PEPTIDES
Honorary Chair:
Chair:
Prof. Carlo Pedone (Napoli)
Prof. Ettore Benedetti (Napoli)
Organizing Committee
E. Benedetti
L. De Luca
B. Di Blasio
R. Fattorusso
V. Ferrari Bravo
G. Morelli
C. Pedone
M. Saviano
Università di Napoli “Federico II”
Consiglio Nazionale delle Ricerche
Seconda Università di Napoli
Seconda Università di Napoli
Consiglio Nazionale delle Ricerche
Università di Napoli “Federico II”
Università di Napoli “Federico II”
Consiglio Nazionale delle Ricerche
Scientific Committee
D. Andreu
E. Benedetti
A.N. Eberle
L. Gierasch
J Martinez
L Moroder
C. Pedone
J. Schneider
C. Toniolo
Universitat Pompeu Fabra, Barcellona, Spain
Università di Napoli “Federico II” - Napoli, Italy
University of Basel, Basel, Switzerland
University of Massachusets, Amherst, USA
Université de Montpllier I, Montpellier, France
Max-Planck-Institute - Münich, Germany
Università di Napoli “Federico II” - Napoli, Italy
University of Delaware, Newark, USA
Università di Padova, Padova, Italy
ELEVENTH NAPLES WORKSHOP ON BIOACTIVE PEPTIDES
Acknowledgments
The Organizing Committee of the Eleventh Naples Workshop on Bioactive Peptides gratefully
acknowledges the support and generous financial assistance of the following Organizations:
Università di Napoli “Federico II”
Dipartimento delle Scienze Biologiche
Centro Interuniversitario di Ricerca sui Peptidi Bioattivi (C.I.R.Pe.B.)
Seconda Università di Napoli
Regione Campania Assessorato Università e Ricerca Scientifica
Istituto di Biostrutture e Bioimmagini del Consiglio Nazionale delle Ricerche
European Peptide Society
aapptec
Bachem AG
INBIOS s.r.l.
Primm s.r.l.
Georg Thieme Verlag
Varian Italia S.p.A.
Waters S.p.A.
ELEVENTH NAPLES WORKSHOP ON BIOACTIVE PEPTIDES
CONTENT
PROGRAMME ................................ 5
YOUNG
INVITED
ORAL
INVESTIGATORS’ LECTURES
LECTURES
...... 9
......................... 21
PRESENTATIONS.....................
41
POSTER PRESENTATIONS .................. 53
LIST
OF
AUTHORS ....................... 143
LIST
OF
PARTICIPANTS .................. 149
Saturday, May 24
Registration
3:00 - 6:30 pm
4:15 - 7:00 pm
The "Murray Goodman" Young Investigators Session
Chairpersons:
Prof. Alex N. Eberle (Basel, Switzerland) - Prof. Alessandra Romanelli (Napoli, Italy)
4:15 - 4:30 pm
Y1
New reagents for the determination of low resolution protein structures
Dr. David Paramelle - Universités Montpellier I, Montpellier, France
4:30 - 4:45 pm
Y2
Synthesis of difficult peptides via depsi-analogues
Dr. Irene Coin - Leibniz-Institute for Molecular Pharmacology, Berlin, Germany
4:45 - 5:00 pm
Y3
Peptide-induced lipid demixing: a new mechanism of action of antimicrobial peptides?
Dr. Ahmad Arouri - Martin Luther University, Halle, Germany
5:00 - 5:15 pm
Y4
A new analgesic peptide: from biophysical screening to in vivo tests
Dr. Marta M.B. Ribeiro - University of Lisbon, Lisbon, Portugal
5:15 - 5:30 pm
Y5
Bioactive peptides as target-selective delivery tools of high relaxivity MRI contrast agents based
on amphiphilic Gadolinium complexes
Dr. Antonella Accardo - University of Napoli "Federico II", Napoli, Italy
5:30 - 5:45 pm
Y6
Application of O-acyl isopeptide method to the synthesis of cyclic peptides
Dr. Jennifer Lécaillon - Universités de Montpellier I et II, Montpellier, France
5:45 - 6:00 pm
Y7
Mechanism of the binding of rBPI 21 with LPS aggregates and membrane model systems
Dr. Marco A. Domingues - University of Lisbon, Lisbon, Portugal
6:00 - 6:15 pm
Y8
Y8 "Cyclic peptides comprising constrained amino acids as inhibitors of integrin-ligand
interaction"
Dr. Soledad Royo - Bielefeld University, Bielefeld, Germany
6:15 - 6:30 pm
Y9
Protein-protein interactions and peptide antagonists
Dr. Daniela Marasco - University of Napoli "Federico II", Napoli, Italy
6:30 - 6:45 pm
Y10
Purification and characterization of an ACE inhibitory peptide from insect protein
Dr. Lieselot Vercruysse - Ghent University, Gent, Belgium
6:45 - 7:00 pm
Y11
Study of the interaction of CPPs with model lipid bilayers by Plasmon Waveguide Resonance
and DSC
Dr. Isabel D. Alves - University Pierre et Marie Curie Paris-6, Paris, France
7:00 - 8:00 pm
Welcome Party
Sunday, May 25
Welcome addresses
8:30 - 8:40 am
MORNING SESSION
8:40 – 10:20 am
Chairpersons:
Prof. Jean Martinez (Montpellier, France) - Dr. Rita Berisio (Napoli, Italy)
Diversity of proteasomal missions: fine tuning of the immune response
Prof. Michael Groll - LMU Munchen, Munchen, Germany
L1
8:40 - 9:05 am
The birth and survival of nascent proteins
Prof. Ada Yonath - Weizman Institute of Science, Rehovot, Israel
L2
9:05 - 9:30 am
Peptides from bench to bedside. (S)low (but effective) throughput methods
Prof. Miguel A. Castanho - University of Lisbon, Portugal
L3
9:30 - 9:55 am
Design and synthesis of C2-symmetric pyrrolidines as HIV protease inhibitors
Prof. Wibke E. Diederich - Philipps University of Marburg, Germany
L4
9:55 - 10:20 am
Coffee Break - sponsored by WATERS S.p.A.
10:20 - 10:50 am
LATE MORNING SESSION
10:50 – 12:30 pm
Chairpersons:
Prof. Evaristo Peggion (Padova, Italy) - Dr. Laura Zaccaro (Napoli, Italy)
The liver "basic" fatty acid-binding proteins
Prof. Hugo L. Monaco - University of Verona, Verona, Italy
L5
10:50 - 11:15 am
The role of turns in the folding of a β-clam protein based on model peptides and protein
engineering
Prof. Lila M. Gierasch - University of Massachusetts, Amherst, USA
L6
11:15 - 11:40 am
Intercellular transfer of infectious prion protein
Prof. Chiara Zurzolo - Institut Pasteur, Paris, France
L7
11:40 - 12:05 am
Amyloidogenic molecules associations - bona fide phenomenon or just an accident
Prof. Paulina Juszczyk - University of Gdansk, Gdansk, Poland
L8
12:05 - 12:30 pm
Lunch Break
12:30 - 2:30 pm
AFTERNOON SESSION
2:30 – 4:50 pm
Chairpersons:
Prof. Claudio Toniolo (Padova, Italy) - Dr. Emilia Pedone (Napoli, Italy)
In silico prediction of sequence-structure-function relationships of peptides
Prof. Robert Brasseur - Centre de Biophysique Moleculaire Numerique, Gembloux, Belgium
L9
Monitoring gene therapy by external mRNA imaging using a Tat-PNA probe
Prof. David Andreu - Pompeu Fabra University, Biomedical Research Park, Barcelona, Spain
L10
Structural studies of large fragments of G-protein coupled receptors
Prof. Oliver Zerbe - University of Zurich, Zurich, Switzerland
O1
Identifying direct binding contacts between a peptide hormone and its receptor by mutual
exchange of functional groups
Prof. Mark Wheatley - University of Birmingham, Birmingham, UK
O2
Development of peptide based vaccines for the treatment of renal cell cancer (IMA901)
Prof. Werner Stuber - immatics GmbH, Tubingen, Germany
O3
Peptide α/310-helix dimorphism: crystal-state evidence for the effect exerted by a
fluoroalcohol
Dr. Marco Crisma - I.C.B.- C.N.R. , Padova, Italy
O4
4:05 - 4:20 pm
Structure-based design of short peptides and small molecules that compete with p53 for
binding to TFIIH
Prof. James G. Omichinski - Université de Montréal, Montréal, Canada
O5
4:20 - 4:35 pm
Structure, function and in vivo activity of a branched antimicrobial peptide
Prof. Luisa Bracci - University of Siena, Siena, Italy
O6
4:35 - 4:50 pm
2:30 - 2:55 pm
2:55 - 3:20 pm
3:20 - 3:35 pm
3:35 - 3:50 pm
3:50 - 4:05 pm
Coffee Break
4:50 - 5:20 pm
LATE AFTERNOON SESSION
Poster Session: discussion of posters with odd number
5:20 - 7:00 pm
Free Evening
7:00 pm
Monday, May 26
MORNING SESSION
8:30 – 10:10 am
Chairpersons:
Prof. Carlo Pedone (Napoli, Italy) Dr. Simona M. Monti (Napoli, Italy)
8:30 – 8:55 am
L11
Oral availability of peptides
Prof. Horst Kessler - Lehrstuhl II TU-Munchen, Munchen, Germany
8:55 - 9:20 am
L12
Design of peptide hydrogels for use in tissue regenerative therapies
Prof. Joel Schneider - University of Delaware, Newark, USA
9:20 - 9:45 am
L13
Solubilizing the insoluble for screening: identifying molecular inhibitors of the JC and BK
viruses
Prof. Dale Mierke - Dartmouth College, Hanover, USA
9:45 - 10:10 am
L14
From peptide to non-peptide for the development of ghrelin receptor ligands
Prof. Jean-Alain Fehrentz Université de Montpellier II, Montpellier, France
10:10 - 10:40 am
Coffee Break
10:40 – 12:25 am
LATE MORNING SESSION
Chairpersons:
Prof. Luis Moroder (Martinsried, Germany) Prof. Stefania Galdiero (Napoli, Italy)
10:40 - 11:05 am
L15
Peptide design for protein-surface recognition
Prof. Ernest Giralt University of Barcelona, Barcelona, Spain
11:05 – 11:30 am
L16
Multiple functionalities of membrane-active peptides
Prof. Anne S. Ulrich - Forschung Szentrum Karlsruhe, Karlsruhe, Germany
11:30 - 11:55 am
L17
Multiple functions associated with the catalytic property of nicotinamide
phosphoribosyltransferase/PBEF/visfatin
Prof. Yuji Kobayashi - Osaka University, Osaka, Japan
11:55 - 12:25 am
L18
Target deconvolution in the postgenomic era
Prof. Georg C. Terstappen - Siena Biotech, Siena, Italy
12:25 - 2:30 pm
Lunch Break
2:30 – 4:10 pm
AFTERNOON SESSION
Chairpersons:
Prof. Manfred Mutter (Lausanne, Switzerland) Prof. Luciana Esposito (Napoli, Italy)
2:30 - 2:55 pm
L19
Targeting cancer cells with neuropeptides platinum complexes
Prof. Aleksandra Misicka - University of Warsaw, Warsaw, Poland
2:55 – 3:10 pm
O7
Acretocins: peptaibiotics from Acremonium crotocinigenum containing the rare 1aminocyclopropanecarboxylic acid
Prof. Hans Brückner University of Giessen, Giessen, Germany
3:10 - 3:25 pm
O8
Exploiting split inteins for the semi-synthesis of proteins and to study the mechanism of
protein splicing
Prof. Henning D. Mootz - Techical University Dortmund, Dortmund, Germany
3:25 - 3:40 pm
O9
The unusual helix stability of a VEGF mimetic peptide
Dr. Luca D. D'Andrea - IBB - CNR, Napoli Italy
3:40 - 3:55 pm
O10
Multiple -sheets molecular dynamics of amyloid formation of two Abl-SH3 domain peptides
Prof. Inta Liepina - Latvian Institute of Organic Synthesis, Riga, Latvia
3:55 - 4:10 pm
O11
Molecular dynamics study of the apoA-I's fragment 104-107 and its Met112 to Ala mutant
Prof. Athanassios Stavrakoudis University of Ioannina, Ioannina, Greece
4:10 - 4:40 pm
Coffee Break
4:40 - 6:40 pm
LATE AFTERNOON SESSION
Poster Session: discussion of posters with even number
8:30 pm
Gala Dinner
YOUNG INVESTIGATORS’ LECTURES - Eleventh Naples Workshop on Bioactive Peptides
New reagents for the determination
of low resolution protein structures
David Paramelle1, Gilles Subra1, David Lascoux2, Christophe Geourjon3, Gilbert Deléage3, Eric
Forest2 et Jean Martinez1
1 Institut des Biomolécules Max Mousseron, UMR 5247, Universités Montpellier I, CNRS, Faculté de Pharmacie, Montpellier Cedex 05
2 Laboratoire de Spectrométrie de Masse des Protéines, Institut de Biologie Structurale, CNRS/CEA/UJF, Grenoble Cedex 1
3 Institut de Biologie et de Chimie des Protéines, Laboratoire de Bioinformatique et de RMN Structurales, Lyon
Determination of three-dimensional protein structure is an important step for the
comprehension of biological processes. The utilization of chemical cross-linkers with mass
spectrometry is an alternative strategy when techniques like NMR and X-ray crystallography
are difficult or impossible to use.1 This method can provide a low resolution three-dimensional
structure of a protein of interest to identify protein folding.
The principal limitation is the detection of modified peptides. To improve their detection, we
developed the utilisation of chemical moiety drastically improving the detection of these
fragments by mass spectrometry MALDI-Tof analysis.2
References
1. SINZ, J. Mass Spectrom. 2003; 38: 1225–1237
2. D.Lascoux et al., Angew. Chem. Int. Ed. 2007 ; 46 : 5594 –5597
3. A. Pashkova, E. Moskovets, B.L. Karger, Anal Chem 2004; 76: 4550-7
4. Pierce Biotechnology, Applications Handbook and Catalog. Pierce: Rockford, IL, 2004
5. Lomant AJ, J.Mol.Biol. 1976 ;104 : 243
10
New Frontiers in the Search of Bioactive Molecules: from Peptides to Drugs
Synthesis of difficult peptides via depsi-analogues
Irene Coin, Peter Schmieder, Michael Bienert and Michael Beyermann
Leibniz-Institute for Molecular Pharmacology, , 13125 Berlin, Germany
Polypeptides prone to fold into β-structures are often difficult and sometimes even impossible
to obtain via chemosynthesis, due to inefficiency of assembly and/or to low solubility in the
media normally used for purification. Such sequences can be more easily obtained through
synthesis of their depsipeptide analogues, by extending the peptide chain, from a suitable
point on, via the β-hydroxyl function of a Ser/Thr residue. The discontinuity introduced in the
regular pattern of amide bonds by the presence of a depsi unit affects the tendency of the
peptide to fold, thus providing in principle an improvement in synthesis efficiency. Moreover,
after cleavage of the peptide from the solid support, each depsipeptide unit provides an
additional ionizable moiety, thereby increasing solubility and facilitating purification. Depsianalogs are isomerized to the native species under essentially physiological conditions
through an O,N-acyl shift, which occurs quantitatively over a short period of time. This
methodology lends itself to the generation of highly soluble prodrugs for medicinal purposes
[1]
and to the study of peptide folding and association for highly aggregating natural systems .
We report about the synthesis of difficult sequences of biological interest achieved through
suitable depsipeptide analogs. In this context many aspects of the technique are elucidated,
like the formation of the ester bond onto solid-phase and the occurrence of epimerization, the
use of preformed depsidipeptide units and the stability of the depsi units during assembly and
work-up procedures. Strategies for prevention of diketopiperazine formation during Fmoc
removal at the second residue following the ester bond are illustrated (use of Bsmoc Nαprotection, use of alternative protocols for Fmoc deblocking)[2].
Application of the depsipeptide technique to the condensation of peptide segments is
demonstrated: under appropriate conditions, coupling of segments bearing a C-terminal
depsipeptide unit occurs quickly and without giving rise to epimerization at the activated
amino acid.[3]
Finally, the first solid phase synthesis of cotransin, a cyclic depsipeptide having high
pharmacological potential, is described.[4]
References
1. a) L.A. Carpino, E. Krause, C.D. Sferdean, M. Schümann, H. Fabian, M. Bienert, M. Beyermann, Tetrahedron Lett. 2004,
45, 7519; b) Y. Sohma, Y. Hayashi, M. Kimura, Y. Chiyomori, A. Taniguchi, M. Sasaki, T. Kimura, Y. Kiso, J. Pept. Sci.
2005, 11, 441; c) S. Dos Santos, A. Chandravarkar, B. Mandal, R. Mimna, K. Murat, L. Saucède, P. Tella, G. Tuchscherer,
M. Mutter, J. Am. Chem. Soc. 2005, 127, 11888
2. I. Coin, R. Dölling, E. Krause, M. Bienert, M. Beyermann, C.D. Sferdean, L.A. Carpino, J.Org.Chem. 2006, 71, 6171
3. a) I. Coin, P. Schmieder, M. Bienert, M. Beyermann, J. Pept. Sci. 2008, 14, 299; b) T. Yoshiya, Y. Sohma, T. Kimura, Y.
Hayashi, Y. Kiso, Tetrahedron Lett. 2006, 47, 7905
4. I. Coin, P. Schmieder, M. Beerbaum, M. Bienert, M. Beyermann, manuscript submitted
11
YOUNG INVESTIGATORS’ LECTURES - Eleventh Naples Workshop on Bioactive Peptides
Peptide-induced lipid demixing: a new
mechanism of action of antimicrobial peptides?
Ahmad Arouri1, Günter Förster1, Luiz C. Salay2, Volker Kiessling2, Lukas Tamm2, Margitta Dathe3
and Alfred Blume1
1 MLU Halle-Wittenberg, Institute of Chemistry, Mühlpforte 1, 06108 Halle, Germany.
2 Department of Molecular Physiology and Biological Physics, Univ. of Virginia, Charlottesville, VA.
3 Research Institute of Molecular Pharmacology, Berlin-Buch, Germany.
In recent years, cationic antimicrobial peptides (CAMPs) have drawn much attention as a
promising solution to overcome the problem of bacterial resistance. So far, all the proposed
scenarios for the mechanism of action of CAMPs are associated with perforating and breaking
down the bacterial membranes. Hereby, we introduce a new possible mechanism based on
the demixing of bacterial membrane lipids.
An extensive demixing phenomenon was observed with a linear and cyclic antimicrobial
peptide, respectively, having the same sequence (RRWWRF)[1]. The cyclization of the peptide
enhanced its antimicrobial activity and selectivity[2]. The peptides interacted specifically with
the PG headgroup and consequently destabilized the DPPG bilayer and fluidized the DPPG
monolayer, abolishing the liquid-expanded to liquid-condensed transition. Similarly, the epifluorescence imaging carried out with supported lipid bilayers demonstrated upon adding the
peptides the formation of protrusions and the disappearance of the liquid condensed domains,
particularly in POPG/POPE and E. coli lipid extract bilayers. As proven by DSC, FT-IR and Xray diffraction, the demixing occurred in DPPG/DPPE and DPPG/DPPE/TMCL lipid
membranes and led to the appearance of two domains; a DPPG-peptide-enriched domain
and a DPPE-enriched domain. However, no domain formation was obtained with
DPPG/DMPC lipid mixtures. Black lipid membrane experiments revealed that the linear
peptide induced channel formation, however rarely and of limited duration, whereas no
channel activity at any voltage was shown with the cyclic peptide despite its enhanced activity.
Moreover, no considerable break-down or micellization of lipid vesicles was observed by
dynamic light scattering.
We therefore propose that the peptide-induced lipid demixing in PG/PE-membranes could be
a further specific effect of CAMPs operating only on bacterial membranes, which contain
mainly PE and PG. These findings indicate that PG/PC membranes are a poor choice as a
model for bacterial membranes.
References
1. Arouri, A.; Dathe, M.; Blume. A. Biochemistry, in revision.
2. Dathe, M.; Nikolenko, H.; Klose, J.; Bienert, M. Biochemistry 2004, 43, 9140 9150.
12
New Frontiers in the Search of Bioactive Molecules: from Peptides to Drugs
A new analgesic peptide: from
biophysical screening to in vivo tests
Ribeiro M B Marta1, Correia D Ana1, Heras Montserrat2, Talleda Monserrat2, Bardají Eduard2, Pinto
Marta3, Tavares Isaura3 and Castanho A Miguel1
1 Instituto de Medicina Molecular – Faculdade de Medicina da U.L., Av. Prof. Egas Moniz, 1649-028, Lisboa, Portugal.
2 LIPPSO, Universitat de Girona, 17071 Girona, Spain.
3 Instituto de Histologia e Embriologia, Faculdade de Medicina da Universidade do Porto, 4200-319 Porto, Portugal.
Kyotorphin was first discovered in 1979 and reported as an endogenous analgesic agent in
the brain. Attempts to utilise it as an analgesic have, however, been unsuccessful due to the
inability of Kyotorphin to cross the Blood-brain-barrier (BBB). BLV200704, a derivative of this
peptide, was designed to overcome this problem.
Biophysical studies were carried out using fluorescence methodologies to characterize the
peptide interaction with lipid-membrane model systems. Partition coefficient quantification
showed a clear preference of BLV200704 towards zwitterionic and anionic fluid lipid bilayers,
which mimics mammal membranes.
Wistar male rats were administered with BLV200704 i.p. and a set of different behavioural
tests were performed to evaluate the anti-nociception efficacy of the peptide: Tail Flick,
Hargreaves, Hot Plate and Formalin. This compound revealed a remarkable efficiency in
models of acute and inflammatory chronic pain, from doses as low as 1.67mg/100g body
mass, with unaffected motor capacities and cardiac pressure.
Additionally, citotoxicity of BLV200704 was evaluated on Chinese hamster long fibroblasts cell
proliferation using three chromogenic methods – MTT, trypan blue exclusion and crystal violet
assays. With doses up to 100 μM for 24h, this compound revealed low potential to affect
either the metabolic activity in the mitochondria or the membrane integrity.
With a clear affinity to mammalian membranes, demonstrated in vivo by the analgesic effect
after i.p. injection, BLV200704 displays the ability to cross the BBB. This, together with the
lack of toxicity, makes BLV200704 a molecule with promising pharmacological application.
13
YOUNG INVESTIGATORS’ LECTURES - Eleventh Naples Workshop on Bioactive Peptides
Bioactive peptides as target-selective delivery
tools of high relaxivity MRI contrast agents based
on amphiphilic Gadolinium complexes
Accardo Antonella, Tesauro Diego, Morisco Anna, Pedone Carlo, Morelli Giancarlo
CIRPeB, Department of Biological Sciences & IBB CNR University of Naples “Federico II”, 80134 Naples (Italy)
Magnetic Resonance Imaging (MRI) is one of the most powerful and non-invasive techniques
for medical diagnosis.[1] Currently, stable Gd(III)- poli(aminocarboxylate) complexes are widely
used as contrast agents (CAs) in MRI.[2] Although MRI gives very resolved images, due to its
very low sensitivity, it needs higher concentration (10-4 M) of contrast agents. In the last years,
several strategies have been proposed to reach the required local concentration of the CAs
on the target organs and to increase the relaxivitiy values. Supramolecular systems such as
micelles[3] or liposomes[4] have also been used. Moreover, the molecular targeting of
supramolecular aggregates containing contrast agents might be attained by the conjugation of
active recognition moieties such as bioactive peptides able to address them on the specific
biological target overexpressed by cancerous cells.[5]
In the present communication, we report an overview on several supramolecular aggregates
(micelles, open bilayers and liposomes), all of them derivatized with bioactive peptides
(Octreotide, CCK8 or [7-14]Bombesin) well exposed on the aggregate surface to promove
binding of the aggregate to target receptors. The supramolecular aggregates are obtained by
starting from different kinds of amphiphilic monomers. Amphiphilic monomers are synthesized
according to SPPS and Fmoc/tBu strategy, aggregates are formulated in water solution by
using sonication and extrusion procedures. Each amphiphilic monomer is formed by a
hydrophilic and a hyrophobic moiety. The hydrophilic part is represented by the gadolinium
complex or by the bioactive peptide. The hydrophobic part is represented by one or more
alkilic chains at eigheen carbon atoms. The different classes of monomers here described (I,
II, III and IV generation) can be distinguisced on the bases of the number of alkilic chains and
the number of the head groups on the hydrophilic moiety (see Figure 1). The relaxivity
behaviour and a detailed physicochemical characterization, by using SANS, DLS and CryoTEM techniques, of peptide exposed supramolecular aggregates are presented.
Figure 1 : Schematic representation of the different classes of monomers
References
1 Weissleder, R.; Mahmood, U.; Molecular Imaging. Radiology, 2001, 316, 219.
2 Toth, I.; Helm, L.; Merbach, A.E.; “Relaxivity of Gadolinium(III) Complexes: Theory and Mechanism” in The Chemistry of
Contrast Agents in Medical Magnetic Resonance Imaging (Eds.: A. E. Merbach, I. Toth), Wiley, Chichester, 2001, pp. 45–
119.
3 Accardo, A.; Tesauro, D.; Roscigno, P.; Gianolio, E.; Paduano, L.; D'Errico, G.; Pedone, C.; Morelli, G.;J. Am. Chem. Soc.,
2004, 126, 3097.
4 Glogard, C.; Stensrud, G.; Hovland, R.; Fossheim, S.L.; Klaveness, J.; Int. J. Pharm. 2002, 233, 131-140.
5 Reubi, J.C., Endocr. Rev. 2003, 24, 389-427
14
New Frontiers in the Search of Bioactive Molecules: from Peptides to Drugs
Application of O-acyl isopeptide method
to the synthesis of cyclic peptides
Jennifer Lécaillon, Pierre Gilles, Gilles Subra, Muriel Amblard, and Jean Martinez
Institut des Biomolécules Max Mousseron, Universités Montpellier I et II, Faculté de Pharmacie,
UMR CNRS 5247, 15 Avenue Charles Flahault, 34000 Montpellier - France.
During the last few years, the O-acyl isopeptide method was the subject of extensive studies
and applications, for the synthesis of peptides containing difficult sequence such as the
amyloid β peptide, prodrug design and native chemoselective ligation and also for
racemization-free segment condensation.[1,2,3] In this study, our aim was to develop an
alternative route for peptide cyclization, which relies on the use of the O-N intramolecular acyl
migration strategy (Figure 1). Cyclic isopeptides of varying cycle sizes (2) including a serine
as a key element to generate an ester bond and to promote racemization-free cyclization were
synthezised. After removing the Nα-serine protecting group, the isopeptides (3) were totally
converted into the native peptides (1) via O-N intramolecular acyl migration under mild basic
conditions.
Figure 1. O-N acyl migration reaction for cyclic peptide synthesis
We demonstrated the influence of the cycle size on this intramolecular migration as well as
the crucial role played by transposition conditions on the control of a hydrolysis side reaction
observed when cycle constraints became significant.
This method presents the main advantage of allowing racemization-free synthesis of cyclic
peptides containing serine residue. Application of this methodology to threonine containing
peptides and to the design of a new strategy for the synthesis of cyclic peptide on solid
support is actually under investigation.
References
1. Sohma Y, Yoshiya T, Taniguchi A, Kimura T, Hayashi Y, Kiso Y. Development of ‘‘O-acyl isopeptide method’’. Biopolymers
2007;. 88: 253–262.
2. Coin, I.; Dolling, R.; Krause, E.; Bienert, M.; Beyermann, M.; Sferdean, C. D.; Carpino, L. A., Depsipeptide methodology for
solid-phase peptide synthesis: circumventing side reactions and development of an automated technique via depsidipeptide
units. J Org Chem 2006, 71, (16), 6171-7.
3. Mutter, M.; Chandravarkar, A.; Boyat, C.; Lopez, J.; Dos Santos, S.; Mandal, B.; Mimna, R.; Murat, K.; Patiny, L.; Saucede,
L.; Tuchscherer, G., Switch peptides in statu nascendi: induction of conformational transitions relevant to degenerative
diseases. Angew Chem Int Ed Engl 2004, 43, (32), 4172-8.
15
YOUNG INVESTIGATORS’ LECTURES - Eleventh Naples Workshop on Bioactive Peptides
Mechanism of the binding of rBPI21 with LPS
aggregates and membrane model systems
Marco M. Domingues, Miguel Castanho, and Nuno C. Santos
Instituto de Medicina Molecular, Faculdade de Medicina da Universidade de Lisboa, Lisboa, Portugal.;
Antimicrobial peptides (AMPs) are important components of innate immunity[1] and possess a
variety of different structures as well as a high positive charge. The peptide rBPI21 is an AMP
with 21 kDa and corresponds to the 193 aminoacids of the N-terminal region of the
bactericidal/permeability-increasing protein (BPI) that is found in neutrophils[2]. This peptide
was shown to be very specific toward Gram-negative bacteria due to high affinity for the
lipopolysaccharide (LPS) molecules that is present in the surface of these bacteria[3]. It is in
Phase III clinical trials against meningococcal sepsis[4]. Our work was focused on the
interaction of the rBPI21 with LPS aggregates and with model membrane systems, specifically
large unilamellar vesicles (LUV) with different lipid composition. For these studies we used
light scattering spectroscopy and fluorescence spectroscopy. Through dynamic light
scattering (DLS) the hydrodynamic diameter (DH) of the LPS aggregates and the LUV
systems in the presence of the peptide were measured. It is shown that the increase of the
peptide concentration promotes a higher aggregation state of both LPS aggregates (Fig.1)
and LUV systems (Fig.2), only in the presence of negatively charged lipids, culminating with
their sedimentation. This phenomenon does not occur with neutral LUV systems made of
phosphatidylcholine. Measurements of the zeta-potential parameter reveal that the increase of
peptide concentration significantly changes the surface charge of LPS aggregates (Fig. 3) and
LUV systems (Fig. 4) toward positive values. Fluorescence spectroscopy was applied to study
the hydrophobic interaction of rBPI21 with LPS aggregates. It was possible to evaluate a blue
shift in tryptophan emission of the peptide in the presence of LPS aggregates. These results
demonstrate that the interaction of the peptide rBPI21 with LPS aggregates and LUV systems
has electrostatic and hydrophobic contributions.
Fig. 1
Fig. 2
Fig. 3
Fig. 4
References
1. Hancock, R.E.W.; Sahl H.G. Nat. Biotechnol. 2006, 24, 1551-1557.
2. Horwitz, A.H.; Leigh, S.D.; Abrahamson, S.; Gazzano-Santoro, H.; Liu, P.S.; Williams, R.E.; Carroll, S.F.; Theofan, G.
Protein Expr. Purif. 1996, 8, 28-40.
3. Haas, C.J.C.; Haas, P.J.; van Kessel, K.P.M.; van Strijp, J.A.G. Biochem. Biophys. Res. Commun. 1998, 252, 492-496.
4. Levin, M.; Quint, P.A.; Goldstein, B.; Barton, P.; Bradley, J.S.; Shemie, S.D.; Yeh, T.; Kim, S.S.; Cafaro, D.P.; Scannon, P.J.;
Giroir, B.P.; rBPI21 Meningococcal sepsis study group Lancet 2000, 356, 961-967.
16
New Frontiers in the Search of Bioactive Molecules: from Peptides to Drugs
Cyclic peptides comprising constrained amino
acids as inhibitors of integrin-ligand interaction
Soledad Royo and Norbert Sewald
Bielefeld University, Department of Chemistry, D-33615 Bielefeld – Germany
Integrins are a family of transmembrane cell surface receptors, which are involved in many
biological processes. They mediate cell-cell and cell-matrix adhesion. The binding of integrins
with their natural ligands is the molecular basis of physiological processes such as cell
adhesion, migration and signal transduction of cells, as well as of patho-physiological
processes. Thus, small molecules that are able to interfere with this integrin-natural ligand
binding process have pharmacological potential in the therapy of cancer and inflammatory
diseases. The amino acid sequence RGD (Arg-Gly-Asp), present on many of the natural
ligands, is a prominent recognition motif of integrin ligands. Synthetic peptides that contain the
RGD sequence have emerged as an excellent starting point for the identification, synthesis
and development of selective integrin ligands.[1]
The affinity and selectivity of the peptide ligands towards different integrins depend strongly
on the secondary structure of the sequence and the overall three-dimensional shape. Since
the three-dimensional structure of most integrins is not yet available, the introduction of local
or global conformational constraints on a rational basis can lead to successful ligands and, at
the same time, provide information on the structural requirements for the pharmacophoric
groups, following a spatial screening approach.[2] Cyclization is frequently used as a method
to reduce the accessible conformational space. Additionally, the incorporation of non-natural
conformationally constrained amino acids, e.g. β-amino acids,[3,4] can greatly affect the
secondary structure of the peptide, in such a way that the synthetic ligands prefer to adopt a
particular conformation. On the other hand, these modifications often imply an increasing
difficulty in peptide synthesis.
The aim of this investigation are small cyclic peptides containing the RGD motif and
constrained amino acids that exhibit well-defined conformational properties.
The present communication describes the synthesis of different cyclic RGD peptides with the
general sequence c-(-Arg-Gly-Asp-Xaa-Yaa-) and the evaluation of their activity as ligands for
the αVβ3 integrin, present on human cells.
Acknowledgments: This work is supported by a Marie Curie Intra-European Fellowship from the 6th Framework Programme.
Refences
1 Meyer, A.; Auernheimer, J.; Modlinger, A.; Kessler, H. Curr. Pharm. Des. 2006, 12, 2723-2747.
2 Haubner R.; Finsinger D.; Kessler H. Angew. Chem. Int. Ed. Engl. 1997, 36, 1374-1389.
3 Urman, S.; Gaus, K.; Yang, Y.; Strijowski, U.; Sewald, N.; De Pol, S.; Reiser, O. Angew. Chem. Int. Ed. Engl. 2007, 46,
3976-3978.
4 Schumann F.; Müller, A.; Koksch, M.; Müller, G.; Sewald N. J. Am. Chem. Soc. 2000, 122, 12009-12010.
17
YOUNG INVESTIGATORS’ LECTURES - Eleventh Naples Workshop on Bioactive Peptides
Protein-protein interactions and peptide antagonists
Daniela Marasco1, Annamaria Sandomenico2, Laura Tornatore2, Francesca Viparelli1, Nunzianna
Doti2, Angela Saporito1, Marco Sabatella1, Simona M. Monti1, Ettore Benedetti 1,2, Carlo Pedone1,2
1
and Menotti Ruvo
1 stituto di Biostrutture e Bioimmagini (IBB), Sez. Biostrutture, CNR, 80134, Napoli, Italy
2 Dipartimento delle Scienze Biologiche, 80134, Napoli, Italy
Protein tridimensional structure is the complex recapitulation of local and distant
intramolecular forces that cooperatively contribute to maintain a finely tuned energetic
equilibrium. Secondary structure motifs and small protein domains might act as building
blocks that often can be isolated and investigated by several techniques to gain structural
insights on the protein global structure and to modulate interactions with external partners. In
this context, peptides derived by protein digestion under suitable conditions and separated by
chromatographic techniques can be usefully utilized in screening assays to identify proteinprotein interaction agonists or antagonists. This simple but effective methodology has been
successfully applied to find out peptide antagonists of several protein-protein interactions,
allowing the identification of inhibitors with nM efficacy[1]. The systems studied include: i) the
CARD of Bcl10 that mediates a protein oligomerisation event responsible of NF-kB activation
and cell proliferation[2]; ii) Gadd45β, a major player of the endogenous NF-kB-mediated
resistance to apoptosis[3]; iii) the complex between PED and PLD1, believed to play a relevant
role in the insulin resistance mechanism in PED-overexpressing tissues[4].
References
1. Tornatore L., Marasco D., et al., 2008, J Mol Biol. 378, 97-111
2. Thome M., 2004, Nat Rev Immun.,4, 348-359.
3. Papa, S., Monti, S., et al., 2007, J Biol Chem 282, 19029-19041.
4. Vigliotta, G., Miele, et al., 2004, Mol Cell Biol 24, 5005-5015.
18
New Frontiers in the Search of Bioactive Molecules: from Peptides to Drugs
Purification and characterization of an
ACE inhibitory peptide from insect protein
Vercruysse Lieselot1, Smagghe Guy2, Matsui Toshiro3, Morel Nicole4, and Van Camp John5
1
2
3
4
5
Ghent University, Department of Crop Protection & Department of Food Safety and Food Quality, 9000 Gent – Belgium.
Ghent University, Department of Crop Protection, 9000 Gent – Belgium.
Graduate School of Kyushu University, Division of Bioresources and Biosciences, Fukuoka 812-8581 – Japan.
Université Catholique de Louvain, Laboratoire de Pharmacologie, 1200 Brussels – Belgium.
Ghent University, Department of Food Safety and Food Quality, 9000 Gent – Belgium.
During the last decades, bioactive peptides received much interest because of their beneficial
effects towards health. The bioactive peptides with an affinity to regulate blood pressure are
generally believed to be inhibitors of the angiotensin I converting enzyme (ACE). ACE leads to
an increase in blood pressure by converting the inactive angiotensin I into the vasoconstrictor
angiotensin II and by inactivating the vasodilator bradykinin[1]. ACE inhibitory peptides are
potentially useful in the dietary treatment of hypertension[2,3]. This study shows that the
enzymatic hydrolysis of the non water soluble protein fraction of an edible insect species, the
cotton leafworm Spodoptera littoralis (Lepidoptera) leads to the release of ACE inhibitory
peptides. A subsequent hydrolysis with pepsin, trypsin and α-chymotrypsin was designed to
simulate the human gastrointestinal digestion process. The gastrointestinal digest, with an
IC50 value of 125 µg/ml, was fractionated using consecutive chromatographic techniques,
including size exclusion chromatography and reverse-phase high performance liquid
chromatography. A new ACE inhibitory tripeptide was purified and identified as Ala-Val-Phe.
The in vitro ACE inhibitory activity assay revealed an IC50 value of 2123 µM for the tripeptide,
however in organ bath experiments with thoracic rat aortic rings, no ACE inhibitory activity
could be detected. By the action of mucosal peptidases, the dipeptide Val-Phe can be
liberated for Ala-Val-Phe. This dipeptide shows a higher ACE inhibitory activity compared to
the tripeptide as the IC50 value is 120 µM. Both the di- and tripeptide are true inhibitors as
preincubation with ACE does not influence the IC50 value. In the organ bath experiments, ValPhe significantly shifts the concentration-response curve evoked by angiotensin I at a
concentration of 5 and 10 mM. No effect of angiotensin II receptors was observed at these
concentrations, indicating that Val-Phe shows ACE inhibitory activity in rat aorta. Further
experiments with spontaneously hypertensive rats are necessary to evaluate the actual
antihypertensive effect of Val-Phe and Ala-Val-Phe.
References
1 Campbell, D.J. The renin-angiotinsin and the kallikrein-kinin systems. Int. J. Biochem. Cell Biol. 2003, 35, 784-791.
2 Kawasaki, T.; Osajima, K.; Yoshida, M.; Asada, K.; Matsui, T.; Osajima, Y. Antihypertensive effect of valyl-tyrosine, a short
chain peptide derived from sardine muscle hydrolyzate, on mild hypertensive subjects. J. Human Hypertens. 2000, 14, 519523.
3 Seppo, L.; Jauhiainen, T.; Poussa, T.; Korpela, R. A fermented milk high in bioactive peptides has a blood pressurelowering effect in hypertensive subjects. Am. J. Clin. Nutr. 2003, 77, 326-330.
19
YOUNG INVESTIGATORS’ LECTURES - Eleventh Naples Workshop on Bioactive Peptides
Study of the interaction of CPPs with model lipid bilayers by
Plasmon Waveguide Resonance and DSC
Isabel D. Alves, Nicole Goasdoué, Isabelle Correia, Solange Lavielle, Sandrine Sagan, Gerard
Chassaing
Université Pierre et Marie Curie-Paris 6, UMR 7613, 75005 Paris, France
Despite the increasing interest in the use of cell penetrating peptides (CPPs) as molecular
carriers, the mechanism by which those molecules enter cells is far from being understood. A
better knowledge of the interaction mechanism of such peptides with the lipid bilayer is
crucial. We have used plasmon-waveguide resonance (PWR) spectroscopy to monitor the
interaction of those peptides with solid-supported lipid bilayers in a concentration and timedependent manner and to monitor the subsequent alterations in the optical properties of the
lipid bilayer [1]. The capacity of PWR to use both p- and s-polarized light allows one to
measure changes in mass but also to monitor alterations in the thickness, uniformity and the
anisotropic properties of the lipid bilayer upon peptide interaction. The interaction of two
and
RL16
(Hcationic
CPPs,
penetratin
(H-RQIKIWFQNRRMKWKK-CONH2)
RRLRRLLRRLLRRLRR-OH) [2], an amphipathic peptide, with lipid bilayers of varied
composition was monitored. The role of electrostatics, fatty acid chain length, level of
unsaturation and bilayer curvature on the peptide/lipid interactions was investigated [3]. Using
differential scanning calorimetry (DSC) and binary mixable lipid mixtures, a recruitment of one
of the lipid component was observed. This information is crucial for a better understanding of
the mechanism of action and internalization of those peptides.
References
1. I. D. Alves, C. Park, V. J. Hruby (2005) Current Prot. Pept. Sci., 293-312.
2. D. Derossi, G. Chassaing, A. Prochiantz, Trends in Cell Biol. 8 (1998) 84-87.
3. I. D. Alves, N. Goasdoue, I. Correia, S ; Aubry, C. Galanth, S. Lavielle, S. Sagan, G. Chassaing, BBA, under revision.
20
INVITED LECTURES - Eleventh Naples Workshop on Bioactive Peptides
Diversity of proteasomal missions:
fine tuning of the immune response
M. Groll, L. Borissenko
Center for Integrated Protein Science, Department Chemie, Technische Universität München, Lichtenbergstrasse 4, D-85747 Garching, Germany
The majority of cellular proteins are degraded by proteasomes within the ubiquitin-proteasome
ATP-dependent degradation pathway. Products of proteasomal activity are short peptides
which are further hydrolysed by proteases to single amino acid. Some peptides though can
escape the degradation, being selected and uptaken by MHC class I molecules for
presentation to the immune system on the cell surface. MHC class I molecules are highly
selective and specific in terms of ligand binding. Variability of peptides produced in living cells
is created by a variety of ways which allow ensuring fast and efficient immune response.
Substitution of constitutive proteasomal subunits with immuno-subunits leads to
conformational changes in the substrate binding channels, resulting in a modified protein
cleavage pattern and consequently, in the generation of new antigenic peptides. The recently
discovered event of proteasomal peptide splicing opens new horizons in the understanding of
additional functions proteasomes apparently possess. Whether peptide splicing is an
occasional side product of the proteasomal activity still needs to be clarified. Both γ-interferon
induced immuno-proteasomes and peptide splicing represent two significant events providing
increased diversity of antigenic peptides for flexible and fine-tuned immune response.
22
New Frontiers in the Search of Bioactive Molecules: from Peptides to Drugs
The birth and survival of nascent proteins
Yonath Ada
Weizmann Institute of Science, Rehovot, Israel
Ribosomes translate the genetic code into proteins in all living cells. They perform the various
tasks involved in the translation most efficiently owing to their spectacular architecture and
their inherent mobility. Peptide bonds are being formed within a striking architectural element,
a highly conserved internal symmetrical region, located at a functionally central junction within
the otherwise non-symmetrical ribosome. The linkage between this region and substrate
mode of binding indicates that ribosomes position their substrate at stereochemistry suitable
for peptide bond formation and subsequent polypeptide elongation.
Nascent chains emerge out of the ribosome through an elongated tunnel that possesses
dynamic properties that facilitate tunnel's interactive participation in gating, elongation arrest,
discrimination and cellular signaling. Initial transient steps in cotranslational folding may occur
within the tunnel, hinting at ribosomal chaperon activity. On the other side, at the tunnel
opening, the first chaperone that encounters the emerging polypeptides, named trigger factor
in eubacteria, minimizes misfolding and aggregation by conformational rearrangements that
expose a hydrophobic region.
Structures of over a dozen antibiotics complexes obtained at clinically relevant concentrations
with ribosomes of eubacteria serving as a pathogen model, illuminated principles of antibiotics
inhibitory action, provided the structural basis for antibiotics selectivity, and revealed
molecular mechanisms of antibiotics resistance. Comparison with antibiotics interaction to
ribosomes from an archaeon sharing properties with eukaryotes showed that whereas the
identity of a single nucleotide determines drug’s binding, proximal nucleotides govern the
therapeutical effectiveness.
23
INVITED LECTURES - Eleventh Naples Workshop on Bioactive Peptides
Peptides from bench to bedside.
(S)low (but effective) throughput methods
Castanho A. Miguel1, Ribeiro M. Marta1, Correia D. Ana1, Heras Montserrat2, Talleda Monserrat2,
Bardají Eduard2, Pinto Marta3 and Tavares Isaura3
1 Instituto de Medicina Molecular, Faculdade de Medicina da Universidade de Lisboa, 1649-028 Lisboa, Portugal
2 LIPPSO, Universitat de Girona, 17071 Girona, Spain.
3 Instituto de Histologia e Embriologia ,Faculdade de Medicina da Universidade do Porto, 4200-319 Porto, Portugal.
Several peptides are in pharma industry pipelines. T20 (Enfuvirtide) was the first to reach
clinical use as HIV-1 fusion inhibitor. Omiganan may be the first rAMP to succeed and enter
clinical use. The accumulated knowledge on peptide chemistry and biophysics favour a
rational design of molecules, as opposed to massive, high throughput methods. BLV200704 is
a short peptide with analgesic properties and low toxicity, conceived under biophysical
reasoning and later proven efficient with behavioural tests in rats, namely tail flick and hot
plate.
Lipophilicity of drugs is a crucial factor to cross the BBB. The aqueous phase/lipid bilayer
partition coefficient was calculated with suspensions of lipid vesicles. The aggregation state of
the drug was studied with fluorescence spectroscopy methodologies. The drug was
administered by i.p. injection in Wistar male rats. Toxicity was evaluated from the plasma
activities of selected enzymes and histological observations of the liver
24
New Frontiers in the Search of Bioactive Molecules: from Peptides to Drugs
Design and synthesis of C2-symmetric
pyrrolidines as HIV protease inhibitors
W. E. Diederich, A. Blum, J. Böttcher, A. Heine, G. Klebe
Dr. Wibke Diederich, Institut für Pharmazeutische Chemie, Philipps-Universität Marburg, Marburg, Germany
Aspartic proteases play an important role in various physiological but also patho-physiological
processes including e.g. Alzheimer's disease, viral (HIV) as well as parasitic infections
(malaria). In case of the HI-virus, HIV-protease has proven to be an invaluable drug target due
to its essential role in the virus’ replication process. Although very potent HIV-protease
inhibitors have already been successfully launched to the market, the continuously increasing
drug resistance towards existing drugs calls for the design of new inhibitors possessing either
a new binding mode or a new mechanism of action.
In a rational, structure-based approach, a new class of inhibitors bearing a 3,4-disubstituted
pyrrolidine moiety was developed. In order to exploit the C2-symmetry of HIV-protease, we
have exclusively designed and synthesized symmetric inhibitors. X-ray structure of the
enzyme-inhibitor complex revealed that the endocyclic protonated amino functionality
establishes strong electrostatic interactions as well as hydrogen bonds to the carboxylates of
the aspartic residues present in the catalytic dyad. Starting from the initial lead which showed
affinity in the low micromolar range, the activity of this new class of HIV protease inhibitors
could be significantly optimized by means of rational structure-based design up to the twodigit nanomolar range for the final inhibitor.
Encouraged by these results, point mutation studies addressing drug-resistant variants were
carried out. The novel pyrrolidine-based inhibitors showed also remarkable nanomolar affinity
against these mutants. Overall, the efficient enantioselective synthesis, the unique binding
mode, and the high affinity against mutants makes this class of compounds promising
candidates for the development of new anti HIV-drugs.
25
INVITED LECTURES - Eleventh Naples Workshop on Bioactive Peptides
The liver “basic” fatty acid-binding proteins
Monaco Hugo L.
Biocrystallography Laboratory - Department of Biotechnology University of Verona, Verona - Italy
Two paralogous groups of liver fatty acid-binding proteins (FABPs) have been described: the
mammalian type liver FABPs and the basic type (Lb-FABPs) characterized in several
vertebrates but not in mammals. The two groups present sequence similarities and share a
highly conserved three-dimensional structure but their specificity and stoichiometry of binding
are different.
Characteristic of the two families is a common fold in which 10 strands of antiparallel beta
sheet surround the hydrophobic ligand binding site. Two short alpha helices, found
topologically between the first and the second strands, are believed to undergo a
conformational change that would create an opening in the otherwise closed beta barrel and
that would allow the ligands to enter or exit the internal cavity
We have determined the three-dimensional structure of the apo and holo forms of the LbFABPs of three species: chicken, axolotl and, more recently, zebrafish. In every case out goal
was to better understand the ligand binding specificity and to compare it with that of the better
known “mammalian type” L-FABPs.
Over the years the binding of many different hydrophobic ligands was tested in the crystals
until the conclusion was reached that this protein family binds best the bile acids. We thus
decided to rename these proteins liver bile acid-binding proteins (L-BABPs).
Chicken and axolotl bind two cholate molecules in the internal cavity whereas zebrafish binds
one.
We have identified in every case the residues that are crucial for binding and, using sitedirected mutagenesis, have changed the stoichiometry of binding in zebrafish from one to two
bile acid molecules.
In all the cases we have studied the apo and holo forms of the protein crystallize in different
space groups and significant changes are observed between the two conformations
References:
1. Scapin, G., Spadon, P., Pengo, L., Mammi, M., Zanotti, G. and Monaco, H.L. (1988) Chicken liver basic fatty acid-binding
protein (pI = 9.0). Purification, crystallization and preliminary X-ray data FEBS Lett. 240, 196-200.
2. Nichesola, D., Perduca, M., Capaldi, S., Carrizo, M.E., Righetti, P.G. and Monaco, H.L. (2004) Crystal structure of chicken
liver basic fatty acid-binding protein complexed with cholic acid, Biochemistry 43, 14072-14079.
3. Capaldi, S., Guariento, M., Perduca, M., Di Pietro, S.M., Santomé, J.A and Monaco, H.L. (2006) Crystal structure of axolotl
(Ambystoma mexicanum) liver bile acid-binding protein bound to cholic and oleic acid. Proteins. 64:79-88.
4. Capaldi, S., Guariento, M., Saccomani, G., Fessas, D., Perduca, M. and Monaco, H.L. (2007) A single amino acid mutation
in zebrafish (Danio rerio) liver bile acid-binding protein can change the stoichiometry of ligand binding. J Biol Chem. 2007
282, 31008-310018.
26
New Frontiers in the Search of Bioactive Molecules: from Peptides to Drugs
The role of turns in the folding of a β-clam protein
based on model peptides and protein engineering
Lila M. Gierasch1,2 and Anna Marie C. Marcelino2
1 University of Massachusetts-Amherst, Department of Biochemistry & Molecular Biology, Amherst, MA 01003 USA
2 University of Massachusetts-Amherst, Department of Chemistry, Amherst, MA 01003 USA
We have explored the roles of reverse turns in the folding of a -clam protein, cellular retinoic
acid binding protein I (CRABP I). This protein is a member of the large and diverse yet
structurally conserved family of intracellular lipid binding proteins (iLBPs). iLBPs bind
hydrophobic ligands in a cavity formed by two orthogonal -sheets each composed of five
strands in anti-parallel up-down architecture. The strands are connected by seven turns, an
omega loop, and a helix-turn-helix. We have shown previously using peptide models that two
of the turns (turns III and IV) have a strong tendency to sample native-like conformations apart
from their protein context (1,2), and we hypothesized that they played a role in nucleating the
formation of the native fold of the protein via locally facilitated long-range interactions. In the
present work, we have carried out alanine-scanning mutagenesis through these two turns,
and examined the impact of these mutations on the stability and kinetics of folding of the
protein. The results show that turn IV forms early in the folding reaction, while turn III forms
late. The topological placement of turn IV has likely led to its selection as a crucial local
feature to insure productive folding of these proteins that might otherwise be vulnerable to
competing aggregation reactions. We can relate these new findings to our previous work on
the folding mechanism of CRABP (3,4) (see figure, turn IV in red, turn III in green) and gain
insight into how sequence encodes the folding information.
Supported by NIH grant GM027616.
References
1. Rotondi, K. S.; Gierasch, L. M. Biochemistry 2003, 42, 7976–7985.
2. Rotondi, K. S.; Gierasch, L. M. Biopolymers 2003, 71, 638–651.
3. Clark, P. L.; Liu, Z. P.; Rizo, J.; Gierasch, L. M. Nat Struct Biol 1997, 4, 883–886.
4. Clark, P. L.; Weston, B. F.; Gierasch, L. M. Fold Des 1998, 3, 401–412.
27
INVITED LECTURES - Eleventh Naples Workshop on Bioactive Peptides
Intercellular Transfer of Infectious Prion Protein
Chiara Zurzolo
Pasteur Institute, Paris, France and Dipartimento di Biologia e Patologia Cellulare e Molecolare, Università degli studi Federico II Napoli, Italy.
Intracerebral inoculation of infectious prion protein PrPSc represents the most efficient route for
transmission of this disease. This mode of transmission represents a significant source of
iatrogenic infections for brain surgery outpatients. However, under normal agricultural and
clinical conditions infectious PrPSc must reach the brain from remote sites such as the
digestive tract or the blood in order to propogate and exert its pathogenic effects. Mechanisms
proposed for intercellular transfer of PrPSc between cells include exosomes and sites of cellcell contact. It is assumed that the initial contact between PrPSc and a host cell must occur at
the plasma membrane. We have adapted a protocol for generating fluorescently labeled
murine PrPSc. Using this tool we were able to visualize the intercellular transfer of PrPSc
through novel stuctures of cell-cell contact in real-time. In addition to the transfer of plasma
membrane components, these structures enable the cross-infection of cells via intercellular
trafficking of cytosolic vesicles.
28
New Frontiers in the Search of Bioactive Molecules: from Peptides to Drugs
Amyloidogenic molecules associations - bona fide
phenomenon or just an accident
P. Juszczyk1, A. Szymanska1, S. Rodziewicz-Motowidło1, E. Jankowska1, A. Kołodziejczyk1, K.
Stachowiak1, Z. Grzonka1, G. Paraschiv2, M. Przybylski2
1 Department of Organic Chemistry, Faculty of Chemistry, University of Gdansk, 80-952 Gdansk, Poland;
2 Laboratory of Analytical Chemistry and Biopolymer Structure Analysis Department of Chemistry, University of Konstanz,78457 Konstanz,
Germany;
The devastating effect of amyloidogenic proteins and peptides is still not fully understood. The
toxicity of amyloid plaques in the cellular environment is also not clear. Numerous
experiments determined many physiological ligands of amyloidogenic proteins or peptides.
These ligands demonstrated interactions with monomeric, oligomeric and/or fibrillar forms of
amyloidogenic proteins. The search for the new molecules binding to amyloid molecules
could help to understand the aggregation pathway, cellular toxicity of amyloids or
neuroprotective properties of protein ligands. The interaction study of the amyloidogenic
proteins or peptides are very difficult and challenging endeavor because of the rapid
aggregation of the components. Therefore, it necessitates the development of sensitive
analytical methods that enable to clarify details of the interactions between amyloidogenic
molecules. Recent developments show that selective proteolytic excision combined with mass
spectrometric peptide mapping (Epitope-Excision-MS) present high potential for the
determination of epitope for antigen-epitope mapping and for the identification of antibody
paratope sequences. In this work we present a novel affinity method for protein-peptide
interaction studies that enabled identification of the interactions between human cystatin C
(HCC) and the amyloid beta peptide (Aβ). The interactions between HCC and Aβ have been
reported by Sastre et. al [1]. We report here the identification of the molecular interaction,
epitope binding sites and primary structure of Aβ recognized by cystatin C. For the
identification of the epitope from Aβ peptide and its paratope binding structure on cystatin C,
proteolytic epitope extraction/excision-MS protocols were applied using different proteolytic
enzymes [2]. Our results might be of paramount importance for the development of new
inhibitors for aggregation processes of both Aβ and human cystatin C. The determination of
the interacting sites could also be useful in designing new tools for AD diagnostics. Our
studies will allow to speculate whether amyloidogenic molecules association is bona fide
phenomenon or just accident.
Acknowledgement: Ministry of Science and Higher Education (grant N204 161 32/4233), University of Gdansk grant to A. S.,
the Deutsche Forschungsgemeinschaft, Bonn, Germany and the European Union (“Ligand Binders to the Human Proteome”).
References:
1 M. Sastre, M. Calero, M. Pawlik, P.M. Mathews, A. Kumar, V. Danlov, S.D. Schmidt, R.A. Nixon, B. Frangione, E. Levy,
(2004) Neurobiol. Aging, 25, 10-33-1043
2. M. Przybylski, R. Stefanescu, R. Iacob, N. Damoc, A. Marquardt, E. Amstalden, M. Manea, I. Perdivara, M. Maftei, G.
Paraschiv, (2007) Adv. Mass Spectrom., in press.
29
INVITED LECTURES - Eleventh Naples Workshop on Bioactive Peptides
Monitoring gene therapy by external
mRNA imaging using a Tat-PNA probe
D. Andreu1, B.G. de la Torre1, C. Fillat2, N. Andreu2, J. Llop3, O. Millan3, J.D. Gispert3, V. Gómez3,
Z. Nikolovski4, A. Pinyot4, J.A. Pascual4 and J. Segura4
1.
2.
3.
4.
Department of Experimental and Health Sciences, Pompeu Fabra University; Biomedical Research Park (PRBB), 08003 Barcelona, Spain
Center for Genomic Regulation, Biomedical Research Park, 08003 Barcelona, Spain
High Technology Institute, Biomedical Research Park, 08003 Barcelona, Spain
Municipal Institute of Medical Research, Biomedical Research Park, 08003 Barcelona, Spain
Gene therapy is on its way to becoming a major medical development in the not too distant
future. Essential to its effectiveness is the achievement of appropriate protein expression
levels in the expected target cells. Non-invasive monitoring of foreign gene expression is
therefore of paramount importance to confirm its successful use or misuse (e.g. gene doping
in sport). External detection of labelled oligonucleotides hybridizing with the messenger RNA
generated by the transferred gene has been proposed as a possible approach to monitoring
gene therapy.
We have performed a pilot study[1] aimed at detecting the expression of foreign erythropoietin
(EPO) gene expression in murine muscle. To this end, hybrid constructs incorporating peptide
nucleic acid (PNA) sequences with predictable antisense binding ability towards unique
murine EPO mRNA, linked to the cell penetrating peptide Tat(48-60), were synthesized. The
penetration and longer half-life of the Tat-PNA in EPO gene-transfected mouse muscle
C2C12 cells relative to untransfected ones was verified by confocal microscopy and single
photon emission computed tomography (SPECT), respectively, using appropriately labelled
fluorescent and radioactive versions of the construct. Demonstration of the down-regulation of
newly expressed EPO in the transfected cells additionally confirmed the penetration and
hybridizing properties of the selected PNA sequence in the synthetic probe.
Figure 1. Differential image intensity (right limb – left limb) in animals
transferred with EPO gene (transfer on right limb) as monitored by
SPECT with two different Tat-PNA probes
References
1. Segura, J., Fillat, C., Andreu, D., Llop, J., Millán, O., De la Torre, B.G., Nikolovski, Z., Gómez, V., Andreu, N., Pinyot, A.,
Gispert, J.D., Pascual, J.A. Ther. Drug Monitor., 2007, 29, 612-8.
30
New Frontiers in the Search of Bioactive Molecules: from Peptides to Drugs
In silico prediction of sequence-structure-function
relationships of peptides
Robert Brasseur and Annick Thomas
Centre de Biophysique Moleculaire Numerique, Gembloux, Belgium
Peptides are versatile molecules i.e. they are frequently polymorphic and adapt their 3D
structures to the environment. Peptides have wide functional capacities: antimicrobial,
fusogenic, carriers… We have developed PepLook an algorithm for calculating 3D structures
from sequence. PepLook is based on a stochastic procedure that iteratively generates large
number of models and ranks them on energy criteria. Evolution of the model during the
iteration starts from random to evoluate to low energy conformations. We amended the
stochastic procedure by a Boltzmanian evolution of the procedure of selection to better catch
the folding mechanism of peptides. Calculations in PepLook take implicitly the medium into
account, either water, hydrophobic or interfacial.
During the calculation we analyze the evolution of molecule energy and contacts. At the end
of the calculation we sort up to 999 models of conformation that are characterized for
polymorphism and
Stability. We shall in our conference show a few example of calculation course, analyze the
polymorphism and its relation to sequence and discuss on the interest of knowing
polymorphism and stability for deciphering the sequence-structure-function relationships of
peptides.
31
INVITED LECTURES - Eleventh Naples Workshop on Bioactive Peptides
Oral availability of peptides
Horst Kessler, Jayanta Chatterjee, Burkhardt Laufer, Eric Biron, Andreas Frank
Department Chemie, TU München, Germany
Oded Ovadia, Chaim Gilon, Amnon Hoffman, Hebrew University, Jerusalem
Oral availability requires stability against enzymatic cleavage in gut, liver and blood serum as
well as transport from gut into the blood. There is a lot of discussion about molecular
requirements to achieve oral availability. Most often Lipinski`s “rule of five” or Veber rules are
used as filter of oral availability. However, these rules are developed by statistical analysis of
available (non-peptidic) drugs.
Peptides are not taken-up into the blood when orally administered. Exceptions are small
dipeptides and some special cases such as cyclosporin. We assumed that cyclosporin is
orally available due to its cyclic structure and its high degree of N-methylated peptide bonds.
To investigate the requirements to convert peptides ivia multiple N-methylation into orally
available drugs the following topics are investigated:
1. A library of all possible N-methylated derivatives of the somatostain-derived VeberHirschmann-peptide[1] is synthesized and checked for activity. The biologically active Nmethylated peptides are checked for bioavialbalilty. One of the active compounds was orally
available[2]
2. A systematic introduction of N-methyl groups in externally oriented peptide bonds in the
case of cyclic peptides that inhibit cell adhesion shows it is possible to design biologically
active derivatives. Their receptor subtype selectivity was influenced by the N-methylation
although oral availability was not achieved[3]
3. A systematic study of a complete library of different N-methylated cyclic alanine peptides
for oral availability was performed[4] Some of the N-methylated peptides are well transported
(mainly paracellularly). To understand the origin we investigated conformational homogeneity,
internal dynamics, intra- and intermolecular hydrogen bonding capability and the hydrophobic
surface.
References:
1. D. F. Veber, R. M. Freidinger, D. S. Perlow, W. J. Paleveda, F. W. Holly, R. G. Strachan, R. F. Nutt, B. H. Arison, C.
Homnick, W. C. Randall, M. S. Glitzer, R. Saperstein, R. Hirschmann, Nature 1981, 292, 55-58.
2. E. Biron, J. Chatterjee, O. Ovadia, D. Langenegger, J. Brueggen, D. Hoyer, H. A. Schmid, R. Jelinek, C. Gilon, A. Hoffman,
H. Kessler, Angew.Chem.Int.Ed. 2008, in press.
3. J. Chatterjee, O. Ovadia, G. Zahn, L. Marinelli, A. Hoffman, C. Gilon, H. Kessler, Multiple N-Methylation by a Designed
Approach Enhances Receptor Selectivity, J.Med.Chem. 2007, 50, 5878-5881.
4. J. Chatterjee, D. Mierke, H. Kessler, J.Am.Chem.Soc.2006, 128, 15164-15172 and Chemistry Eur. J. 2008, 14, 1508-1517.
32
New Frontiers in the Search of Bioactive Molecules: from Peptides to Drugs
Design of peptide hydrogels for use
in tissue regenerative therapies
Joel Schneider
University of Delaware, Department of Chemistry and Biochemistry.
We are developing peptide-based hydrogels, heavily hydrated materials that are finding use
as extracellular matrix substitutes and in the delivery of therapeutics (e.g. small molecules,
biomolecules, and cells). Specifically, we have designed “smart” peptides that undergo solgel phase transitions in response to biological media enabling minimally invasive delivery of
the material in-vivo. When dissolved in aqueous solutions, these peptides exist in an
ensemble of random coil conformations rendering them fully soluble. The addition of an
exogenous stimulus results in peptide folding into -hairpin conformation. This folded
structure undergoes rapid assembly into a highly crosslinked hydrogel network whose
nanostructure is defined and controllable. This mechanism, which links intramolecular peptide
folding to self-assembly, allows temporally resolved material formation. Peptides can be
designed to fold and assemble affording hydrogel in response to changes in pH or ionic
strength, the addition of heat or even light. In addition to these stimuli, DMEM cell culture
media is able to initiate folding and consequent self-assembly. DMEM-induced gels are
cytocompatible towards NIH 3T3 murine fibroblasts, mesenchymal stem cells, hepatocytes,
osteoblasts and chondrocytes. As an added bonus, many of these hydrogels possess broad
spectrum antibacterial activity suggesting that adventitious bacterial infections that may occur
during surgical manipulations and after implantation can be greatly reduced. Lastly, when
hydrogelation is triggered in the presence of a therapeutic, gels become impregnated and can
serve as a delivery vehicle. A unique characteristic of these gels is that when an appropriate
shear stress is applied, the gel will shear-thin, becoming an injectable low viscosity gel.
However, after the application of shear has stopped, the material quickly self-heals producing
a gel with mechanical rigidity nearly identical to the original hydrogel. This attribute allows
therapeutic-impregnated gels to be delivered to target tissues via syringe where they quickly
recover complementing the shape of the tissue defect. If cells have been impregnated into the
gel, this shear-thin delivery method is a convenient way to introduce cells to wound sites.
33
INVITED LECTURES - Eleventh Naples Workshop on Bioactive Peptides
Solubilizing the insoluble for chemical library screening:
molecular inhibitors of the JC and BK viruses.
Dale F. Mierke
Dept. Chemistry, 6128 Burke Hall, Dartmouth College, Hanover NH 03755.
I will be partaking in a program of screening chemical libraries using both high throughput and
NMR-based methods targeting the viral coat protein of two common human viruses, JCV and
BKV. Using homology models for the VP1 protein of these viruses, we have established the
binding pocket for sialic acid, the first step in viral entry into the cell [1,2]. These determinants
for receptor binding have been transferred to a soluble protein template allowing for
biophysical characterization and amenable for compound library screening. The development
of this soluble platforms and preliminary results from our screening effort will be presented.
References
1. Dugan, A.S., et al. [2007] J. Virology 88, 11798-808.
2. Gee, G.V., et al. [2004] J. Biol. Chem. 279, 49172-6.
34
New Frontiers in the Search of Bioactive Molecules: from Peptides to Drugs
From peptide to non-peptide for the
development of ghrelin receptor ligands
J.A. Fehrentz1, A. Moulin1, A.L. Blayo1, L. Demange1, Didier Gagne1, J. C. Galleyrand1, V. Locatelli2,
A. Torsello2, D. Perrissoud3, J. Martinez1
1 IBMM, UMR 5247, CNRS, Universités de Montpellier 1 et 2, France ;
2 Dipartimento di Medicina Sperimentale, Ambientale e Biotecnologie Mediche, Università degli Studie di Milano-Bicocca, Italy;
3 Æterna Zentaris GmbH, Frankfurt, Germany.
We previously described a potent Growth Hormone Secretagogue (GHS), compound JMV
1843 which was a pseudopeptide. This compound is an agonist of GHS Receptor-1a able to
act by oral route and to stimulate GH secretion in man. It is actually in clinical phase III for GH
deficiency diagnosis in man. The discovery of Ghrelin, the natural ligand of GHSR-1a, focused
the attention on Ghrelin receptor antagonists. Indeed the central actions of Ghrelin include
stimulation of appetite and GH secretion. Such properties support the hope that ghrelin
receptor antagonists could be useful for the treatment of obesity. However for an efficient antiobesity activity, a ghrelin antagonist should counteract the orexigenic effect of ghrelin but not
the GH secretagogue effect, since GH deficiency is frequently associated with increased
adiposity.
Starting from a triazole scaffold, we have designed and prepared a series of novel small
molecules with high binding affinity for the cloned human GHS-R1a and we have investigated
their effects on food intake and GH secretion in animal models. The systematic screening of
over 250 novel compounds for their ability to displace radiolabelled ghrelin and to activate or
to inhibit calcium uptake in cells transiently expressing GHS-R1a led to the characterization of
several compounds acting as GHS-R1a ghrelin full agonists, partial agonists or antagonists.
Our results showed that non-peptide compounds characterized as in vitro GHS-R1a ghrelin
antagonist or partial agonist were able to inhibit food intake without altering in vivo GH
secretion. The dissociated effect of the novel ghrelin receptor ligands on food intake and GH
secretion supports the role of different subtypes or signaling pathways of the ghrelin receptor
in the control of these functions. Thus this report supports the feasibility of a specific
pharmacological modulation of the ghrelin effect on appetite.
35
INVITED LECTURES - Eleventh Naples Workshop on Bioactive Peptides
Peptide design for protein-surface recognition
Ernest Giralt1,2
1 Institute for Biomedical Research, Barcelona Science Park, 08028 Barcelona – Spain.
2 University of Barcelona, Department of Organic Chemistry, 08028 Barcelona – Spain.
Both from a basic science perspective as well as from a drug design point of view there is no
doubt that proteins can be considered as privileged targets for binding of small ligands. In this
context the design of ligands able to disrupt protein-protein interactions is emerging as an
even more relevant issue. However, the design of protein-surface binders and, specially, the
design of ligands able to bind tightly and selectively to hydrophilic protein-surface patches is a
very challenging task. In our laboratory, these last years we have been trying to get some
insight in the principles that govern these molecular recognition processes using peptides as
models for the entire protein receptors. Protein-protein interactions are usually mediated
through large areas, ca. 600 square Å, that have complementary shape and charge. So, in
our opinion, medium-size peptide compounds can be very appropriate candidates to modulate
this kind of interactions. Of course, before a generalized use of peptides as therapeutic agents
the metabolic stability and bioavailability issues must be solved, but there are very recent and
spectacular advances in this area. The general methodology that is currently used in our
laboratory for the design of new peptides able to recognize protein-surface patches is based
on three consecutive steps: i) peptide-ligand design; ii) solid-phase synthesis of strongly
focused peptide libraries; and iii) ligand screening. We report here on the different steps
involved in such an approach including virtual screening using evolutionary algorithms and
exhaustive use of NMR methods.
36
New Frontiers in the Search of Bioactive Molecules: from Peptides to Drugs
Multiple functionalities of membrane-active peptides
Anne Ulrich, Johannes Reichert, Parvesh Wadhwani, Jochen Bürck, Erik Strandberg, Sergiy Afonin,
Ulrich Sternberg, Stephan Grage, Marina Berditsch, Christian Mink, Serge Ruden
Institute of Organic Chemsitry, Karlsruhe, Germany.
There are many different types of membrane-active peptides with designated functions: (a)
antimicrobial peptides kill bacteria by selectively permeabilizing their lipid membranes; (b)
cytotoxic peptides are lytic also against eukaryotic cells; (c) cell penetrating peptides are used
to carry cargo across biomembranes in a non-leaky fashion; and (d) fusogenic peptides are
able to perturb and thereby merge the lipid bilayers of two cells or cellular compartments with
one another. There are no specific sequence homologies or structural characteristics to
differentiate between these kinds of peptides, as they are commonly just named according to
their function. Nevertheless, many of these peptides tend to be amphiphilic (to interact with
the lipid bilayer) as well as cationic (to interact with negatively charged lipids or extracellular
components). Here, we have tested and compared peptides from different functional classes,
to see whether they would exhibit any activity also with regard to one of the other functions,
namely antimicrobial activity (MIC test), activity against eukaryotes (hemolysis assays), and
vesicle fusion (FRET lipid mixing assay, and light scattering). We could confirm that many
antimicrobial peptides have hemolytic side effects, and that some cell penetrating peptides
can also be used as antibiotics, as expected from their similar characteristics. Interestingly,
we found that membrane fusion was massively initiated by certain peptides, irrespective of
their functional class, whilst other petides were completely inactive. The ability to induce
vesicle fusion could be correlated to a good degree with observations by circular dichroism.
Those peptides which undergo a strong conformational change upon binding to a lipid bilayer
also tend to exhibit the highest fusion activity, while others that retain their secondary
structure or are pre-aggregated in buffer are not at all fusogenic. These observations suggest
that irrespective of the nominal peptide class and irrespective of its actual pre- or post-fusion
structure, the driving force for fusion appears to be derived from the energy released by the
conformational change of the peptide upon binding to the membrane.
37
INVITED LECTURES - Eleventh Naples Workshop on Bioactive Peptides
Multiple functions associated with the catalytic property of
nicotinamide phosphoribosyltransferase/PBEF/visfatin
Yuji Kobayashi1,2 Takuya Yoshida2 Ryo Takahashi2, Shota Nakamura2, and Takashi Nakazawa3
1 Osaka University of Pharmaceutical Sciences, Division of Rational Drug Design, Nasahara, Takatsuki, Osaka 569-1094, Japan,
2 Graduate School of Pharmaceutical Sciences, Osaka University, Suita, Osaka 565-0871, Japan
3 Department of Chemistry, Nara Women’s University, Nara 630-8506, Japan,
Nicotinamide phosphoribosyltransferase (NMPRTase) catalyzes the synthesis of nicotinamide
mononucleotide (NMN) from nicotinamide (NM) and 5’-phosphoribosyl-1’-pyrophosphate
(PRPP) in the NAD biosynthesis pathway. NMPRTase was once called pre-B cell colonyenhancing factor (PBEF) because it was originally identified as a growth factor for early stage
B cells, and has recently attracted further attention as visfatin, named after its identity as a
cytokine secreted by visceral fat tissues, reported to have an insulin-like function.
The approaches to address the intriguing issue of the relationship between a simple
phosphoribosyltransferase activity and a variety of physiological functions of this enzyme
include structural and kinetic studies such as X-ray analysis and NMR spectroscopy, hoping
that the regulation of the enzyme activity based on the interaction with its substrate or inhibitor
at the molecular level could possibly allow a rational design of drugs including peptides. At
least two groups have reported the X-ray structure of this enzyme complexed with FK866, an
anti-cancer drug, showing that FK866 binds to the enzyme at the same site as the substrates
(PRPP and NM) normally occupy. Instead of finding a new evidence suggesting the existence
of a link to physiological functions other than NMPRTase activity, our kinetic study in
conjunction with X-ray analysis revealed that the catalysis of phosphoribosyltransferase
activity is reversible to attain an equilibrium state where the formation of PRPP and NM is
more favourable than their reaction to yield NMN and inorganic pyrophosphate (PPi), on the
contrary to our expectation. Furthermore, the reaction rates for both directions of the
reversible reaction were represented by second-order rate constants, indicating that the
reactions undergo in the SN2 mechanism, unlike most of other phosphoribosyltransferases.
This mechanism is consistent with the X-ray structure of the enzyme with two respects: (i)
there is no negatively charged side-chain group that can stabilize the oxocarbonium ion
intermediate involved inherently in the SN1 mechanism, and (ii) several free water molecules
are found in the active site where rapid degradation of the oxocarbonium ion by the reaction
with water should be strictly avoided, while no degradation product, 5-phosphoribose, is
detected in the reaction mixture.
The reversibility of the NMPRTase activity prompted us to speculate the connection of this
enzyme with its functions as PBEF and visfatin. That is, the formation of NMN due to the
NMPRTase activity is associated with the NAD biosynthesis, while its reverse reaction
produces NM, which is also an important metabolite not to be exhausted by the reaction for
NAD biosynthesis. The equilibrium found in NMPRTase activity can effectively regulate the
physiological balance of NM and NMN. Considering that the functions of PBEF and visfatin
appear at the specific organs or cells, we suggest that NMPRTase regulates other enzymes
or proteins (receptors), such as CD38, by locally enhancing or attenuating NM as their potent
effector.
We will review these studies and discuss the NMPRTase activity.
38
New Frontiers in the Search of Bioactive Molecules: from Peptides to Drugs
Target deconvolution in the postgenomic era
Terstappen Georg C.
Siena Biotech S.p.A., Discovery Research, 53100 Siena-Italy
In times of reduced productivity coupled to increasing costs within the pharmaceutical
industry, current ‘reductionistic’ target-based drug discovery has come under pressure and
recently led to the renaissance of a more holistic approach that involves screening small
organic molecules for phenotypic changes elicited in mammalian cells and model organisms.
The retrospective identification of the molecular targets underlying the observed phenotypic
responses ― termed target deconvolution — is important for elucidating biological
mechanisms of disease, will aid rational drug design and enable efficient structure–activity
relationship studies in a chemical optimization program by configuration of target-specific
assays.
A wide range of experimental strategies can in principle be applied to the identification of
targets mediating phenotypic effects and the choice will often mainly be influenced by the
properties of the small molecule.
Methods leading to direct identification of targets typically exploit the affinity between the small
organic molecule and its target protein. These methods include affinity chromatography,
three-hybrid systems, phage and mRNA display, protein and ‘reverse-transfected’
microarrays, and biochemical suppression.
Methods based on comprehensive DNA microarray or proteomics analyses can aid target
deconvolution by investigating the mode of action of an active small molecule. In a more
indirect way, application of these technologies can also lead to the identification of the
molecular targets.
The final aim of target deconvolution is not only the identification of biological targets that
directly interact with the small molecule, but also demonstration that their modulation is
associated with functional effects detectable in the phenotypic assay. ‘Authenticity’ of targets
can be confirmed by functional studies employing a variety of methods such as RNAi and
overexpression.
Since phenotype-based drug discovery has regained momentum, target deconvolution is an
important aspect of current drug discovery.
39
INVITED LECTURES - Eleventh Naples Workshop on Bioactive Peptides
Targeting cancer cells with neuropeptides platinum complexes
Misicka Aleksandra1, Głowinska Agnieszka 1, Kosson Piotr 2, Kubiak Nina 2, Szaniawska Bozena 2,
Lipkowski W. Andrzej2
1 University of Warsaw, Faculty of Chemistry, 02-093 Warsaw, Poland,
2 Medical Research Centre, Polish Academy of Sciences, 02-106 Warsaw, Poland
Most powerful and useful anticancer agents belong to cisplatin derivatives. Cisdiaminedichloplatinum analogs are responsible for the inhibition of DNA transcription what
leads to cell death. Cisplatin binds strongly to DNA in regions containing several guanine
units, forming Pt-DNA links within strands. Through disrupting base-pairing guanine to
cytosine cross-links lead to unwinding of the DNA. Regarding many side effects and
disadvantages, including high toxicity, various anticancer drugs with different mechanisms of
action are combined to form effective antitumour tool[1]. As a result chemotherapy agents work
against both types of cells, destroying cancer and normal type ones. Therefore more selective
delivery system of platinum to cancer cells is still needed.
Based on the evidence of the presence of δ- and µ-opioid receptor types in carcinoma cells[2]
we proposed to use opioid peptides as selective carriers for delivering platinum ions to the
cancer cells. We designed hybride molecules which combine two fragments. One part of the
molecule contains the opioid pharmacophore and the other fragment is designed to form a
complex with platinum ion. Such molecule can serve not only as carrier for platinum, but also
give a strong analgesic effect. As a result such hybride molecule should express analgesic
properties and provide anticancer activity.
Tyr-D-Ala-Gly-Phe-NH-NH←Phe←AA-PtCl2
opioid fragment
complexing fragment of Pt(II)
The opioid fragment of our hybride molecules is based on the biphalin analogue (Tyr-D-AlaGly-Phe-NH-NH-Phe), which is known as a strong antinociceptive agent[3]. The other
fragments, designed to form complexes of Pt(II) could be described as cisDDP analogues. Nterminal cysteine, histidine or metionine residues has been selected to coordinate Pt(II),
because these amino acids are able to coordinate platinum ion by S,N, or N,N atoms.
H
N
OH
N
Cl
Pt
H
N
H2N
O
O
CH3
N
H
H
N
O
NH Cl
O
NH
NH
NH
O
O
Tyr-D-Ala-Gly-Phe-NH-NH-Phe-His(N,N)PtCl2
The binding affinity at the opioid receptors and effect on the proliferation of the human
glioblastoma cells of the synthesized compounds will be discussed.
Acknowledgement: Project supported by EU grant Normolife (LSHC-CT-2006-037733)
References
1. Torigoe T. et. al., Cur. Med. Chem.-Anti-Cancer Agents, 2005, 5, 15-27
2. Madar I. et. al., Journal of Nuclear Medicine, 2007, 48, 207-213
3. Lipkowski AW., et. al., Bioorg. Med. Chem. Lett. 1999, 2763-2766
40
ORAL PRESENTATIONS - Eleventh Naples Workshop on Bioactive Peptides
Structural studies of large fragments
of G-protein coupled receptors
O. Zerbe1, C. Zou1, S. Kumaran1, L. Cohen3, Jeff. Becker2, F. Naider3
1 Institute of Organic Chemistry, University of Zurich, CH-8057 Zurich
2 Department of Microbiology, University of Knoxville, Tennessee, USA
3 Department of Chemistry, College of Staten Island, CUNY, USA
We present our structural studies of larger fragment of G-protein coupled receptors. One
system of interest is the Ste2p receptor, for which fragments containing the 7th TM helix (1)
as well as TM1-C1-TM2-E1 have been biosynthetically produced by our collaborators Prof.
Naider and Becker. The Ste2p receptor presents a yeast GPCR that is activated by binding
the α-factor. Signaling by Ste2p results in growth arrest and gene regulation in preparation for
sexual conjugation of yeast cells.
The second system to be presented comprises constructs derived from the Y4 receptor, a
GPCR targeted by peptides from the NPY family, and in particular by the pancreatic
polypeptide (PP). Herein, we present data on constructs comprising the N-terminal
(extracellular) domain and on a construct that contains the N-terminal domain fused to TM1C1-TM2.
The polypeptides have been expressed in isotopically labelled form in E. Coli. We introduce
methods to produce the double-TM domain proteins without the requirement to be fused to
another protein, and also present a strategy for purification. Their structures in solution are
determined in the presence of phospholipid micelles using high-resolution NMR techniques.
NMR is also utilized to establish the way these polypeptides are embedded in the detergent
micelles. The resonances of both backbone and sidechain resonances have been almost
completely assigned using triple-resonance NMR experiments in the case of the TM1-TM2
construct from the Ste2p receptor. This challenging work needed optimization of procedures
in all aspects of the work, e.g. in labeling procedures but also in the spectroscopic work. In
particular sample conditions and the detergent had to be extensively optimized. Finally, we
have been able to find conditions under which 15N,1H-correlation spectra of good to very good
quality can be acquired after extensive optimization of sample conditions, and with the usage
of detergent mixtures in the case of the Y4 receptor fragments. The present data now reveal
secondary structure, and details of tertiary structure start to emerge.
Progress and problems of the spectroscopy of large GPCR fragments in membrane mimetics
will be reviewed. It will be discussed, in which way structures possibly differ from those of the
entire receptors.
References
1. NMR studies in DPC of a fragment containing the seventh transmembrane helix of a GPCR from Saccharomyces
cerevisiae, Biophys. J., 93, 467-482.
2. Studies of the structure of the N-terminal domain from the Y4 receptor, a G-protein coupled receptor, and its interaction with
hormones from the NPY family, submitted.
42
New Frontiers in the Search of Bioactive Molecules: from Peptides to Drugs
Identifying direct binding contacts between a peptide hormone
and its receptor by mutual exchange of functional groups
Wheatley Mark, and Wootten Denise
University of Birmingham, School of Biosciences, Edgbaston, Birmingham, B15 2TT, UK.
The primary event in generating an intracellular signal by a peptide hormone is the formation
of a specific complex between the hormone and its corresponding receptor, expressed in the
plasma membrane of target tissues. It is of fundamental importance to map this ligand binding
site at the molecular level and to define the binding contact partners formed between residues
in the hormone and residues in the receptor. Such information will increase our understanding
of receptor activation and also aid rational drug design in the future.
G-protein-coupled receptors (GPCRs) form the largest receptor family in the human genome,
with 80% of GPCRs belonging to Family A (rhodopsin-like). The neurohypophysial
nonapeptide hormone vasopressin (AVP) binds to three GPCR subtypes (V1aR, V1bR and
V2R) belonging to Family A, with the majority of physiological effects being mediated by the
V1aR subtype. Mutagenesis studies have identified key residues in the V1aR for high affinity
AVP binding. These residues are dispersed in their distribution and are located in the Nterminus[1], transmembrane helices (TMs)[2] and extracellular loops[3,4] of the V1aR.
Wild-type AVP has glycinamide at position-9. To identify the V1aR residue that provides direct
binding contacts for the C-terminus of AVP, we exploited a series of AVP analogues which
differed in their C-terminal functional group or peptide backbone length: (i) wild-type AVP (Cterminus=CONH2), (ii) AVP-free acid (C-terminus=COOH) and (iii) [β-Ala9]AVP (Cterminus=CONH2 & -CH2 longer backbone compared to AVP) in conjunction with mutant V1aR
constructs containing Gln (R=CH2CH2CONH2), Glu (R=CH2CH2COOH) or Asp
(R=CH2COOH). These combinations of ligand and receptor mutation allowed functional
groups to be ‘swapped’ between the ligand and the receptor. In addition, ligand backbone or
receptor residue side-chain could be increased/decreased by one –CH2 in a reciprocal
manner. Functional interaction between the various AVP analogues and the V1aR mutant
constructs was characterised pharmacologically by (i) radioligand binding assays and (ii)
second messenger generation (inositol trisphosphates accumulation).
Our data allow us to identify for the first time, a single V1aR residue which forms the binding
contact for the C-terminus of the natural agonist AVP.
Supported by grants to MW from The Wellcome Trust and the BBSRC.
References
1. Hawtin, S.R., Wesley, V.J., Simms, J., Argent, C.H., Latif, K. and Wheatley, M. (2005) Mol. Endocrinol. 19, 2871-2881.
2. Mouillac, B., Chini, B., Balestre, M.-N., Elands, J., Trump-Kallmeyer, S., Hoflack, J., Hibert, M., Jard, S., and Barberis, C.
(1995) J. Biol. Chem. 270, 25771-25777.
3. Hawtin, S.R., Simms, J., Conner, M., Lawson, Z., Parslow, R.A., Trim, J., Sheppard A. and Wheatley, M. (2006) J. Biol.
Chem. 281, 38478-38488.
4. Conner, M., Hawtin, S.R., Simms, J., Wootten, D., Lawson, Z., Conner, A., Parslow, R.A. and Wheatley, M. (2007) J. Biol.
Chem. 282, 17405-17412.
43
ORAL PRESENTATIONS - Eleventh Naples Workshop on Bioactive Peptides
Development of peptide based vaccines for
the treatment of renal cell cancer (IMA901)
Stüber Werner, Lewandrowski Peter, Frisch Jürgen and Singh Harpreet
immatics GmbH, Paul-Ehrlich Strasse 15, 72076 Tübingen - Germany
IMA901 is a multiple peptide vaccine for the treatment of renal cancer (RCC). The tumorassociated peptides (TUMAPs) contained in IMA901 were identified by immatics directly from
primary renal cells (= primary RCC tumor tissue samples), selected regarding their overexpression in RCC and proven to be immunogenic using in vitro T-cell assays.
IMA901 consists of 10 individual peptides (10 TUMAPs) and non-active ingredients which are
used as excipients of the pharmaceutical presentation of IMA901.
All 10 peptides are synthesized by conventional Fmoc chemistry. The sequences of the
peptides will be presented and synthetic issues will be discussed. In the final formulation of
IMA901 578 µg of each peptide plus excipients are filled into glass vials and lyophilized. The
challenges of the production of multi peptide drugs will be discussed. Such challenges
comprise the synthesis, the production of the formulation as well as the analyses of the final
presentation of IMA901.
Results of the phase 1 trial in 28 vaccinated RCC patients showed that (1) IMA901 was safe,
(2) multiple T-cell responses to vaccinated peptides correlated with favourable clinical
outcome and (3) patients with a lower percentage of regulatory T cells (Tregs) were more
likely to develop a vaccine-induced multiple T-cell response.
44
New Frontiers in the Search of Bioactive Molecules: from Peptides to Drugs
Peptide α/310-helix dimorphism: crystal-state
evidence for the effect exerted by a fluoroalcohol
Marco Crisma1, Michele Saviano2, Alessandro Moretto1, Bernard Kaptein3, Quirinus B.Broxterman3,
and Claudio Toniolo1
1 Institute of Biomolecular Chemistry, Padova Unit, CNR, Department of Chemistry, University of Padova, 35131 Padova, Italy
2 Institute of Biostructures and Bioimaging, CNR, 80134 Naples, Italy
3 DSM Pharmaceutical Products, Advanced Synthesis, Catalysis and Development, MD 6160 Geleen, The Netherlands
The Aib-rich, membrane-active, antibiotic alamethicin, although largely α-helical, is
characterized by a significant degree of plasticity in terms of intramolecular H-bonding pattern
as 310-helical segments of varying length and location have been also crystallographically
documented[1,2]. Being accompanied by a significant molecular elongation, a transition from αhelix to 310-helix, beside affecting the membrane-spanning capability of a helical peptide, may
represent the first step towards a molecular switch based on these two conformational states.
These observations prompted us to investigate systematically the equilibrium between α- and
310-helices, beginning from simplified peptide sequences formed exclusively by the same Cαtetrasubstituted α-amino acid. From our previous studies it was already known that among the
chiral residues of this class the β-branched Cα-methyl-L-valine [L-(αMe)Val] is that with the
most pronounced bias toward the right-handed 310-helix[3]. Circular dichroism (CD)
experiments on the Nα-acylated homo-heptapeptide alkylamide Ac-[L-(αMe)Val]7-NHiPr (Ac,
acetyl; NHiPr, isopropylamino) clearly showed that it undergoes a fast, solvent-driven,
reversible α-helix / 310-helix equilibrium. More specifically, according to the CD patterns this
peptide is overwhelmingly folded in the α-helix conformation in HFIP (1,1,1,3,3,3hexafluoropropan-2-ol) solution, whereas it essentially adopts the 310-helix conformation in the
less polar methanol solution. Crystallographic analysis showed that Ac-[L-(αMe)Val]7-NHiPr is
completely 310-helical when its crystals are grown from a methanol solution. By contrast, it is
folded in the α-helical conformation when crystallized from HFIP. In this latter case, two cocrystallized solvent molecules bind to the three C-terminal peptide (or amide) carbonyl
functions not involved in the C=O ••• H-N intramolecular H-bonding network. The two
structures represent an unambiguous example of a solvent-driven α/310-helix dimorphism for a
peptide molecule in the crystal state, and provide clues for a deeper understanding of the
interactions of HFIP with helical peptides[4].
References
1. Fox, R.O.; Richards, F.M. Nature 1982, 300, 325.
2. Crisma, M.; Peggion, C.; Baldini, C.; MacLean, E.J.; Vedovato, N.; Rispoli, G.; Toniolo, C. Angew. Chem. Int. Ed. 2007, 46,
2047.
3 Polese, A.; Formaggio, F.; Crisma, M.; Valle, G.; Toniolo, C.; Bonora, G. M.; Broxterman, Q. B.; Kamphuis, J. Chem. Eur. J.
1996, 2, 1104.
4 Crisma, M.; Saviano, M.; Moretto, A.; Broxterman, Q.B.; Kaptein, B.; Toniolo, C. J. Am. Chem. Soc. 2007, 129, 15471.
45
ORAL PRESENTATIONS - Eleventh Naples Workshop on Bioactive Peptides
Structure-based design of short peptides and small
molecules that compete with p53 for binding to TFIIH.
James G. Omichinski1, Laura Zaccaro2, Chantal Langlois1, Paola Di Lello1, Jérôme Baffreau1,
Caroline Mas1, Annarita Del Gatto2, Emma Langella2, Michele Saviano2, Pascale Legault1 and Carlo
2
Pedone .
1 Département de Biochimie, Université de Montréal, Montréal, Canada.
2 Istituto di Biostrutture e Bioimmagini- CNR, Napoli- Italy.
In eukaryotes, activators enhance transcription through protein/protein interactions involving
their transactivation domains (TADs). A wide range of protein targets for TADs have been
identified both in vivo and in vitro, and they include several general transcription factors (TBP,
TFIIB, TFIIH), components of the mediator complex (MED15, MED25), histone
acetyltransferase complexes (CBP/p300, Tra1) and components of ATP-dependent
nucleosome remodeling complexes (Swi1, Snf5). Through these interactions with multiple
partners, activators are able to enhance transcription at multiple stages of the transcription
process, including nucleosome disassembly, pre-initiation complex formation, promoter
clearance and/or transcription elongation.
Given their occurrence in a number of crucial transcriptional regulatory proteins, the most
extensively studied TADs are those that contain acidic TADs and two extremely important
proteins that contain acidic TADs are the human tumour suppressor protein p53 and the
Herpes Simplex Virion (HSV) protein 16 (VP16). Given the presence of long repetitive
stretches of acidic amino acids, acidic TADs are generally disordered in the free state and this
has been demonstrated for both the p53TAD and the VP16TAD. Using a combination of
NMR spectroscopy, isothermal titration calorimetery (ITC) and site-directed mutagenesis
studies we have recently characterized the interaction of both the p53TAD and VP16TAD with
a common target. This common target was the Pleckstrin Homology (PH) domain of the
Tfb1/p62 (yeast/human) subunit of TFIIH. The NMR structures of the Tfb1/VP16TAD complex
and the Tfb1/p53TAD complex demonstrate that both p53 and VP16 form 9-residue α-helices
in complex with Tfb1. Comparison of the VP16/Tfb1and p53/Tfb1 structures clearly
demonstrates how the viral activator VP16 mimics numerous aspects of the mammalian
activator p53. Despite their remarkable similarity, important differences are observed
between the p53TAD and VP16TAD recognition of Tfb1, and our results suggest that selected
TADs such as p53 have evolved so that phosphorylation events play an important role in their
regulation. The Tfb1/p53 and Tfb1/VP16 structures are an important step towards developing
a sequence code for acidic TADs binding to the PH domain of Tfb1/p62. Such detailed
structural information is absolutely essential to design molecules that mimic transcription
activators such as p53.
Given the fact the PH domain of Tfb1/p62 also binds small molecule phosphoinositides
(PI(5)P) , we are currently preparing small molecules and peptide mimics that bind to the PH
domain of Tfb1/p62 and compete with PI(5)P, VP16 and p53 for binding to Tfb1/p62. We will
present the structures of the p53/Tfb1 and VP16/Tfb1 complexes and discuss structural
studies with additional peptides from acidic activation domains. In addition, we will discuss
structural and biophysical characterization of two families of small molecules that compete
with p53 for binding to Tfb1/p62 as wells as peptide analogs of p53 that are stabilized by
helical capping motifs.
46
New Frontiers in the Search of Bioactive Molecules: from Peptides to Drugs
Structure, function and in vivo activity of a
branched antimicrobial peptide.
Luisa Bracci, Alessandro Pini, Chiara Falciani, Sara Iozzi, Jlenia Brunetti, Silvia Pileri, Barbara Lelli,
Andrea Bernini and.Neri Niccolai.
University of Siena, Department of Molecular Biology, 53100 Siena – Italy
The growing emergency of multi-drug resistant bacteria is a global concern: a number Grampositive and Gram-negative bacteria have developed resistance against most traditional, as
well as new generation antibiotics. Therefore, the demand for new antibiotics urges
researchers and pharmaceutical companies to consider new antimicrobial agents. Among
these, antimicrobial peptides turned out to be particularly interesting, in consideration of their
peculiar mechanism of action, which is specifically targeted to bacterial membrane.
Nonetheless, pharmaceutical companies have shown a general reluctance to the
development of peptide drugs, which can be explained by a number of problems related to
development of peptide as drugs, including their short half life produced by rapid proteolysis.
We selected by phage display against E.coli, a non-natural peptide sequence, which showed
a strong antimicrobial activity against Gram-negative bacteria[1]. This peptide was synthesized
in a Multiple Antigen Peptide (MAP) form, which we had previously demonstrated to induce
general resistance to proteolysis, rendering peptides more suitable for therapeutic
applications[2,3]. The antimicrobial branched peptide (M6) was characterized for its activity
against a number of bacteria, including many multi drug resistant isolates and showed very
promising MIC against infectious pathogens of clinical interest. We also demonstrated that M6
shows a poor toxicity for eukaryotic cells, it binds LPS and it does not produce appreciable
haemolysis even upon prolonged incubation. Moreover, we evaluated M6 acute toxicity and
we also demonstrated that it is not immunogenic upon repeated injections in animals[4].
We report here on M6 in vivo activity in models of sepsis induced in mice by E. coli and P.
aeruginosa. These experiments showed that the peptide can prevent animal death and can
neutralize sepsis symptoms when used in doses comparable to traditional antibiotics and
compatible with a clinical use.
These results make the branched M6 peptide a strong candidate for the development of a
new antibacterial drug.
References
1 Pini et al. Antimicrob Agents Chemother. 2005; 49: 2665-72.
2 Bracci et al. J Biol Chem. 2003; 278: 46590-5.
3 Falciani et al. Chem Biol Drug Des. 2007; 69: 216-21
4 Pini et al. J Pept Sci. 2007; 13: 393-9.
47
ORAL PRESENTATIONS - Eleventh Naples Workshop on Bioactive Peptides
Acretocins: peptaibiotics from Acremonium crotocinigenum
containing the rare 1-aminocyclopropanecarboxylic acid
Brückner Hans and Kirschbaum Jochen
Interdisciplinary Research Centre (IFZ), University of Giessen, 35392 Giessen - Germany
Screening of filamentous fungi for the production of Aib and Iva had revealed that
Acremonium crotocinigenum (CBS 217.70) was a potent producer of peptides containing
these non-protein amino acids which are characteristic markers for peptaibiotics[1,2]. The
mold was cultured submers in a malt extract medium. A peptide mixture named acretocin
(ACR) was isolated from the culture broth by XAD and Sephadex LH-20 chromatography.
Comparison of ACR with efrapeptin (EFR) from strains of Tolypocladium[3,4] by TLC provided
identical RF-values and similar HPLC-elution profiles. Individual peptides of ACR were
fractioned using semi-preparative HPLC on a Kromasil KR100 column (150 mm x 10 mm i.d.,
particle size 3.5 µm; column temperature 40°C). For elution a binary gradient consisting of
MeCN/MeOH/water with addition of 0.1% TFA and a flow rate of 2.5 ml/min was used. The
individual peptides of ACR were analyzed by direct infusion ESI-MS (LCQ-System, Thermo
Electron Corp.) as well as on-line analytical HPLC-ESI-MS. The molecular weights of the ACR
peptides were 1604 Da, 1618 Da, 1632 Da, 1646 Da and 1660 Da, whereas the molecular
masses of EFR components were 1606 Da, 1620 Da, 1634 Da, 1648 Da and 1662 Da. The
difference of 2 Da indicated the presence of either didehydroaminobutyric acid or 1aminocyclopropanecarbonic acid (Acc) in ACR. The latter was found in structurally related
[5]
neoefrapeptins . Amino acid analysis of total hydrolysates of ACR and comparison with
reference amino acids on ‘Chirasil-Val’ revealed the presence of achiral Aib, Acc, β-Ala, and
Gly as well as L-Leu, L-Pip, and D-Iva. No L-Iva was detected in ACR peptides. This is in
contrast to EFR-peptides containging L-Iva. Characteristic fragment ions of ACR count for a
total mass difference (Δ) of 140 Da in AA positions nos. 8 and 9. In EFR the AA positions nos.
8 and 9 represent the sequence Gly-Aib, the sum counting for a mass difference of 142 Da.
Therefore, in ACR the mass difference of 2 Da is also located in this domain representing the
sequence Gly-Acc. The sequences of EFR-F and ACR peptides 1-6 are presented in the
figure. Comparison of the MS data from ACR and EFR indicates that both peptaibiotics have
the identical C-terminal heterocyclic residue indicated by ‘Y’ in the figure.
EFR
F
ACR
1a
1b
2
Y
1
2
3
Ac Pip Aib Pip
Ac
Ac
Ac
Ac
Ac
5 Ac
6 Ac
4
Aib
5
Aib
D-Iva
Aib
D-Iva
D-Iva
D-Iva
D-Iva
D-Iva
Aib
Aib
D-Iva
Aib
D-Iva
Aib
D-Iva
6
7
8
9 10 11 12 13 14
15 16 (Da)
Leu β-Ala Gly Aib Aib Pip Aib Ala Leu L-Iva Y 1648
(%)
Pip
Pip
Pip
Pip
Pip
Pip
Pip
Aib
Aib
Aib
Aib
Aib
Aib
Aib
Pip
Pip
Pip
Pip
Pip
Pip
Pip
Leu
Leu
Leu
Leu
Leu
Leu
Leu
β-Ala
β-Ala
β-Ala
β-Ala
β-Ala
β-Ala
β-Ala
Gly
Gly
Gly
Gly
Gly
Gly
Gly
Acc
Acc
Acc
Acc
Acc
Acc
Acc
Aib
Aib
Aib
Aib
Aib
Aib
Aib
Pip
Pip
Pip
Pip
Pip
Pip
Pip
Aib
Aib
Aib
Aib
Aib
Aib
Aib
Gly
Gly
Gly
Gly
Gly
Ala
Ala
Leu
Leu
Leu
Leu
Leu
Leu
Leu
Aib
D-Iva
Aib
D-Iva
D-Iva
D-Iva
D-Iva
Y
Y
Y
Y
Y
Y
Y
1618
1618
1632
1632
1646
1646
1660
7.1
26.0
31.1
20.8
15.0
Figure. Sequences of efrapeptin F (EFR, from Eli Lilly Company) in comparison to acretocin (ACR) peptides 1-6
listed according to their elution order from HPLC; Ac, acetyl; Aib, -aminoisobutyric acid (2-methylalanine); Acc,1aminocyclopropanecarboxylic acid; Iva, isovaline (2-ethylalanine); Pip, pipecolic acid; Y, N-peptido-1-isobutyl-2[1pyrrol-(1,2-a)-pyrimidinium-2,3,4,6,7,8-hexahydro]ethylamine. Ala, Leu and Pip are of the L-configuration.
Molecular weights (Da) and relative amounts (%) of ACR peptides are given.
References
1 Kirschbaum, J.; Slavickova, M.; Brückner, H., In: Flegel, M., Fridkin, M., Gilon, C., Lebl, M. and Slaninova, J. (Eds. )
Peptides 2004 (Proceedings of the Third International and Twenty-Eight European Peptide Symposium, September 5-10,
2004, Prague, Czech Republic), Kenes Int. Geneva, 2005, 415-416.
2 Degenkolb, T.; Kirschbaum, J.; Brückner, H. Chem. Biodiv. 2007, 4, 1052-1067.
3 Bullough, D.A.; Jackson, C.G.; Henderson, P.J.F.; Cottee, F.H.; Beechey, R.B.; and Linnett P.E. Biochem. Int., 1982, 4,
543- 549.
4 Krasnoff, S.B.; Gupta, S.; Leger, R.J.S.; Renwick, J.A.A.; Roberts, D.W. J. Invertebrate Pathol, 1991, 58, 180-188.
5 Fredenhagen, A.; Molleyres, L.-P.; Böhlendorf, B.; Laue, G. J. Antibiot. 2006, 59, 267-280.
48
New Frontiers in the Search of Bioactive Molecules: from Peptides to Drugs
Exploiting split inteins for the semi-synthesis of proteins
and to study the mechanism of protein splicing
Henning D. Mootz
Technische Universitä Dortmund, Fakultät Chemie – Chemische Biologie, 44227 Dortmund – Germany.
Protein splicing is an autocatalytic reaction, in which an internal protein domain, the intein,
excises itself out of a precursor protein and concomitantly links the two flanking sequences,
the exteins, with a native peptide bond. In split inteins, the intein domain is divided into two
parts that undergo fragment association followed by protein splicing in trans. Thus, the extein
sequences joined in the process originate from two separate molecules. Due to this feature,
the recombinant part of a protein of interest can be expressed as a fusion protein with one
split intein fragment, while the complementary fragment of the split intein is synthesized as
part of a synthetic peptide, including various types of chemical modifications. The specificity
and sequence promiscuity of split inteins make this approach a generally useful tool for the
preparation of semi-synthetic proteins. In contrast to chemical ligation procedures, like native
chemical ligation and expressed protein ligation, the incorporation of a thioester group and an
amino terminal cysteine into the two polypeptides to be linked is not necessary.
We have recently introduced the split Ssp DnaB mini-intein, which enables the chemoenzymatic synthesis of N-terminally modified semi-synthetic proteins (see Figure 1). The Nterminal intein fragment consists of only 11 native amino acids, which has greatly facilitated
preparation of the synthetic part by solid-phase peptide synthesis (1). The remaining 143
amino acids of C-terminal intein fragment are expressed as a fusion protein with the desired
recombinant protein component. The splicing reaction can be performed under native
conditions and at protein and peptide concentrations in the low micromolar range. We have
applied this methodology to link a synthetic peptide pentamer, including a fluorescein moiety,
to the N-terminus of the 12 kD protein thioredoxin and the 31 kD protein β-lactamase,
resulting in the fluorophore-labelled products with total yields of up to 70% after 24h,
exhibiting full enzymatic activity. A detailed structure-function analysis of the synthetic Nterminal intein fragment will be presented (2), which provides the basis for a further
exploitation of this system for applications in protein semi-synthesis. Using mutants blocked in
protein splicing, we have also identified aberrant formation of a thiazoline ring as a sideproduct of the N,S-acyl shift in the initial step of protein splicing.
Figure 1: Semi-synthetic protein trans-splicing
References
1 C. Ludwig, M. Pfeiff, U. Linne, H. D. Mootz, Angew. Chem. Int. Ed. 2006, 45, 5218-5221
2 C. Ludwig, D. Schwarzer, H. D. Mootz, manuscript in preparation.
49
ORAL PRESENTATIONS - Eleventh Naples Workshop on Bioactive Peptides
The unusual helix stability of a VEGF mimetic peptide
Luca D. D’Andrea1, Donatella Diana2, Barbara Ziaco1, Giorgio Colombo3, Guido Scarabelli3,
Alessandra Romanelli4, Carlo Pedone1, Roberto Fattorusso2
1
2
3
4
CNR, Istituto di Biostrutture e Bioimmagini, 80134 Napoli - Italy
Seconda Università di Napoli, Dipartimento di Scienze Ambientali, 81100 Caserta - Italy
CNR, Istituto di Chimica del Riconoscimento Molecolare, 20131 Milano –Italy
Università di Napoli “Federico II”, Dipartimento delle Scienze Biologiche, 80134 Napoli - Italy
Understanding helix stability and formation is a prerogative to elucidate mechanism of protein
folding and design helix peptide with specific activity. Peptide helix is a simple model system
in which various contributions to helix formation can be dissected and understood
qualitatively. Many strategies have been pursued to design peptide helices and notable
results have been achieved even with very short sequences, but mainly these methods rely
on the use of non natural amino acids or introducing constraints. In this communication, we
report the stability characterization, via CD, NMR and MD studies, of a designed, α-helical,
15-mer peptide, composed only of natural amino acids, which activates the VEGF-dependent
angiogenic response[1]. This peptide shows an unusual thermal stability whose structural
determinants have been determined. Two factors, the N-terminal region and an hydrophobic
interaction i, i+4, are found as playing a mayor role of this remarkable stability[2]. These results
could have implication in the field of protein folding and in the design of helical structured
scaffolds for the realization of peptides to be applied in chemical biology
References
1 D'Andrea L.D.; Iaccarino G.; Fattorusso R.; Sorriento D.; Carannante C.; Capasso D.; Trimarco B.; Pedone C. Proc. Natl.
Acad. Sci. USA 2005, 102, 14215-14220.
2 Diana D.; Ziaco B.; Colombo G.; Scarabelli G.; Romanelli A.; Pedone C.; Fattorusso R.; D'Andrea L.D. Chem. Eur. J., 2008,
10.1002/chem.200800180
50
New Frontiers in the Search of Bioactive Molecules: from Peptides to Drugs
Multiple β-sheets molecular dynamics of amyloid
formation of two Abl-SH3 domain peptides
Inta Liepina1, Salvador Ventura2, Cezary Czaplewski3 and Adam Liwo3
1 Latvian Institute of Organic Synthesis, Riga, LV1006, Latvia,
2 Institut de Biotecnologia i de Biomedicina, Universitat Autonoma de Barcelona, E-08193 Bellaterra, Spain,
3 Faculty of Chemistry, University of Gdansk, 80-952 Gdansk, Poland
Short-peptide sequences drive protein aggregation in amyloid fibril [1-2]. Aggregation-prone
region from Abl-SH3 domain of Drosophila DLSFMKGE (MK), and homologous human region
DLSFKKGE (KK) were extracted. Two multisheet systems consisting of (1): six antiparallel
flat β-sheets of ten strands of MK (10x6xMK), (2) six antiparallel flat β-sheets of ten strands of
KK (10x6xKK), surrounded by water were subjected to molecular dynamics (MD), Amber 8.0.
The MD revealed that:
1 10x6xMK beta sheet stack is stable (Fig1.), but 10x6xKK beta-sheet stack is not.
2 10x6xMK beta-sheet is stable because of hydrophobic interactions of metioninephenilalanine and leucine of the neighbouring sheets. Met, Phe, Leu make a
hydrophobic core for the stack of beta-sheets.
3 During MD run the Met, Phe, Leu of neighbouring sheets act as conformational switch
moving beta sheets by two amino acid step towards each other.
4 Replacement of Met by Lys destroys the hydrophobic core, which is the stability factor
of the beta sheets stack. 10x6xKK system maintains beta sheets, but loses
interactions between beta sheets.
5 The calclulacions of six beta sheets confirm the conclusion drawn for single sheet
systems: parallelly placed beta-sheets stabilize each other [3].
Fig. 1. Multisheet system of DLSFMKGE peptides is stable.
Acknowledgments This work was supported by NATO Collaborative Linkage Grant LST.CLG.979807, by stipend for I.L. from
the “Kasa im. Jozefa Mianowskiego” Fund, Poland, by Latvian Science Council Grant 05.1768, calculations were carried out at
the Gdansk Academic Computer Centre TASK
References
1. Ventura, S., Zurdo, J., Narayanan, S., Parreno, M., Mangues, R., Reif, B., Chiti, F., Giannoni, E., Dobson, C.M., Aviles, F.X.
and Serrano L. Short amino acid stretches can mediate amyloid formation in globular proteins: the Src homology 3 (SH3)
case. Proc. Natl. Acad. Sci. U S A 2004, 101, 7258-7263.
2. Ventura S., Lacroix E.and Serrano L Insights into the origin of the tendency of the PI3-SH3 domain to form amyloid fibrils.
Journal of Molecular Biology 2002, 332, 1147-1158.
3. Liepina I., Ventura S., Czaplewski C., Liwo A. Molecular dynamics study of amyloid formation of two Abl-SH3 domain
peptides Journal of Peptide Science, 2006, 12, 780-789.
51
ORAL PRESENTATIONS - Eleventh Naples Workshop on Bioactive Peptides
Molecular dynamics study of the apoA-I’s
fragment 104-107 and its Met112 to Ala mutant
2
1
Athanassios Stavrakoudis1,2, Maria Darvaris , Maria Sakarellos-Daitsiotis , Constantinos
1
Sakarellos and Alexandros Tselepis
1 Department of Economics, University of Ioannina, GR-45110, Ioannina, Greece
2 Department of Chemistry, University of Ioannina, GR-45110, Ioannina, Greece
Apolipoprotein A-I (apoA-I)[1] plays an important role in reverse cholesterol transport has antioxidant and anti-inflammatory activities. ApoA-I is a helical protein and the majority of its
amphipathic α-helices are assigned to the class A or Y depending on the distribution of the
positively and negatively charged residues on the polar face[2]. This work presents a
conformational study of the fragment 104-107 of apoA-I, part of the helix 4. In order to explore
the conformational properties of the native sequence of apoA-I we performed molecular
dynamics (MD) simulations in explicit water, using the NAMD package. We also simulated the
Met112Ala mutant peptide in order to explore the contribution of this amino acid to the
structural and biological properties of this part of helix 4. The initial coordinates of the model
were extracted from the crystal structure of apoA-I. Simulations were last for 50 ns and
produced 50000 frames for data analysis. Secondary structure analysis revealed the
conservation of the initial α-helical conformation for both peptides during the whole MD
trajectory. The i←i+4 hydrogen bond network was also found well formed. Electrostatic
interactions with Lys107 and Glu110 or Glu111 side chains were found to stabilize the helix in
both of cases. Anyway, in the Met112 case, another strong coulombic interaction between
Lys106 and Glu110 was also recorded. Another interesting interaction in this trajectory was
the close contact of Met112 and Trp108 side chains. This hydrophobic interaction contributed
significantly to the helix stabilization. This interaction also appeared in the Ala112 case, where
a C-H…aromatic type hydrogen recorded.
Figure 1. Last frame of the 50 ns molecular dynamics trajectory. Met112 peptide
(left) and Ala112 peptide (right) are shown with stick representation (hydrogen
atoms have been omitted). They both retain the α-helical conformation.
References
1 Brouillette, C.G., and Anantharamaiah, G.M. Structural models of human apolipoprotein A-I. Biochim Biophys Acta 1256:
103-129 (1995).
2 Beaufils, C., Alexopoulos, C., Petraki, M.P., Tselepis, A.D., Coudevylle, N., Sakarellos-Daitsiotis, M., Sakarellos, C., and
Cung, M.T. 2007. Conformational study of new amphipathic alpha-helical peptide models of apoA-I as potential
atheroprotective agents. Biopolymers 88: 362-372.
52
POSTER PRESENTATIONS - Eleventh Naples Workshop on Bioactive Peptides
Synthesis and conformational studies of
octapeptides containing four ΔPhe residues
Latajka Rafal1, Jewginski Michal2, Makowski Maciej2 and Kafarski Pawel2
1 Department of Bioorganic Chemistry, Faculty of Chemistry, Wroclaw University of Technology, 50-370 Wroclaw, Poland
2 Institute of Chemistry, University of Opole, Oleska 48, 45-052 Opole, Poland
Dehydropeptides, the compounds which inclusive one or more dehydroamino acid residues,
are very interesting as an object of conformational studies. Presence of double bond between
Cα and Cβ and two neighboring peptide bonds lead to coupling of π electrons, which not only
influence on side chain but also on all peptide conformation1-5. Full knowledge about relation
between presence of dehydroamino acid and peptide's conformation is necessary to predict
biological properties of new designed peptides..
In this presentation we summarized the results of synthesis and conformational investigations
of octapeptides containing four dehydrophenylalanine residues in peptide chain. The general
formula of studied compounds is Boc-Gly-ΔX-Gly-ΔX-Gly-ΔX-Gly-ΔX-OMe, where X=ΔPhe
with different possible combinations of its isomers.
The structural investigations were based on NMR measurements (standard 2D techniques
and 1D experiments, typical for detection of hydrogen bonding) and theoretical calculations.
Conformational preferences of investigated systems were obtained on base of ROESY and
NOESY experiments and calculations by use of X-PLOR.
References
1. Fuzery AK, Csizmadia IG , J. Molecular Structure (Theochem) 2000, 501-502¸539
2. Palmer DE, Pattaroni Ch, Nunami K, Chadha RK, Goodman M, Wakamiga T, Fukase K, Horimoto S, Kitazawa M, Fujita H,
Kubo A, Shiba T, J. Am. Chem. Soc. 1992, 114, 5634
3. Rajashankar KR, Chauhan VS, Ramakumar S, Int. J. Peptide Protein Res. 1995, 46, 487
4. Kaur P, Uma K, Balaram P, Chauhan VS, Int. J. Peptide Protein Res. 1989, 33, 103
5. Busetti V, Crisma M, Toniolo C, Salvadori S, Balboni G, Int. J. Biol. Macromolecule 1992, 14, 23
54
New Frontiers in the Search of Bioactive Molecules: from Peptides to Drugs
New and selective radiolabeled αvβ3
antagonist as tracer in tumor diagnosis
A. Del Gatto1, L. Zaccaro1, S. De Luca1, F. Iommelli2, A. Zannetti2, S. Del Vecchio2, M. Salvatore2, C.
Pedone1, M. Saviano1
1 Istituto di Biostrutture e Bioimmagini, CNR, Dipartimento delle Scienze Biologiche, Università di Napoli"Federico II", Napoli;
2 Istituto di Biostrutture e Bioimmagini, CNR, Dipartimento di Scienze Biomorfologiche e Funzionali Napoli.
Angiogenesis, the process whereby new capillaries are formed by outgrowth from existing
microvessels, is required for tumor growth and metastasis. The transmembrane cell-surface
receptor αvβ3 has recently received increasingly attention, because of the critical role in tumor
associated angiogenesis and metastasis formation. The restricted expression of integrin αvβ3
during tumor growth, invasion, and metastasis presents an interesting target for both detection
and treatment of solid tumors. Targeting αvβ3 with radiolabelled ligands may provide
information about the receptor status and enable the planning and the monitoring of
therapeutic approaches. Recently we developed a novel αvβ3 antagonist that showed a high
selectivity for the receptor. Adhesion assays, competitive binding assays and cross-linking
experiments performed in human erythroleukemia K562 cells, stably cotransfected with cDNA
of alpha(v) or alpha(IIb) and beta(3), demonstrated the high selectivity for αvβ3 integrin (1,2).
Starting from these evidences, RGDechi was covalently bound to the chelating agent DTPA
able to give stable complexes of radionuclides, such as 111In. The final goal has been to obtain
a radiolabelled compound to be used in nuclear medicine as a diagnostic and therapeutic
agent. In particular DTPA-RGDechi has been labeled with 111In and used in SPECT for
diagnostic purpose.
References
1. A. Del Gatto et al. Journal of Medicinal Chemistry (2006), 49, 3416-3420.
2. A. Del Gatto, et al. PCT Int. Appl. (2007)
55
POSTER PRESENTATIONS - Eleventh Naples Workshop on Bioactive Peptides
A comparative study between computational prediction and
biophysical characterization of antibacterial peptides
Linser Sebastian1, Rzeszutek Agnieszka1, Shental-Bechor Dalit2, Ben-Tal Nir2, Funari S. Sérgio3 and
Willumeit Regine1
1 GKSS Research Center, Department WPS, 21502 Geesthacht, Germany.
2 Tel Aviv University, Department of Biochemistry, Tel Aviv, Israel.
3 Hasylab/Desy, 22603 Hamburg, Germany.
The uprising resistance of pathogenic bacteria against treatments with conventional antibiotics
emerged an acute search for alternatives. One class of promising alternatives are naturally
occurring antimicrobial peptides and derivatives thereof. We present a comparative study of
computational modelling of peptide properties with structural characterization of the interaction
(by Small Angle X-Ray Scattering (SAXS), Surface Plasmon Resonance (SPR) and Circular
Dichroism (CD).) and antibacterial or haemolytic activity of three peptides (NK-CS, NKCS-[LP]
and NKCS-[AA]).
All peptides were active against Escherichia coli (Gram negative) and Staphylococcus
carnosus (Gram positive) bacterial cultures, but the haemolytic properties against human red
blood cells were found to be poor and indicated the peptides’ selectivity.
CD studies of the peptide secondary structure confirmed the computational prediction of
peptide helicity. The antibacterial activity can be correlated with a change of the hexagonal
phase transition temperature of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine
(POPE) as determined by SAXS. The THII was increased by the peptides and this promotes a
positive curvature of the membranes. We assume that this curvature finally leads to the
disruption of the model membranes.
The calculated peptide membrane affinity is not directly related in a linear way with the
antibacterial activity. The reason for this might be aggregation as shown by SPR. In summary
an overall helical structure, electrostatic and hydrophobic parameters as well as strong
amphipaticity are good measures to describe antibacterial peptide interaction.
56
New Frontiers in the Search of Bioactive Molecules: from Peptides to Drugs
Correlations between peptide geometry and conformation
revealed by quantum-mechanics calculations and statistical
analyses of structural databases
Esposito Luciana1, Pedone Carlo1,2, Vitagliano Luigi1, and Improta Roberto1
1. Istituto di Biostrutture e Bioimmagini, C.N.R., I-80134 – Napoli, Italy.
2. Dipartimento delle Scienze Biologiche, Università Federico II, I-80134 Napoli, Italy.
Over the last decade, the number of high- or ultra-high-resolution protein crystal structures
has increased dramatically[1,2]. Statistical analyses of geometric and conformational
parameters of residues in this extended and very accurate structural database are revealing
fine details[3-5]. Deviations of peptide bonds from planarity have been extensively investigated
but the identification of clear trends for the distortion derived from experimental data has been
controversial[6,7]. We have previously analysed the planarity of the peptide group by surveying
a dataset of atomic resolution protein structures[3,4,7]. We demonstrated that the values of the
ω dihedral angle are strictly correlated to the values of the ψ adjacent angle[7]. In order to
propose an explanation for this experimental trends as well as to investigate the influence of
local effects, we have carried out quantum-mechanics calculations on different peptide model
systems. Calculations have been performed by density functional theory methods either in
gas phase or in solvent, modelled by using the PCM model. In particular, optimization of ω
angle and geometry have been performed while setting (ϕ,ψ) dihedral angles to fixed values
which cover the most populated regions of the Ramachandran map. Computations are able to
reproduce the conformational dependence of θC and Δω planarity deviations emerged from
experimental data. In addition, the examination of bond distances and angles involving mainchain atoms reveals subtle conformational dependences hardly detected in experimental Xray structures. When comparing the ψ conformational dependence of CO/CN distances and
Δω angles it is evident that the resonance model is not adequate to describe these trends.
Results of the searching of the small molecule CSD database and ultrahigh-resolution protein
structure dataset indicate good agreement with the computational findings. Altoghether the
highlighted conformational trends propose a new picture for deviations of peptide geometry
from “ideal” values. These trends may also be relevant for a future revisitation of both protein
refinement restraints and molecular mechanics force-field parameters.
References
1 Lecomte, C.; Guillot, B.; Muzet, N.; Pichon-Pesme, V.; Jelsch, C. Cell. Mol. Life Sci. 2004, 61, 774-782.
2 Esposito, L.; Vitagliano, L.; Mazzarella, L. Protein and Peptide Letters 2002, 9, 95-105.
3 Esposito, L.; Vitagliano, L.; Zagari, A; Mazzarella, L. Protein Eng. 2000, 13, 825-828.
4 Esposito, L.; Vitagliano, L.; Zagari, A.; Mazzarella, L. Protein Sci. 2000, 9, 2038-2042.
5 Jaskolski, M.; Gilski, M.; Dauter, Z.; Wlodawer, A. Acta Crystallogr D Biol Crystallogr. 2007, 63, 611-620.
6 MacArthur, M. W.; Thornton, J. M. J. Mol. Biol. 1996, 264, 1180–1195.
7 Esposito, L.; De Simone, A.; Zagari, A.; Vitagliano, L. J. Mol. Biol. 2005, 347, 483-487.
57
POSTER PRESENTATIONS - Eleventh Naples Workshop on Bioactive Peptides
BioShuttle as a carrier for temozolomide
transport into prostate cancer cells
Pipkorn Rüdiger1, Waldeck Waldemar2, Didinger Bernd3, Koch Mario1, Mueller Gabriele2, Wiessler
Manfred4, and Braun Klaus4
1
2
3
4
German Cancer Research Center, Peptide Synthesis Unit, Heidelberg, - Germany.
German Cancer Research Center, Dept. Biophysics of Macromolecules, Heidelberg, - Germany.
University of Heidelberg, Radiation Oncology, Heidelberg, - Germany.
German Cancer Research Center, Dept. Molecular Toxicology, Heidelberg, - Germany.
If metastatic prostate cancer (PCa) gets resistant to antiandrogen therapy, there are few
treatment options, because PCa is not sensitive to cytostatic agents[3]. Temozolomide (TMZ)
an oral applicable chemotherapeutic substance has been proven to be effective and tolerated
especially for brain tumors[2] with occasional moderate toxicity. Unfortunately TMZ was
inefficient in the treatment of symptomatic progressive hormone-refractory PCa. This may
have different reasons like the inauspicious plasma half-life of TMZ, non adapted application
schemata and as a result, an insufficient bioavailability. To improve the situation, we built a
TMZ-BioShuttle directed against cathepsin B (CTSB) mRNA expressed in PCa cells. This
complex carries a transmembrane transporter module (CPP) which is cleavably linked to an
antisense-peptide nucleic acid (PNA) against exon 1 in the CTSB mRNA. This in turn is
covalently-bonded to a substrate for CTSB and a nuclear localization sequence (NLS)
harbouring the cargo TMZ (Figure 1). The PNA recognizes the cytoplasmic CTSB mRNA. The
formation of the PNA/RNA hybrid is described in Fig. 2. It is not a substrate for RNaseH and
results in a cell-specific retention of the TMZ-cargo in the cytosol of CTSB-expressing cells.
Finally, after the CTSB-mediated cleavage the NLS-sequence is separated and activated for
[1]
an importin-mediated transfer of the TMZ into the nucleus of target cells .
Figure 1
Figure 2
References
1. Pipkorn, R.; Waldeck, W.; Spring, H.; Jenne, J. W.; Braun, K. Delivery of substances and their target-specific topical
activation. Biochim Biophys Acta 2006 May;1758(5):606-610.
2. Stupp, R.; Dietrich, P. Y.; Ostermann, K. S.; Pica, A.; Maillard, I.; Maeder, P.; Meuli, R.; Janzer, R.; Pizzolato, G.; Miralbell,
R.; Porchet, F.; Regli, L.; de Tribolet, N.; Mirimanoff, R. O.; Leyvraz, S. Promising survival for patients with newly diagnosed
glioblastoma multiforme treated with concomitant radiation plus temozolomide followed by adjuvant temozolomide. J Clin
Oncol 2002 Mar;20(5):1375-1382.
3. van Brussel, J. P.; Busstra, M. B.; Lang, M. S.; Catsburg, T.; Schroder, F. H.; Mickisch, G. H. A phase II study of
temozolomide in hormone-refractory prostate cancer. Cancer Chemother Pharmacol 2000;45(6):509-512.
58
New Frontiers in the Search of Bioactive Molecules: from Peptides to Drugs
Tumor necrosis factor-α neutralizing antibodies
induced by autovaccination using
glycolaldehyde-modified peptide conjugates
De Vendel Jolanda1, Bavoso Alfonso1, Ostuni Angela1, Bracalello Angelo1, Shcheglova Tatiana2,
Vizzuso Domenica2, Makker Sudesh2, and Tramontano Alfonso 2
1. University of Basilicata, Department Chemistry, 85100 Potenza-Italy
2. University of California, Medical School, Department of Pediatrics , 95616 Davis, CA-USA
The pro-inflammatory cytokine TNF-α is overexpressed in diverse inflammatory states,
including rheumatoid arthritis. Anti-TNF therapies have demonstrated remarkable success in
management of arthritis and other chronic inflammatory diseases [1,2]. The possibility to elicit
autoantibodies to TNF-α (beneficial autoimmunity) has been proposed as an
immunotherapeutic approach to neutralize systemic TNF[3]. Studies using a DNA vaccination
strategy in rat adjuvant arthritis mapped several epitopes for natural anti-TNF-α
autoantibodies in the C-terminal portion of the sequence. We previously showed that
aldehyde-tagged peptide conjugates representing immunogenic epitopes of the pyrogenic
superantigen toxic shock syndrome toxin –1, could induce anti-toxin neutralizing antibodies
without the need of potent adjuvants[4]. In the present study we followed a similar approach to
generate potentially neutralizing antibodies against TNF-α.
A number of unmodified and N-terminal palmitoylated peptides corresponding to the natural
TNF-α linear epitopes were synthesized using conventional solid phase synthesis. Synthetic
peptides having a (GlyLys)x3 extension at the C-terminus were also prepared in order to
provide residues for aldehyde incorporation by Amadori modification. Initially we compared
the immune responses in mice to unconjugated peptides and lipopeptides injected in PBS. No
significative anti-TNF response was observed in mice immunized with any of the peptides or
their glycolaldehyde-modified derivatives.
In further experiments we examined BSA conjugates of peptides GDLLSAEVNLPKC- NH2
(TNF8) and GDLLSAEVNLPKG(GK)3C- NH2 (TNF8M) as possible vaccines, which could be
enhanced by glycolaldehyde modification. Conjugation of TNF8M produced only insoluble
products. Lewis rats immunized with unmodified TNF8-BSA and glycolaldehyde-modified
TNF8-BSA-ga in alum developed low-titer antibodies. However responses in the group
injected with TNF8-BSA-ga were about 2-fold greater and were enhanced by booster
immunization. IgG purified from antisera of this group demonstrated significant TNF
neutralizing activity in L929 cytotoxicity assay. These results suggest that aldehyde-tagged
peptide conjugates could bypass tolerance to support beneficial autoimmunity against
inflammatory cytokines. This approach could be superior to other vaccine strategies that take
advantage of innate immune mechanisms to avoid the need for strong adjuvants.
References
1. Maini, R.N., M. Elliott, F.M. Brennan, R.O. Williams, and M. Feldmann. 1994. Targeting TNF alpha for the therapy of
rheumatoid arthritis. Clin Exp Rheumatol 12 Suppl 11:S63-66.
2. Andreakos, E.T., B.M. Foxwell, F.M. Brennan, R.N. Maini, and M. Feldmann. 2002. Cytokines and anti-cytokine biologicals
in autoimmunity: present and future. Cytokine & Growth Factor Reviews 13:299-313.
3. Dalum, I., D.M. Butler, M.R. Jensen, P. Hindersson, L. Steinaa, A.M. Waterston, S.N. Grell, M. Feldmann, H.I. Elsner, and
S. Mouritsen. 1999. Therapeutic antibodies elicited by immunization against TNF-alpha. Nat Biotechnol 17:666-669.
4. Bavoso, A., A. Ostuni, J. De Vendel, F. Pollaro, F. Armentano, T. Knight, S. Makker, and A. Tramontano. 2006. Aldehyde
modification of peptide immunogen enhances protein-reactive antibody response to toxic shock syndrome toxin-1. J Pept
Sci 12:843-849.
59
POSTER PRESENTATIONS - Eleventh Naples Workshop on Bioactive Peptides
Anti-inflammatory activity of a lactoferricin-derived peptide
Chelu Flori1, Icriverzi Madalina1, Trif Mihaela1, Louise Strömbeck2, Inger Mattsby-Baltzer2, Anca
Roseanu1
1 Institute of Biochemistry, Ligand-Receptor Interaction Dept., Bucharest, Romania
2 University of Gothenburg, Department of Clinical Bacteriology, Gothenburg, Sweden
Lactoferricin (Lfcin), is an antimicrobial peptide derived by pepsin digestion of lactoferrin (Lf),
a multifunctional innate-defense protein in milk. Lfcin contains a large portion of the functional
domain of Lf and in many cases it not only retains the properties of the native protein, but is
more active. Due to the broad biological effects, Lfcin or peptides derived from it are of current
interest for practical applications. However one of the barriers for the therapeutic use is their
stability in vivo and much effort are undertaken to design more stable and efficient peptides.
By down-sizing a sequence of the Lfcin corresponding to the alpha-helix region in the Lf
molecule, a peptide consisting of amino acid residues 19-31 was obtained. The peptide was
evaluated for its ability to affect the release of pro-inflammatory cytokines TNF alpha, IL-6 and
IL-8 induced by endotoxins (LPS) in macrophage-like cells (THP-1).
A significant inhibition of the cytokine responses was observed regardless whether LPS was
added 15 min before or after the addition of peptide, indicating that pre-neutralisation of LPS
by the peptide was not a major cause of the observed inhibitory effects. Analyses of
phosphorylated MAPkinases (p38, pERK1/2) and pNFκB by Western blotting revealed that
the presence of peptide reduced the levels of the phosphorylated proteins. The results
suggest that the peptide is not only endowed with a strong antimicrobial activity but also
expresses an anti-inflammatory capacity by interfering with the NFκB and some MAPK
signalling pathways.
Acknowledgments. This work was supported by Romanian Academy Grant (GAR 2008) and a Swedish Institute Project.
60
New Frontiers in the Search of Bioactive Molecules: from Peptides to Drugs
Serum stability and interactions of short antimicrobial peptides
Nguyen T. Leonard1, Chau K. Johnny1, Perry A. Nicole1, de Boer Leonie2, Zaat A.J. Sebastian2, and
Vogel J. Hans1
1 University of Calgary, Dept. of Biological Sciences, T2N 1N4 Calgary – Canada.
2 University of Amsterdam, Dept. of Microbiology at the Centre of Infection and Immunity, 1105 AZ Amsterdam – The Netherlands.
Most antimicrobial peptides that are currently in the pharmaceutical pipeline are geared
towards topical applications, which inherently limit their effectiveness to local infections.
Systemic distribution of an antimicrobial peptide in a patient through oral or intravenous
administration would be more desired. To better understand the pharmacokinetics of free
peptide drugs in circulation, we have measured the stability of a series of short antimicrobial
peptides in human serum. The peptide sequences were derived from hexameric stretches rich
in Trp and Arg residues from bovine lactoferricin and an optimized peptide previously
identified from a combinatorial library. The results show that although C-terminal amidation is
advantageous for antimicrobial activity, this cap does not provide significant protection for the
peptide from being digested. Conversely, N-terminal acetylation of the hexapeptides
increases their stability but does not improve their antimicrobial activity. Cyclization of the
peptides, either through head-to-tail backbone ligation or by disulfide bonding between added
terminal cystines, greatly increased the serum stability lifetimes, some with half-lives greater
than nine hours. However, this effect of cyclization is considerably less prominent for larger
peptides with sequences lengths greater than ten amino acids. The possible interaction
between these peptides and human serum albumin, the most abundant protein in blood
plasma, was also investigated by isothermal titration calorimetry. These interactions may
effectively shield the peptides from degradation and aid in their circulation throughout the
body. This work begins to establish the important principles that should be considered for
short antimicrobial peptides to be developed as systemic antimicrobial agents.
61
POSTER PRESENTATIONS - Eleventh Naples Workshop on Bioactive Peptides
Chiral nucleopeptides for biotechnological
applications: synthesis and hybridization studies
Roviello Giovanni1, Musumeci Domenica2, Castiglione Mariangela1, De Cristofaro Andrea1,
D’Alessandro Cristian1, Pedone Carlo1, Bucci Enrico2 and Benedetti Ettore3
1 CNR, Ist. di Biostrutture e Bioimmagini, 80134 Napoli – Italy.
2 Bionucleon srl, 80131 Napoli – Italy.
3 Università Federico II, Dip. delle Scienze biologiche, 80134 Napoli – Italy.
The development of new analogs able to bind DNA and RNA sequences with good sequencespecificity and thermal stability has gained a great scientific interest in recent years bearing to
a number of artificial nucleotidic molecules, with various modifications both in backbone and
nucleobases, that in some cases presented hybridization properties even better than natural
oligonucleotides.
Among these, PNA, with an artificial pseudopeptide backbone in place of the sugar-phosphate
one, emerged as the most powerful DNA analogs forming complexes (double and triple
helices) with a great thermal stability and sequence specificity[1].
Nevertheless, some characteristics of PNA like water-solubility and strand-orientation
selectivity during binding need to be improved.
B
O
HN
H
H
N
l
n
OH
*
m
O
l=1,2..
n=0,1..
m=5,6..
One way to address these requirements is to introduce chiral residues, possibly with charged
groups, in the PNA skeleton. Our attention in this field has focused on the realization of chiral
nucleoaminoacids, suitably protected for solid phase oligomerization, and the realization of
chiral nucleopeptides based on various diamino carboxylic acids also in combination with
achiral PNA. In this work we present the synthesis, purification and characterization of both a
novel chiral nucleoaminoacid and the corresponding nucleopeptide, as well as some
preliminary results on its hybridization ability towards complementary oligonucleotides in view
of a possible use in biomedical applications.
References
1. Nielsen, P.E., Egholm, M., Berg, R.H. and Buchardt, O. Science 1991, 254, 1497-1500.
62
New Frontiers in the Search of Bioactive Molecules: from Peptides to Drugs
Fallaxin: Correlation of antibacterial activity with
hydrophobicity <H> and mean hydrophobic moment <μH>
Lerche Sandra1, Frimodt-Møller Niels2, and Hansen Paul R.3
1 Faculty of Life Sciences, Department of Natural Sciences, 1871 FC, Denmark.
2 Statens Serum Institut, Center for Antimicrobials and Infection Control, 2300 S, Denmark.
3 Faculty of Life Sciences, Department of Natural Sciences, 1871 FC, Denmark.
Fallaxin a 25-mer antibacterial peptide, has recently been isolated from the West Indian
mountain chicken frog “leptodactylus fallax”. Fallaxin has been shown to inhibit the growth of
primarily Gram-negative bacteria including E. coli, K. pneumoniae and P. aeruginosa. Fallaxin
shows almost no toxicity towards erythrocytes with a haemolytic activity of HC50>200 μM[1]. In
order to identify a possible relationship between antibacterial and haemolytic activity with the
structural parameters mean hydrophobicity <H> and mean hydrophobic moment <μH>, we
performed a complete alanine scan of fallaxin as well as N and C-terminal truncated
analogues. A total of 66 analogues were synthesised and analysed. Fallaxin and analogues
were tested for antibacterial activity against methicilin-susceptible S.aureus (MSSA), E.coli
and K.pneumoniae. The cytotoxicity of the fallaxin analogues against human erythrocytes was
assessed in a hemolytic activity assay. Eisenbergs consensus scale was used to calculate
both the mean hydrophobicity <H> and mean hydrophobic moment <µH>[2].
Fallaxin and derivatives was synthesised manually using standard Fmoc-chemistry on a
TentaGel S RAM resin. Protected amino acids were coupled in three-fold excess employing a
protocol with HATU/DIEA (1:1.5) activation, and NMP as solvent. Final cleavage and
deprotection were carried out using reagent L: TFA, TIS, DTT and water (88:2:5:5) for 2h.
Following synthesis, the peptides were purified by RP-HPLC and characterised using MALDI
TOF MS and amino acid analysis. The peptides were tested for antibacterial activity against
S.aureus ATCC 33591, E.coli ATCC 25922 and K.pneumoniae ATCC 700603. The
haemolytic activities were determined against human erythrocytes.
We were able to group the active (MIC ≤ 25μM) and inactive analogues (MIC > 25μM)
according to mean hydrophobicity <H> and mean hydrophobic moment <μH>. The majority of
the active fallaxin analogues, except for two analogues, had a <H> ranging from -0,0256 to 0,0088 and a <μH> ranging from 0,00766 to 0,1164. The less active analogues incl. the
inactive C- and N-terminal truncated analogues (MIC >100μM) had <H> and <μH> values
outside this range. Based on previous studies, on antibacterial peptides and hydrophobicity,
we expected some of the truncated analogues to show antibacterial activity [3]. However, our
results show that the mean hydrophobicity and mean hydrophobic moment alone cannot be
used as a general tool to predict antibacterial activity of a peptide. These parameters are only
useful when comparing analogues derived from the same peptide.
References
1 Nielsen, S.L. et al 2007. Structure activity study of the antibacterial peptide fallaxin. Protein Sci. 16:1969-1976
2 Eisenberg, D. 1984. Three-dimensional structure of membrane and surface proteins. Ann. Rev. Biochem. 53: 595-623.
3 Kiyota, T., Lee, S., Sugihara, G. 1996. Design and synthesis of amphiphilic alpha-helical modelpeptides with systematically
varied hydrophobic-hydrophilic balance and their interaction with lipid- and bio-membranes. Biochemistry 35: 13196-204
63
POSTER PRESENTATIONS - Eleventh Naples Workshop on Bioactive Peptides
A flow cytometric method to detect internalization
of antimicrobial peptides in Gram-negative bacterial cells
Monica Benincasa, Sabrina Pacor, Chiara Pelillo, Renato Gennaro, Marco Scocchi
University of Trieste, Dept. of Life Sciences, 34127 Trieste – Italy.
Antimicrobial peptides inactivate bacteria via different mechanisms of action, some of which
are mediated by internalization of the peptides without any apparent membrane damage.
Immunoelectron transmission microscopy and confocal microscopy are the techniques used
to directly verify peptide uptake. However, these assays are quite complex to perform and the
results are sometimes ambiguous.
Here we propose a simple and rapid flow cytometric method based on the use of
fluorescently-labeled peptides and of the extracellular quencher Trypan Blue to discriminate
between a bacterial cell surface and cytoplasmic localization of the tested molecules. To his
aim, we used BODIPY-labeled peptides showing different modes of action. These included
some fragments of Bac7, a proline-rich peptide known to penetrate bacterial and eukaryotic
cells without membrane damage[1,2], and polymyxin B, a peptide antibiotic that binds to LPS
and to the cell membranes. By using this approach coupled to flow cytometric analysis, we
showed that the fluorescence intensity of E. coli and S. typhimurium cells treated with subinhibitory BODIPY-Bac7 concentrations did not decrease despite extensive washing and
addition of the quencher Trypan blue. In contrast, the fluorescence of cells treated with
BODIPY-polymyxin B, as well as that of bacteria treated with a fluorescein-labeled anti LPS
antibody, were promptly and almost totally quenched by addition of Trypan Blue. These
results confirm the suitability of this method to rapidly infer the localization of labeled
molecules in an accessible or inaccessible compartment in the treated bacterial cells
References
1. Podda E., Benincasa M., Pacor S., Micali F., Mattiuzzo M., Gennaro R., Scocchi M. Dual mode of action of Bac7, a prolinerich antibacterial peptide. (2006) Biochim. Biophys. Acta. 1760, 1732-1740.
2. Mattiuzzo M., Bandiera A., Gennaro R., Benincasa M., Pacor S., Antcheva N. and Scocchi M. Role of the Escherichia coli
Sbma in the antimicrobial activity of proline-rich peptides (2007) Mol. Microbiol. 66, 151-163.
64
New Frontiers in the Search of Bioactive Molecules: from Peptides to Drugs
Molecular modelling of the interactions of peptides
containing Tyr residue and β-cyclodextrin
Czaplewski Cezary, Juszczyk Paulina, Romankiewicz Justyna, and Wiczk Wiesław
University of Gdańsk, Faculty of Chemistry, 80-952 Gdańsk – Poland.
Cylcodextrins are cyclic oligomers of 1,4-linked,α-D-glucose monomers that have a
hydrophilic exterior and a hydrophobic center. This enables cyclodextrins to form inclusion
complexes with various organic molecules[1]. Aromatic amino acid residues bind to βcyclodextrin with deep penetration of the cyclodextrin cavity. In case of oligopeptides binding
depends on the peptide conformation.[2] The formation of β-cyclodextrin inclusion complexes
with the tyrosine residues within three oligopeptides was investigated using steady-state
fluorescence spectroscopy and molecular dynamic simulations. The oligopeptides consist of
eighteen amino acids and the tyrosine residues are located at the position 1, 2 or 4. Selected
sequences are fragments of NOTCH receptors.[3] (NOTCH1=YKIEAVQSETVEPPPPAQ,
NOTCH2=TLSYPLVSVVSESLTPER, NOTCH3=PYPLRDVRGEPLEPPEPS). Out of three
peptides only NOTCH3 binds strongly to β-cyclodextrin with binding constant similar to that of
AcTyrNHMe. The binding of cyclodextrins with phenolic compounds involves nonspecific van
der Waals and hydrophobic interactions and depends on accessibility of tyrosine sidechain.
References
1 Rekharsky, M.V.; Inoue, Y. Chem. Rev. 1998, 98, 1875-1917.
2 Bekos, E.J.; Gardella Jr, J.A.; Bright, F.V. J.Incl. Phenom. Mol. Rec. Chem. 1996, 26, 185-195
3 Baron, M. Semin. Cell & Dev. Biol. 2003, 14, 113-119.
65
POSTER PRESENTATIONS - Eleventh Naples Workshop on Bioactive Peptides
Identification of Cripto-Alk4 inhibitors through
the screening of combinatorial peptide libraries
D. Marasco1, S. Ponticelli2, S. De Falco2, C. Pedone1, G. Minchiotti2 and M. Ruvo1
1 Institute of Biostructures and Bioimaging, CNR, Napoli, Italy.
2 Institute of Genetics and Biophysics ‘Adriano Buzzati-Traverso’, CNR, Napoli, Italy.
Cripto is a membrane-bound growth factor that performs a variety of functions, including stem
cell differentiation and cell growth. Among the others, Cripto is able to regulate stem cell fate
and is highly over-expressed in many tumor tissues [1,2].
Several distinct transduction pathways are activated by Cripto, the most important of which
involves the activin receptor complex comprising Alk4 and the ActRIIB in particular the binding
of Cripto to Alk4 is a key event leading to cell differentiation and transformation [3].
Thus disruption of the Cripto-Alk4 complex is believed to be a valuable way to restore proper
receptor activities in Cripto-overexpressing tissues.
Here we report on the screening of combinatorial libraries of synthetic tetrameric peptides for
the selection of Cripto-Alk4 antagonists. Tetrameric tripeptide libraries have been assembled
around a polylysine scaffold using 30 non natural amino acids as building blocks, following the
Mix-Split approach, and iteratively deconvoluted using a competition ELISA-based assay;
whereby Cripto antagonists have been subsequently selected and characterized. One
tetrameric tripeptide was identified that efficiently disrupts the Cripto-Alk4 interaction and
binds to Cripto with a nM affinity as demonstrated by direct-binding ELISA and SPR analyses.
Comparative data with peptide analogs show that the selected tripeptide is highly specific and
that its multimeric structure is strictly required to efficiently bind the target.
References
1 Minchiotti G. Oncogene 2005; Nodal-dependant Cripto signaling in ES cells: from stem cells to tumor biology 24, 5668–
5675.
2. Adkins HB, et al. Antibody blockade of the Cripto CFC domain suppresses tumor cell growth in vivo. J Clin Invest
2003;112(4):575-587.
3. Gray PC, et al. Cripto forms a complex with activin and type II activin receptors and can block activin signaling. Proc Natl
Acad Sci U S A 2003;100(9):5193-5198.
66
New Frontiers in the Search of Bioactive Molecules: from Peptides to Drugs
Influence of solvent polarity on dehydropeptide’s conformation
Jewginski Michal1, Latajka Rafal 2, Makowski Maciej 1, Krezel Artur 3, Kafarski Pawel 1,2
1 Institute of Chemistry, University of Opole, 45-052 Opole, Poland
2 Faculty of Chemistry, Wroclaw University of Technology, 50-370 Wroclaw, Poland
3 Department of Protein Engineering, Faculty of Biotechnology, University of Wroclaw, Wroclaw, Poland
As it is known peptides could adopts different three-dimensional structure depending on
environmental conditions, especially on type of solvent. In this communication will be
described influence of three different solvents on dehydropeptide’s conformation. It should
reveal conformational preference of dehydropeptides in non-polar, medium polar and polar
solvents. Moreover determination of influence of peptide chain size on conformational stability
will be presented. Results for four peptides will be reported (Fig. 1)
H
O
O
C
H2
H3C
O
CH3
H3C
OMe
CH
N
H
O
O
H 3C
H
N
N
H
O
H 3C
No3. Boc-Gly-ΔZPhe-Phe-Gly-ΔZPhe-Phe-OMe
H
H
H3 C
H 3C
O
N
H
H2
C
H
N
N
H
C
H2
O
H 3C
CH
N
H
O
H 2C
OCH3
H3C
C
H2
CH3
H2
C
H
N
N
H
O
CH
N
H
O
O
H2 C
H2C
O
O
O
O
O
O
O
H
H2 C
No1. Boc-Gly-ΔZPhe-Phe-OMe
CH3
OCH3
CH
N
H
O
H2 C
O
O
H
N
N
H
CH
N
H
C
H2
CH3
H2
C
H
N
N
H
H2C
O
O
O
H
H
N
OCH3
CH
N
H
N
H
O
O
H
No2. Boc-Gly-Gly-ΔZPhe-Phe-OMe
No3. Boc-Gly-ΔZPhe-Phe-Gly-ΔZPhe-Phe-OMe
Fig. 1 Structures of investigated peptides
Mainly experimental method used in research was nuclear magnetic resonance. The
investigations were based on standard measurements (2D techniques and 1D experiments,
typical for detection of hydrogen bonding) and theoretical calculations. Conformational
preferences of investigated systems were obtained on base of ROESY and NOESY
experiments and calculations by use of X-PLOR and quantum chemical calculation.
Additionally CD spectroscopy was used to confirm proposed conformation.
67
POSTER PRESENTATIONS - Eleventh Naples Workshop on Bioactive Peptides
Synergism between temporins in killing of gram-negative bacteria
Mangoni M. Luisa1, Rosenfeld Yosef 2, Barra Donatella1 and Shai Yechiel2
1. Dept. of Biochemical Sciences, University of Rome La Sapienza, 00185 Rome- Italy
2. Dept. of Biological Chemistry, the Weizmann Institute of Science, 76100 Rehovot- Israel
The increasing emergence of multidrug-resistant microbes has urgently required the
discovery of new antibiotics with a new mode of action, and naturally occurring antimicrobial
peptides (AMPs) represent promising candidates[1]. In Amphibia, temporins are among the
shortest (10 to 16 residues) AMPs, with up to ten isoforms within the same specimen[2]. In
this study, we show that temporins A (FLPLIGRVLSGIL-NH2) and B (LLPIVGNLLKSLLNH2), which are only weakly active on Gram-negative bacteria, can synergize, when
combined each with temporin L (FVQWFSKFLGRIL-NH2), to overcome the bacterial
resistance imposed by the lipopolysaccharide (LPS)-outer membrane which forms a very
efficient barrier, in Gram-negative bacteria, against a variety of hydrophilic and hydrophobic
molecules. Furthermore, this effect is highly dependent on the type of LPS. More specifically,
we compared the ability of temporins and their combinations to inhibit the growth of
Escherichia coli O111:B4, E. coli O26:B6 (with a shorter LPS-carbohydrate chain) and three
different cell-wall defective mutant strains of E. coli D21, which have lost increasing amounts
of sugar residues in their LPS backbone. Interestingly, there is a clear synergism for
temporins A+L and B+L against E. coli O111:B4, whereas an additive interaction is found
toward the other bacterial strains. This suggests that the loss of a synergistic effect between
temporins on Gram-negative bacteria is parallel to the shortening of the LPS-polysaccharide
chain length of the target microorganism. To understand the underlying mechanism, we
investigated the effect of LPS from E. coli O111:B4 and O26:B6 on the organization of
temporins, alone and when mixed one with each other. Our data suggest that the synergism
between temporins is related to the ability of temporin L to prevent the oligomerization of A
and B when in contact with the longer LPS O111:B4, thus allowing their traslocation across
[3]
the bacterial cell wall into the target cytoplasmic membrane (Fig. 1) .
Fig.1. Schematic representation of a possible mechanism for the synergism (panel
A) or non-synergism (panel B) between temporin A and L, when bound to LPS
O111:B4 (panel A) and O26:B6 (panel B) layer of Gram-negative bacteria
References
1 Zasloff, M. 2002. Nature. 415: 389-395
2 Mangoni, M.L. 2006. Cell Mol Life Sci. 63: 1060-1069
3 Rosenfeld, Y., Barra, D. Simmaco, M. Shai Y. and Mangoni ML. 2006. J Biol Chem. 281: 28565-74
68
New Frontiers in the Search of Bioactive Molecules: from Peptides to Drugs
What happens at HLA/peptide/antibody interface? A computer
simulation study of MAGE.A1 peptide/MHC/antibody complex
Athanassios Stavrakoudis1,2 and Maria Sakarellos-Daitsiotis2
1 Department of Economics, University of Ioannina, GR-45110, Ioannina, Greece
2 Department of Chemistry, University of Ioannina, GR-45110, Ioannina, Greece
It is widely accepted that the adaptive immune system employs two defence routes in
response to pathogenic substances: a) proteolytic fragments of antigenic proteins are
presented by class I or II major histocompatibility (MHC) molecules to T cell receptors (TCRs)
and b) humoral immune response which involves interaction of (in most of the cases)
unprocessed antigens with antibodies. A very interesting procedure that it has been recently
applied by researchers is the direct interaction of TCR like antibodies with peptide/MHC
complexes. The first crystal structure of such a complex between MHC/peptide/antibody
molecules has been recently published[1]. The antibody fragment Fab-Hyb3 employed a
distinct diagonal mode of binding the peptide/MHC complex. In order to further investigate to
dynamical properties of the binding and the individual contribution of the peptide’s residues to
this immunological synapse we performed large scale molecular dynamics (MD) of the whole
complex solvated in explicit water. The NAMD 2.6 package was used as MD engine with
parameters taken from CHARMM27 force field. After equilibration steps the system (consisted
of 140970 atoms) was passed to the production phase under NPT/PBC/PME conditions for 10
ns. MD results showed that the antibody interacts directly with the MHC molecule and has
only few contacts the peptide. It was also found that the antibody has relative diagonal
approach to the MHC face and most of their interactions involve the α1 chain of the MHC
molecule. This work is the first attempt of computer modeling of such complexes and provides
insightful information about this recently discovered binding mode.
Figure 1. MHC molecule (light grey in ribbons), antibody (dark grey with ribbons) and
peptide (sticks) as found at the last frame of molecular dynamics simulations.
References
1 Hülsmeyer M, Chames P., Hillig R.C., Stanfield R.L., Held G., Coulie P.G. Alings C., Wille G, Saenger W., Uchanska-Ziegler
B., Hoogenboom H.R, Zieglerd A.: A Major Histocompatibility Complex Peptide-restricted Antibody and T Cell Receptor
Molecules Recognize Their Target by Distinct Binding Modes. Journal of Biological Chemisry: 280 2972-2980 (2005)
69
POSTER PRESENTATIONS - Eleventh Naples Workshop on Bioactive Peptides
New bradykinin antagonists – influence of C-terminal
modifications on their pharmacological properties
Małgorzata Śleszyńska1, Anna Kwiatkowska1, Dariusz Sobolewski1, Jiřina Slaninovà2, Tomasz
Wierzba3, Bernard Lammek1, Adam Prahl1
1 Faculty of Chemistry, University of Gdańsk, 80-952 Gdańsk, Poland
2 Institute of Organic Chemistry and Biochemistry, Academy of Sciences of Czech Republic, Prague, Czech Republic
3 Department of Physiology, Medical University of Gdańsk, 80-211 Gdańsk, Poland
The first report on practically effective bradykinin (BK) antagonists for B2 receptors was
published in 1984. The key to conversion of bradykinin into an antagonist was replacement of
Pro7 with an aromatic D-amino acid; D-Phe was first used. However, our studies
demonstrated that the D-amino acid residue at position 7 of the bradykinin antagonist, until
recently considered to be necessary for B2 antagonism, can be replaced by suitable L-amino
acid or achiral residue or, together with the amino acid occupying position 8, by a sterically
restricted dipeptide unit. Having all this in mind we synthesized and bioassayed two new
analogues of bradykinin. The peptides were designed by substitution of position 7 of
bradykinin B2 receptor antagonist ([D-Arg0,Hyp3,Thi5,8,D-Phe7]BK), previously described by
Stewart’s group, with structural isomer of proline: β2-iso-Pro or its homologue: β3-homo-Pro.
It’s worth emphasizing that position 7 in bradykinin molecule is occupied by proline residue.
Our previous results demonstrated the importance of the position in the peptide chain into
which the sterically restricted 1-aminocyclohexane-1-carboxylic acid residue (Acc) was
inserted. These findings prompted us to investigate how introduction of L-pipecolic acid
residue (L-Pip) in position 7 or 8 of Stewart’s antagonist will affect pharmacological properties
of resulting compounds. In comparison to the Acc residue, the ring of L-pipecolic acid also
consists of six atoms, but includes the nitrogen atom.
Bearing in mind that acylation of the N-terminus of several known B2 blockers with a variety of
bulky groups has consistently improved their antagonistic potency in the rat blood pressure
assay, the aforementioned four analogues were also synthesized in the N-acylated form with
1-adamantaneacetic acid (Aaa).
The activity of eight new analogues was assessed in isolates rat uterus and in rat blood
pressure test.
70
New Frontiers in the Search of Bioactive Molecules: from Peptides to Drugs
An antibacterial peptide with stability
toward proteolytic degradation
Marta De Zotti1, Fernando Formaggio1, Claudio Toniolo1, Lorenzo Stella2, Mi-Hyun Kim3, Yoonkyung
Park3, and Kyung-Soo Hahm4
1
2
3
4
Institute of Biomolecular Chemistry, Padova Unit, CNR, Department of Chemistry, University of Padova, 35131 Padova, Italy
Department of Chemical Sciences and Technologies, University of Rome “Tor Vergata”, 00133 Rome, Italy
Research Center for Proteineous Materials, Chosun University, 501-759 Gwangju, Korea
Department of Cellular Molecular Medicine, Chosun University, 501-759 Gwangju, Korea
Peptaibols are members of a unique class of membrane-active compounds of fungal origin.
More recently, a variety of peptaibols was sequenced with a fatty acyl moiety linked to the Nterminal amino acid. Because of the lipophilic character of their N-terminus, these peptides
are referred to as lipopeptaibols[1]. Their amino acid sequences range from 6 to 10 residues
and the fatty acyl moieties from 8 to 16 carbon atoms. The amino acid sequence of trichogin
GA IV[2], the prototype of lipopeptaibols, is as follows:
nOct-Aib1-Gly-Leu-Aib-Gly5-Gly-Leu-Aib-Gly-Ile10-Lol
where Aib is α-aminoisobutyric acid and Lol is leucinol.
In this work the antibacterial, antifungal, and hemolytic activities of trichogin GA IV and
several synthetic analogues were investigated. The natural peptide exhibits a specific activity
against S. aureus and only a marginal hemolytic effect. Interestingly, trichogin GA IV is active
also against several methicillin-resistant S. aureus strains. Studies on synthetic analogues
demonstrated that substitution of the C-terminal Lol by Leu-OMe or replacement of one Aib
residue by the EPR-label TOAC, also a Cα-tetrasubstituted α-amino acid, does not perturb
significantly the biological activity of the peptide. On the other hand, removal of three or seven
N-terminal residues eliminated alla types of bioactivity. Moreover, investigations of enzymatic
(with endopeptidase, pepsin, and chymotripsin) degradation showed that trichogin GA IV and
its above mentioned analogues are endowed with a remarkable resistance to proteolysis. To
check whether the proteolytic stability of trichogin GA IV is related to the presence of the three
non-coded, sterically hindered, helicogenic Aib residues[3] we are currently replacing them
with Leu residues in a set of novel synthetic analogues.
The present results indicate that trichogin GA IV could represent an interesting lead
compound for the realization of new, selective, and protease-resistant antibacterial drugs.
71
POSTER PRESENTATIONS - Eleventh Naples Workshop on Bioactive Peptides
Development of quality specifications for peptide drugs.
B. De Spiegeleer1, V. Vergote1 and C. Burvenich2
1 Drug Quality and Registration (DruQuaR) group, Department of Pharmaceutical Analysis, Faculty of Pharmaceutical Sciences, Ghent University,
B-9000 Ghent, Belgium
2 Department of Physiology and Biometrics, Faculty of Veterinary Medicine, Ghent University, B-9820 Merelbeke, Belgium.
Peptides show great pharmaceutical potential as active drugs in different therapeutic areas
like allergy, anti-infection, oncology, obesity, etc… and as functional excipients in drug
delivery systems to overcome tissue and cellular membrane barriers. The development of a
peptide toward a pharmaceutical compound poses however unique challenges: the rational
development of its quality specifications is one of the major issues in this process.
Current international regulatory guidelines and pharmacopoeia covering the quality of
medicines do not consistently encompass pharmaceutical peptides in an unequivocal way:
some explicitly exclude peptides from their scope (e.g. ICH Q3A), while others are more
ambiguous and/or include peptides (e.g. ICH 6A). Technical guidances have been developed
or are under development in preparation of updated and/or new pharmacopoeial monographs.
Next to these regulatory guidelines, we will also assess the current peptide quality
specifications for existing drugs, as applied in pharmacopoeial monographs and finished drug
products, and for investigational compounds used in biomedical research. Specific peptide
issues in stability-indicating method development and validation, stability and impurity
qualification will be highlighted.
Finally, general guidance will be proposed to establish rational quality specifications during
the peptide drug development process.
72
New Frontiers in the Search of Bioactive Molecules: from Peptides to Drugs
Membrane interactions governing antimicrobial peptide activity
Manuel N. Melo1, Rafael Ferre2, Ana D. Correia1, Lidia Feliu2, Marta Planas2, Eduard Bardají2 and
Miguel Castanho1
1 University of Lisbon, Institute of Molecular Medicine, 1649-028 Lisbon – Portugal.
2 Laboratori d’Innovació en Processos i Productes de Síntesi Orgànica, Campus Montilivi, E-17071 Girona – Spain.
The interaction of the antimicrobial peptides (AMPs) omiganan (H-ILRWPWWPWRRK-NH2)
and BP100 (H-KKLFKKILKYL-NH2) with model bilayers was characterized. The activity and
selectivity of these peptides could be attributed to a strong preference towards anionic
membrane model systems, which mimic bacterial membranes. In addition, cytotoxicity
assessments using eukaryotic models are being carried out to further understand the overall
low haemolytic activity of these peptides. Regarding the interactions with bacterial membrane
models, there were marked differences in the interaction patterns, as well as in functional
properties of the peptides at high peptide:lipid ratios. These differences occurred for both
peptides, despite their being unrelated in sequence and in occurrence in nature. Such events
at high membrane coverage could represent the equivalent at the molecular scale of the
conditions at which the antimicrobial activity of the peptides is triggered. Although the
lipid:peptide ratios at these transitions are lower than 10 phospholipids per peptide molecule,
the plausibility of this hypothesis was demonstrated taking into account an estimate of the
amount of lipid per bacterium, and the bacterial concentration in minimum inhibitory
concentration (MIC) assays. According to the partition constants obtained towards bacterial
membrane models, these peptides are expected to reach, at the MIC, precisely those high
concentrations in the membrane. In addition, surface charge neutralization was shown to
occur in these conditions. Activity at high membrane coverage is thus likely not only for these
peptides but also for any peptide displaying high membrane affinity and micromolar MICs,
which is common amongst AMPs.
73
POSTER PRESENTATIONS - Eleventh Naples Workshop on Bioactive Peptides
Biochemical properties of amide
derivatives of pyridyl- and tolylporphyrins
J. Habdas1, B. Boduszek2
1 University of Silesia, Institute of Chemistry, 40-006 Katowice - Poland
2 Wroclaw University of Technology, Department of Organic Chemistry, 50-370 Wroclaw - Poland
Porphyrins are important compounds not only because of their significance in nature (e.g.
haem, chlorophyll, vitamin B12) but also because of their application as active compounds in
medicine. For about three decades porphyrin derivatives have been the main group of
photosensitizers for the PDT method of diagnosis and cancer therapy[1,2]. In our earlier papers
we presented amide derivatives of tolylporphyrins I as effective photosensitizers[3,4], and
recently porphyrin derivatives of peptidyl phosphonates II as compounds with inhibitory
activity towards aminopeptidaseN[5,6]. The synthesised compounds were prepared with the
use of DCC as a coupling agent of carboxyporphyrins with amine acids or peptidyl
phophonates, respectively. The desired properties, hydrophilic or amphiphilic, were achieved
by using as initial porphyrins phenyl- or pyridylporphyrins.
I
II
We have explained the good photodynamic and inhibitory properties of the above mentioned
derivatives with the presence of amide bonds in the synthesized molecules which makes their
structure more similar to cell components e.g. membrane.
References
1 Bonnett R., Chem. Soc. Rev., 19 (1995)
2 Habdas J., Arthropods, Chemical, Physiological and Enviromental Aspects, Ed. D. Konopińska, Wroclaw 2002, p.244
3 Drzewiecka A., Urbańska K., Matuszak Z., Pineiro M., Arnout L.G., Habdas J., Ratuszna A., Stochel G., Acta Biochim. Pol.,
1, 277 (2001)
4 Habdas J., Annals Pol. Chem. Soc., Vol. 2, 88 (2003)
5 Habdas J., Boduszek B., Phosphorus, Sulfur Silicon 180, 2039 (2005)
6 Habdas J., Boduszek B., Heteroatom Chemistry, 1, 107 (2008)
74
New Frontiers in the Search of Bioactive Molecules: from Peptides to Drugs
The 1-18 fragment of esculentin-1b: an attractive
antimicrobial peptide from the skin of Rana esculenta
Marcellini H.G. Ludovica, Di Giacomo Daniele and Mangoni Maria Luisa
Dipartimento di Scienze Biochimiche and Unità di Diagnostica Molecolare Avanzata, Università La Sapienza di Roma, Azienda Ospedaliera S.
Andrea, Rome-00189-Rome-Italy
In the past two decades, numerous families of genetically encoded antimicrobial peptides
(AMPs), from all living organisms, have been described [1,2]. They are conserved components
of the innate immune response, and represents the first host defence line against microbial
infections. Alghough AMPs can show a marked variation in size, sequence and structure,
most of them are polycationic and fold into an amphipathic helical or beta-sheet structure, a
feature which aids their interaction and insertion into microbial membranes that are believed
to be the principal target for their killing mechanism. Before reaching the negatively-charged
bacterial membrane, AMPs need to bind, via electrostatic interactions, the anionic
components of the microbial cell surface and diffuse through the cell wall. Among the natural
sources for AMPs, the granular glands of amphibian skin are one of the richest storehouses.
Intense research focusing on AMPs from frog skin is currently devoted to the elucidation of
their mode(s) of action. However, very little is known about their effects on intact bacteria.
Here we report on Esculentin 1-18 [Esc(1-18)], a linear peptide covering the first 18 N-terminal
residues of the full length peptide esculentin-1b from the skin of Rana esculenta. As described
in a recent paper, this peptide retains the antimicrobial activity of the longer esculentin-1b
[3]
against a wide range of microorganims, with negligible effects on mammalian erythrocytes .
However, to expand our knowledge on the molecular mechanism underlying the antimicrobial
activity of Esc(1-18) on Gram-negative bacteria, we investigated the effect of this peptide on
Escherichia coli ATCC 25922, by studying its: i) killing kinetic; ii) ability to permeate both
artificial and bacterial membranes; iii) capacity to synergize with conventional antibiotics; iv)
effect on cell morphology by means of scanning electron microscopy. These studies have
shown that Esc(1-18) increases the permeability of the bacterial inner membrane in a dosedependent manner, without destroying the cell's integrity and without causing a total
membrane disruption (Figure 1). Notably, its antimicrobial activity is not significantly affected
by the ionic strength of the incubation medium and can be partially preserved also in the
presence of 70% human serum.
A
B
Figure 1. Scanning electron microscopy of E. coli cells before (A) and after peptide’s treatment (B)
References
1 Zasloff, M. (2002) Nature. 415, 389-395
2 Brown, K. L., and Hancock, R. E. (2006) Curr Opin Immunol. 18, 24-30
3 Mangoni, M. L., Fiocco, D., Mignogna, G., Barra, D., and Simmaco, M. (2003) Peptides. 24, 1771-1777
75
POSTER PRESENTATIONS - Eleventh Naples Workshop on Bioactive Peptides
Aminophosphonates and their derivatives the
new receptors for metal ions and amino acids.
Mlynarz Piotr 1, Ptak Tomasz 1, Czernicka Anna 1, Berlicki Łukasz 1, Schroeder Grzegorz 2 and
Kafarski Paweł 1
1 Wroclaw University of Technology, Faculty of Chemistry, 50-370 Wroclaw, Poland.
2 University of A. Mickiewicz in Poznan, Faculty of Chemistry, 60-780 Poznan, Poland.
Aminophosphonates have been found to be not only good enzyme inhibitors but also a
promising group of metal ion chelators. In our recent papers the interaction of aminophosphonate with amino acids and mono- and divalent metal ions were described [1,2].
In this work the supramolecular properties of two new receptors: tetra-2-methoxyethyl aminobis(phenylo-methanephosphonate) and 4-aminomethyl-phenylmethylphosphonate towards
amino acids and metal ions binding were studied. The formation of molecular complexes
between these host molecules and amino acids (Lys and Arg), and polyamines were found by
1
H and 31P NMR (longitudinal relaxation T1, spin diffusion) spectroscopy and ESI-MS
spectrometry. Moreover, crown ether-like esters of the phosphonate form of stable complexes
with monovalent and divalent metal cations. The performed studies allow to use both
receptors immobilized on electronic cantilevers to construct analytical tools for investigated
molecules detection.
References
1 Mlynarz P., Czernicka A., Rydzewska A., Schroeder G., Kafarski P., J. Mol. Struc. 2008, 875, 130-134.
2 Mlynarz P., Majkut-Olbert A., Sliwinska S., Schroeder G., Bankowski B., Kafarski P., J Mol. Struc. 2008, 873, 173-180.
76
New Frontiers in the Search of Bioactive Molecules: from Peptides to Drugs
Membrane interactions of the human
antimicrobial cathelicidin LL37
Morgera Francesca1,2, Vaccari Lisa 2, Antcheva Nikolinka1, Pacor Sabrina1 and Tossi Alessandro1
1 University of Trieste, Life Sciences Department, 34127 Trieste, Italy
2 Elettra Synchrotron Light Laboratory, Area Science Park, 34012 Trieste, Italy.
An important family of Host Defence Peptides (HDPs), the Cathelicidins, have been identified
in mammalian and non-mammalian vertebrate species[1]. They are important components of
both the innate and adaptive immune systems, with a direct capacity to inactivate microbes at
the level of mucosal surfaces. In mammals, some of these molecules have also been found to
act as immune modulators and mediate healing processes. These features make them
promising tools for the development of multifunctional agents for the treatment of infection.
Cathelicidins are characterized by a conserved pro-region carrying highly variable
antimicrobial sequences, a property that seems to affect their structural and aggregational
behaviour in different environments, and their capacity to interact with biological membranes,
leading to differential biological effects on prokaryotic or eukaryotic cells. LL37 is the only
cathelicidin-derived HDP in humans, identified in plasma and airway surface fluids, and it
shows a broad-spectrum antimicrobial activity in vitro[1,2]. However, this activity seems to be
quite medium sensitive and accompanied by a relative cytotoxicty, possibly related to its
tendency to aggregate in specific conditions[3,4]. The structural and aggregational behaviour of
LL37 and selected orthologous primate peptides[4], was investigated by biochemical and
biophysical methods, such as CD in different buffers, SDS micelles and membrane-like
environments (anionic or zwitterionic phospholipid liposomes) and FTIR spectroscopy in
transmission mode. Moreover, the behaviour in a lipid surrounding was further probed by dye
release from liposomes, by Attenuated Total Reflection (ATR)-FTIR spectroscopy on
supported lipid monolayers and by atomic force microscopy (AFM) that allowed an
assessment of the morphological effects on lipid order. These findings were correlated with
biological data from antimicrobial, cell lysis and cytofluorimetric assays, giving an interesting
picture of the possible mode of action of these helical peptides, and highlighting the
importance of biochemical parameters such as structuring and dimerization/oligomerization
processes in the selective interaction with biological membranes.
References
1 Zanetti, (2004) J Leukoc Biol 75(1): 39-48.
2 Bals et al, (2003) Cell Mol. Life Sci. 60, 711–720.
3 Johansson et al, (1998) J. Biol. Chem. 273, 3718 – 3724.
4 Zelezetsky et al, (2005) Biochem J. Aug 15;390(Pt 1):177-88.
77
POSTER PRESENTATIONS - Eleventh Naples Workshop on Bioactive Peptides
Quantitative analyses: CPP uptake in 4 different cell lines
Prisca Boisguerin, Judith Mueller, Ines Kretzschmar, Rudolf Volkmer,
Institut für Medizinische Immunologie, Charité - Universitätsmedizin Berlin, 10115 Berlin, Germany.
The major limitation in utilizing macromolecules for basic research or/and therapeutic
applications is the inability of them to diffuse across the cellular membrane. Cell-penetrating
peptides (CPPs)[1,2] are short peptides that are able to penetrate cell membranes and
translocate different cargoes into cells. In the last years many research articles are published
but to our best knowledge no single systematic study has been carried out since nowadays.
Alternatively, information can only by gathered piece by piece from different sources. For that
raison, we decided to start with an analytical screen of the CPP specificity in cell lines. We
used 22 different CPPs which are all being published before and were selected based on their
classification in protein-derived peptides, model peptides, and designed peptides. Here, we
present the first analytical screen of 22 FITC-labelled CPPs in four cell lines (MDCK, HEK293,
HeLa and Cos-7). Furthermore, we dissected the influences of different conditions such as
protease inhibitors, incubation conditions, endocytosis inhibitors, temperature and cytotoxicity.
The cellular uptakes of each condition are monitored by microplate reader (cell lyses) or by
confocal microscopy.
By comparing our results, we could clearly demonstrate that the differences in cellular uptake
depending on the used CPP. Furthermore, we show that in particular cases, cellular uptake
could be increased by varying of the incubation conditions. Altogether, we always
recommended a detail analyse of the used CPPs, cell lines and working conditions before
functional assay to ensure biological activity of the CPP[3].
Financial support from Charité and the Deutsche Forschungsgemeinschaft (VO885/3-1), is
gratefully acknowledged.
References
1 S. Deshayes, M. C. Morris, G. Divita, F. Heitz, Cell Mol Life Sci 2005, 62, 1839.
2 P. Jarver, U. Langel, Biochim Biophys Acta 2006, 1758, 260.
3 J. Mueller, I. Kretzschmar, R. Volkmer, P. Boisguerin, Submitted Bioconjugate Chemistry 2008
78
New Frontiers in the Search of Bioactive Molecules: from Peptides to Drugs
Evidence for an elongated dimeric structure of
heparin-binding haemagglutinin from M. tuberculosis
Esposito Carla1, Ruggiero Alessia1, Pethoukov Maxim V.,2 Svergun Dmitri I.,2, Pedone Carlo,1,3
Pedone Emilia1 and Berisio Rita 1
1 Istituto di Biostrutture e Bioimmagini, C.N.R., I-80134 – Napoli, Italy.
2 European Molecular Biology Lab, Hamburg, c/o DESY, 22603 Hamburg, Germany.
3 Dipartimento delle Scienze Biologiche – Sezione di Biostrutture, Università degli Studi di Napoli “Federico II”, I-80134 – Napoli, Italy.
Mycobacterium tuberculosis (MTB) is one of the most devastating human microbial
pathogens. It invades and multiplies in both phagocytes and epithelial cells. In MTB,
adherence to epithelial cells is mediated by the heparin-binding haemagglutinin adhesin,
HBHA[1]. This protein binds to heparan sulphate proteoglycans on the surface of epithelial
cells and is, therefore, responsible for extra-pulmonary dissemination of tuberculosis[2].
Binding to target epithelial cells involves the C-terminal lysine-rich domain of the protein,
which is exposed at the mycobacterial cell surface[3]. However, despite the interest of HBHA
both as a potential antigen against tuberculosis and as a diagnostic tool, no structural data are
so far available, nor detailed information on the protein oligomerization state have yet been
provided. We here present a biophysical characterization and the low resolution structure by
Small Angle X-ray Scattering (SAXS) of both the full length HBHA and a truncated form, here
denoted as HBHAΔC, which lacks the C-terminal heparin-binding domain (residues 161199)[4].
We have cloned, expressed and purified both HBHA and HBHAΔC to study their molecular
properties and oligomerisation, a key event for bacterial haemagglutination[5]. Circular
dichroism studies have provided an experimental evidence that HBHA presents a coiled coil
domain, like predicted by sequence analysis[6]. To validate this result, we performed chemical
denaturation, using urea as a denaturating agent, of HBHAΔC. The urea denaturation profile
of HBHAΔC is characteristic of a two-state helix-coil transition with a peculiarly low urea Cm, a
characteristics which is typical of coiled coil systems.
By performing cross-linking experiments using both glutaraldehyde and bis-sulfosuccinnimidylsuberate (BS3) as cross-linking agents, we evidenced that HBHA is dimeric in solution. Small
Angle X-ray Scattering (SAXS) experiments on both HBHA and HBHAΔC confirmed the
dimeric nature of these proteins[5]. The experimental Rg and Dmax values showed that HBHA
exhibits an elongated shape. The increase of both Rg and Dmax of the full-length protein
compared to HBHAΔC suggests peripheral location of the C-terminal of HBHA. This is
consistent with the role attributed to HBHA C-termini in epithelial cell adhesion. Furthermore,
our data suggest that, like for other known systems, the coiled-coil nature of the N-terminal
region of HBHA is responsible for dimerization, which may be regarded as a process that
leads to enhanced affinity to epithelial cells and improves cell adhesion.
References
1. Pethe, K., S. Alonso, F. Biet, G. Delogu, M. J. Brennan, C. Locht, and F. D. Menozzi. 2001. The heparin-binding
haemagglutinin of M. tuberculosis is required for extrapulmonary dissemination. Nature 412:190-4.
2. Franco D. Menozzi, Rainer Bischoff, Emmanuelle Fort, Michael J. Brennan, and Camille Locht. 2006. Molecular
characterization of the mycobacterial heparin-binding hemagglutinin, a mycobacterial adhesion. PNAS 1998:95;1262512630.
3. Menozzi, F. D., R. Bischoff, E. Fort, M. J. Brennan, and C. Locht. 1998. Molecular characterization of the mycobacterial
heparin-binding hemagglutinin, a mycobacterial adhesin. Proc Natl Acad Sci U S A 95:12625-30.
4. Svergun, D. I. 1999. Restoring low resolution structure of biological macromolecules from solution scattering using simulated
annealing. Biophys J 76:2879-86.
5. Esposito, C., Pethoukov, M. V., Svergun, D. I., Ruggiero, A., Pedone, C., Pedone, E. and Berisio, R. Identification of a
heparin-binding hemagglutinin present in mycobacteria. J Bac in press.
6. Delogu, G., and M. J. Brennan. 1999. Functional domains present in the mycobacterial hemagglutinin, HBHA. J Bacteriol
181:7464-9.
79
POSTER PRESENTATIONS - Eleventh Naples Workshop on Bioactive Peptides
The identification and characterisation of fusogenic
domains in Herpesvirus glycoprotein B molecules
Falanga Annarita1, Galdiero Stefania1, Vitiello Mariateresa2, Cantisani Marco1, Kampanaraki
Aikaterini2, Raieta Katia2, Browne Helena3, Pedone Carlo1, Galdiero Massimiliano2
1 University of Naples “Federico II”, Department of Biological Sciences & CIRPEB, 80134, Naples, Italy
2 II University of Naples, Department of Experimental Medicine,80138, Naples, Italy
3 University of Cambridge, Department of Pathology, Division of Virology, Cambridge UK
The molecular mechanism of entry of herpesviruses requires a multicomponent fusion
system. Virus entry and cell-cell fusion of Herpes simplex virus (HSV) requires four
glycoproteins: gD, gB and gH/gL. The role of gB has remained elusive until recently when the
crystal structure of Herpes simplex virus type 1 (HSV-1) gB became available. Glycoprotein B
homologues represent the most highly conserved group of herpesvirus glycoproteins,
however despite the high degree of sequence and structural conservation, differences in
posttranslational processing are observed for different members of this virus family. Whereas
gB of HSV is not proteolytically processed after oligomerization, most other gB homologues
are cleaved by a cellular protease into subunits that remain linked via disulfide bonds.
Proteolytic cleavage is common for activation of many other viral fusion proteins, therefore it
remains difficult to envisage a common role for different herpesvirus gB structures in the
fusion mechanism. We have selected Bovine herpesvirus type 1 (BoHV-1) and HSV-1 as
representatives viruses expressing cleaved and uncleaved gBs, and have screened their
amino acid sequences for regions of highly interfacial hydrophobicity. Synthetic peptides
corresponding to such regions were tested for their ability to induce the fusion of large
unilamellar vesicles and to inhibit herpesvirus infection. These results highlight that several
regions of the gB protein are involved in the mechanism of membrane interaction.
80
New Frontiers in the Search of Bioactive Molecules: from Peptides to Drugs
New analogs of antiherpes virus
drugs – synthesis and biological activity
Ivanka G. Stankova1 , Tatyana Dzimbova2 and Stoyan A. Shishkov3
1 Department of Chemistry, South-West University ‘’Neofit Rilski’’, 2700 Blagoevgrad, Bulgaria
2 nstitute of Molecular Biology, Bulgarian Academy of Sciences
3 St. Kl. Ohridski Sofia University, Faculty of Biology , Laboratory of Virology, Sofia 1164, Bulgaria
Acyclovir, 9-[(2-hydroxyethoxy)methyl]guanine (ACV) is an acyclic guanine nucleoside
analogue that is widely used clinically as an antiherpetic agent. Its limited absorption in
humans after oral administration prompted the search for prodrugs [1-3].
The objective of this work has been synthesis a new amino acid ester of acyclovir-valine,
alanine, leucine containing thiazole ring and glycine containing thiazole, oxazole, thiazolylthiazole ring and to evaluate their activity against herpes simplex type I and II in vitro.
References
1. Beauchamp, L. M., Orr, G. F., de Miranda, P., Burnette, T. and Krenitsky T., A., 1992, Amino acid ester prodrugs of aciclovir.
Antiviral Chemistry  Chemotherapy, 3 (3), 157-164.
2. Beauchamp LM and Krenitsky TA, Drugs Future, 1993, 18: 619-628, Acyclovir prodrugs: the road to valaciclovir.
3. Anand BS, Katragadda S and Mitra AK, J. Pharmacol. Exp. Ther., 2004, 311(2): 659 – 667, Pharmacokinetics of Novel
Dipeptide Ester Prodrugs of Acyclovir after Oral Administration: Intestinal Absorption and LiverMetabolism.
81
POSTER PRESENTATIONS - Eleventh Naples Workshop on Bioactive Peptides
Characterisation of bioactive peptides produced
by cyanobacteria blooms in Italian lakes
Ferranti Pasquale1, Fabbrocino Serena2, Nasi Antonella1, Bruno Milena3, Serpe Luigi2, and Gallo
Pasquale2
1 Department of Food Science, University of Naples "Federico II", 80055 Portici (NA) (Italy).
2 Istituto Zooprofilattico Sperimentale del Mezzogiorno, 80055 Portici (NA) (Italy).
3 Istituto Superiore di Sanità, 00161 Rome, Italy.
Cyanobacteria, known also as blue-green algae (BGA), are prokaryotic organisms widely
distributed in surface waters, producing blooms and scums. These phenomena in lakes and
freshwaters are seasonal, caused by several species belonging to the genera Microcystis,
Planktotrix, Anabaena, Nostoc, Oscillatoria, the most frequently observed. Some BGA genera
produce peptide biotoxins, that can be toxic to wildlife, domestic livestock and even to humans;
not all cyanobacteria strains are toxic within one species. Microcystins (MCs), the most
abundant and dangerous toxins produced by cyanobacteria, are cyclic heptapeptides of about
1000 g mol-1, showing the general structure cyclo(D-Ala(1)–L-X(2)–D-MeAsp(3)–L-Z(4)–
Adda(5)–D-Glu(6)–Mdha(7)). As the presence of cyanobacteria in Italian freshwaters is
common in the last years, it is urgent to monitor the risks for consumer’s health deriving from
possible intake of biotoxins from contaminated foods.
In addition, cyanobacteria produce non-toxic peptides, some of which show remarkable
bioactivities and have therefore attracted the attention of the pharmaceutical industry. Herein,
we present a peptidomic method for profiling peptide biotoxins based on the combined use of
MALDI-TOF-MS for rapid peptide detection and nanoLC/ESI-MS/MS for structural
characterization and quantitative analysis. The different analytical systems were compared to
evaluate their performances and reliability in monitoring environmental outbreaks. The method
was applied to the analysis of water and algae samples from Lake Averno, near Naples and
from other freshwater sources, as a consequence of a cyanobacteria blooms, as well as to fish
and livestock tissues from the same areas. In this way, we characterised the complete peptide
pattern of biotoxins produced, which allowed an evaluation of the possible health risk related to
their presence in the lake, and to plan strategies of monitoring and intervention. Finally, the
structural characterisation could can lead to a deeper definition of the structure-bioactivity
relationship in these peptides.
82
New Frontiers in the Search of Bioactive Molecules: from Peptides to Drugs
Purification and identification of new
bioactive peptides from leguminous
Salem Elkahoui1, Ines Karkouch1, Thierry Jouenne2, Pascal Cosette2 And Ferid Limam1
1 Laboratoire Interactions Légumineuses-Microorganismes (LILM), Centre de Biotechnologie, Borj-Cédria, BP-901, 2050 Hammam-lif, Tunisia.
2 Laboratoire Polymères, Biopolymères, Membranes , UMR CNRS 6522, Faculté des Sciences de Rouen, 76821 Mont-Saint-Aignan, France.
In recent years, the growing number of drug resistant infectious diseases points to the
necessity to research new sources of therapeutic substances from natural sources, such as
plants. In particular, leguminous seeds could constitute a source of new bioactive peptides
resulting from the hydrolysis of their proteins.
The protein extract of dried leguminous seeds, was evaluated for antimicrobial activity against
Gram-positive and Gram-negative human pathogenic bacteria and twelve phytopathogen
fungi. The total protein extract did not show any antimicrobial activity. However, after
hydrolysis of this extract by protease 1, an antibacterial activity was obtained only against
Staphylococcus aureus strain. Antifungal activity was detected against Fusarium oxysporum.
Protease 2 hydrolysed-extract showed high antibacterial activity against all tested human
pathogenic strains especially against Staphylococcus aureus and low antifungal activity
against phytopathgen fungi.
Purification of the bioactive peptides from protease 1 hydrolysed-extract was carried out by
HPLC. Identification of these peptides was performed using microsequencing, masse
spectrometry MALDI-TOF (prOTOF 2000TM) and LC/MS/MS.
83
POSTER PRESENTATIONS - Eleventh Naples Workshop on Bioactive Peptides
Singular properties of HspA, the GroES
homologue from Helicobacter pylori
Scannella Alessandra1, Dathan Nina2, Loguercio Salvatore1, Monti Simona M.2, Pucci Piero 3,
Flagiello Angela3, Terradot Laurent4 and Adriana Zagari1,2
1
2
3
4
Università di Napoli “Federico II”, Dip.delle Scienze Biologiche, Napoli, Italy.
CNR, Istituto di Biostrutture e Bioimmagini, Napoli, Italy.
Università di Napoli “Federico II”, Dip. di Chimica Organica e Biochimica, Napoli, Italy.
European Synchrotron Radiation Facility, Grenoble Cedex 9, France.
Helicobacter pylori is a gram negative bacterium that colonises the human stomach. It is
present in more than half of the world’s population and causes major diseases such as
gastritis, peptic ulcers and stomach cancer [1].
Helicobacter pylori produces an unusual GroES homologue protein referred as to HspA (Heatshock protein A). Besides its classical co-chaperone activity, HspA plays additional roles
being involved in nickel binding. It also exhibits an extended subcellular localization, ranging
from cytoplasm to bacterial cell surface. In fact, unlike its homologue proteins, HspA is highly
immunogenic being also present in the extra cellular media [2]. For this reason it is considered
a target for new therapeutic strategies [3] HspA consists of two domains: an N-terminal domain
(domain A, residues 1-90), that is homologous with other GroES bacterial proteins and a Cterminal domain (domain B, residues 91-118), which other GroES-like proteins lack [4].Domain
B is unique to HspA and contains 8 histidines and 4 cysteines which have been suggested to
be involved in nickel binding. We have produced the recombinant HspA and its mutants
Cys94Ala and Cys94Ala/Cys111Ala and have identified the disulphide bridge pattern of the
protein [5]. By combining biochemical methodologies with mass spectrometry we found that
the cysteines (two from domain A and four from domain B) are engaged in three disulphide
bonds between residues Cys51/Cys53, Cys94/Cys111 and Cys95/Cys112 [6].
Our results suggest that two of the disulphide bridges, located in the B domain, force the Cterm domain to adopt a unique closed loop structure that would be optimal for binding to 2 Ni
ions as suggested by the different redox environments that the protein experiences inside and
outside the bacterial cell. A large scale production of HspA and Cys to Ala mutants of HspA
for structural studies is currently in progress.
References
1 Covacci, A., Telford, J.L.,del Giudice, G., Parsonnet, J., Rappuoli, R. (1999). Helicobacter pylori virulence and genetic
geography. Science 284, 1328-1333.
2 Vanet, A. and Labigne, A. (1998). Evidence for specific secretion rather than autolysis in the release of some Helicobacter
pylori proteins. Infect Immun 66, 1023-7.
3 Ferrero R. and Labigne A. (1995). The GroES homolog of Helicobacter pylori confers protective immunity against mucosal
infection in mice. Microbiology 92, 6499-503.
4 Kansau, I., Guillain, F., Thiberge, J.M. and Labigne, A. (1996). Nickel binding and immunological properties of the C-terminal
domain of the Helicobacter pylori GroES homologue (HspA). Mol Microbiol 22, 1013-23.
5 Loguercio, S., Dian, C., Flagello, A., Scannella A., Pucci, P., Terradot, L. and Zagari, A. (2008). Unique properties of
HspA, a GroEs homologu,e from Helicobacter pylori, submitted
6. Amoresano, A., Pucci, P., Duro, G., Colombo, P., Costa, M.A., Izzo, V., Lamba, D. and Geraci, D. (2003). Assignment of
disulphide bridges in Par j 2.0101, a major allergen of Parietaria judaica pollen. Biol Chem 384, 1165-72.
84
New Frontiers in the Search of Bioactive Molecules: from Peptides to Drugs
An analysis of deletion mutants of the PLD1 D4 domain defines
short regions within the PLD1 interacting with PED/PEA15:
implications for the development of peptides-specific antagonist.
N. Doti1,2, D. Marasco1, M. Sabatella1, S. M. Monti1, N. Dathan1, F. Viparelli1, A. Cassese3, C. Miele3, C.
Pedone1 and M. Ruvo1
1 Institute of Biostructures and Bioimaging, CNR, 80134, Naples, Italy.
2 Biochemical and Biophysical Dept. Naples Second University, 80138, Naples, Italy.
3 Endocrinology and Experimental Oncology Institute (IEOS), CNR, 80131, Naples, Italy.
Phosphoprotein enriched in astrocytes 15 (PEA15) (also known as phosphoprotein enriched
in diabetes (PED)) is a small protein widely produced in different tissues and highly conserved
among mammals [1]. Several studies have revealed that it regulates multiple cellular functions
by binding components of major intracellular transduction pathways. Recent reports have also
revealed that PED/PEA15 binds to and enhances phospholipase D (PLD1) stability, resulting
in increased intracellular levels of diacylglycerol [2], deregulating protein kinase C (PKC)
signalling and generating resistance to insulin action on glucose transport [3]. Thereby,
disrupting the interaction between PED/PEA15 and PLD1 by a cell-penetrating compound
represents a novel strategy for improving insulin sensitivity in target cells. The expression of
D4 domain, (the shortest PED/PEA15-interacting region of PLD1 (residues 712-1074) [2]) in
L6 skeletal muscle cells stably overexpressing PED/PEA15 (L6PED/PEA-15) reduces
PED/PEA15 interaction with PLD1 [4], suggesting that the D4 region could bind PED/PEA15
preventing its interaction with the full length PLD1 and restoring insulin action [4]. Aim of the
present study is the identification of D4 crucial residues for PED/PEA15 interaction and the
development and refinement of specific antagonists. We expressed a series of soluble
truncated D4 domains fused at the N-terminus with MBP (Maltose Binding Protein),
determining whether D4α, D4β e D4γ, like the D4 wild type (wt), binds to PED/PEA15 by
carrying out dose-response ELISA assays. Remarkably, only D4α exhibited an efficacy similar
to the whole D4, and functional data in PED/PEA15-overexpressing 293-Y1 support this
evidence. To further investigate the D4 residues involved in PED/PEA15 binding, we prepared
a set of overlapping synthetic peptides covering the entire D4α. These peptides were used in
competition ELISA assays using PED/PEA15 and D4. Data indicate that fragments of the Cterminus region of D4α, efficiently block the interaction in a dose dependent way, in particular
suggesting an active role of the region 762-801 in PED binding. Furthermore their
conformational properties were analyzed by CD spectroscopy. Collectively, our results
suggest that the N-terminus region of D4 encompassing residues [762-801] is involved in
PED/PEA15 recognition and, currently, cellular experiments are underway.
References
1. Danziger N, et al (1995) J. Neurochem 64:1016–1025
2. Zhang, Y., et al (2000) The Journal of Biological Chemistry 275:35224-35232.
3. Miele C., et al (2007) The Journal of Biological Chemistry VOL. 282, No. 44, pp. 31835–31843.
4. Viparelli, F. et al (2008) The Journal of Biological Chemistry (in revision).
85
POSTER PRESENTATIONS - Eleventh Naples Workshop on Bioactive Peptides
Structural characterization of a membrane interacting
region of Herpes simplex virus type 1 glycoprotein H
Galdiero Stefania1, Falanga Annarita1, Vitiello Mariateresa2, Raiola Luca3, Fattorusso Roberto3,
Browne Helena4, Pedone Carlo1, Isernia Carla3, Galdiero Massimiliano2
1
2
3
4
University of Naples “Federico II”, Department of Biological Sciences & CIRPEB, 80134, Naples, Italy
II University of Naples, Department of Experimental Medicine,80138, Naples, Italy
II University of Naples, Department of Environmental Science, 81100, Caserta, Italy
University of Cambridge, Department of Pathology, Division of Virology, Cambridge UK
Herpes simplex virus (HSV), the prototype of the alphaherpesviruses, is a human pathogen
that infects epithelial cells before spreading to the peripheral nervous system to establish a
life-long latent infection. It is an enveloped DNA virus, and must therefore fuse its membrane
with a cellular membrane to establish infection.
The minimal fusion machinery in HSV is composed of gD, gB, and gH/gL, which are all
essential for the entry process.
In HSV the candidates for driving membrane fusion are gB and gH.
The fusogenic properties of gB are well established, in fact, the crystal structure of HSV-1 gB
showed that the protein displays features of both class I and class II membrane-fusion
proteins.
Indeed, although no three-dimensional crystal structure of gH/gL from HSV-1 is available, gH
has also been considered as a fusion effector.
We have previously identified a set of regions in the gH ectodomain able to induce membrane
fusion in a model system of synthetic membranes, namely gH220-262, gH381-420, gH493537, gH626-644 and gH776-802. Here we explored in more details the domain gH626-644
that showed to be the shortest and most active segment in artificial liposome fusion assays.
We applied biochemical and biophysical assays to a set of mutants of the synthetic peptide to
analyse its characteristics and taken together, the observation reported, point to the possibility
that gH might operate by actively using the sequence gH626-644 to perturb the lipid bilayer of
apposing membranes and its content of ellipticity and aromatic residues is of fundamental
importance.
86
New Frontiers in the Search of Bioactive Molecules: from Peptides to Drugs
Antihypertensive properties of the
dipeptide Val-Tyr in rat thoracic aortas
Vercruysse Lieselot1, Morel Nicole2, Van Camp John3, Szust Justyna4, and Smagghe Guy5
1
2
3
4
5
Ghent University, Department of Crop Protection & Department of Food Safety and Food Quality, 9000 Ghent – Belgium.
Université Catholique de Louvain, Laboratoire de Pharmacologie, 1200 Brussels – Belgium.
Ghent University, Department of Food Safety and Food Quality, 9000 Ghent – Belgium.
Ghent University, Department of Crop Protection & Department of Food Safety and Food Quality, 9000 Ghent – Belgium.
Ghent University, Department of Crop Protection, 9000 Ghent – Belgium.
Antihypertensive peptides derived from food proteins are biologically active peptides with a
potential role in prevention and treatment of hypertension. These peptides are proven to be in
vitro angiotensin converting enzyme (ACE) inhibitors, but the actual antihypertensive
mechanisms in vivo are still unclear. The aim of this research was to clarify the
antihypertensive mechanisms of the dipeptide, Val-Tyr. Val-Tyr was first identified as an in
vitro ACE inhibitory peptide derived from sardine muscle hydrolysed by Bacillus licheniformis
alkaline protease[1]. The in vivo antihypertensive activity in spontaneously hypertensive rats as
well as in human subjects has been reported, although the underlying mechanisms are not
fully elucidated[2,3]. Organ bath experiments with rat aortic rings were used to investigate five
important antihypertensive mechanisms, corresponding with the mode of action of the main
antihypertensive drugs used today. We confirmed ACE inhibitory activity in the organ baths.
Stimulation of the aortic rings with angiotensin I after incubation of the rings with 5 mM Val-Tyr
resulted in a decreased contraction, indicating ACE inhibition. The pD2-values (M) of 0 and 5
mM Val-Tyr were significantly different, 8.60 ± 0.04 and 7.96 ± 0.05, respectively.
Furthermore, we studied the effect of Val-Tyr on angiotensin II receptors, adrenergic
receptors, Ca2+ channels and NO dependent vasodilation in aortic rings, but no activity
towards these four mechanisms was detected. In conclusion, our results indicate the
specificity of the activity of the antihypertensive peptide Val-Tyr. Five important
antihypertensive mechanisms were tested, but only in vivo ACE inhibition could be detected[4].
References
1. Matsufuji, H.; Matsui, T.; Seki, E.; Osajima, K.; Nakashima, M.; Osajima, Y. Angiotensin I-converting enzyme inhibitory
peptides in an alkaline protease hydrolyzate derived from sardine muscle. Biosci. Biotechnol. Biochem. 1994, 58, 22442245.
2. Matsufuji, H.; Matsui, T.; Ohshige, S.; Kawasaki, T.; Osajima, K.; Osajima, Y. Antihypertensive effects of angiotensin
fragments in SHR. Biosci. Biotechnol. Biochem. 1995, 59, 1398-1401.
3. Saito, Y.; Wanezaki, K.; Kawato, A.; Imayasu, S. Antihypertensive effects of peptide in sake and its by-products on
spontaneously hypertensive rats. Biosci. Biotechnol. Biochem. 1994, 58, 812-816.
4. Vercruysse, L.; Morel, N.; Van Camp, J.; Szust, J.; Smagghe, G. Antihypertensive mechanism of the dipeptide Val-Tyr in rat
aorto. Peptides 2008, 29, 261-267.
87
POSTER PRESENTATIONS - Eleventh Naples Workshop on Bioactive Peptides
Natural membrane permeabilization induced by alamethicin and
its analogues under strictly physiological conditions
Marta De Zotti1, Fernando Formaggio1, Claudio Toniolo1, Natascia Vedovato2, Martina Infanti2,
Alberto Milani2, and Giorgio Rispoli2
1 Institute of Biomolecular Chemistry, Padova Unit, CNR, Department of Chemistry, University of Padova, 35131 Padova, Italy
2 CNISM and Department of Biology and Evolution, Section of Physiology and Biophysics, University of Ferrara, 44100 Ferrara, Italy
The biophysical characteristics and membrane-pore formation dynamics of synthetic, naturally
occurring, peptaibols were investigated by using isolated rod outer segments (OS) of reptilia
and amphibia recorded in whole-cell configuration. These peptides were applied to (and
removed from) the OS in ~50 ms with a computer-controlled microperfusion system. Once
blocked the main OS endogenous conductance (the cGMP channels) with saturating light, the
OS membrane resistance was mostly >5 GΩ. Peptides were applied and removed by
switching forth and back the OS from a stream of control solution (containing 120 mM K+) to a
stream of the same solution but containing the peptide, while holding the OS to constant
voltage (Vh). Patch pipettes were filled with the same perfusion solution in order to drive the
current just with Vh (generally –20 mV). Fast application of 1 µM synthetic alamethicin F50/5
produced a current after ~0.21 s from the solution exchange (called Delay) that activated
mono-exponentially (time constant τa~0.26 s) to a maximal amplitude (Imax) of ~700 pA.
Peptide removal caused the current to return to 0, with a non-measurable Delay, and again
mono-exponentially (time constant τd~0.31 s), showing the full reversibility of the
permeabilization process. Since the hydrophilic Gln residues at positions 7, 18 and 19 are
supposed to face the pore lumen, form hydrogen-bonded rings, and play a key role in channel
stability and ion permeation, they were substituted totally or partially with side-chain esterified
Glu [Glu(OMe)] residues. The spin-labeled analogue of [Glu(OMe)7,18,19] alamethicin F50/5,
bearing a 2,2,6,6 tetramethylpiperidine-1-oxyl-4-amino-4-carboxylic acid (TOAC) at position
16, [TOAC16, Glu(OMe)7,18,19], was also tested. Imax, Delay, τa and τd values of the current
produced by a 1 µM solution of the two latter peptides were respectively similar, ~8-fold, ~16fold and ~6-fold larger than those of alamethicin F50/5. Interestingly, the current exhibited a
large increase in noise. No difference was found between the recordings obtained with a 1 µM
solution of the [Glu(OMe)18,19] analogue and alamethicin F50/5 itself. All peptides tested
produced single channel events of different sizes at concentrations <250 nM: the most
probable one was ~50 pS for alamethicin F50/5 and its [Glu(OMe)18,19] analogue, while it was
10-fold larger (~500 pS) for the [Glu(OMe)7,18,19] and [TOAC16, Glu(OMe)7,18,19] analogues.
These results strongly suggest that the hydrophilic Gln residues at positions 7, 18 and 19 of
alamethicin F50/5 are not key factors for pore formation, but the substitution of Gln at position
7 produced larger pores with a lower probability of formation than those typical of alamethicin
F50/5. For all peptides tested, the current-to-voltage characteristics (obtained with voltage
ramps) showed a strong inward rectification. Moreover, the current was carried equally well by
monovalent and divalent cations.
In general, pore assembly and disassembly were observed as very fast and cooperative
events. The large variability of pore-forming properties of all these simple peptides makes
them an ideal tool for our understanding of the biophysical properties of channel and
transporter proteins and for the development of novel, membrane-active compounds.
88
New Frontiers in the Search of Bioactive Molecules: from Peptides to Drugs
Design and synthesis of Vascular Endothelial Growth Factor
receptors ligands
Auriemma Sara1, Diana Donatella2, Basile Anna3, Turco Caterina3, Pedone Carlo1, Fattorusso
Roberto2 and D’Andrea Luca D.2
1 CNR, Istituto di Biostrutture e Bioimmagini, 80134 Napoli – Italy.
2 Seconda Università di Napoli, Dipartimento di Scienze Ambientali, 81100 Caserta - Italy.
3 Università di Salerno, Dipartimento di Scienze Farmacologiche, 84084 Fisciano (SA) - Italy.
Angiogenesis is a physiological process that permits the formation of new blood vessel
starting from old ones. In same cases the angiogenic process becomes pathological and it
brings to different diseases, in particular to the tumoral cell growth. There are many molecular
systems involved in its regulation and the VEGF is considered to be the main responsible of
the angiogenic switch. VEGF is a homodimeric protein belonging to the family of cysteine knot
growth factors, and it binds with high affinity two tyrosine kinase receptors Flt-1 and KDR.
Recently has been reported that the VEGF homolog, Placenta Growth Factor (PlGF), which
binds only to Flt1 receptor, is able to modulate the angiogenic response. Solid state structure
of the complex between VEGF and domain 2 of Flt1[1] (VEGF/Flt1D2) and PlGF and Flt1D2 [2]
revealed the interacting regions of the two growth factors. In order to find new active
biomolecules which can modulate the interaction of VEGF and PlGF with their own receptors,
we designed and synthesized peptides mimicking the interacting region 79-92 of VEGF and
87-100 of PlGF, which in the natural protein adopt a β-hairpin conformation. Based on the Xray structure of the complex VEGF/Flt1D2 we have designed peptides stabilizing the native
fold and keeping the VEGF interacting residues. The synthesis of peptides were performed by
SPPS on a Wang resin using a standard Fmoc/tBu strategy.
The designed peptides were characterized by NMR in water solution and preliminary tested to
assess their biological properties.
References
1 Wiesmann et al. Cell ( 1997), 91, 695-704.
2 Christinger et al. J. Biol. Chem. (2004), 279, 10382-10388
89
POSTER PRESENTATIONS - Eleventh Naples Workshop on Bioactive Peptides
Stimulation of antiangiogenic activity of PSA by peptides
Koistinen Hannu1, Aaltonen Johanna1, Laakkonen Pirjo2, Närvänen Ale3 and Stenman Ulf-Håkan1
1 University of Helsinki, Department of Clinical Chemistry, 00014 University of Helsinki, Finland.
2 University of Helsinki, Molecular Cancer Biology Research Program and Institute of Biomedicine, 00014 University of Helsinki, Finland.
3 University of Kuopio, Department of Biosciences and AIV Institute, 70211 Kuopio, Finland.
Human umbilical vein endothelial cells (HUVECs) form tubular networks when cultured on top
of the Matrigel basement membrane preparation, reflecting the ability of the cells to form
blood vessels. Using this model, we have previously shown that PSA (also known as KLK3)
inhibits endothelial cell tube formation[1], indicating reduced angiogenic potential. Furthermore,
we have shown that the antiangiogenic activity of PSA is related to its enzymatic activity[1,2].
We have developed peptides that stimulate the enzymatic activity of PSA towards a small
colorimetric substrate. These peptides also enhance the antiangiogenic activity of PSA,
supporting our hypothesis that enhanced PSA-activity by our peptides could be used to
reduce tumor angiogenesis and, thus, to reduce tumor growth.
References
1 Mattsson JM, Valmu L, Laakkonen P, Stenman U-H, Koistinen H. Structural characterization and anti-angiogenic properties
of prostate-specific antigen isoforms in seminal fluid. Prostate, in press.
2 Koistinen H, Wohlfahrt G, Mattsson JM, Wu P, Lahdenperä J, Stenman U-H. Novel small molecule inhibitors for prostate
specific antigen. Prostate, in press.
90
New Frontiers in the Search of Bioactive Molecules: from Peptides to Drugs
Vascular Endothelial Growth Factor (VEGF) and its receptors: key
regulators of angiogenesis
Rossella Di Stasi1, Dominga Capasso2, Donatella Diana3, Roberto Fattorusso3, Carlo Pedone1,2 and
Luca D. D’Andrea1
1 Istituto di Biostrutture e Bioimmagini, CNR, 80134 Napoli – Italy.
2 Università di Napoli “Federico II”, Dipartimento delle Scienze Biologiche, 80134 Napoli – Italy.
3 II Università di Napoli, Dipartimento di Scienze Ambientali, 81100 Caserta – Italy.
Angiogenesis is a remodeling process characterized by the sprouting of new blood vessels
from pre-existing ones. It occurs during embryogenesis and to a limited extent in the adult, for
example in the female reproductive system, in physiological wound healing and in
pathological disease processes such as cancer[1]. Vascular endothelial growth factor (VEGF)
is a homodimeric protein and has been characterized as a prime regulator of angiogenesis
and vasculogenesis; when cells lose the ability to control the synthesis of VEGF, angiogenic
disease ensues[2]. In vitro studies show that VEGF is a potent and specific angiogenic factor
involved in the development of the vascular system and in the differentiation of endothelial
cells[3]. VEGF biological function is mediated through binding to two receptor tyrosine kinases:
the kinase domain receptor (KDR) and the Fms-like tyrosine kinase (Flt-1), which are
localized on the cell surface of various endothelial cell types. This binding activates signal
transduction and can regulate both physiological and pathological angiogenesis[4]. VEGF and
its receptors are in fact overexpressed in pathological angiogenesis, making this system a
potential target for therapeutic and diagnostic applications[5, 6].
The extracellular portion of VEGF receptors is comprised of 7 immunoglobulin-like domains
that is a common feature of membrane-anchored proteins; deletion studies have shown that
the ligand binding function resides within the first three domains of Flt-1 and in domains 2 and
3 of KDR. Both VEGF receptors contain several putative N-glycosylation sites and apparent
molecular weights of the mature proteins suggest that both receptors are extensively
glycosylated. Anyway, glycosylation is not a prerequisite of high affinity binding of VEGF to its
receptors[7]. Actually, no structural data are known on the extracellular portion of these
receptors except for the second domain of Flt-1[8]. So, our aim is the cloning and the
expression of part of extracellular domains of both VEGF receptors for structural
characterization and to be used in interaction studies with peptide ligands or small organic
molecules.
References
1 Folkman, J. (1999), Nat. Med. 1, 27-31.
2 Ferrara, N. (2002) Semin. Oncol. 29, 10-14.
3 Ferrara, N. (1989) Biochem. Biophys. Res. Commun. 161, 851-858.
4 Neufeld, G. et al. (1999), FASEB J., 13: 9-22.
5 Ferrara, N. and Davis-Smyth, T., (1997), Endocrinol. Res. 18, 4-25.
6 Ferrara, N. (2000), Curr. Opin. Biotechnol. 11, 617-624.
7 Millauer, B. et al. (1993), Cell 72, 835-846.
8 Wiesmann et al. ( 1997), Cell, 91: 695-704
91
POSTER PRESENTATIONS - Eleventh Naples Workshop on Bioactive Peptides
Enediyne-bridged dipeptide mimetics
Jerić Ivanka and Gredičak Matija
Ruđer Bošković Institute, Division of Organic Chemistry and Biochemistry, 10002 Zagreb –Croatia
The discovery of enediyne anticancer antibiotics in the late 1980s has arouse a broad interest
for this new family of compounds with, up to that time, unknown structures and intrigue
mechanism of action.[1] Till know, their chemical structure, in-site triggered activation, and
bioactivity profile were intensively studied. Also, an extensive search for new enediyne-related
structures have been activated, and this group is now enriched with numerous examples of
cyclic and acyclic enediynes, metalloendiynes, structures equipped with pH-dependent or
photo-triggering devices. Interestingly, there is a very limited number of enediyne-amino acid
or enediyne-peptide structures, although is well documented that enediyne-polyamine
conjugates and enediyne-lysine conjugates exhibit high affinity towards DNA, the site of action
of enediyne compounds.
Recently, we have described a synthetic route towards enediyne-bridged amino acid motifs,
as a general strategy for the preparation of C→N bridged -peptide units comprising the
enediyne hinge.[2] This procedure has been further improved and applied to a number of
enediyne bridged dipeptide units (Scheme 1).
O
Boc HN
N
H
O
O
R
H2 N
Boc HN
1a-f
Cl
+
R
Pd/Cu, piperidine
HO
O
H
N
HO
3a-f
OH
2
TFA/H2O (9/1)
Boc
N
1,3 a R = -CH2-C6H4-OBoc
b R = -CH2-C6H5
c R = -(CH2)4-NHBoc
d R = -CH(CH3)2
e R = -CH3
f R=H
N
H
R
O
THF
O
Boc N
N
H
4a-f
4
a R = -CH2-C6H4-OH
b R = -CH2-C6H5
c R = -(CH2)4-NH2
d R = -CH(CH3)2
e R = -CH3
f R=H
(YeG)
(FeG)
(KeG)
(VeG)
(AeG)
(GeG)
Compounds were prepared in a straightforward manner and characterized by spectroscopic
and mass spectrometry methods. Further, thermal reactivity of synthesized enediyne
compounds towards the Bergman cycloaromatization (BC) were studied by differential
scanning calorimetry.
References
1 Nicolaou, K., C.; Smith, A., L.; Yue, E., W. Proc. Natl. Acad. Sci. U.S.A. 1993, 90, 5881-5888
2 Jerić, I.; Chen, H.-M. Tetrahedron Lett. 2007, 48, 4687-4690.
92
New Frontiers in the Search of Bioactive Molecules: from Peptides to Drugs
Branched peptides for cancer cells targeting
Chiara Falciani1, Jlenia Brunetti1, Alessandra Cappelli1, Barbara Lelli1, Silvia Pileri1, Alessandro Pini1,
Luisa Lozzi1, Stefano Menichetti2, Chiara Pagliuca2, Lapo Bencini3, Renato Moretti3, Luisa Bracci1
1 University of Siena, Department of Molecular Biology, 53100 Siena – Italy
2 University of Florence, Department of Organic Chemistry, 50019 Firenze – Italy .
3 Azienda Ospedaliero-Universitaria di Careggi, SOD Chirurgia Generale Firenze– Italy .
Innovative chemotherapy through selective tumor targeting agents, has not fulfilled the
promising results that were envisaged since many years ago. Antibodies and, more recently,
small molecules, have been used as targeting agents, and both have limits and advantages[1].
We demonstrated here that oligo-branched peptides can be efficient tumor targeting agents
that conjugate advantages of antibodies and small molecules. They allow multimeric binding
with a molecular weight much lower than antibodies and can be easily synthesized and
chemically modified, like the small molecules[2]. We produced tetra-branched peptides
targeting the neurotensin receptors, which are over-expressed in a number of human tumors.
Tetra-branched peptides were used either for spotlighting tumor cells or for killing them, by
simply exchanging the functional moiety that can be coupled to a conserved targeting core.
The approach resulted very promising for the personalized therapy of tumors that overexpress neurotensin receptors, like colon, pancreas and prostate carcinoma, and might be
applied to many different tumor targets.
References
1 Collins I, Workman P. New approaches to molecular cancer therapeutics. Nat Chem Biol. 2006, 2(12):689-700.
2 Falciani C, Fabbrini M, Pini A, Lozzi L, Lelli B, Pileri S, Brunetti J, Bindi S, Scali S, Bracci L. Synthesis and biological activity
of stable branched neurotensin peptides for tumor targeting. Mol Cancer Ther. 2007; 6(9):2441-8.
93
POSTER PRESENTATIONS - Eleventh Naples Workshop on Bioactive Peptides
Structure, function and in vivo activity
of a branched antimicrobial peptide.
Luisa Bracci, Alessandro Pini, Chiara Falciani, Sara Iozzi, Jlenia Brunetti, Silvia Pileri, Barbara Lelli,
Andrea Bernini and Neri Niccolai.
University of Siena, Department of Molecular Biology, 53100 Siena – Italy
The growing emergency of multi-drug resistant bacteria is a global concern: a number Grampositive and Gram-negative bacteria have developed resistance against most traditional, as
well as new generation antibiotics. Therefore, the demand for new antibiotics urges
researchers and pharmaceutical companies to consider new antimicrobial agents. Among
these, antimicrobial peptides turned out to be particularly interesting, in consideration of their
peculiar mechanism of action, which is specifically targeted to bacterial membrane.
Nonetheless, pharmaceutical companies have shown a general reluctance to the
development of peptide drugs, which can be explained by a number of problems related to
development of peptide as drugs, including their short half life produced by rapid proteolysis.
We selected by phage display against E.coli, a non-natural peptide sequence, which showed
a strong antimicrobial activity against Gram-negative bacteria[1]. This peptide was synthesized
in a Multiple Antigen Peptide (MAP) form, which we had previously demonstrated to induce
general resistance to proteolysis, rendering peptides more suitable for therapeutic
applications[2,3]. The antimicrobial branched peptide (M6) was characterized for its activity
against a number of bacteria, including many multi drug resistant isolates and showed very
promising MIC against infectious pathogens of clinical interest. We also demonstrated that M6
shows a poor toxicity for eukaryotic cells, it binds LPS and it does not produce appreciable
haemolysis even upon prolonged incubation. Moreover, we evaluated M6 acute toxicity and
we also demonstrated that it is not immunogenic upon repeated injections in animals[4].
We report here on M6 in vivo activity in models of sepsis induced in mice by E. coli and P.
aeruginosa. These experiments showed that the peptide can prevent animal death and can
neutralize sepsis symptoms when used in doses comparable to traditional antibiotics and
compatible with a clinical use.
These results make the branched M6 peptide a strong candidate for the development of a
new antibacterial drug.
References
1 Pini et al. Antimicrob Agents Chemother. 2005; 49: 2665-72.
2 Bracci et al. J Biol Chem. 2003; 278: 46590-5.
3 Falciani et al. Chem Biol Drug Des. 2007; 69: 216-21
4 Pini et al. J Pept Sci. 2007; 13: 393-9.
94
New Frontiers in the Search of Bioactive Molecules: from Peptides to Drugs
Synthesis and conformational analysis of homo-oligopeptides of
a novel amino acid, α,α-dicyclopropylglycine
Takashi Yamada1, Shio Fujioka1, Shin Gohda1, Takashi Murashima1, Toshifumi Miyazawa1, and
Yasuko In 3
1 Konan University, Department of Chemistry, 658-8501 Kobe – Japan.
2 Osaka University of Pharmaceutical Sciences, 569-1094 – Japan.
α,α-Disubstituted glycines represent useful tools to make the peptide backbone conformation
into a well-defined secondary structure. The conformational preferences of various α,αdisubstituted glycines have been extensively studied both in solution and in the solid state.
For α,α-dimethylglycine (Aib) the folded 310- and α-helical structures are more stable than the
extended structure. On the contrary, for α,α-diethylglycine (Deg) and α,α-dipropylglycine
(Dpg) with substituents at the Cβ atoms, the extended C5
conformation seems to be more stable. Previously, we reported the
crystal structure of a homo-dipeptide (Z-Dcp-Dcp-OMe) of a novel
α,α-disubstituted glycine, α,α-dicyclopropylglycine (Dcp)[1]. The
preliminary study showed that the Dcp residue, similarly to Aib,
H2N C COOH
prefers folded structures. With the aim of clarifying such propensity
of Dcp, we have undertaken synthesis and a conformational analysis
of Dcp homo-oligopeptides.
The tri- and tetrapeptides of Dcp (Z-Dcp-Dcp-Dcp-OMe and Z-DcpDcp
Dcp-Dcp-Dcp-OMe) were synthesized in moderate yields by using
the EEDQ (N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline) method
and the CMPT (2-chloro-1-methylpyridinium p-toluenesulfonate) /
AC9M (Acid Captor 9M: 9-methyl-3,4-dihydro-2H-pyrido[1,2-a]pyrimidin-2-one) method. The
X-ray crystal structural analysis has clarified that the Dcp homo-tetrapeptide adopts a 310 helix (Fig. 1). In addition, NMR analysis has shown that the preferred conformation of the
same peptide in chloroform solution is consistent with the crystal structure. The Dcp residue
has moderate reactivity, and thus it may be one of promising conformationally constrained
building blocks.
Fig. 1. Crystal structure of Z-Dcp-Dcp-Dcp-Dcp-OMe.
References
1 De Simone, G., Lombardi, A., Galdiero, S., Nastri, F., Di Costanzo, L., Gohda, S., Sano, A., Yamada, T., Pavone, V.,
Biopolymers, 2000, 53, 182-188.
95
POSTER PRESENTATIONS - Eleventh Naples Workshop on Bioactive Peptides
Modulation of activity and anxiety by cortexin polypeptide
extract and its synthetic analog cortagen in mice
Walter Adriani1, Oleg Granstrem2, Emilia Romano1, Giovanni Laviola1
1 Section of Behavioral Neurosciences, Dept. Cell Biology & Neurosciences, Istituto Superiore di Sanità, Rome, Italy.
2 Dept. Neurology & Neurosurgery, I.P. Pavlov State Medical University, St. Petersburg, Russia.
Objective. Cortexin is a polypeptide extract, which is used in clinics for its positive effects on
memory, attention, and cortical processes[1]. A synthetic analog of cortexin, Cortagen (i.e. AlaGlu-Asp-Pro peptide), was developed. Both are able to stimulate neural growth in vitro,
presumably in association with neurotrophic factors. We assessed, in outbred CD-1 mice, the
potential psychoactive effects of both cortexin and cortagen, using the elevated plus maze
(EPM) and the locomotor activity habituation (LAH) paradigms. In Exp. I, mice were injected
with cortexin (0, 0.25, 0.50, or 1.00 mg/kg i.p.) and tested in the EPM (acute) and the LAH
(sub-chronic response). In Exp. II, a separate set of mice were injected with cortagen (0, 0.01,
0.03, or 0.10 mg/kg i.p.) or a selected reference dose of cortexin, and tested in the LAH (acute
and sub-chronic response), and in the EPM (sub-chronic response).
Results. Convincing evidence of anxyolitic effects was found for acute cortexin treatment at
the 0.25 and 1.00 mg/kg dosages. Indeed, mice spent an elevated amount of time in the open
arms of the EPM and showed a shorter latency to enter them, together with fewer riskassessment postures, compared to controls. The cortexin 0.25 mg/kg was selected as
reference dose for Exp. II, since it had no locomotor effects over a sub-chronic regimen (4
days), whilst the 1.00 mg/kg dose led to the development of hyperactivity. When comparing to
this cortexin reference, we found only one active dose of cortagen (i.e. 0.03 mg/kg): it was
able to enhance locomotion, both upon acute and after sub-chronic treatment, also having few
modulatory effects on anxiety-related behavior. Conversely, following the sub-chronic regimen
(5 days), the reference dose of cortexin turned out to produce hyperlocomotor and anxiogenic
effects.
Conclusion. The cortexin can decrease levels of anxiety when given acutely, but it may
sensitize towards an anxiogenic kind of arousal following repeated treatment. Conversely,
both upon acute and after sub-chronic administration, the cortagen leads to motor stimulation
with no side effects on emotional-affective profiles. Such kind of behavioral stimulation may
find room for beneficial employment in the treatment of neuro-psychiatric diseases, like e.g.
major depression. Like cortexin, cortagen may indeed have nootropic, neuroprotective and
neurotrophic effects in humans. Peptides are active in very low non-toxic dosages with no
side effects. According to literature[1], these compounds are worth of deeper investigation
because of their promising role for therapy.
This work was supported by Geropharm Ltd, Russia, and by the European Mind and Metabolism
Association
References
1. Tsyganov VN, Bogoslovskii MM (2004). Influence of cortexin on memory and attention [in Russian]. Voen Med Zh. 325: 315, 80.
96
New Frontiers in the Search of Bioactive Molecules: from Peptides to Drugs
Pathophysiological changes of gram-negative bacterial
infection can be reproduced by a synthetic peptide mimicking
loop L7 sequence of Haemophilus influenzae porin
Vitiello Mariateresa1, Galdiero Marilena1, D’Amico Michele1, Di Filippo Clara1, Cantisani Marco2,
Finamore Emiliana1, Benedetti Ettore2 and Galdiero Stefania2
1 Department of Experimental Medicine - II University of Naples – 80138 Naples, Italy
2 Department of Biological Sciences & CIRPEB - University of Naples “Federico II” – 80134 Naples, Italy
Activation of the coagulation and fibrinolytic systems is an important manifestation of the
systemic inflammatory response of the host to infection. Several in vivo models have been
used to dissect the molecular mechanisms that contribute to these dysfunctions by bacteria
and bacterial products but many aspects remain poorly understood. In this study we examined
the in vivo effect of the synthetic peptide corresponding to loop L7 from Haemophilus
influenzae type b (Hib) porin compared with the effect of the entire protein to evaluate its role
on the coagulative/fibrinolytic cascade and the circulating markers of endothelial injury.
Plasma was obtained from rats injected intravenously with loop L7, Hib porin or a scrambled
peptide and tested for fragment 1+2 (F1+2), tissue-type plasminogen activator (tPA),
plasminogen activator inhibitor type I (PAI-1) antigen, von Willebrand factor (vWF) and soluble
E-selectin (sE-selectin). The coagulative/fibrinolytic cascade was impaired as shown by PAI-1
level increased. Concomitantly, E-selectin, a marker of endothelial injury, was also
significantly elevated. In addition either loop L7 or Hib porin injection induced hyperglycaemia
and inflammatory cytokine production. The data were correlated with hemodynamic functions
(significant reduction of blood pressure and increase of heart rate). The results indicate that,
in our experimental model, loop L7 plays an essential role in the pathophysiologic events
observed during gram-negative infection. These findings may have implications for the
development of alternative therapies to counteract excessive inflammatory responses during
septic shock.
97
POSTER PRESENTATIONS - Eleventh Naples Workshop on Bioactive Peptides
Polypetide antagonists of IgE-FcεRI interaction
Sandomenico A.1,2, Marasco D.2, Monti S.M. 2, Dathan N.2, Benedetti E. 1,2, Pedone C.1,2, and Ruvo M2.
1 Dipartimento delle Scienze Biologiche, 80134, Napoli, Italy
2 . Istituto di Biostrutture e Bioimmagini (IBB), CNR, 80134, Napoli, Italy
The human high affinity IgE receptor, hFcεRI, found on the surface of mast cells and
basophils, is believed to mediate allergic diseases, anaphylaxis and asthma through binding
of IgE itself. Antagonizing the interaction between IgE and the receptor is thereby a useful
approach to block the early events of allergic diseases and also suppress the associated
symptoms. hFcεRI contains four distinct polypeptide chains: an α chain, a β chain and a dimer
of γ chains. The extracellular portion of the α chain binds with high affinity to the Fc region of
the IgE (KD=10-9 M), whereas the β and γ chains are responsible of down-stream signal
propagation through phosphorylation of their intracytoplasmatic immunoreceptor tyrosinebased activation motifs (ITAM). The crystallographic structure of αFcεRI-FcIgE shows that the
N-terminal region of the FcεRI-α chain is made up of two Ig-like domains called D1 e D2 that
form an acute angle, like an inverse V, and that the convex surface, comprising the D2
domain, constitutes the binding interface with the IgE. Nevertheless, the D1 domain, though
not being directly involved in the binding with the IgE structurally contributes to the high affinity
maintenance[2]. In the attempt to identify specific FcεRI antagonists, we have designed small
receptor-mimicking peptides that bind to soluble IgE with low affinity (10-4-10-5 M) and are also
able to inhibit the IgE-receptor interaction. Three different peptides, called IgEtrap, 1 loop and
2 miniloop show binding for the IgE with KD of 2.4±0.5*10-5, 6.5±0.5*10-4 M and 5.7±0.5*10-4
M, respectively. However, despite the low affinity, the peptides exhibit a high specificity for this
class of immunoglobulins, as they don’t bind to IgG and IgA.
To perform these studies and to try to obtain a receptor variant useful as soluble antagonist,
we have also undertaken the recombinant expression of the receptor in E.Coli. Both the D1D2 and the D2 domains were expressed in BL21(DE3) cells. While the refolding of the D1-D2
domain is still underway, the D2 domain alone was readily renatured (CD spectrum consistent
with an all-beta protein) and used in binding and competition assays with the synthetic
peptides. The recombinant His6-D2 fusion protein exhibits a KD of 3.6±1*10-6 (by Biacore
assay), a value that is in agreement with that estimated by ELISA assay (about 4µM). This
relatively low affinity compared to the full length receptor, is probably due to the lack of the
adjacent D1 domain that stabilizes the whole structure by keeping in place key contact
residues on the D2 portion.
98
New Frontiers in the Search of Bioactive Molecules: from Peptides to Drugs
1.4-DHP-lipid forms a tubular micellae
Inta Liepina1,2, Gunars Duburs1, Cezary Czaplewski3, Alex Bunker2
1. Latvian Institute of Organic Synthesis, Riga, LV1006, Latvia
2. Drug Design Tecnology Center, University of Helsinki, Helsinki 00014, Finland,
3. Faculty of Chemistry, University of Gdansk, 80-952 Gdansk, Poland
1,1’-{[3,5-bis(dodecyloxycarbonyl)-4-phenyl-1,4-dihydropyridin-2,6-diyl]dimethylene}
bispyridinium dibromide (1,4-DHP lipid) is a gene transfection agent[1,2]. 1,4-DHP lipid
structure was calculated with ab initio quantum mechanics to obtain the charges for molecular
dynamics (MD) with AMBER 8.0 force field. 1,4-DHP-lipid molecules were subjected to MD
from the initial structure of a periodic lipid bilayer-water box, with a small amount of excessive
water on the lipid edges to ensure the mobility of lipid molecules. After 14 ns of MD simulation
the lipid molecules with the fatty acid tails started to squeeze from one bilayer layer to another
one. After 35 ns few lipid molecules turned with their charged heads to the side of the lipid
bilayer and after 100 ns a profound tubular micelle structure began to form. The tubular
micelle structure becomes more perfect during the course of simulation of 300 ns (Fig).
Conclusion is that one of the gene transfection agent 1,4-DHP lipid structures is a tubular
micellae, and we could expect that such the micellaes are capable to form lipoplex for the
DNA transfection.
Fig. 1,4-DHP lipid tubomicellae side view and top view.
Acknowledgements. This work was supported by Stipend of Finland Academy of Sciences for IL, and by Latvian Science
Council Grant 05.1768.
Calculations were performed on computers of the Gdansk Academic Computer Center TASK.
References
1 Z.Hyvonen, A.Plotniece, I.Reine, B.Chekavichus, G.Duburs, A.Urtti Novel cationic amphiphilic 1,4-dihydropyridine
derivatives for DNA delivery. Biochim. Biophys. Acta, (2000) 1509, 451 – 466.
2 Z.Hyvonen, S. Ronkko, M.-R. Toppinen, I. Jaaskelainen, A.Plotniece, A.Urtti . Dioleoyl phosphatidylethanolamine and PEGlipid conjugates modify DNA delivery mediated by 1,4-dihydropyridine amphiphiles. Journal of Controlled Release, (2004)
99, 177-190.
99
POSTER PRESENTATIONS - Eleventh Naples Workshop on Bioactive Peptides
Mild electrophilic S-trifluoromethylation of cysteine side
chains in α- and β-peptides: synthesis and application
Stefania Capone1, Iris Kieltsch2, Antonio Togni2 and Dieter Seebach1
1. Department of Chemistry and Applied Biosciences. ETH-Zürich, Hönggerberg, HCI, CH-8093, Zürich, Switzerland
2. Laboratory of Organic Chemistry & Laboratory of Inorganic Chemistry ETH-Zürich, Hönggerberg, HCI, CH-8093, Zürich, Switzerland
The introduction of trifluoromethyl groups into drug candidates is of particular interest in
medicinal chemistry[1]. Effects of an SCF3 group on biological activities of organic molecules
such as aryl- or alkyl thioethers are also well known[2]. We were interested to see whether
analogous effects can be found when cysteine thiol groups in peptides are trifluoromethylated.
The electrophilic trifluoromethylation is a challenging synthetic problem. However, recently
one of our groups has developed hypervalent iodine(III)–CF3 reagents A and B[3] that can lead
to S-trifluoromethylation of simple cysteine esters (either N-protected or unprotected) in high
yield under very mild reaction conditions[3b]. We now applied the trifluoromethylation to α- and
β-peptides containing cysteine side chains. The reaction proceeds smoothly with excellent
yields and good selectivity, also in the presence of free amino (1b, 2b), carboxylic acid (3-5)
and hydroxy (5) groups. If desired, the thus introduced CF3 groups can be readily removed
under reducing conditions (Na/NH3).
O
F3C I
F3C I
O
O
R 1 HN
CO 2Me
SCF3
N
H
O
MeO2 C
A
1a: R1=Boc
1b: R1=H2+TFA-
B
O
SCF3
BocHN
O
Ph
2a: R1=Boc
2b: R1=H2+TFA-
Ph
O
O
O
O
O
HO
O
O
H2N
N
H
H
N
HO
O
N
H
O
HO
O
H
N
N
H
H
N
O
N
H
H
N
O
N
H
H
N
O
N
H
HO
HO
H
N
O
OH
4
NH
6a: disulfide, R3 = H
H
N
N
H
R2S
O
N
H
R3
Ph
O
H
N
O R1S
NH
O
Ph
5
H2N
OH
BocHN
3
Ph
O
O
O
N
H
NH2
F3CS
F3CS
F3CS
Ph
NHR 1
H
N
(Octreotide)
O
O
O
N
H
HO
NH 2
H
N
6b: R1 = R2 = R3 = H
6c: disulfide, R3 = CF3
6d: R1 = R2 = CF3; R3 = H
6e: R1 = R2 = R3 = CF3
O
NH 2
Since we also wanted to learn to which extent the biological activity is affected upon
replacement of a thiol by an SCF3 group in a peptide, we applied the trifluoromethylation for
modification of the drug Octreotide (Sandostatin®). The disulfide moiety of Octreotide was
reduced, and the resulting dithiol 6b treated with reagents A or B. The reaction was not
selective, producing mixtures of derivatives 6c-e, two of which contain a trifluoromethylated
tryptophan residue. The three new compounds (6c-e) are currently subjected to biological
tests to find out how the CF3 groups influence the activity as compared to Octreotide.
References
1 (a) Special issue on “Fluorine in the Life Sciences”, ChemBioChem 2004, 5, 557 – 726. (b) Fluorine-containing Amino
Acids, Synthesis and Properties; Kukhar, V. P., Soloshonok, V. A., Eds.; J. Wiley and Sons: New York, 1995. (c) Biomedical
Aspect of Fluorine Chemistry; Filler, R., Kobayashi, Y., Eds.; Kodansha Ltd.; Tokyo, Elsevier Biomedical: Amsterdam, 1982.
(d) Asymmetric Fluoroorganic Chemistry, Synthesis, Application and Future Directions; Ramachandran, P. V., Eds.;
American Chemical Society: Washington, DC, 2000. (e) Organofluorine Compounds in Medicinal Chemistry and
Biochemical Applications; Filler, R., Kobayashi, Y., Yagupolskii, L. M., Eds.; Elsevier: Amsterdam, 1993. (f) Fluorine in
Bioorganic Chemistry; Welch, J. T., Eswarakrishnan, S., Eds.; Wiley, New York, 1991.
2 (a) Kolomeitsev, A. A., Chabanenko, K. Yu., Röschenthaler, G.-V., Yagupolskii, Yu. L. Synthesis 1994, 145-146. b) Tang,
R.-Y., Zhong, P., Lin, Q.-L. Journal of Fluorine Chemistry 2007, 128, 636–640 and cited references.
3 (a) Eisenberger, P., Gischig, S., Togni, A. Chem. Eur. J. 2006, 12, 2579-2586. (b) Kieltsch, I., Eisenberger, P., Togni, A.
Angew. Chem. Int. Ed. 2007, 46, 754 –757.
100
New Frontiers in the Search of Bioactive Molecules: from Peptides to Drugs
New glutathionyl conjugates of caffeic acid and its
esters: an assessment of their potential as
antioxidant and antinitrosating agents
Lettieri Gregory1, De Lucia Maria2, Panzella Lucia2, Napolitano Alessandra2 and d’Ischia Marco2.
1 Erasmus student from Univ. Paul Cezanne Aix-Marseille III
2 Univ. Naples Federico II, Dept of Organic Chemistry and Biochemistry, 80126 Naples-Italy
Caffeic acid and its ester chlorogenic acid (5-caffeoyl-D-quinic acid) belong to a group of
dietary polyphenolic micronutrients of plant origin endowed with antinflammatory and
chemopreventive properties against colorectal cancer and other tumors of the digestive
tract.[1] Most of the effects of these compounds have been attributed to their protective action
on oxidative stress in vivo and their potent antinitrosating properties, preventing carcinogenic
nitrosamine formation, mutation and nitrosative DNA damage.[2,3] Search for novel non-toxic
derivatives of polyphenolics of natural origin with enhanced antioxidant and antinitrosating
activities is therefore the focus of current interest in food, pharmaceutical and dermo-cosmetic
industry. Among candidate prototypes, glutathionyl derivatives are of particular interest [4]
because they are involved in the physiological metabolism of phenols and occur commonly in
wine and other food, e.g. 2-S-glutathionylcaftaric acid.
We report here the preparation of a conjugation product of caffeic acid with glutathione (GSH),
1, and three GSH adducts of chlorogenic acid (2a-c). These were obtained in good yields (up
to 70%) by one-step oxidation of the phenolic compounds in the presence of glutathione,
followed by HPLC purification. A variety of oxidizing agents of biological relevance including
tyrosinase, peroxidase/H2O2, proved able to carry out the reaction.
NH2
H
N
HOOC
N
H
O
S
1
HO
HO
HOOC
O
COOH
O
R1
HO
OH
O
HO
OH
OH
R3
R2
NH2
O
OH
H
N
HOOC
GS=
O
N
H
O
COOH
S
2a: R1= GS, R2,R3= H
2b: R1, R2= GS, R3=H
2c: R1, R2, R3= GS
The antioxidant properties of the mono adducts 1 and 2a were assayed by 2,2-diphenyl-1picrylhydrazyl (DPPH) radical scavenging assay in comparison with the parent compounds.
Both 1 (51% inhibition) and 2a (45% inh.) proved significantly more effective than caffeic acid
(35% inh.) and chlorogenic acid (30% inh.). Scavenging of nitrosating species was assayed
by two currently used test reactions, namely the diaminonaphthalene (DAN) assay and the
inhibition of nitrotyrosine (NT) formation. A significant reduction of the activity in the DAN
assay was observed for 1 with respect to caffeic acid, and a similar trend was obtained for 2a.
In NT inh the efficiency order was chlorogenic acid > caffeic acid>1>2a.
Interpretation of the effects of the alkylthio residue of GSH on the antioxidant and
antinitrosating properties of these o-diphenolic compounds will be discussed.
References
1. Kris-Etherton, P.M. et al Am. J. Med. 2002, 113, Suppl. 9B 71S-88S.
2. d’Ischia, M.; Panzella, L.; Manini, P.; Napolitano A. Curr. Med. Chem. 2006, 13, 3133-3144.
3. Roche M.; Dufour, C.; Mora, N.; Dangles, O. Org. Biomol Chem. 2005, 3, 423-430.
4. Lozano, C. et al. FEBS Lett. 2005, 579, 4219-4225.
101
POSTER PRESENTATIONS - Eleventh Naples Workshop on Bioactive Peptides
New insights on DABA-based nucleopeptides
Castiglione Mariangela1, Roviello Giovanni1, Musumeci Domenica2, Cesarani Annalisa1, Benedetti
Ettore3, Bucci Enrico2 and Pedone Carlo1
1 CNR, Ist. di Biostrutture e Bioimmagini, 80134 Napoli – Italy.
2 Bionucleon srl, 80131 Napoli – Italy.
3 Università Federico II, Dip. delle Scienze biologiche, 80134 Napoli – Italy.
In continuing our research on ODN analogs suitable for a wide variety of biomedical and
bioengineering applications, here we report novel studies relative to a chiral nucleopeptide
with a diaminobutyric acid (DABA) backbone. In particular, in this work we describe the
synthesis of the nucleoaminoacid monomer possessing the D stereochemistry, performed in
analogy to that we recently published on the L-DABA derivative[1], and the oligomerization
using both enantiomers to form the alternate D,L- nucleopeptide.
O
N
N
O
H
N
NHR
NH
O
N
O
O
H
H
N
N
O
N
O
O
H
N
N
H
O
NH
O
N
HN
O
N
O
H
O
O
N
H
O
H
N
NH
HN
RHN
O
O
N
O
H
O
H
O
O
N
N
H
HN
O
O
N
O
This oligomer, characterized by ESI-MS and CD spectroscopy, was studied for its ability to
bind not only complementary DNA, but also a free nucleobase in order to develop
supramolecular structures, based on hydrogen bonding and controllable in a thermoreversible
fashion. This kind of structures is of particular interest for the development of new
nanomaterials with many desirable properties as well as of new ODN-analogues for
biotechnological applications.
References
1. Roviello, G.N. et al. J. Pept. Sci. 2006, 12, 829-835; and Roviello, G.N. et al. , Nucleos. Nucleot. Nucleic Acids 2007, 26,
1307-1310.
102
New Frontiers in the Search of Bioactive Molecules: from Peptides to Drugs
Analogues of AVP and its agonists modified in the
N-terminal part of the molecule with homoproline isomers
Anna Kwiatkowska1, Dariusz Sobolewski1, Adam Prahl1, Jiřina Slaninová2, and Bernard Lammek1
1 Faculty of Chemistry, University of Gdańsk, 80-952 Gdańsk, Poland;
2 Institute of Organic Chemistry and Biochemistry, Academy of Sciences of the Czech Republic, 16610 Prague, Czech Republic.
During the past decade, the incorporation of unnatural non-proteinogenic α-amino acids into
peptides has emerged as a novel and promising approach in peptide synthesis. The
conformationally restricted amino acid derivatives are of particular interest.
We have designed ten new analogues of arginine vasopressin (AVP) modified in the Nterminal part of the molecule with pipecolic acid (Pip) isomers, a non-proteinogenic α-imino
acid, also know as homoproline. This modification, apart from reducing the flexibility, also
changed the character of a fragment of the molecule from aromatic to aliphatic. We
synthesized the following analogues: [L-Pip2]AVP (I), [Mpa1,L-Pip2]AVP (II), [D-Pip2]AVP (III),
[Mpa1,D-Pip2]AVP (IV), [L-Pip3]AVP (V), [Mpa1,L-Pip3]AVP (VI), [D-Pip3]AVP (VII), [Mpa1,DPip3]AVP (VIII), [Mpa1,L-Pip3,Val4,D-Arg8]VP (IX), [Mpa1,D-Pip3,Val4,D-Arg8]VP (X).
Ten new analogues of AVP (I-X) were synthesized manually, using Fmoc chemistry.
Surprisingly enough, peptides I-IV demonstrated a high sensitivity to trifluoroacetic acid (TFA)
used for deprotection and cleavage of the synthesized peptides from the resin. The products
of fragmentation are presented in Table 1.
Table 1. TFA-catalyzed hydrolysis of peptides I-IV during TFA cleavage from the resin.
[L-Pip2]AVP
I
Molecular iona
Products of cleavage
calc.
found
1032.2 931.2 H-Cys + L-Pip-Phe-Gln-Asn-Cys-Pro-Arg-Gly-NH2
[Mpa1,L-Pip2]AVP
II
1017.2 820.2
Mpa-L-Pip + Phe-Gln-Asn-Cys-Pro-Arg-Gly-NH2
[D-Pip ]AVP
III
1032.2 931.2
H-Cys + D-Pip-Phe-Gln-Asn-Cys-Pro-Arg-Gly-NH2
[Mpa1,D-Pip2]AVP
IV
1017.2 820.3
Mpa-D-Pip + Phe-Gln-Asn-Cys-Pro-Arg-Gly-NH2
Peptide
2
a
Mass spectra of the peptides were recorded on a MALDI TOF mass spectrometer.
For analogues II and IV, we propose a mechanism of cleavage via formation of an oxazolonelike intermediate from a species protonated at the Pip residue by nucleophillic attack of an
adjacent carbonyl group.[1] In regard to peptides I and III protonation of the free N-terminal
amino group results in both decreased electron density (-I effect) on the carbonyl oxygen of
the Cys1 residue and in formation of a hydrogen bond between the N-terminus and that
oxygen. Consequently, the acid-promoted hydrolysis of the amides can serve as a model for
cleavage of the peptide bond in analogues I and III.[2]
Our results, while not impressive in terms of biological activities of the reported analogues,
offer important information on structure – activity relationships and response of the L- or DPip-containing peptides to standard TFA cleavage conditions.
References
1. Urban, J., Vaisar, T., Shen, R. and Lee, M. S. Int. J. Pept. Protein Res. 47, 182-189 (1996).
2. Brown, R., Bennet, A. and Ślebocka-Tilk, H. Acc. Chem. Res. 25, 482-488 (1992).
103
POSTER PRESENTATIONS - Eleventh Naples Workshop on Bioactive Peptides
From amyloid fibers to toxic oligomers:
insights from molecular dynamics simulations
De Simone Alfonso1,2, Esposito Luciana2, Pedone Carlo2,3 and Vitagliano Luigi2
1 Department of Chemistry,University of Cambridge, Lensfield Road CB2 1EW, Cambridge UK
2 Istituto di Biostrutture e Bioimmagini, C.N.R., 80134 – Napoli, Italy
3 Dipartimento delle Scienze Biologiche, Unibersità Federico II, 80134 Napoli, Italy.
The insurgence of severe neurodegenerative disease is frequently associated with insoluble
amyloid fibrils by proteins and peptides that often possess a globular fold in their precursor
state. A deep understanding of the protein misfolding process requires detailed information on
the factors stabilizing/destabilizing the globular state (1) and on the structural features of
amyloid fibrils. The structural characterization of these fibrils has long been hampered by the
very low solubility and by the non-crystalline nature of these aggregates.
Recently, high-resolution structures of several amyloid-like peptides (2-3) offered an atomic
detailed model for amyloid-like fibrils, namely cross-β spine steric zipper. In order to obtain
further insights into the determinants of amyloid fibers structure/formation and to analyze the
effects of crystal packing on aggregate structures, we employed molecular dynamics (MD)
simulations on a variety of different models arranged in a cross-β spine structure (4-7). The
analysis of large aggregates formed by the peptide GNNQQNY shows that these assemblies
can assume twisted β-sheeted structures. The evolution of pairs of sheets separated by a wet
interface during the simulation has additionally provided interesting information on the
structure of larger aggregates (4). These analyses, extended to polyglutamine fragments (5-6)
and to the human prion fragment SNQNNF (7), have clearly indicated that the steric zipper
motif is the basic scaffold of amyloid fibers formed by proteins directly involved in
neurodegenerative diseases.
In recent years, new evidences shed light on the role of small soluble amyloid precursors as
the actual toxic species (8). However, little is known about these particular aggregates since
experimental limitations prevent their characterization. To construct the conformational free
energy of amyloid precursor aggregates, we carried extensive replica exchange molecular
dynamics simulations on small aggregates of GNNQQNY. Analyses of small-sized assemblies
provided information on the structural properties of possible intermediate states along the fiber
formation pathway (9). The data evidenced new insights into the structural basis of amyloid
oligomers formation and toxicity (9). Our model is also able to explain the toxicity of
monomeric polyglutamine peptides (5).
References:
1 De Simone, A.;. Dodson, G. G; Verma C; S.; Zagari, A.; Fraternali, F. PNAS. 2005, 102, 7535-7540.
2 Nelson, R.; Sawaya, M.R.; Balbirnie, M.; Madsen, A.O.; Riekel, C.; Grothe, R.; Eisenberg. D. Nature. 2005. 435, 773-778.
3 Sawaya, M. R., Sambashivan, S.; Nelson, R.; Ivanova, M.I.; Sievers, S.A.; Apostol, M.I.; Thompson, M.J.; Balbirnie, M.;
Wiltzius, J.J.; McFarlane H. T.; Madsen, A.O.; Riekel, C.; Eisenberg. D. Nature. 2007, 447, 453-457.
4 Esposito, L.; Pedone, C.; Vitagliano, L. PNAS. 2006, 103, 11533-11538.
5 Esposito, L.; Paladino, A., Pedone, C.; Vitagliano, L. Biophysical J. 2008, In press.
6 Colombo, G.; Meli, M.; De Simone, A.; Proteins. 2008, 70, 863-872.
7 De Simone, A.; Pedone, C.; Vitagliano, L. Biochem Biophys Res Commun. 2008, 366, 800-806.
8 Chiti, F.; Dobson, C.M. Annu Rev Biochem. 2006, 75, 333-366.
9 De Simone, A; Esposito, L.; Pedone, C.; Vitagliano, L. Biophysical J. 2008, In press.
104
New Frontiers in the Search of Bioactive Molecules: from Peptides to Drugs
Imaging of αvβ3 integrin expression with a
18F-labeled RGD peptide and microPET
Sommella Jvana1, Zannetti Antonella1, Iommelli Francesca1, Papaccioli Angela1, Panico M. Rosaria1,
Del Gatto Annarita2, De Simone Mariarosaria2, Zaccaro Laura2, Saviano Michele2, Pedone Carlo2,
and Salvatore Marco3
1 Istituto di Biostrutture e Bioimmagini, CNR, Dipartimento di Scienze Biomorfologiche e Funzionali, 80131 Napoli
2 Istituto di Biostrutture e Bioimmagini, CNR, Dipartimento delle Scienze Biologiche, Università di Napoli “Federico II”, 80134 Napoli
3 Università di Napoli “Federico II”, Dipartimento di Scienze Biomorfologiche e Funzionali, 80131 Napoli
The αvβ3 integrin is a cell adhesion receptor involved in angiogenesis, tumor cell migration
and metastatic dissemination. The tripeptide sequence RGD binds to αvβ3 but also interacts
with other integrins. We developed and characterized a novel cyclized RGD pentapeptide
covalently linked by a spacer to an echistatin domain that showed a high selectivity for αvβ3
integrin and not for αvβ5 [1]. The aim of this study was to asses whether this chimeric RGDechi
peptide labeled with 18F is suitable for selective in vivo imaging of αvβ3 receptor expression.
RGDechi was labeled with one-step procedure using 18F via N-succinimidyl-4[18F]fluorobenzoate through the lysine side chain ε-amino group. U87MG human
glioblastoma cells and A431 human epidermoid cells endogenously expressing high levels of
αvβ3 and αvβ5, respectively, were used to develop xenografts in nude mice. Imaging studies
were then performed using microPET/CT scanner (eXplore Vista PET/CT GE). Mice were
anaesthetized and 200 microCi of 18F-RGDechi were i.v. injected through the tail vein and
microPET images were obtained after 1h .The data were acquired in list mode and images
were reconstructed by using an iterative 2D-OSEM reconstruction algorithm that provided
transaxial, coronal and sagittal slices.
Nude mice bearing U87MG tumor xenografts, overexpressing αvβ3, showed a high tumor
uptake of 18F-labeled RGDechi whereas no tumor uptake of the tracer could be observed in
A431 tumor xenografts overexpressing αvβ5. Our findings indicate that αvβ3 overexpression
can be selectively visualized by using 18F radiolabeled RGDechi.
References
1. A. Del Gatto et al. Journal of Medicinal Chemistry (2006), 29(11), 3416-3420.
105
POSTER PRESENTATIONS - Eleventh Naples Workshop on Bioactive Peptides
Upgrade of SPOT synthesis to prepare soluble
peptides and phosphorylated peptide libraries
Bernhard Aÿ, Prisca Boisguerin, Ines Kretzschmar, Christiane Landgraf, and Rudolf Volkmer
Institut für Medizinische Immunologie, Charité - Universitätsmedizin Berlin, 10115 Berlin, Germany.
Discovering new targets for vaccines and novel methodologies in vaccination are current
research goals in immunology[1,2]. Besides the well-known combinatorial approaches[3], huge
arrays of sequence-based peptides (e. g. virus proteome based) are powerful tools for
proteome-wide T-cell epitope mapping studies, which deliver new targets for vaccine
development. For that reason, we should have the possibility to easily synthesize 100 to 1000
different peptides with a few µmol’s. Therefore, we developed three different methods for the
fully automated SPOT synthesis:
1. 1,1’-carbonyldiimidazole (CDI) can be used as an activating reagent for cellulose
membrane esterification with all 20 L-amino acids by fully bathing the membrane with
the solution[4].
2. Using 1,1’-carbonyl-di-imidazole (CDI) or 1,1’-carbonyl-di-(1,2,4-triazole) (CDT) as
activators, we could couple the C-terminal amino acid spot-wise[5].
3. Within our third new method, we used hydroxymethylphenoxyacetic acid (HMPA) as
an acidic-labile linker in SPOT synthesis.
1. Taking together, these methods for the synthesis of cleavable peptides are an
important step towards the generation of soluble peptides for the in vivo screening of
cellular interactions.
Another highlight of peptide libraries is the screen of posttranslational modification in proteins
by phosphorylation at serine, threonine, and tyrosine residues which plays a role in eukaryotic
cell cycle regulation, cell differentiation, apoptosis, or cytoskeletal regulation[6,7]. Positive
regulation (switch on) by phosphorylation is known for protein interaction domains such as
PTB, SH2, and 14–3–3 domains requiring phosphorylated ligands for binding. Negative
regulation (switch off) was first described for a WW domain where the domain/ligand
interaction is disrupted by phosphorylated amino acids. Unfortunately, there is as yet no
simple experiment directly revealing the effect of negative regulation in vivo. However, in vitro
comparison of phosphorylated versus non-phosphorylated peptide libraries can potentially
reveal both positive and negative regulation of protein domain functions. For that reason, we
tested analytically and quantitatively the coupling efficiency of different activation reagents.
The improved protocol for coupling phosphorylated amino acids is thereafter demonstrated
using phospho-libraries to determine the regulation mechanism of PDZ domains and the
binding properties of WW domains.
Financial support from Charité and the Deutsche Forschungsgemeinschaft (FOR 299, VO885/3-1 and SFB/TR 19), is gratefully
acknowledged.
References
1 A. S. De Groot, Drug Discovery Today 2006, 11, 203.
2 S. J. Paston, I. A. Dodi, J. A. Madrigal, Hum Immunol 2004, 65, 544.
3 A. G. Beck-Sickinger, G. Jung, in Combinatorial Peptide and Nonpeptide libraries (Ed.: G. Jung), VCH Verlagsgesellschaft,
Weinheim, 1996, p. 79.
4 B. Ay, M. Streitz, P. Boisguerin, A. Schlosser, C. C. Mahrenholz, S. D. Schuck, F. Kern, R. Volkmer, Biopolymers 2007, 88,
64.
5 B. Ay, R. Volkmer, P. Boisguerin, Tetrahedron Letters 2007, 48, 361.
6 T. Hunter, Cell 1995, 80, 225.
7 T. Hunter, Cell 2000, 100, 113.
106
New Frontiers in the Search of Bioactive Molecules: from Peptides to Drugs
Identification and characterization of a
new form of oncosuppressor KCTD11
Luciano Pirone1, Stefania Correale1, Vincenzo Alterio1, Sonia Di Gaetano1, Luigi Vitagliano1,
Giuseppina De Simone1, Marta Moretti2, Enrico De Smaele2, Lucia Di Marcotullio2, Alberto Gulino2,
Carlo Pedone1 and Emilia Pedone1
1 Istituto di Biostrutture e Bioimmagini, 80134, Italia.
2 Dipartimento di Medicina Sperimentale, Università di Roma La Sapienza, 00185, Italia.
Hedgehog (Hh) signaling is suggested to be a major oncogenic pathway in medulloblastoma,
which arises from aberrant development of cerebellar granule progenitors. Hh signaling is
regulated by multiple E3 ubiquitin ligases, that process the downstream transcription factors
Gli via dual Cullin-based ubiquitin-dependent pathways[1]. Interestingly, Cullin3 has been
shown to require proteins containing BTB (Broad Complex, Tramtrack and Bric a Brac)
domain to target substrates. Recently a human BTB protein has been identified, KCTD11 [2, 3,
4]
, which displays allelic deletion as well as significantly reduced expression in
medulloblastoma. Data in vivo have suggested that KCTD11 binds Cullin3 promoting the
degradation of Gli1 and inhibiting the transactivation of the Hedgehog target genes. Although
the available literature data clearly show the role of KCTD11 as oncosuppressor factor, its
biochemical properties remain to be defined. In this context we have undertaken a structural
and functional study on KCTD11 protein. We report here the amplification by PCR of the
human genomic region coding for KCTD11, its cloning and overexpression in Escherichia coli
in soluble form, as a fusion product with Maltose Binding Protein (MBP) and a N-terminal Histag. The protein has been purified to homogeneity by three purification steps utilizing its Nickel
affinity and molecular weight properties. Gel-filtration cromatography allowed to stucturally
characterize KCTD11 as an homotetramer in agreement with in silico analysis. Our attention
has been focused on the BTB/POZ domain of KCTD11. Bioinformatic analysis have allowed
to identify the exact extension of this domain. It has been cloned, expressed as a fusion
product with Thioredoxin-A (TrxA) and a N-terminal His-tag, purified in soluble form, and
structurally characterized. The homogeneous protein has been used for crystallization
experiments. Site-directed mutagenesis has been used to validate the ionic interactions as
responsible of the tetrameric form of the BTB/POZ domain of KCTD11. In conclusion our
results lead to identify a new BTB/POZ domain and suggest a possible role of the BTB/POZ
domain tetramerization in KCTD11 biological function.
References
1 Di marcotullio L, Ferretti E, Greco A, De Smaele E, Screpanti I, Gulino A. Cell cycle. 6, 4: 1-4 (2007)
2 Gallo R, Zazzeroni F, Alesse E, Mincione C, Borello U, Buanne P, D'Eugenio R, Mackay AR, Argenti B, Gradini R,
Screpanti I, Gulino A. J.Cell Biol. 158, 731 (2002).
3 Di Marcotullio L, Ferretti E, De Smaele E, Argenti B, Mincione C, Zazzeroni F, Gallo R, Masuelli L, Napolitano M, Maroder
M, Modesti A, Giangaspero F, Screpanti I, Alesse E, Gulino A. Proc. Natl. Acad. Sci. USA 100, 7331 (2004).
4 Ferretti E, De Smaele E, Di Marcotullio L, Screpanti I, Gulino A. Trends Mol Med. 11, 12: 537-45. Epub (2005).
107
POSTER PRESENTATIONS - Eleventh Naples Workshop on Bioactive Peptides
Designing trehalose-conjugated peptides for the inhibition of
Alzheimer’s Aβ oligomerization and neurotoxicity.
Pappalardo Giuseppe1, De Bona Paolo2, Giuffrida Maria Laura3, Attanasio Francesco1, Copani
Agata3,1, Pignataro Bruno4, Cataldo Sebastiano4 and Rizzarelli Enrico2
1
2
3
4
National Research Council, Institute of Biostructures and Bioimaging, 95125 Catania – Italy.
University of Catania, Department of Chemical Sciences, 95125 Catania – Italy.
University of Catania, Department of Pharmaceutical Sciences, 95125 Catania – Italy.
University of Palermo, Department of Physical chemistry “F. Accascina”, 90133 Palermo – Italy.
Alzheimer’s disease (AD) is a neurodegenerative disorder characterized by loss of memory
and language skills, damaged cognitive function and altered behaviour.[1]
Besides these clinical symptoms, the central histopathological feature of AD is the presence
of extracellular senile plaques, found in the hippocampus and neocortex, associated with
synaptic loss and cell death.[2] The principal protein component of these plaques is the βamyloid peptide (Aβ), a 39-42 residues peptide fragment generated by the proteolysis of
cellular amyloid precursor protein (APP).[3,4]
It is now believed that early stages of aggregation of Aβ in the brain, initiate a cascade of
events that result in neuronal cell death and leads to cognitive decline.[5]
Inhibiting Aβ self-oligomerization might, therefore, provide a useful approach to treating and
controlling the pathogenic pathways underlying AD.
Several small molecules capable of binding to Aβ have been identified, among these
trehalose and the pentapeptide LPFFD have been reported to have effects on the aggregation
as well as on Aβ neurotoxicity.[6,7] We hypothesized that the conjugation of trehalose with the
pentapeptide LPFFD would result in new compounds with higher affinity for Aβ, thereby acting
as new effective inhibitors of Aβ’s cellular toxicity.
In this communication we report the synthesis and the spectroscopic characterization of three
new trehalose conjugates with the LPFFD peptide. All the synthesized compounds were
tested as inhibitors of both Aβ’s fibrillogenesis and toxicity toward pure cultures of rat cortical
neurons. In addition, the effects of these glycopeptides on the morphology of Aβ aggregates
were analyzed by AFM microscopy.
References
1. Arendt, T.; Bigl, V.; Tennstedt, A.; Arendt, A.; Neurosci. Lett. 1984, vol 48(1), 81-85.
2. Selkoe, D. J.; Neuron. 1991, vol 6: 487–498.
3. Miller, D. L.; Papayonnopoulus, I. A.; Styles, J.; Bobin, S. A.; Lin, Y. Y.; Biemann, K.; Iqbal, K.; Arch. Biochem. Biophys.
1993, vol 301, 41-52.
4. Yankner, B. A.; Nat. Med. 1996, vol 2, 850-852.
5. Cleary, J. P.; Walsh, D. M.; Hofmeinster, J. J.; Shankar, G. M.; Kuskowski, M. A.; Selkoe, D. J.; Ashe, K. H.; Nat. Neurosci.
2005, vol 8(1), 79-84.
6. Soto, C.; Sigurdsson, E. M.; Morelli, L.; Kumar, R. A.; Castano, E. M.; Frangione, B.; Nat. Med. 1998, vol 4, 822–826.
7. Liu, R.; Barkhordarian, H.; Emadi, S.; Park C. B.; Sierks, M. R.; Neurobiol. Dis. 2005, vol 20,74-81.
108
New Frontiers in the Search of Bioactive Molecules: from Peptides to Drugs
Membrane active peptides selective for
phosphatidylserine as novel cancer therapeutics
Manavbasi Yasemin, Zweytick Dagmar and Lohner Karl
Institute of Biophysics and Nanosystems Research, Austrian Academy of Sciences, 8042, Graz, Austria
Chemotherapeutic agents, commonly used as anti-cancer drugs, have severe side effects,
also affecting healthy human cells. Natural antimicrobial peptides, and their derivatives, which
are also referred as host defense peptides, have gained interest as potential anti-cancer
agents. Under normal conditions, due to the asymmetric distribution of plasma membrane
lipids across the bilayer, mammalian cells comprise phosphatidylserine (PS) only in the inner
leaflet. In the case of malignant transformation inner leaflet PS can move to the outer leaflet
and act as a surface marker. The elevated surface level of negatively charged PS on various
tumour cells makes these cells susceptible to killing by cationic membranolytic peptides such
as, NK-2 [1].The aim of this study is to develop short peptide sequences still acting selectively
towards PS exposed on cancer cells without damaging healthy cells. In order to use this new
strategy fighting against cancer with PS-specific peptides it is necessary to analyze the lipid
composition of mammalian cancer and non cancer cell membranes. Further as a basis for
peptide activity studies the biophysical characteristics of cancer cell membranes and healthy
counterparts with respect to lipid composition were determined by investigation of liposomal
mimics composed of phosphatidylcholine and/or phosphatidylserine by DSC and X-ray.
These model systems were also used for an initial screening of the activity of a series of
peptides. Fluorescence spectroscopy was applied to test the release of fluorescence marker
molecules from liposomes composed of solely PS, PC or PC/PS mixtures in the presence of
various concentrations of peptides. Data revealed that some NK-2 derived peptides have a
high affinity towards PS causing significant leakage of liposomal content, whereas healthy
mammalian cell mimicking PC liposomes were not affected. Optimized peptides resulting from
these experiments will be used for in-vitro studies on prostate cancer cell lines.
References
1. Scröder-Borm H., Bakalova R,. Andrä J. The NK-lysin derived peptide NK-2 preferentially kills cancer cells with increased
surface levels of negatively charged phosphatidylserine FEBS Letters 579 (2005) 6128-6134
109
POSTER PRESENTATIONS - Eleventh Naples Workshop on Bioactive Peptides
Carbonic Anhydrase inhibitors: bioreductive
nitro-containing sulfonamides with selectivity
for targeting the tumor associated isoforms IX and XII
Katia D’Ambrosio1, Rosa-Maria Vitale2, Jean-Michel Dogné3, Bernard Masereel3, Alessio Innocenti4,
Andrea Scozzafava4, Claudiu T. Supuran4 and Giuseppina De Simone1
1
2
3
4
Istituto di Biostrutture e Bioimmagini-CNR, 80134 Napoli, Italy
Istituto di Chimica e Biomolecolare-CNR, 80078, Pozzuoli, Italy.
Drug Design and Discovery Center, University of Namur, 5000 Namur, Belgium.
Laboratorio di Chimica Bioinorganica, Università degli Studi di Firenze, I-50019 Sesto Fiorentino (Firenze), Italy.
Carbonic anhydrase, the catalyst for the interconversion between carbon dioxide and
bicarbonate, is an essential enzyme all over the phylogenetic tree, being present in Bacteria,
Archaea and Eukaryotes. In humans, the CA-catalyzed reaction is involved in respiration and
transport of CO2/bicarbonate between metabolizing tissues and lungs, pH and CO2
homeostasis, electrolyte secretion in a variety of tissues/organs, biosynthetic reactions, bone
resorption, tumorigenicity, and many other physiological and pathological processes.[1] Many
of the CA isozymes involved in these processes among the 15 known in humans, are
important therapeutic targets with the potential to be inhibited or activated to treat a wide
range of disorders.[1]
The main class of CA inhibitors (CAIs) is constituted by unsubstituted sulfonamides and their
bioisosteres (sulfamates, sulfamides, etc.), which bind to the Zn2+ ion of the enzyme, by
substituting the non-protein zinc ligand to generate a tetrahedral adduct and participating in
various other favourable interactions with amino acid residues situated in the active site.[1,2] A
critical problem in the design of these inhibitors is related to the high number of isoforms, their
rather diffuse localization in many tissues/organs, and the lack of isozyme selectivity.[1]
Hypoxia, a condition associated with many types of cancers, triggers a strong overexpression
of at least two CA isozymes, i.e. CA IX and XII.[2,3] CA IX belongs to the very active human
CAs, its catalytic properties for the CO2 hydration reaction being comparable with those of the
highly evolved catalyst CA II, whereas CA XII is slightly less effective [1,3]. As for all
mammalian CAs, CA IX and XII are susceptible to inhibition by anions and
sulfonamides/sulfamates/sulfamides. The almost exclusive localization of CA IX (and to a less
degree of CA XII) in tumors, make these proteins attractive targets for the design of
conceptually novel anti-tumor therapies.
In a preceding work we reported hypoxia-activatable sulfonamides incorporating disulfide
functionalities.[4] Here we extend the earlier work to sulfonamides incorporating nitro moieties
and report the inhibitory activity of a series of such aromatic derivatives against the
physiologically relevant isoforms I, II and the tumor-associated ones CA IX and XII. X-Ray
crystallography and molecular modeling were employed for rationalizing some of our results.[5]
References
1 Supuran C. T. Nat. Rev. Drug Discov. 2008, 7, 168-181.
2 Thiry A.; Dogné J. M.; Masereel B.; Supuran C. T. Trends Pharmacol. Sci. 2006, 27, 566-573.
3 Pastorekova S.; Kopacek J.; Pastorek J. Curr. Top. Med. Chem. 2007, 7, 865-878.
4 De Simone G.; Vitale R.M.; Di Fiore A.; Pedone C.; Scozzafava A.; Montero J.L.; Winum J.Y.; Supuran C. T. J. Med. Chem.
2006, 49, 5544-5551.
5 D’Ambrosio K.; Vitale R.M.; Dogné J.M.; Masereel B.; Innocenti A.; Scozzafava A.; De Simone G.; Supuran C.T. J. Med.
Chem. 2008, in press
110
New Frontiers in the Search of Bioactive Molecules: from Peptides to Drugs
Preliminary docking studies on fibril aggregation inhibitory
activity of the β-sheet breaker peptides.
Maria G. Chini, Mario Scrima, Anna M. D’Ursi, and Giuseppe Bifulco.
University of Salerno, Department of Pharmaceutical Sciences, 84084 Fisciano (SA) - Italy.
β-sheet breaker peptides have been shown to inhibit fibrillogenesis and disassemble
preformed fibrils of β-amyloid (Aβ), characteristic of Alzheimer’s disease, both in vitro,[1] and in
vivo.[2] The beta-amyloid plaques, consisting of dense deposits of protein (β-amyloid) derived
by the amyloid precursor protein (APP), and cellular material, are highly ordered with
predominant β-pleated sheet structure, and they accumulate outside and around nerve cells.
We studied the inhibition of the fibrillogenesis process, through the molecular docking,
between the β-sheet breaker peptide (Ac-L1-V2-(NMet)F3-F4-A5-NH2) and β-amyloid. For our
calculations, we used as target the NMR structure of β-amyloid (Aβ1-42) solved by Luhrs[3] and
co-workers in 2005. To improve the molecular docking calculations, we performed a full
geometry and energy optimization of the ligand by quantum mechanical (QM) methods at DFT
level in vacuo, using the mPW1PW91 functional and the 6-31G(d) basis set (Gaussian 03
software). Minimum energy conformer obtained was used as input for docking calculation by
Autodock 3.0.5 software.[4]
a)
b)
Figure 1: a) 3D structure of β-sheet breaker peptide obtained by QM methods. b) 3D model of the interaction between β- sheet
breaker (purple) and the β-amyloid (1-42).
We used molecular docking as a rapid, interactive method to study the inhibition
fibrillogenesis process by β-sheet breaker peptide step by step. In particular, after a validation
of the method (docking two fibrils of Aβ1-42), our ultimate purpose is to find the complex (1:1,
1:2, 1:3 and 1:4 target:ligand respectively) that blocks the aggregation of fibrils. In Figure 1 is
shown the first result of our procedure, where the small peptide perfectly reproduces the trend
of the amyloid, folding as a β-sheet. The ligand interacts with hydrophobic region of the target
that runs from L17 to A21, and plays an essential role in the fibril formation inhibition.
Moreover, the calculated complex is stabilized by aromatic and hydrophobic interactions, and
by H-bonds between the backbone of the β-sheet breaker and the one of the β-amyloid.
References
1 Soto, C.; Sigurdsson, E. M.; Morelli, L.; Kumar, R. A.; Castano, E. M.; Frangine, B. Nat. Med. 1998, 4(7), 822-6.
2 Soto, C.; Kascsak, R. J.; Saborio, G. P.; Aucouturier, P.; Wisniewski, T.; Prelli, F.; Kascsak, R.; Mendez, E.; Harris, D. A.;
Ironside, J.; Tagliavini, F.; Carp, R. I.; Frangine, B. Lancet 2000, 355(9199), 192-7.
3 Luhrs, T.; Ritter, C.; Adrian, M.; Riek-Loher, D.; Bohrmann, B.; Dobeli, H.; Schubert, D.; Riek, R. Proc. Natl. Acad. Sci.
U.S.A. 2005, 102, 17342-7.
4 Morris, G. M.; Goodsell, D. S.; Halliday, R. S.; Huey, R.; Hart, W. E.; Belew, R. K.; Olson, A. J. J. Comput. Chem. 1998 19,
1639-1662.
111
POSTER PRESENTATIONS - Eleventh Naples Workshop on Bioactive Peptides
Vasoactive metabolites: synthesis peptide analogues
and evaluation of their cardiovascular activity
Veveris Maris, Keivish Tatiana, Kalvinsh Ivars, Polevaya Ludmila
Latvian Institute of Organic Synthesis, Riga, LV-1006, Latvia
The renin-angiotensin system (RAS) is recognized as the most powerful signaling system for
controlling sodium balance, body fluid volumes and blood pressure. Its primary effector
hormone, angiotensin II (Ang II), not only mediates immediate physiological effects of
vasoconstriction and blood pressure regulation, but is also implicated in inflammation,
endothelial dysfunction, atherosclerosis, hypertension, and congestive heart failure. Recent
studies indicate that such Ang II metabolites as Ang III or Ang-(1-7) and Ang IV also
accomplish cardiovascular, central and renal functions [1]. Some of these effects are AT1
receptor dependent but others most likely acts as counteracting factors to Ang II actions, in
order to remove the imbalance created by the predominating stimulation of vasoconstricting
AT1 receptors. Ang-(1-7), the product of ACE2 activity, is an endogenous ligand for the G
protein-coupled receptor mas. Since Ang IV is an endogenous agonist of insulin-regulated
aminopeptidase (IRAP) and an inhibitor of the IRAP catalytic activity, its in vivo effects might
interact with the uptake of glucose [2].
In the present study, analogues of some vasoactive peptides were prepared applying both the
classical method of peptide synthesis in solution and solid phase method on MBHA- or FmocPAL-PEG-PS-resins using standard Fmoc (N-(9-fluorenyl)-methoxycarbonyl) protocol. Crude
products were purified by preparative reversed-phase on a Vydac C18 column /a Waters
HPLC system or on Hitachi l-6200 Intelligent Pump with Grace Vydac Protein and Peptide
semipreparative column. The identity of peptides was verified by MALDI-TOF MS (Voyager –
DE PRO Biospectrometry workstation (PE Applied Biosystems).
Biological activity of synthesized peptides - VF-9, TR-6, LP-mc and TP-H7 was evaluated in
vitro and in vivo. Ang IV (10-11 to 10-8 M/kg) was used as a reference compound. In
experiments on anesthetized male Wistar rats blood glucose level and hemodynamic
parameters were measured. Intra venous administration of investigated peptides induced
more or less pronounced two-phase change of mean arterial blood pressure and peripheral
resistance. Most of peptides lowered arterial blood pressure. The decreasing potency of
investigated peptides (dose-effect activity) on blood pressure were: TR-6 > LP-mc > VF-9.
Peptides Ang IV and TP-H7 induced dose dependent but short lasting increase in mean
arterial blood pressure and peripheral resistance, which usually accompanied with more
prolonged but less expressed vasodilatation. The some time heart rate changes less
significantly. Only VF-9 evoked sustained bradycardia. Investigated peptides possessed only
marginal effect on blood glucose level in venous blood.
The obtained results demonstrate that investigated peptides revealed activity on
cardiovascular system – blood pressure and peripheral vascular resistance. These peptides
might be used as template to develop novel cardiovascular drugs.
References
1 Chansel D., Ardaillou R. (1998) Active metabolites derived from angiotensin II. Nephrology 19: 427-432.
2 Chai SY., Fernando R., Peck G. et all. (2004) The angiotensin IV/AT4 receptor. Cell. Mol. Life Sci. 61: 2728-37.
112
New Frontiers in the Search of Bioactive Molecules: from Peptides to Drugs
Viral fusion peptides induce several signal transduction pathways
activation that are essential for IL-10 and β-interferon production
Raieta Katia1, Galdiero Marilena1, Vitiello Mariateresa1, Falanga Annarita2, Finamore Emiliana1,
Kampanaraki Aikaterini1, Benedetti Ettore2 and Galdiero Stefania2
1 Department of Experimental Medicine - II University of Naples – 80138 Naples, Italy;
2 Department of Biological Sciences & CIRPEB - University of Naples “Federico II” – 80134 Naples, Italy
Signal transduction pathways convey external stimuli generated at the cell surface into the cell
nucleus in order to initiate a program of gene expression that is typical of a particular stimulus.
Virus interactions with cell surface receptors can elicit two types of signals, conformational
changes of viral particles, and intracellular signals triggering specific cellular reactions. Many
enveloped viruses, upon receptor binding, can trigger a fusion reaction between the viral
membrane and a cellular membrane, which in some cases can happen at a neutral pH on the
plasma membrane. The fusion reactions are directed by fusion proteins that undergo
consistent structural modifications that lead to the exposure of small stretches of hydrophobic
aminoacids, the fusion peptides, which interact with the opposing lipid bilayer and are involved
in the initial stages of virus penetration. Since specific ligand-interactions may result in cellular
activation we analysed whether fusion peptides binding to the cell surface were sufficient to
induce early activation of signalling pathways. Thus, we selected and synthesized a set of
known fusion peptides from different virus families from Class I, Class II and the newly
identified Class III fusion proteins.
Focusing on the earliest events of the viral life cycle, we have demonstrated that hydrophobic
domain of fusion proteins from different viruses are able to induce several transduction
pathways that lead to the early cytokine production (such as IL-10 and IFN-β) in the host cell.
Our experimental results represent a starting point to investigate with deeper details the
mechanisms of signal transmission pathway due to viral infections.
113
POSTER PRESENTATIONS - Eleventh Naples Workshop on Bioactive Peptides
Conformational studies of Temporin A and Temporin L: Design,
synthesis and biological activities of new analogues.
Paolo Grieco1 Stefania Malfi1, Alfonso Carotenuto1, Luigia Auriemma1, Maria Rosaria Saviello1,
Isabel Gomez-Monterrey1, Maria Luisa Mangoni2, Pietro Campiglia3, Cristina Marcozzi1, Alessia
Bertamino3, Ettore Novellino1.
1 Department of Chimica Farmaceutica e Tossicologica, University of Naples “Federico II”, Italy
2 II Facoltà di Medicina e Chirurgia, University of Rome “La Sapienza”, Italy
3 Department of Scienze Farmaceutiche, University of Salerno, Italy.
Temporins A and L[1] are antimicrobial peptides isolated from the skin of Red European frog
“Rana temporaria”. Temporins are active against a broad spectrum of microorganism:
Temporin A (FLPLIGRVLSGIL-NH2) is preferentially active against Gram-positive bacterial
strains; Temporin L (FVQWFSKFLGRIL-NH2) has the highest activity among all temporins
against fungi, and bacteria, including resistant Gram-negative strains, but it shows haemolytic
activity too.
We investigated the preferential conformation of TL and TA in SDS and DPC solutions and on
the bases of the results we designed and synthesized new TA and TL analogues (Table 1) to
the aim to understanding the exact mechanism of the action and finding a new potent
antimicrobial agent without haemolytic activity.
TA
TL
Gln3-TA
Pro3-TL
H-Phe1-Leu2-Pro3-Leu4-Ile5-Gly6-Arg7-Val8-Leu9-Ser10-Gly11-Ile12-Leu13-NH2
H-Phe1-Val2-Gln3-Trp4-Phe5-Ser6-Lys7-Phe8-Leu9-Gly10-Arg11-Ile12-Leu13-NH2
H-Phe1-Leu2-Gln3-Leu4-Ile5-Gly6-Arg7-Val8-Leu9-Ser10-Gly11-Ile12-Leu13-NH2
H-Phe1-Val2-Pro3-Trp4-Phe5-Ser6-Lys7-Phe8-Leu9-Gly10-Arg11-Ile12-Leu13-NH2
Table 1. Sequences of analyzed peptides
References
1 Carotenuto, Alfonso; Malfi, Stefania; Saviello, Maria Rosaria; Campiglia, Pietro; Gomez-Monterrey, Isabel; Mangoni, Maria
Luisa; Marcellini Hercolani Gaddi, Ludovica; Novellino, Ettore; Grieco, Paolo. “A Different Molecular Mechanism Underlying
Antimicrobial and Hemolytic Actions of Temporins A and L” Journal of Medicinal Chemistry (In Press)
114
New Frontiers in the Search of Bioactive Molecules: from Peptides to Drugs
New α-MSH analogues with improved candidacidal activity
Paolo Grieco1, Luigia Auriemma1, Stefania Malfi1, Alfonso Carotenuto1, Maria Rosaria Saviello1,
Cristina Marcozzi1, Isabel Gomez-Monterrey1, Pietro Campiglia2, Alessia Accettola1, Ettore
Novellino1, Anna Catania3.
1 Department of Chimica Farmaceutica e Tossicologica, University of Naples “Federico II”, Italy
2 Department of Scienze Farmaceutiche, University of Salerno, Italy
3 Divisione di Medicina Generale, IRCCS Ospedale Maggiore di Milano, Milan, Italy
α-Melanocyte stimulating hormone (α-MSH) is an endogenous linear tridecapeptide with
potent antiinflammatory effects. It was demonstrated that α-MSH and its C-terminal sequence
Lys-Pro-Val (α-MSH [11-13]) have antimicrobial effects against two major and representative
pathogens: Staphylococcus aureus and Candida albicans. In an attempt to improve the
candidacidal activity of α-MSH and to better understand the peptide structure-antifungal
activity relations, we designed and synthesized novel peptide analogs. Because previous data
suggested that the peptide [DNal-7, Phe-12]-α-MSH(6-13) has greater candidacidal activity
than α-MSH and is the most potent of the analogs tested in the past, this compound has
became our lead [1,2]. From this lead compound we have synthesized a new library of
peptides where we have replaced the glycine in position 10 with unconventional amino acids.
Here, we report preliminary results on candidacidal activity and conformational studies.
References
1 Grieco, P.; Rossi, C.; Colombo, G.; Carlin, A.; Gatti, S.; Novellino, E.; Lama, T.;. Lipton, J.M.; Catania, A. Journal of
Medicinal Chemistry, 48, 1384-1388, 2005
2 Carotenuto, A.; Saviello, M.R.; Auriemma, L.; Campiglia, P.; Catania, A.; Novellino, E.; Grieco P. Chemical Biology & Drug
Design, 69, 68-74, 2007
115
POSTER PRESENTATIONS - Eleventh Naples Workshop on Bioactive Peptides
Peptaibiomics: towards a myriad of bioactive
peptides containing Cα-dialkylamino acids?
Degenkolb Thomas, Brückner Hans
Interdisciplinary Research Centre (IFZ), University of Giessen, 35392 Giessen – Germany
Fungi are generally regarded as a literally infinite resource of bioactive secondary metabolites
displaying remarkable structural diversity. Peptide antibiotics constitute a considerable part of
these metabolites. During the past two decades, a constantly growing group of peptide
antibiotics, the peptaibiotics, has started to gain particular interest because of their unique
bioactivities and conformations[1]. Besides well-known membrane-modifying or channelforming activities, neuroleptic, anti-HIV integrase 1, insecticidal, nematicidal and antiprotozoal
effects have been reported as well as inhibition of amyloid β-peptide formation, mimicking of
transforming growth factor-β (TGF-β)-like activity and inhibition of histone deacetylases
(HDAC’s). Peptaibiotics are defined as linear or cyclic polypeptide antibiotics which (i) have a
molecular weight between 500 and 2,200 Dalton, thus containing 4–21 residues; (ii) show a
high content of the marker α-aminoisobutyric acid (Aib); (iii) are characterized by the presence
of other non-proteinogenic amino acids and/or lipoamino acids; (iv) possess an acylated Nterminus, and (v) , in the case of linear peptides, have a C-terminal residue that in most of
them consists of a free or acetylated amide-bonded 1,2-amino alcohol. The C-terminus might
also be an amine, amide, free amino acid, 2,5-dioxopiperazine, or sugar alcohol. A
remarkable number of species and strains from 20 genera of fungi have been investigated for
the presence of peptaibiotics. Many more species have been shown to produce the
characteristic Cα-dialkylamino acids – α-aminoisobutyric acid (Aib), L- and/or D-isovaline (Iva)
–, being indicative for this group of peptide antibiotics[2, 3].
The discovery of microheterogeneity is to be considered as a first milestone in peptaibiotic
research[4]. Recently, the advanced method of “peptaibiomics” was introduced to characterize
the “peptaibiome” that is defined as the entirety and dynamics of peptaibiotics produced by a
selected strain under defined culture conditions[5]. Characterization of the peptaibiome is
accomplished by SPE followed by HPLC/ESI-CIDMSn[2,5,6,7,8]. To date, more than 850
individual sequences of peptaibiotics are described in literature[2,3]. Of those, we have
characterized about 200 new sequences within the past two years by applying the
peptaibiomic approach[2,5,6,7,8]. Notably, most of the fungi investigated are common in soil or
on decaying plant material but some of them also occupy ecological niches. Owing to the
ubiquity and biodiversity of their producers, we predict that a myriad of new sequences of
peptaibiotics will be discovered within the next decade!
References
1 Toniolo, C; Benedetti, E. Trends Biochem. Sci. 1991, 16, 350–353.
2 Degenkolb, T.; Kirschbaum, J.; Brückner, H. Chem. Biodivers. 2007, 4, 1052–1067.
3 Degenkolb, T.; Gams, W. Brückner, H. Chem. Biodivers. 2008, 5, accepted.
4 Przybylski, M; Dietrich, I.; Manz, I.; Brückner H. Biomed. Mass Spectrom. 1984, 11, 569–582.
5 Krause, C.; Kirschbaum, J., Brückner, H. Amino Acids 2006, 30, 435–443.
6 Degenkolb, T.; Gräfenhan, T.; Nirenberg, H. I.; Gams, W.; Brückner, H. J. Agric. Food Chem. 2006, 54, 7047-7061.
7 Degenkolb, T.; Gräfenhan, T.; Berg, A.; Nirenberg, H. I.; Gams, W.; Brückner, H. Chem. Biodivers. 2006, 3, 593–610
8 Degenkolb, T.; Dieckmann, R.; Nielsen, K. F.; Gräfenhan, T.; Theis, C.; Zafari, D.; Chaverri, P; Ismaiel, A.; Brückner, H.; von
Döhren, H.; Thrane, U.; Petrini, O.; Samuels, G. J. Mycol. Prog. 2008, 7, accepted
116
New Frontiers in the Search of Bioactive Molecules: from Peptides to Drugs
Conformational preferences of peptides from Haemophilus
influenzae P2 Porin loop-7 by a novel bioinformatic analysis tool.
Molnar Anca Florina1, Galdiero Stefania2, Benedetti Ettore2, and Amodeo Pietro1
1 National Research Council (CNR), Institute of Biomolecular Chemistry (ICB), I-80072 Pozzuoli (NA) - Italy.
2 University “Federico II” of Naples, Department of Biological Science, Division of Biostructure and CIRPEB, I-80134 Naples - Italy.
In Gram-negative bacteria, porins are the most represented of the outer membrane proteins
and allow the diffusion through the outer membrane of small hydrophilic solutes. In addition to
their pore function, they serve as receptor for bacteriophages and bacteriocins, and in the
pathogenic species, they also appear to be targets of the immunological system. Porins
induce many cellular responses, including cellular activation, cytokine release and
immunological effects.
Loop-7 from porin P2 of Haemophilus influenzae (TSVDQGEK) is responsible for most of the
activation of Raf/MEK1-MEK2/MAPK kinases cascade elicited by the whole protein[1]. In this
view, it represents both an useful model system for the mechanisms involved in infection
insurgency, and a promising template to develop antimicrobial agents and synthetic vaccines.
Peptides derived from loop-7 have previously shown[2] a critical dependence upon exact
sequence, length, termination and structural constraints, thus any attempt to design analogs
with increased stability and/or structural rigidity has failed so far.
To gain information about structure-activity relationships and to guide the design of a new
generation of loop-7 analogs, conformational analysis of loop-7 was attempted[2]. Traditional
analysis, based on molecular dynamics simulations, does not suggest clear conformational
preferences for the isolated peptide in water, while a limited, manual and qualitative
bioinformatic search on homolog sequences of loop-7 showed that, whenever the Gly
underlined in loop-7 sequence was conserved inside the motif, the identified homologs all
occurred in protein loops and clusterized into two structural families that where qualitatively
classified, on the basis of their backbone arrangements, as “U-shaped” and “V-shaped”.
However, to adequately support this hypothesis, and to obtain more solid structural
information aimed at designing new loop-7 analogs, a more exhaustive and rigorous analysis
was required.
So, a new bioinformatic tool was designed, to process the output of several PROSITE[3]
pattern searches for loop-7 homologs in the PDB structural database. The tool was aimed at:
a) characterizing several structural relationships (like distances and angles among backbone
and sidechain atoms) occurring in homolog peptides; b) performing a cluster analysis of these
parameters, to identify any recurring structural pattern and, eventually, classify into structural
homolog families the selected motifs.
This tool allowed a systematic exploration of PDB database with different homology patterns,
resulting in the identification of new loop-7 homolog peptides. After structural analysis and
clustering, the two previously identified conformational families were confirmed and
unambiguously defined on the basis of characteristic distance patterns. The spread in
interresidue distances observed within each family has been used to guide the design of new
conformationally-restrained loop-7 analogs, presently under active investigation.
References
1 Galdiero S., Capasso D., Vitiello M., D’Isanto M. Pedone C., Galdiero M. (2003) Infect. Immunology, 71: 2798-2809.
2 Galdiero S.,Vitiello M., Amodeo P., D’Isanto M., Cantisani M., Pedone C., Galdiero M. (2006) Biochemistry, 45: 4491-4501.
3 http://www.expasy.ch/prosite
117
POSTER PRESENTATIONS - Eleventh Naples Workshop on Bioactive Peptides
Antimicrobial Proline-rich peptides from
secretory parotid granules of pig (Sus scrofa)
A. Vitali1, C. Fanali2, R. Longhi3, S. Conti4, L. Polonelli4, R. Inzitari2 , I. Messana5, T. Cabras5, B.
Manconi5 and M. Castagnola2
1
2
3
4
5
Institute for the Chemistry of Molecular Recognition, National Research Council (C.N.R.), Sez. Roma, 00168 Roma, Italy
Institute of Biochemistry and Clinical Biochemistry, Faculty of Medicine, Catholic University, , Rome I-00168, Italy
Institute for the Chemistry of Molecular Recognition, National Research Council (C.N.R.), Sez. Milano, Milano, Italy
Department of Pathology and Laboratory Medicine, University of Parma, 43100 Parma, Italy
Department of Sciences Applied to Biosystems, University of Cagliari, Cittadella Universitaria, Monserrato I-09042, Cagliari, Italy
A group of proline-rich peptides have been isolated and characterized by means of LC-MSMS spectrometry, from secretory parotid granules of pig [1]. Their primary structure have been
elucidated allowing their synthesis. One of the most recognized roles of proline-rich peptides
is to be part of the innate immune system in animals expressing antimicrobial activities [2]. In
this view six structurally related peptides, have been synthesized and challenged with human
pathogenic bacteria and fungi, comprehending clinical isolated strains of Candida albicans,
Cryptococcous neoformans, Aspergillus fumigatus, Pseudomonas aeruginosa and
Staphyolococcus aureus. With regard to the antimicrobial activity, these peptides showed
diverse killing capabilities in terms of EC50 of growth inhibition, with respect to the different
tested species.
The secondary structure of these peptides was also investigated employing CD and FTIR
spectroscopy techniques. The data obtained showed that all the peptides considered shared a
similar conformational arrangement, likely due to a mixed contribute of unordered and
Polyproline II structural motifs; the only exception derived from a small peptide of 881 Da
which did not show any distinct structural conformation.
The data presented lead to link the biological activity both with their whole secondary structure
arrangement and with the presence of specific primary sequence differences.
References
1. Fanali. C. et al. J. Sep. Sci. 2008, 31, 516 – 522
2. Otvos L. jr. Cell Mol Life Sci. 2002, 59, 1138-1150
118
New Frontiers in the Search of Bioactive Molecules: from Peptides to Drugs
Efficacy of HIV fusion inhibitor Sifuvirtide
related to its ability to adsorb on rigid membranes
Henri G. Franquelim1, Luís M.S. Loura2, Nuno C. Santos1, and Miguel A.R.B. Castanho1
1 Instituto de Medicina Molecular – Faculdade de Medicina da U.L., 1649-028, Lisboa, Portugal.
2 Faculdade de Farmácia da Universidade de Coimbra, 3000-295 Coimbra, Portugal.
Sifuvirtide, a 36 amino acid negatively charged peptide, is a novel and promising HIV fusion
inhibitor, presently in clinical trials[1]. Because of the aromatic amino acid residues of the
peptide, its behaviour in aqueous solution and the interaction with lipid-membrane model
systems (large unilammelar vesicles) were studied by using mainly fluorescence spectroscopy
techniques Various biological and nonbiological lipid-membrane compositions were analyzed,
and atomic force microscopy was used to visualize phase separation in several of those
mixtures. Results showed no significant interaction of the peptide, neither with zwitterionic
fluid lipid membranes (liquid-disordered phase), nor with cholesterol-rich membranes (liquidordered phase). Fluorescence quenching using acrylamide and lipophilic probes was carried
out to study the location of the peptide in the membrane models. In the gel-phase DPPC (1,2dipalmitoyl-sn-glycero-3-phosphocholine) membrane model, an adsorption of the peptide at
the surface of these membranes was observed and confirmed by using energy-transfer
experiments. These results indicate a targeting of the peptide to gel-phase domains relatively
to liquid-disordered or liquid-ordered phase domains. Our results were compared with
previous studies performed in similar membrane model systems with HIV fusion inhibitors
[2]
[3]
enfuvirtide and T-1249 . The larger affinity and selectivity of sifuvirtide toward the more rigid
areas of the membranes, where most of the receptors are found, may help explain the
improved clinical efficiency of sifuvirtide, by providing a local increased concentration of the
peptide at the fusion site.
0,06
B
A
Trp Trp
Trp Trp
SIFUVIRTIDE
SIFUVIRTIDE
0,05
Trp Trp
0,04
SIFUVIRTIDE
0,03
Trp Trp
0,02
0,01
0,00
0,0000
0,0001
0,0002
0,0003
0,0004
DPPC Bilayer
Energy Transfer Efficiency
0,07
FRET
44 Å
SIFUVIRTIDE
Trp
W ≈ 32.4Å
DPH
DPH/Å2
Fig.1 – Energy transfer from Trp residues of sifuvirtide (donor) to DPH (acceptor) in DPPC vesicles (3mM). (A) FRET
efficiencies obtained at different proportions of DPH. (B) Schematic representation of the FRET assay suggests an adsorption of
sifuvirtide on DPPC gel bilayers. Results suggest that the peptide is adsorbed at the surface of DPPC membranes. R0 ≈ 30Å; W =
32.4Å; xL (fraction in lipid) ≈ 0.25.
References
1 He Y., Xiao Y., Song H., Liang Q., Ju D., Chen X., Lu H., Jing W., Jiang S., Zhang L. J. Biol. Chem., 2008, Feb 26 [Epub
ahead of print].
2 Veiga, S.; Henriques, S.; Santos, N.C.; Castanho, M. Biochem. J., 2004, 377, 107-110.
3 Veiga, A.S.; Santos, N.C.; Loura, L.M.; Fedorov, A.; Castanho; M.A.R.B. J. Am. Chem. Soc., 2004, 126, 14758-14763
119
POSTER PRESENTATIONS - Eleventh Naples Workshop on Bioactive Peptides
PNA zipper: inducing dimerization of the basic
region of a bZip protein by a PNA duplex.
Pensato S.1; Saviano M.2; Renda M.3, Leccia F.3,Pedone, P.V.3; Pedone C.1 and Romanelli A.1
1 Facoltà di Scienze Biotecnologiche, Università di Napoli. 80134 Napoli-Italy.
2 Istituto di Biostrutture e Bioimmagini, 80134 Napoli- Italy.
3 Dipartimento di Scienze Ambientali, Seconda Università di Napoli-81100 Caserta- Italy
The sequence specific DNA binding of gene-regulatory proteins is often mediated by dimeric
proteins, which recognize DNA as homodimers or heterodimers. The basic leucine zipper
(bZip) DNA binding proteins are transcriptional regulatory proteins consisting of a coiled coil
leucine zipper dimerization domain and a highly charged basic region responsible for the DNA
binding. 1 Several efforts have been devoted to the synthesis of minimized bZip analogues,
able to bind DNA. The dimerization of the basic region (BR) has been achieved in several
ways, for example by host-guest complexes of β-cyclodextrin and adamantane, by
photoresponsive devices as azobenzene, by Fe(II) complexes, by disulfide bond between
extra cysteines. 2 In this work we present a new tool for the dimerization of the basic region of
bZip proteins based on the use of a short PNA duplex. We used the PNA duplex as
dimerization tool in consideration of its high thermal, chemical and enzymatic stability, which
will reveal useful for its future use as a decoy. The PNA dimerizer was linked to the BR
peptide by a native chemical ligation reaction. The peptide, corresponding to the basic region
of the GCN4 protein, was obtained by solid phase synthesis with an extra Lys residue at the
C-terminus. The Lys side chain was selectively deprotected on the resin and reacted with the
monobenzylthioester of the succinic acid to give the C-terminus thioester peptide. The peptide
was reacted with complementary PNA strands bearing a cysteine respectively at the C and
the N terminus to give the PNA-peptide conjugate. Formation of the PNA duplex induces
dimerization of the BR peptides, to give a molecule able to bind its target DNA. CD spectra of
the PNA-peptide single strand and of the dimeric (PNA-peptide)2 show the pronounced helical
tendency of the ligated molecules. Following complexation with the 19 mer DNA sequence the
helical content increases. The ability of binding DNA was further confirmed by EMSA
experiments.
Figure: Schematic representation of the PNA zipper-BR peptide complexed to DNA
References
1 Ellenberger, T.E.; Brandl, B.J.; Struhl, K. Cell (1992), 71, 1223.1237
2 Sato S.; Hagihara, M.; Sugimoto, K.; Morii, T. Chem. Eur. J. (2002), 8, 5067-5071
120
New Frontiers in the Search of Bioactive Molecules: from Peptides to Drugs
Insights into the catalytic mechanism of the Carbonic Anhydrase
family from the crystal structure of human isoform XIII
Anna Di Fiore1, Simona Maria Monti1, Mika Hilvo2, Seppo Parkkila2, Vincenza Romano1, Andrea
Scaloni3, Carlo Pedone1, Andrea Scozzafava4, Claudiu T. Supuran4 and Giuseppina De Simone1
1
2
3
4
Istituto di Biostrutture e Bioimmagini-CNR, 80134 Napoli, Italy
Institute of Medical Technology and School of Medicine, University of Tampere, Tampere, Finland
Laboratorio di Proteomica e Spettrometria di Massa, ISPAAM-CNR, 80147 Napoli, Italy
Laboratorio di Chimica Bioinorganica, Università degli Studi di Firenze, I-50019 Sesto Fiorentino (Firenze), Italy.
Carbonic anhydrases (CAs) are ubiquitous metalloenzymes, which catalyze the reversible
hydration of carbon dioxide to the bicarbonate ion. These proteins are present in prokaryotes
and eukaryotes, and are encoded by four evolutionarily unrelated gene families: the α-CAs,
the β-CAs, the γ-CAs and the δ-CAs.[1] Human CAs belong to the alpha class; fifteen isoforms
are presently known, among which twelve are catalytically active (CAs I-IV, CA VA-VB, CAVIVII, CA IX and CAs XII-XIV), whereas the CA-related proteins (CARPs) VIII, X and XI are
devoid of any catalytic activity.
The α-CA isozymes are widely distributed in many tissues and organs in mammals. Since at
these sites CAs play a crucial role in various physiological processes, they have recently
become interesting targets for pharmaceutical research. However, most of the available CAdirected pharmacological agents are still far from being optimal drugs. They present various
non-desired side-effects, mainly because of their lack of selectivity for the different CA
isozymes. Thus, developing isozyme-specific or, at least, organ-directed CA inhibitors should
be highly beneficial in obtaining novel classes of drugs. Prospects for achieving such a goal
have not been very optimistic, because of the high similarity observed between various
isozymes. However, recently, a plethora of X-ray crystallographic studies on different
isozymes and CA-inhibitor complexes has provided a scientific basis for the rational drug
design of more selective enzyme inhibitors.[1]
CA XIII is the most recently reported and characterized CA isozyme in humans. It is
selectively expressed among other tissues in the reproductive organs, where it may control
pH and ion balance regulation, ensuring thus proper fertilization conditions. In this study, we
report on the X-ray crystallographic characterization of human CA XIII in the unbound state
and in complex with the CA inhibitor acetazolamide. A detailed comparison of the new
structural data with those previously reported for other CA isozymes provided additional
insights into the catalytic properties of the members of this protein family. On the basis of the
information reported here, novel prospects for the design of isozyme-specific CA inhibitors are
proposed.[2]
References
1 Supuran, C. T. Nat. Rev. Drug Discov. 2008, 7, 168-181.
2 Di Fiore A., Monti S.M., Hilvo M., Parkkila S., Romano V., Scaloni A., Pedone C., Scozzafava A., Supuran C.T., De Simone
G. Proteins 2008, in press
121
POSTER PRESENTATIONS - Eleventh Naples Workshop on Bioactive Peptides
Determination of antimicrobial peptide location
inside lipid bilayers by combined fluorescence spectroscopy and
molecular dynamics simulations
Lorenzo Stella1, Gianfranco Bocchinfuso1, Giacinto Grande1, Barbara Orioni1, Mariano Venanzi1, JinYoung Kim2, Yoonkyung Park2,3, Kyung-Soo Hahm2,3, Marta De Zotti2, Fernando Formaggio4,
Claudio Toniolo4, and Antonio Palleschi1
1.
2.
3.
4.
Dipartimento di Scienze e Tecnologie Chimiche, Università di Roma “Tor Vergata”, 00133 Roma, Italy
Research Center for Proteineous Materials (RCPM), Chosun University, 501-759 Gwangju, South Korea
Department of Cellular Molecular Medicine, Chosun University, 501-759 Gwangju, South Korea
Institute of Biomolecular Chemistry, Padova Unit, CNR, Department of Chemistry, University of Padova, 35131 Padova, Italy
Several bioactive peptides, such as antimicrobial, cell penetrating or fusogenic peptides, exert
their biological function by interacting with cellular membranes. Therefore, structural data on
the location of these molecules inside lipid bilayers are very important for a detailed
understanding of their mechanism of action. While it is difficult to apply the most powerful
structural techniques (X-ray diffraction and NMR) to membrane systems, fluorescence
spectroscopic methods are particularly suited to the study of peptide-membrane association,
but give only low-resolution information on peptide position in the lipid bilayer. Molecular
dynamics simulations, on the other hand, can provide a very detailed picture of the peptidemembrane interaction, but need to be validated by quantitative comparison with experimental
data.
We exploited several fluorescence approaches, together with MD simulations, to the
investigation of two antimicrobial peptides: the lipopeptaibol trichogin GA IV, and PMAP-23, a
member of the cathelicidin family. To perform the spectroscopic studies, a variety of peptide
analogues containing a single fluorophore was synthesized. Carboxyfluorescein leakage
experiments showed that their membrane-perturbing activity is comparable to that of the
parent peptide, suggesting that our derivatizations do not perturb significantly the peptide
behaviour.
Fluorescence spectra, depth-dependent quenching experiments, and peptide-translocation
assays were employed to determine the location of the two peptides inside lipid bilayers, in
particular as a function of peptide/lipid ratio. Molecular dynamics simulations were performed
by a “minimum bias” approach, starting from a random mixture of water, lipid and peptide, and
following the spontaneous self-assembling of the lipid bilayer. The final membrane-bound 3Dstructure is perfectly consistent with the experimental results and in quantitative agreement
with the position of the fluorescent labels determined by depth-dependent quenching
experiments. For both peptides investigated, the atomic details of MD simulations provide new
insights on the mechanism of membrane destabilization.
Acknowledgements: With the support of the Ministry of Education, University and Research, and the Ministry of Foreign
Affairs of Italy.
122
New Frontiers in the Search of Bioactive Molecules: from Peptides to Drugs
Switching peptide bioactivity from cell-penetrating to
antimicrobial: the case of Pep-1 and Pep-1-K
Lorenzo Stella1, Sara Bobone1, Alessandro Piazzon1, Barbara Orioni1, Mariano Venanzi1, Song Yub
Shin2,3, Jae-Il Kim4, Kyung-Soo Hahm2,3
1
2
3
4
Dipartimento di Scienze e Tecnologie Chimiche, Università di Roma Tor Vergata, Roma, Italy.
Research Center for Proteineous Materials (RCPM), Chosun University, South Korea.
Department of Cellular Molecular Medicine, Chosun University, South Korea
Gwangju Institute of Science and Technology, Gwangju, South Korea
Cell-penetrating peptides (CPPs) are cationic oligopeptides able to translocate across
biological membranes without perturbing them, overcoming the impermeable nature of the
lipid bilayer. Antimicrobial peptides (AMPs), on the other hand, perturb the stability of bacterial
membranes, leading to cell death. Since the two peptide classes share several characteristics
(charge, amphipathicity, helicity, length), slight modifications in the primary sequence could
change peptide activity from membrane-penetrating to lytic.
Pep-1-K [KKTWWKTWWTKWSQPKKKRKV] is a new antimicrobial peptide derived from the
widely studied CPP Pep-1 [KETWWETWWTEWSQPKKKRKV], or “Chariot”, known for its
ability to carry large cargos across biological membranes. Pep-1-K was obtained from Pep-1
by substituting the three Glu residues with Lys residues, to increase its cationic character and
affinity for negatively charged bacterial membranes. Pep-1-K exhibited potent antimicrobial
activity, with MICs in the low μM range. Cellular studies showed that the bactericidal activity of
Pep-1-K involves dissipation of the biological transmembrane potential, suggesting a poreformation mechanism [1].
The five Trp residues in the Pep-1-K sequence allowed a spectroscopic characterization of its
interaction with model membranes. These studies showed that Pep-1-K has a higher affinity
for charged membranes than Pep-1, and that it is able to induce significant vesicle
aggregation. Depth-dependent quenching experiments showed that Pep-1-K lies close to the
membrane surface, parallel to it.
Leakage experiments, performed with liposomes loaded with carboxyfulorescein, yielded a
negative response, showing no dye leakage. However, studies performed by entrapping ion
sensitive fluorophores in lipid vesicles indicated that Pep-1-K causes ion leakage, in
agreement with the transmembrane potential dissipation previously observed in cellular
studies. Therefore, the membrane perturbation induced by Pep-1-K appears to be sufficient to
induce the flow of ions but not of larger solutes.
Acknowledgements: with the support of the Ministry Foreign Affairs, and the Ministry of Education, University and Research of
Italy.
References
1 W. L. Zhu, H. Lan, I.S. Park, J. I. Kim, H. Z. Jin, K.S. Hahm , S.Y. Shin, “Design and mechanism of action of a novel
bacteria-selective antimicrobial peptide from the cell-penetrating peptide Pep-1”. Biochem. Biophys. Res. Comm., 2006 349:
769–774
123
POSTER PRESENTATIONS - Eleventh Naples Workshop on Bioactive Peptides
Determination of antimicrobial peptide location inside lipid
bilayers by combined fluorescence spectroscopy and molecular
dynamics simulations
Lorenzo Stella1, Gianfranco Bocchinfuso1, Giacinto Grande1, Barbara Orioni1, Mariano Venanzi1, JinYoung Kim2, Yoonkyung Park2,3, Kyung-Soo Hahm2,3, Marta De Zotti2, Fernando Formaggio4,
Claudio Toniolo4, and Antonio Palleschi1
1
2
3
4
Dipartimento di Scienze e Tecnologie Chimiche, Università di Roma “Tor Vergata”, 00133 Roma, Italy
Research Center for Proteineous Materials (RCPM), Chosun University, 501-759 Gwangju, South Korea
Department of Cellular Molecular Medicine, Chosun University, 501-759 Gwangju, South Korea
Institute of Biomolecular Chemistry, Padova Unit, CNR, Department of Chemistry, University of Padova, 35131 Padova, Italy
Several bioactive peptides, such as antimicrobial, cell penetrating or fusogenic peptides, exert
their biological function by interacting with cellular membranes. Therefore, structural data on
the location of these molecules inside lipid bilayers are very important for a detailed
understanding of their mechanism of action. While it is difficult to apply the most powerful
structural techniques (X-ray diffraction and NMR) to membrane systems, fluorescence
spectroscopic methods are particularly suited to the study of peptide-membrane association,
but give only low-resolution information on peptide position in the lipid bilayer. Molecular
dynamics simulations, on the other hand, can provide a very detailed picture of the peptidemembrane interaction, but need to be validated by quantitative comparison with experimental
data.
We exploited several fluorescence approaches, together with MD simulations, to the
investigation of two antimicrobial peptides: the lipopeptaibol trichogin GA IV, and PMAP-23, a
member of the cathelicidin family. To perform the spectroscopic studies, a variety of peptide
analogues containing a single fluorophore was synthesized. Carboxyfluorescein leakage
experiments showed that their membrane-perturbing activity is comparable to that of the
parent peptide, suggesting that our derivatizations do not perturb significantly the peptide
behaviour.
Fluorescence spectra, depth-dependent quenching experiments, and peptide-translocation
assays were employed to determine the location of the two peptides inside lipid bilayers, in
particular as a function of peptide/lipid ratio. Molecular dynamics simulations were performed
by a “minimum bias” approach, starting from a random mixture of water, lipid and peptide, and
following the spontaneous self-assembling of the lipid bilayer. The final membrane-bound 3Dstructure is perfectly consistent with the experimental results and in quantitative agreement
with the position of the fluorescent labels determined by depth-dependent quenching
experiments. For both peptides investigated, the atomic details of MD simulations provide new
insights on the mechanism of membrane destabilization.
Acknowledgements: With the support of the Ministry of Education, University and Research, and the Ministry of Foreign
Affairs of Italy.
124
New Frontiers in the Search of Bioactive Molecules: from Peptides to Drugs
Host-pathogen adhesion at atomic level
Vitagliano Luigi1, Ruggiero Alessia1, Ciccarelli Luciano1, Pedone Carlo1,2 and Berisio Rita1
1 Istituto di Biostrutture e Bioimmagini, C.N.R., I-80134 – Napoli, Italy
2 Dipartimento delle Scienze Biologiche, Unibersità Federico II, I-80134 Napoli, Italy.
Most pathogens express fibrous adhesive virulence organelles that mediate targeting to the
sites of infection. The biogenesis of most of these fibres is driven by a complex protein
secretion system denoted as “chaperone–usher pathway” (1). The final architecture of the
fibrous organelle (pili or capsulae) depends on subunit:subunit interactions, which may lead to
different structures, like rigid pili, or capsulae. In both cases, fibres are constituted by subunits
(PapE, PapK in P pili and Caf1 in capsulae) which adopt a non-canonical imuno-globulin-like
(IG-like) fold, with a missing C-terminal strand and an N-terminal region which does not
interact with the rest of the protein (1-3). The folding of pilin subunits is assisted by a
chaperone, which donates the missing strand via a strand-donor mechanism. The stranddonor mechanism is vital to pilin-pilin assembly.
In order to obtain a detailed picture at atomic level of the molecular events related to this
process, we undertook molecular dynamics studies of the non-canonical immuno-globulin-like
PapE from the uropathogenic E. coli (2). Notably, the equilibrated structure of unliganded
PapE, which is difficult to characterise experimentally, displays unexpected features. Indeed,
a significant rearrangement of the local structure of the groove, which hosts the
complementary strands, is observed. This reorganisation, characterised by the formation of
several new hydrogen bonds, leads to a closure of the groove that likely makes pilin
polymerisation more difficult. These data suggest that chaperone release and pilin-pilin
association must be concerted processes and that chaperone plays an important role in
preventing pilin transitions towards states that are not prone to polymerise (2).
These analyses were extended to the F1 capsular antigen of the pathogen Yersinia pestis (4).
MD data indicate that the mechanism identified for the P pili E. coli (2) also operates for this
system. Using as starting model the structure of complex Caf1’-Caf1M-Caf1’’ (3) we also build
a stable dimeric state of Caf1 subunits. On this basis, a model for the capsula was generated
(4).
Since the mechanism of adhesion of other bacteria, e.g. Staphylococcus epidermidis, to
human fibrinogen and collagen shares some similarities with the protein of chaperone–usher
pathway we extended our computations to these systems. Preliminary data suggests similarity
and differences between the proteins involved in adhesion and in fibrillogenesis processes.
References
1 Remaut, H.; Rose, R. J.; Hannan, T. J.; Hultgren, S. J.; Radford, S. E.; Ashcroft, A. E.; Waksman, G. Mol Cell. 2006. 22,
831-842.
2 Vitagliano, L.; Ruggiero, A.; Pedone, C.; Berisio, R. Journal of Molecular Biology. 2007, 367, 935-41.
3 Zavialov, A. V.; Berglund, J.; Pudney, A. F.; Fooks, L. J.; Ibrahim, T. M.; MacIntyre, S.; Knight, S. D. Cell 2003, 113, 587596.
4 Vitagliano, L.; Ruggiero, A.; Pedone, C.; Berisio, R.. 2008, Submitted.
125
POSTER PRESENTATIONS - Eleventh Naples Workshop on Bioactive Peptides
Revival of M. tuberculosis from latency
Ruggiero Alessia1, Pedone Emilia1, Squeglia Flavia1, Pedone Carlo1,2, Wilmanns Matthias and
Berisio Rita1
1 Istituto di Biostrutture e Bioimmagini, C.N.R., I-80134 – Napoli, Italy
2 Dipartimento delle Scienze Biologiche, Unibersità Federico II, I-80134 Napoli, Italy.
The interaction between M. tuberculosis and the human host after infection may manifest itself
as a chronic disease or as a latent (or dormant) infection, a state capable to evade host
responses. The probability of reactivation from dormancy is strongly affected by the type of
host immune response and it is significantly enhanced in immuno-compromised patients, e.g.
suffering from AIDS. Understanding and controlling the entry and exit from dormancy is
important in the development of new anti-tubercular therapies.
Resuscitation of dormant bacteria is promoted by resuscitation-promoting-factors, Rpfs, which
are secreted from slowly replicating bacteria close to dormant bacteria. These proteins are
thought to cleave peptidoglycans which constitute the cell wall(1). Cell wall cleavage could
alter cell wall mechanical properties and favour cell division and/or release anti-dormancy
factors (1). Although these proteins are targets for antibiotics and key proteins for the
formulation of vaccines, available structural information are scarce.
We have determined the first crystal structure of a Resuscitation Promoting Factor, RpfB
(2,3). Beside aiming at a better understanding of the mechanism of exit from dormancy in M.
tuberculosis, the comprehension of the structural features associated to Rpf and RipA
activity/inhibition will provide the bases for the identification of molecules (pro-latency
molecules) able to restrict bacterial life to the latent, non-dangerous, state.
References
1 Keep, N.H.; Ward, J.M.; Cohen-Gonsaud, M.; Henderson, B. Trends Microbiol, 2006, 14: 271-276.
2 Ruggiero, A; Tizzano, B; Geerlof, A; Pedone, E; Pedone, C; Wilmanns, M.; Berisio, R. Acta Crystallogr Sect F Struct Biol
Cryst Commun. 2007, 63, 870-873.
3 Ruggiero, A; Tizzano, B; Pedone, E; Pedone, C; Wilmanns, M.; Berisio, R. Submitted.
126
New Frontiers in the Search of Bioactive Molecules: from Peptides to Drugs
Insights into the dimeric structure of
Gadd45β and its interaction with MKK7
Monti S.M.1, Tornatore L. 1,2, Vitale R. M. 3, Dathan N. 1, Marasco D. 1, Papa S. 4; Franzoso G. 4;
Benedetti E. 2, and Ruvo M. 1
1.
2.
3.
4.
Istituto di Biostrutture e Bioimmagini (IBB), CNR, via Mezzocannone, 16, 80134, Napoli, Italy
Dipartimento delle Scienze Biologiche, via Mezzocannone, 16, 80134, Napoli, Italy
Istituto di Chimica Biomolecolare (ICB), CNR, Via Campi Flegrei, 34, 80078 Pozzuoli, (NA), Italy.
Department of Immunology at Hammersmith, Division of Investigative Science, Imperial College, London, London W12 ONN, UK.
The NF–κB/rel transcription factors are crucial regulators of cell survival, inflammation and
immune response. Activation of NF-κB antagonizes programmed cell death (PCD) induced by
death receptors, including tumor necrosis factor–receptor (TNF-Rs), while their suppression
promotes apoptosis by several mechanisms. One of these involves the blockade of the JNK
cascade through a mechanism regulated by the expression of Gadd45β, a member of the
Gadd45 family of stress-inducible factors, therefore Gadd45β is now believed to be an
important molecular link between the NF-κB response and the JNK pro-apoptotic pathway.
This mechanism has been largely investigated[1, 2] and it has been found that Gadd45β
strongly interacts with MKK7, an upstream JNK activator, inhibiting its kinase activity. Indeed,
Gadd45β binds to crucial residues within the kinase catalytic pocket, including the ATPbinding residues Lys149, thus acting as an endogenous inhibitor. Thus it represents an
important target for developing selective anti-inflammatory and anti-tumor drugs. For this
purpose, it would be of utmost importance to know the 3D structure of both Gadd45β and
MKK7 and the molecular details underlying their interaction. Since all the attempts to obtain
the 3D structure by X-ray crystallography have so far failed, we have undertaken an accurate
structural study of the complex by using homology modeling and a large set of data based on
co-immunoprecipitation and limited proteolysis and alkylation analyses. The structure of
Gadd45b, modeled using as template the spliceosomal protein 15.5K, suggests that the
protein is constituted by a series of alternating alpha helices and beta-strands which form an
alpha-beta-alpha sandwich fold fully consistent with the experimental data on both Gadd45β
alone and its complex with the kinase[3]. Homodimerization of Gadd45β has also been
investigated by using in competitive ELISA assays peptidic fragments coming from Gadd45β
extensive digestion and purified by HPLC. Results show that peptides corresponding to the
predicted helix 1 and predicted helix 5 are able to markedly reduce Gadd/Gadd selfassociation thus suggesting their involvement in recognition. Gadd45β interaction with MKK7
has also been investigated and we found that the large homodimerization surface is not
involved in MKK7 recognition[4].
References
1 De Smaele, E., Zazzeroni, F., Papa, S., Nguyen, D. U., Jin, R., Jones, J., Cong, R. & Franzoso, G. (2001). Induction of
Gadd45beta by NF-kappaB downregulates pro-apoptotic JNK signalling. Nature 414, 308-13.
2 Papa, S., Zazzeroni, F., Bubici, C., Jayawardena, S., Alvarez, K., Matsuda, S., Nguyen, D. U., Pham, C. G., Nelsbach, A.
H., Melis, T., De Smaele, E., Tang, W. J., D'Adamio, L. & Franzoso, G. (2004). Gadd45 beta mediates the NF-kappa B
suppression of JNK signalling by targeting MKK7/JNKK2. Nat Cell Biol 6, 146-53.
3 Papa, S., Monti, S. M., Vitale, R. M., Bubici, C., Jayawardena, S., Alvarez, K., De Smaele, E., Dathan, N., Pedone, C.,
Ruvo, M. & Franzoso, G. (2007). Insights into the structural basis of the GADD45beta -mediated inactivation of the JNK
kinase, MKK7/JNKK2. J Biol Chem 2 an endogenous inhibitor. It thus represents an important target 82, 19029-19041.
4 Tornatore L, Marasco D, Dathan N, Vitale RM, Benedetti E, Papa S, Franzoso G, Ruvo M, Monti S M. (2008) Gadd 45beta
forms a homodimeric complex that binds tightly to MKK7 J Mol Biol. 378, 97-111.
127
POSTER PRESENTATIONS - Eleventh Naples Workshop on Bioactive Peptides
Antimicrobial peptides to modulate
infectious diseases: defensins.
Cantisani Marco1,2, Galdiero Stefania1, Scudiero Olga2, Vitello Mariateresa3, Galdiero Massimiliano3,
Castaldo Giuseppe2, Salvatore Francesco2, Pedone Carlo1
1,. University of Naples “Federico II”, Department of Biological Sciences & CIRPEB, 80134, Naples, Italy
2. University of Naples “Federico II” Department of Biochemistry and Medical Biotechnologies, Faculty of Biotechnological Sciences, & CEINGE,
80131, Naples, Italy
3. II University of Naples, Department of Experimental Medicine,80138, Naples, Italy
Defensins, antimicrobial peptides, form part of our innate immune system; two classes are
found in humans, alpha- and beta-defensins. Epithelial cells secrete human beta defensins
(hBDs). They kill bacteria and fungi in a quite aspecific way and/or by pore formation in the
target membrane; they also exert chemotactic properties towards immature dendritic cells and
T-cells, coupling the human innate immune response to the cellular response. A properly
operating immune system is necessary in the prevention and elimination of infections, like
lung infections, or viral infections. Since defensins are expressed in the airways and since
they have chemotactic and antimicrobial properties, they are good candidate modifier genes
for lung diseases.
It is well known that, the antibacterial activity of beta-defensin 1, 2 and 4 is modulated by the
salt concentrations; the high levels of NaCl observed, for example in the respiratory layer of
cystic fibrosis patients, can inhibit the activity of beta-defensins, contributing to chronic
infections and bacterial colonisation. On the other hand, the antibacterial activity of human
beta defensin 3 is not modulated by the salt concentration of the medium. To increase our
insight into the pathophysiology of beta defensins and to clarify the potential role of these
peptides as therapeutic targets, we performed in vitro tests to evaluate the antimicrobial
(against Pseudomonas aeruginosa), pro-chemotactic (toward neutrophils) and the antiviral
activity (against herpes simplex virus) of beta-defensin variants obtained by chemical
synthesis.
128
New Frontiers in the Search of Bioactive Molecules: from Peptides to Drugs
New peptide antagonists / agonists as
selective ligands for CXCR4 receptor
Monfregola Luca1, Salapete Filomena1, Scala Stefania2, Amodeo Piero3, Vitale Rosa Maria3,
De Luca Stefania4
1.
2.
3.
4.
University of Naples “Federico II”, Department of Biological Sciences, 80134 Naples - Italy
National Institute for the Study and Cure of Cancer “G.Pascale”, 80131 Naples – Italy
National Institute of Research ICB- CNR, 80078 Pozzuoli (NA) – Italy
National Institute of Research IBB- CNR, 80134 Naples – Italy
The CXC chemokine receptor 4 (CXCR4) plays a role in recruitment of leukocytes to sites of
inflammation and to secondary lymphoid organs [1-3]. Its ligand, CXCL12, is a highly efficient
chemotactic factor for T cells, monocytes, pre-B cells, dendritic cells, and hematopoietic
progenitor cells [4]. Numerous evidences demonstrated that CXCL12 supports survival or
growth of a variety of normal or malignant cell types, including hematopoietic progenitors,
germ cells, leukemia B cells, and carcinoma cells [5-10]. The distinct pattern of chemokine
receptor expression by neoplastic cells has a critical role in determining the site(s) of
metastatic spread. Taken together, CXCR4 receptor is thought to represent an important
therapeutic target. Thus, a library of peptide analogues derived from a specific domain of the
endogenous ligand CXCL12 has been designed and synthesised. Biological activities of the
synthetic compounds have been evaluated by several assays.
References
1. Aiuti, A., Webb, I. J., Bleul C., Springer, T., Gutierrez-Ramos, J. C.. J. Exp. Med. 1997. 185: 111-120
2. Gupta, S. K., Lysko, P. G., Pillarisetti, K., Ohlstein, E., Stadel, J. M. J. Biol. Chem. 1998. 273: 4282-4287
3. Ma, Q., Jones, D., Borghesani, P. R., Segal, R. A., Nagasawa, T., Kishimoto, T., Bronson, R. T., Springer, T. A. Proc. Natl.
Acad. Sci. 1998. 95: 9448-9453
4. Baggiolini, M. Chemokines and leukocyte traffic. Nature. 1998. 392, 565-56
5. Zhou Y, Larsen PH, Hao C, Yong VW. J Biol Chem. 2002. 277: 49481-49487.
6. Geminder H, Sagi-Assif O, Goldberg L, et al. J Immunol. 2001. 167:4747-4757.
7. Zeelenberg IS, Ruuls-Van Stalle L, Roos E. Cancer Res. 2003. 63:3833-3839.
8. Wang J, Sun Y, Song W, Nor JE, Wang CY, Taichman RS. Cell Signal. 2005. 17:1578-1592.
9. Scotton CJ, Wilson JL, Scott K, et al. Cancer Res. 2002. 62:5930-5938.
10. Muller A, Homey B, Soto H, et al. Nature. 2001;410:50-56.
129
POSTER PRESENTATIONS - Eleventh Naples Workshop on Bioactive Peptides
Human prion α2-helix-related peptides: role of
metal cation in protein folding and stability
Luisa Ronga1, Pasquale Palladino1, Carole Dubois1, Raffaele Ragone2, Ettore Benedetti1 and
Filomena Rossi1
1 Università degli Studi di Napoli “Federico II”, Dipartimento delle Scienze Biologiche & CIRPeB, 80134 Naples - Italy
2 Seconda Università di Napoli, Dipartimento di Biochimica e Biofisica & CRISCEB, 80138 Naples- Italy
The transformation of the cellular prion protein (PrPC) into the infectious form (PrPSc) is
implicated in the fatal transmissible spongiform encephalopathies [1]. The normal function of
PrPC remains unknown, although its conservation in different species infers that it has some
relevance in basic physiological processes. PrPC has been proposed to be involved in several
functions such as copper and/or zinc ion transport or metabolism [2,3].
The metal binding region of PrP resides in the flexible N-terminal tail. In fact, most of the
major metal-dependent responses are linked to this region, which contains four repeats of the
highly conserved octapeptide PHGGGWGQ. PrP constructs lacking the metal binding region
fail to undergo endocytosis efficiently. However, based on the formation of different Cu2+
complexes with blocked and free C- and N-termini analogues of the peptide fragment 180–
193 (VNITKQHTVTTTT), which almost entirely encompasses the PrP α2-helix, Brown and coauthors [4] have suggested that a copper(II) binding site is located on the His187 residue of
the structured C-terminal domain. They have also proposed that the anchoring imidazole
residue drives the metal coordination environment towards a common binding motif in
different regions of the prion protein. As the α2-helix possesses chameleon conformational
behavior [5] and gathers several disease-associated point mutations [1], it is a suitable model
to investigate both structural determinants of PrPC misfolding and rational structure-based
drug design of compounds able to block or prevent prion diseases.
The intriguing structural properties of this protein domain, as well as the influence that a
disease-associated mutation can have on its relative stability, prompted us to perform CD and
steady-state fluorimetry structural investigations on N-terminal fluoresceine derivatized
peptides corresponding to the sequences: Fluo-βANNFVHDC(Met)VNITIKQHTVTTTTKGNH2, Fluo-PrP[173-195], Fluo-βANNFVHNC(Met)VNITIKQHTVTTTTKG-NH2 Fluo-PrP[173195]D178N, in water and in presence of increasing amounts of metal cations. Peptide titration
with metal cations provided further evidence that the main target of metal interaction is likely
located elsewhere in the protein, where structural restraints opposing binding are less
effective.
Acknowledgments: C. Dubois thanks the Erasmus Project: Universitè Paul Cezanne Aix-Marseille III and University “Federico
II” of Naples for the stage.
References
1 S.B. Prusiner Proc. Natl. Acad. Sci. USA (1998) 95, 13363-13383.
2 D.R. Brown et al. J. Neurochem. (2003) 87, 353-363.
3 A.G. Kenward et al. Biochemistry (2007) 46, 4261-4271.
4 D.R. Brown et al. Biochemistry (2004) 98, 133-143.
5 B. Tizzano et al. Proteins (2005) 59, 72-79
130
New Frontiers in the Search of Bioactive Molecules: from Peptides to Drugs
Effect of cholesterol on Aβ (25-35)-membrane interaction
investigated by EPR spectroscopy.
Anna Maria D’Ursi2, Gerardino D’Errico1, Anna Ramunno2, Pietro Campiglia2, Ettore Novellino3
1. Dipartimento di Chimica, Università di Napoli “Federico II”, via Cintia, Napoli, Italy.
2. Dipartimento di Scienze Farmaceutiche, University of Salerno, 84084- Fisciano Italy;
3. Dipartimento di Chimica Farmaceutica e Tossicologica, University of Napoli, - Napoli Italy;
Alzheimer disease (AD) is the most common form of senile dementia, affecting up to 15
million individuals worldwide. Because of the ongoing increase in life expectancy, by 2050 we
can expect approximately 25% of people living in the Western hemisphere to be over 65 years
of age, one third of whom are likely to develop AD. From a morphological viewpoint, the main
features of AD are: i. accumulation of extracellular amyloid plaques, triggered by the
aggregation of the β-amyloid peptides (Aβ), in the brains of affected individuals; ii. presence of
intracellular neurofibrillary tangles, which consist mainly of aggregates forms of the
microtubule stabilizing protein tau. In the past years amyloid plaques were generally regarded
as the cause of the cognitive disorder. More recently, the relevance of soluble protofibrillar
oligomeric forms of Aβ has been recognised.
In the Aβ peptides sequence, the residues from 29 to the C terminus belong to the
transmembrane domain of the precursor protein, and consequently these peptides are
expected to favourably interact with lipid membranes. In the present work we focus our
attention on the interaction between a cytotoxic fragment of Aβ, i.e., Aβ (25-35), and
phospolipid bilayers of different composition and net charge.
Particularly, our interest is in the effect of cholesterol on the peptide insertion into a lipid
bilayer. Cholesterol is an essential component of animal cellular membranes and is well
known for influencing the membrane fluidity, permeability and capacity.
Electron Paramagnetic Resonance (EPR) spectroscopy, by using spin-labelled substances,
has proved to be a fruitful experimental approach to the study of the interactions between
peripheral as well as integral proteins and membranes. In this work, we investigate the Aβ
(25-35)-membrane interaction using EPR probes. Both the effect of the bilayer charge and the
presence of cholesterol have been investigated, in the attempt to clarify the molecular bases
of the peptide-membrane interaction.
The results show in membranes containing more less than 20% cholesterol w/w, the insertion
of Aβ (25-35) is favoured, while higher cholesterol contents lead to a release of the peptide
from the lipidic bilayer.
131
POSTER PRESENTATIONS - Eleventh Naples Workshop on Bioactive Peptides
Synthesis and immunological activity of β3-phenylalaninesubstituted cyclolinopeptide A analogs
Biancamaria Farina1, Krzysztof Kaczmarek2, Paweł Zubrzak2, Stefan Jankowski2, Michał Zimecki3,
Piotr Suder4, Janusz Zabrocki2, Michele Saviano1, Roberto Fattorusso5 and Ettore Benedetti1
1 Istituto di Biostrutture e Bioimmagini, CNR and Dipartimento di Chimica Biologica, Universitả degli Studi di Napoli “Federico II”, 80134, Napoli,
Italy
2 Institute of Organic Chemistry, Faculty of Chemistry, Technical University of Łódź, 90-924 Łódź, Poland
3 Institute of Immunology and Experimental Therapy, Polish Academy of Sciences, 53-114 Wroclaw, Poland.
4 Faculty of Chemistry and Regional Laboratory, Jagiellonian University, 30-060 Kraków, Poland.
5 Dipartimento di Scienze Ambientali, Seconda Università di Napoli, 81100, Caserta, Italy.
Cyclolinopeptide A (CLA), a natural highly hydrophobic cyclic nonapeptide: cyclo(–Pro1-Pro2Phe3-Phe4-Leu5-Ile6-Ile7-Leu8-Val9-), isolated from linseed oil, was found to possess a strong
immunosuppressive activity comparable in low doses with that of cyclosporine A (CsA) with a
mechanism that depends on the inhibition of the interleukin-1 and interleukin-2 action. The
determination of the x-ray structure and the NMR structural analysis of CLA, gave the basis to
study the biological, structural, and conformational properties of CLA and related compounds.
In solution, at room temperature, CLA exhibits marked conformational mobility leading to the
presence of a mixture of several conformers. A suitable NMR analysis of CLA has been
carried out in CDCl3 at low temperature (214 K). In these conditions CLA exists as a single
“frozen” conformer, whose structure is consistent with that found in the solid state. It has been
postulated that the following structural features are important for the immunosuppressive
activity of CLA: presence of the tetrapeptide Pro-Pro-Phe-Phe sequence containing the ProPro cis amide bond and the “edge-to-face” interaction and distance between the aromatic
rings.
In order to evaluate the role and significance of “edge-to-face” interaction in a process of
molecular recognition by receptors, we have synthesized three linear precursors and three
cyclic analogues of CLA, in which one or both phenylalanine residues have been replaced by
β3-phenylalanine residues.
1. Ile6-Ile7-Leu8-Val9-Pro1-Pro2-β3Phe3-Phe4-Leu5
2. Ile6-Ile7-Leu8-Val9-Pro1-Pro2-Phe3-β3Phe4-Leu5
3. Ile6-Ile7-Leu8-Val9-Pro1-Pro2-β3Phe3-β3Phe4-Leu5
4. cyclo(Ile6-Ile7-Leu8-Val9-Pro1-Pro2-β3Phe3-Phe4-Leu5-)
5. cyclo(Ile6-Ile7-Leu8-Val9-Pro1-Pro2-Phe3-β3Phe4-Leu5-)
6. cyclo(Ile6-Ile7-Leu8-Val9-Pro1-Pro2-β3Phe3-β3Phe4-Leu5-)
An NMR characterization was carried out on compound 4 in CD3CN/H2O mixture to study the
influence of the β3Phe residue on the three dimensional structure of the CLA analogue. The
linear and cyclic CLA analogues, containing β3-phenylalanine, were tested in the humoral and
cellular immune response in vivo assays in mice. The activities of the peptides were
compared with cyclosporine A, as the reference drug.
132
New Frontiers in the Search of Bioactive Molecules: from Peptides to Drugs
A NMR fragment-based approach for the identification of
PED/PEA15 binders
Biancamaria Farina1,4, Laura Zaccaro1, Luciano Pirone1,4, Emilia Pedone1, Francesca Viparelli1,
Menotti Ruvo1, Claudia Miele3, Francesco Beguinot3, Carlo Pedone1, 4, Roberto Fattorusso2
1
2
3
4
Istituto di Biostrutture e Bioimmagini, CNR and Dipartimento di Chimica Biologica, Universitả degli Studi di Napoli “Federico II”, 80134, Napoli, Italy
Dipartimento di Scienze Ambientali, Seconda Università di Napoli, 81100, Caserta, Italy.
Dipartimento di Biologia e Patologia Cellulare e Molecolare “L. Califano”, Napoli, 80131 – Italy
Università degli Studi di Napoli, Napoli 80134 – Italy
Nuclear Magnetic Resonance is a powerful tool in the discovery of novel bioactive molecules.
In particular, the search for strongly binding ligands that inhibit protein-protein interactions
constitutes an active and challenging field of research in drug discovery.
In this work we have implemented some of the NMR tools to target the system, PEA15/PED
(Phosphoprotein Enriched in Diabetes or in Astrocytes). PED is a 15 KDa cytosolic protein
that, through protein-protein interactions, is involved in multiple cellular functions including
apoptosis, phospholipase D expression, ERK activation and promotion of resistance to insulin
in type II diabetes[1,2]. Therefore, the development of molecules able to bind PED and inhibit
its interaction with its binding partners is a crucial step in the design and the development of
new drugs.
We have performed a screening of a previously designed molecular fragments library using
an unlabelled sample of PED using protein-ligand magnetisation transfer, such as
waterLOGSY[3] and STD[4]. Several frameworks able to weakly bind PED have been identified.
In particular, one pair of molecules have resulted to bind PED in close proximity and the
structural details of their interaction with PED have been characterized by Inter-Ligand NOE
(ILOE) experiments and chemical shift mapping by means of 15N-1H HSQC.
References
1. Vigliotta, G.; Miele, C.; Santopietro, S.; Portella, G.; Perfetti, A.; Maitan, M. -A.; Cassese, A.; Oriente, F.; Trencia, A.; Fiory,
F.; Romano, C.; Tiveron, C.; Tatangelo, L.; Troncone, G.; Formisano, P.; Beguinot, F. Mol. Cell. Biol. 2004, 24, 5005-5015.
2. Zhang, Y.; Redina, O.; Altshuller, Y. -M.; Yamazaki, M.; Ramos, J.; Chneiweiss, H.; Kanaho, Y. and Frohman, M. -A. J. Biol.
Chem., 2000, 275, 35224-35232.
3. Dalvit, C.; Pevarello, P.; Tato, M.; Veronesi, M.; Vulpetti, A.; Sundstrom, M. J. Biomol NMR, 2000, 18 , 65-68.
4. Mayer, M.; Meyer, B. Angew. Chem. Int. Edn. Engl. 1999, 38, 1784-1788.
133
POSTER PRESENTATIONS - Eleventh Naples Workshop on Bioactive Peptides
Inside the PXXP motif. A combined NMR and CD
study of hexapeptides belonging to the
N-terminal domain of Chicken prion protein
Luca Raiola1, Carla Isernia1, Roberto Fattorusso1, Gaetano Malgieri1, Giuseppe Pappalardo2,
Adriana Pietropaolo3 and Diego La Mendola2
1. Dipartimento di Scienze Ambientali, Seconda Università di Napoli, 81100 Caserta, Italy
2. CNR-Istituto di Biostrutture e Bioimmagini, 95125 Catania, Italy
3. Dipartimento di Scienze Chimiche, Università di Catania, 95125 Catania, Italy
Prion diseases result from the conversion of a normal glycoprotein PrPC into a misfolded and
pathogenic isoform (PrPSc). Although the prion protein is expressed in different species, the
diseases have been so far observed only in mammals. Mammal and chicken prion proteins
both contain repeated regions in the N-terminal portion: mammal octarepeats (PHGGGWGQ)
contain a high number of glycines while chicken hexarepeats (PHNPGY) are characterized by
a high number of prolines. The presence of the PXXP motif in the chicken prion protein
suggests that this region may possess a defined secondary structure. Here, we study, by
means of NMR and CD spectroscopies, a series of hexapeptides encompassing the
sequence of the repeat region.
134
New Frontiers in the Search of Bioactive Molecules: from Peptides to Drugs
The NMR structure of an antimicrobial decapeptide
Neri Niccolai, Chiara Falciani, Alessandro Pini, Luisa Bracci, Alexandros Athanasopoulos, Vincenzo
Venditti, Ottavia Spiga and Andrea Bernini
University of Siena, Department of Molecular Biology, 53100 Siena - Italy
Antimicrobial peptides are a new family of antibiotics that have stimulated research and
clinical interest [1]. Most antibacterial peptides are components of the innate immunity of
animals and plants against microbial infections [2,3]. Here we present a structural investigation
in solution of QKKIRVRLSA, L6, a linear decapeptide which exhibits strong antibacterial
activity [4,5]. As expected, the bioactive peptide is fully disordered in water at physiological
conditions. Conversely, in the presence of SDS micelles the conformational equilibrium of L6
is totally shifted towards the formation of a regular α helix, as unambiguously suggested by
the observed NOE network.
In order to correlate L6 activity and structure, the orientation of the helix in respect to the
membrane surface was investigated by analysing paramagnetic perturbation profiles of 1HTOCSY signal attenuations obtained for L6 in the presence of Gd(III) DTPA-BMA.
The observed pattern of paramagnetic attenuations of CαH correlations is consistent only with
an horizontal alignment of the decapeptide on the surface of the SDS micelle, see Fig. 1.
The sterical features of the L6 – vesicle interaction will drive the design of new analogs of
increased antimicrobial activity.
Fig. 1. The decapeptide QKKIRVRLSA laying on the surface of a SDS vesicle, as
suggested by the obtained paramagnetic attenuation profile of -1H-TOCSY signals
References:
1. Wu, M., E. Maier, R. Benz, and R. E. Hancock. 1999. Mechanism of interaction of different classes of cationic antimicrobial
peptides with planar bilayers and with the cytoplasmic membrane of Escherichia coli. Biochemistry 38:7235–7242.
2. Zasloff, M. 2002. Antimicrobial peptides of multicellular organisms. Nature 415:389–395.
3. Boman, H. G. 1995. Peptide antibiotics and their role in innate immunity. Annu. Rev. Immunol. 13:61–92.
4. Pini A, Giuliani A, Falciani C, Runci Y, Ricci C, Lelli B, Malossi M, Neri P, Rossolini GM, Bracci L. 2005 Antimicrobial activity
of novel dendrimeric peptides obtained by phage display selection and rational modification. Antimicrob Agents Chemother.
497:2665-2672.
5. Pini A, Giuliani A, Falciani C, Fabbrini M, Pileri S, Lelli B, Bracci L. 2007 Characterization of the branched antimicrobial
peptide M6 by analyzing its mechanism of action and in vivo toxicity. J Pept Sci.
135
POSTER PRESENTATIONS - Eleventh Naples Workshop on Bioactive Peptides
Molecular recognition between neurotrophins
and their receptors: a molecular dynamics approach
Stanzione Francesca1, Esposito Luciana1, Paladino Antonella1,2, Morelli Giancarlo1,3, Pedone Carlo1,3
and Vitagliano Luigi1
1 Istituto di Biostrutture e Bioimmagini, C.N.R., I-80134 – Napoli, Italy
2 Istituto di Scienze Alimentari, C.N.R. I-83100 – Avellino, Italy
3 Dipartimento delle Scienze Biologiche, Università Federico II, I-80134 Napoli, Italy.
Neurotrophins (NTs) play a major role in the differentiation, survival, and maintenance of
nervous cells. A particular attention is currently devoted to the development of mimetics
endowed with NT-like actions since their potential as therapeutic has been demonstrated by
several independent investigations (1,2). Therefore, a deep understanding of the structural
determinants of their function and specificity is required. Extensive crystallographic studies
have provided a detailed picture of NTs in their unliganded states and in complex with
receptors (Trk and p75) (3-6). On the other hand, limited data are available on their dynamic
properties. Here we present a detailed molecular dynamics investigation aimed at identifying
the role of the intrinsic conformational preferences of the N-terminal regions of NTs in the NTTrk recognition and specificity. These investigations have been extended by the analysis of
the dynamical properties of the complex between the nerve growth factor (NGF) and the low
affinity receptor p75.
Our data clearly indicate that intrinsic conformational preferences of the N-terminal regions of
NGF and NT4 play an important role in the binding process to their tyrosine kinase receptors
(TrkA and TrkB). We also identified the residues responsible for the distinct behavior of NGF
and NT4 N-termini. On this basis, we suggest that the design of peptide mimetics of these
fragments with aminoacids endowed with enhanced preferences for these structural motifs is
likely to yield high affinities for these receptors. Depending on their monomeric or dimeric
state, these peptides may act as NT antagonist or agonist.
The analyses of the dynamical properties of the complex between NGF and p75 have
provided clear indications on the role played by the residues located at protein-protein
interface. Our data clearly support the idea that electrostatic interactions play a major role in
the stabilization of the complex.
References
1 Chao, M.V. Nature Rev. Neurosci. 2003, 4, 299-309.
2 Colangelo, A.M.; Bianco, M.R.; Vitagliano, L.; Cavaliere, C.; Cirillo, G.; De Gioia, L.; Diana, D.; Colombo, D.; Redaelli, C.;
Zaccaro, L.; Morelli, G.; Papa, M.; Sarmientos, P.; Alberghina, L.; Martegani, E. J Neurosci. 2008, 28, 2698-2709.
3 Wiesmann, C.; Ultsch, M.H.; Bass, S.H.; de Vos, A.M. Nature. 1999, 401, 184-188.
4 Wehrman, T.; Hem X.; Raab, B.; Dukipatti, A.; Blau, H.; Garcia, K.C. Neuron. 2007, 53, 25-38.
5 Wiesmann, C.; de Vos, A.M. Cell Mol Life Sci. 2001, 58, 748-759.
6 He, X.L.; Garcia, K.C. Science. 2004, 304, 870-875.
136
New Frontiers in the Search of Bioactive Molecules: from Peptides to Drugs
Unveiling the structural bases of collagen
stability: computational approaches
Berisio Rita1, De Simone Alfonso1,2, Improta Roberto1 and Vitagliano Luigi1
1 Istituto di Biostrutture e Bioimmagini, C.N.R., I-80134 – Napoli, Italy
2 Department of Chemistry,University of Cambridge, Lensfield Road CB2 1EW, Cambridge UK
Unveiling sequence-stability and structure-stability relationships is a major goal of protein
chemistry and structural biology. Despite the enormous efforts devoted, answers to these
issues remain elusive. In principle, collagen represents an ideal system for such investigations
due to its simplified sequence and regular structure. However, the definition of the molecular
basis of collagen triple helix stability has hitherto proved to be a difficult task. Particularly
puzzling is the decoding of the mechanism of triple helix stabilization/destabilization induced
by imino acids. Although the propensity-based model (1,2), which correlates the propensities
of the individual iminoacids with the structural requirements of the triple helix, is able to
explicate most of the experimental data, it is unable to predict the rather high stability of
peptides embedding (Gly-Hyp-Hyp) triplets (3-5). Starting from the available X-ray structures
of this polypeptide (6,7), we carried out an extensive quantum chemistry analysis of the
mutual interactions established by hydroxyproline residues located at the X and Y positions of
the Gly-X-Y motif. Our data clearly indicate that the opposing rings of these residues establish
significant van der Waals and dipole-dipole interactions that play an important role in triple
helix stabilization (8). These findings suggest that triple helix stabilization can be achieved by
distinct structural mechanisms. The interplay of these subtle but recurrent effects dictates the
overall stability of this widespread structural motif.
We extended the analysis of collagen stability by performing molecular dynamics simulations
to analyze triple helix hydration in regions characterized by different imino/aminoacid
contents. Data emerged from MD simulations show that (a) MD simulations can reliably
reproduce the hydration sites identified experimentally, (b) water molecules bound to regions
with a different amino/iminoacid content exhibit diversified residence times, (c) the local
environment of the water-bridges located in the aminoacid–rich regions strongly influence
their binding to the peptide. MD results also suggest that, in aminoacid rich regions, the
stabilizing effects of Arg and Hyp residues on collagen triple helix also depend on watermediated interactions. On this basis, we propose that the mechanism of triple helix
stabilization is sequence-dependent (9). MD simulations have also been extended to stable
collagen-like motifs lacking iminoacids (10).
References
1 Vitagliano, L.; Berisio, R.; Mazzarella, L.; Zagari, A. Biopolymers. 2001, 58, 459-464.
2 Vitagliano, L.; Berisio, R.; Mastrangelo, A.; Mazzarella, L.; Zagari, A. Protein Sci. 2001, 10, 2627-2632.
3 Berisio, R.; Granata, V.; Vitagliano, L.; Zagari, A. J. Am. Chem. Soc. 2004, 126, 11402-11403.
4 Mizuno, K.; Hayashi, T.; Peyton, D.H.; Bachinger, H.P. J. Biol. Chem. 2004, 279, 38072-38078.
5 Doi, M.; Nishi, Y.; Uchiyama, S.; Nishiuchi, Y.; Nishio, H.; Nakazawa, T.; Ohkubo, T.; Kobayashi, Y. J. Pept. Sci. 2005 11,
609-616.
6 Schumacher, M.; Mizuno, K.; Bachinger, H.P. J. Biol. Chem. 2005, 280, 20397-20403.
7 Kawahara, K.; Nishi, Y.; Nakamura, S.; Uchiyama, S.; Nishiuchi, Y.; Nakazawa, T.; Ohkubo, T.; Kobayashi, Y. Biochemistry.
2005, 44, 15812-15822
8 Improta, R.; Berisio, R.; Vitagliano, L. Protein Sci. 2008, In press.
9 De Simone, A.; Vitagliano, L.; Berisio, R. Biochem Biophys Res Commun. 2008, Submitted.
10 Mohs, A.; Silva, T.; Yoshida, T.; Amin, R.; Lukomski, S.; Inouye, M.; Brodsky, B. J Biol Chem. 2007, 282,29757-65.
137
POSTER PRESENTATIONS - Eleventh Naples Workshop on Bioactive Peptides
Synthesis and conformational analysis of a cyclic peptides
obtained via i-to-i+4 Intramolecular side-chain-to-side-chain
Azide–Alkyne 1,3-Dipolar Cycloaddition
Mario Scrima1, Sonia Cantel2, Alexandra Le Chevalier-Isaad3,4, Jay J. Levy5, Richard D. DiMarchi5,
Paolo Rovero3,6, Jose A. Halperin,7, Anna Maria D'Ursi1, Anna Maria Papini3,4 and Michael Chorev2,7
1
2
3
4
5
6
7
Dipartimento di Scienze Farmaceutiche, Salerno, I-84084, Italy;
University Laboratory for Translational Research, Harvard Medical School, Cambridge, MA 02139, USA;
Laboratory of Peptide & Protein Chemistry & Biology, Polo Scientifico e Tecnologico, University of Firenze, I-50019 Sesto Fiorentino, Italy;
Dipartimento di Chimica Organica, Polo Scientifico e Tecnologico, University of Firenze, Sesto Fiorentino, I-50019, Italy;
Department of Chemistry, Indiana University, 800 E. Kirkwood, Bloomington, IN 47405, USA;
Dipartimento di Scienze Farmaceutiche, University of Firenze, Via Ugo Schiff 3, Polo Scientifico e Tecnologico, Sesto Fiorentino, I-50019, Italy;
Department of Medicine, Brigham and Women's Hospital, Boston, MA 02115, USA.
Intramolecular side chain-to-side chain cyclization is an established approach to achieve
stabilization of specific conformations and a recognized strategy to improve resistance
towards proteolytic degradation. We recently synthesized the 1,4-[1,2,3]triazolyl-containing
cyclopeptide [Ac-Lys-Gly-Xaa(&1)-Ser-Ile-Gln-Yaa(&2)-Leu-Arg-NH2][(&1(CH2)4-1,4-[1,2,3]triazolylCH2&2)] by solution phase CuI-catalyzed 1,3-dipolar cycloaddition of the linear precursor AcLys-Gly-Nle(ε-N3)-Ser-Ile-Gln-PrG-Leu-Arg-NH2 including side chains modifications with ωazido and ω-alkynyl functions[1]. The cyclopeptide was derived from a highly helical and potent
i-to-i+4 side chain-to-side chain lactam-containing antagonist of parathyroid hormone-related
peptide (PTHrP), [Lys13(&1),Asp17(&2),Tyr34]hPTHrP(7-34)NH2 [2]. The side chains topology of
the triazolyl-containing cyclopeptide, was found to be very similar to the one in the lactamcontaining cyclopeptide, preserving an analogous spatial orientation of the side chains, which
are considered to be important for the biological activity. Herein, we report the synthesis and
the conformational analysis of a series of triazolyl-containing cyclopeptides obtained by
intramolecular CuI-catalyzed click reaction between ω-alkynyl- and ω-azido-containing αamino acid residues incorporated in positions i and i+4. The series of cyclopeptides [Ac-LysGly-Xaa(&1)-Ser-Ile-Gln-Yaa(&2)-Leu-Arg-NH2][(&1(CH2)n=1-41,4-[1,2,3]triazolyl-(CH2)n=1-4&2)]
comprises different sizes of 1,4-disubstituted triazolyl-containing rings, which vary in the
location and orientation of the triazolyl moiety. Conformational analysis by NMR of the most
representative triazolyl-containing cyclopeptides shows that the size of the ring and orientation
of the triazolyl moiety play important roles in reproducing the topological features of the lactam
portion in the bioactive parent cyclopeptide.
References
1 Cantel S, Le Chevalier-Isaad A., Scrima M., Levy J. J., DiMarchi R D., Rovero P., Halperin J. A., D'Ursi A. M., Papini A. M.,
Chorev M. J. Org. Chem., in press
2 Mierke DF, Maretto S, Schievano E, DeLuca D, Bisello A, Mammi S, Rosenblatt M, Peggion E, Chorev M. Biochemistry.
1997 Aug 26;36(34):10372-83.
138
New Frontiers in the Search of Bioactive Molecules: from Peptides to Drugs
Semi-synthesis of consensus tetratricopeptide
repeat proteins for folding studies
De Rosa Lucia1,4, Romanelli Alessandra1, Regan Lynne2, Cortajarena Aitziber L.2, Haran Gilad3 &
D’Andrea Luca D.4
1
2
3
4
Università di Napoli “Federico II”, Dipartimento delle Scienze Biologiche, 80134 Napoli –Italy
Yale University, Department of Molecular Biophysics & Biochemistry, 06520 New Haven –Connecticut
Weizmann Institute of Science, Department of Chemical Physics, 76100 Rehovot – Israel
Istituto di Biostrutture e Bioimmagini, CNR, 80134 Napoli – Italy
The tetratricopeptide repeat (TPR) is a 34-amino acid α-helical motif that occurs in over 300
different proteins from all kingdoms of life. In this type of repeat proteins three to sixteen or
more TPR motifs occur in tandem arrays and function to mediate protein-protein interaction in
a myriad of biological processes, such as cell cycle regulation, transcriptional control,
mitochondrial and peroxisomal protein transport, neurogenesis, protein folding and cancer [1].
The repetitive and elongated nature of TPR proteins causes them to differ radically in their
construction from normal globular proteins; all repeat proteins, like TPR proteins, are
dominated by short-range and regularised interaction, whereas globular proteins exhibit
complex topologies that frequently have numerous long-range interactions [2, 3].
Recently, the Regan lab has designed idealized, consensus TPR modules which can be
combined to create TPR proteins. This proteins were named Consensus TPR numer of
repeats (e.g. CTPR2, CTPR3) [4]. We want investigate the folding mechanism of repeat
proteins using the consensus TPR comprised of different number of tandem repeats by
ensemble and single molecule approaches, like fluorescence resonance energy transfert
(FRET). This latter method provides a means by which to analyze every intermediates of the
folding process that are very difficult to identify in ensemble experiment due to their
coexistence with fully unfolded and partially folded species [5]. FRET experiments require
homogenous double labelled proteins and we design CTPR variants in which fluorescence
donor and acceptor pairs are incorporated at different positions within the protein. In order to
obtain the CTPR proteins labelled with the fluorescent dyes in homogenous and purified form,
we use a semi-synthetic strategy which combines the solid phase peptide synthesis and the
protein expression techniques for protein production [6]. The strategy we propose here
represents a very useful means for labelling proteins site-specifically and bi-functionally in
high yield.
139
POSTER PRESENTATIONS - Eleventh Naples Workshop on Bioactive Peptides
Hydrophobic effect in the self-assembling
of ionic-complementary peptides
D’Auria Gabriella1,2, Vacatello Manuela1, Falcigno Lucia1,2, Luigi Padano1, Calvanese Luisa1,
Gambaretto Roberta3, Dettin Monica3 and Paolillo Livio1,2
1. University of Naples ‘Federico II’, Dept. of Chemistry, 80126 Naples-Italy.
2. Institute of Biostructures and Bioimaging, CNR, 80134 Naples-Italy.
3. University of Padua, Dept. of Chemical Process Engineering, 35131 Padua-Italy.
Self-assembling is the spontaneous organization of molecules into supramolecular systems
with a well defined structure stabilized by non covalent interactions. There are various
examples of self-assembled systems in nature such as the DNA, enzymes, cellular
membranes and fibrous proteins. Great interest in defining the molecular mechanisms
underlying these aggregative processes lies in the opportunity to use them for clinical and
nanotechnological purposes. Protein aggregation is at the base of many neurological
diseases known as amyloidosis and protein/peptide aggregates represent excellent
biomaterials for tissue engineering. Furthermore these studies aim at understanding the very
principles of protein folding.
A series of amphiphilic ionic-complementary peptides made of 16 and 8 residues derived from
the peptide EAK16, known in literature [1] to self-assemble into beta structured macroscopic
membranes, have been studied via CD, NMR and small angle neutron scattering (SANS).
These EAK16 analogues were designed to have different hydrophilic and hydrophobic
composition in order to understand the role of these factors in driving the assembly. We have
previously reported on the 16-mer series which had shown that the more hydrophobic
sequences have the highest propension for beta aggregation and that a difference in the
hydrophilic composition can totally impair the self-assembling process. [2,3,4] Therefore the
driving force in guiding the assembly is the exclusion of water from the apolar surfaces. The
analogous analysis on the related 8-mer peptides reported here shows how the hydrophobic
effect has a fundamental role not only at a tertiary structural level but also in stabilizing the
right secondary structure for the subsequent assembly process.
The more hydrophobic 8-mer sequences show the highest propension for the extended
structure. The hydrophilic composition also has an effect on the secondary structure of these
peptides with the longer chained residues favouring the extended conformation. Once again
this phenomenon seems to be governed by the hydrophobic effect as longer chain residues
have higher apolar surfaces and are therefore more hydrophobic. The tendency to the
extended conformation of the 8-mer peptides very well correlates with the self-assembling
propensity of the related 16-mer peptides.
References
1 Zhang S, Holmes T, Lockshin C, Rich A. Proc Natl Acad Sci 1993; 90, 3334– 3338.
2 D’Auria G, Vacatello Ma, Falcigno L, Oliva R, Gambaretto R, Dettin M, Di Bello C, Paolillo L. First European Conference on
Chemistry for Life Sciences 2005 Rimini-Italy
3 D’Auria G, Vacatello Ma, Falcigno L, Oliva R, Gambaretto R, Dettin M, Di Bello C, Paolillo L. GDRM 2006 Vietri sul Mare
(SA)-Italy
4 D’Auria G, Vacatello Ma, Falcigno L, Oliva R, Gambaretto R, Dettin M, Di Bello C, Paolillo L. J Pept Sci 2006; 12 S1 publ. on
line.
140
New Frontiers in the Search of Bioactive Molecules: from Peptides to Drugs
Insights on the interaction between
human Cripto and its receptors
Daniela Marasco1, Luisa Calvanese2, Angela Saporito1, Manuela Vacatello2, Romina Oliva3,
Gabriella D’Auria1,2, Carlo Pedone1, Livio Paolillo1,2, Lucia Falcigno1,2, and Menotti Ruvo1
1. Istituto di Biostrutture e Bioimmagini del CNR, 80134, Napoli.
2. Dipartimento di Chimica, Università Federico II, Complesso Universitario MSA, 80126, Napoli.
3. Dipartimento di Scienze Applicate, Università degli Studi di Napoli "Parthenope" - 80134 Napoli.
The human protein Cripto is the founding member of the extra-cellular EGF-CFC growth
factors, which are composed of two adjacent cysteine-rich domains: the EGF-like and the
CFC [1,2]. Cripto displays all the features of an oncogene being able to support survival,
transformation, migration and proliferation in a large variety of cell lines [3-5]. Cripto interacts
with ALK4 receptor and is necessary for both Nodal recruitment to the ALK4/ActRIIB receptor
complex and for Nodal signalling [6,7]. It has also been reported that the EGF-like domain binds
to Nodal while the CFC domain binds to ALK4 [5,7,8]. Although both Cripto domains are
involved in its tumorigenic activity, the CFC domain appears to play a crucial role. Indeed,
through this domain, Cripto interferes with the onco-suppressive activity of Activins, either by
blocking the Activin receptor ALK4 [5,9] or by binding soluble Activins [10]. Structural mapping of
functional sites of ALK4 could provide insights into the mechanisms underlying the Criptodependent tumour formation and progression and could pave the way to the design of
antagonists. As there is currently no direct structural information for the interaction between
CFC and ALK4, we have used a combined NMR and computational approach to investigate
the molecular basis of their recognition.[11]. To this aim we have prepared the human CFC,
residues 112-150, by chemical synthesis and carried out an NMR structural characterization.
By using the NMR 3D-structures of hCFC and an homology model of ALK4 extracellular
domain (ECD), we propose a structural model of the human CFC in complex with ALK4-ECD
receptor, built using macromolecular docking assisted by known experimental data. Such
model should provide a structural basis for understanding the primary interaction of CFC with
its receptor and help in designing useful antagonists.
In addition, the EGF-like domain of human Cripto, residues 75-113, has also been prepared
by chemical synthesis and refolded in order to achieve the network of disulfide bridges.
Comparative binding analysis have then been carried out using both synthetic domains and
the recombinant ALK4 and ActRIIB receptors. These assays, performed by the SPR
technique, have shown that the synthetic human CFC does bind to the immobilized ALK4 with
a KD in the low µM range, while, as expected, it does not recognize ActRIIB. In contrast with
previous findings, the EGF-like also binds to ALK4 with an affinity comparable to hCFC and
does not interact with the ActRIIB receptor. These data open new perspectives for the
understanding of the intricate network of interactions that regulate the formation of this
important complex of receptors, co-receptors and ligands.
References
1. Shen MM. J Clin Invest 2003;112(4):500-502.
2. Salomon DS, Bianco C, Ebert AD, Khan NI, De Santis M, Normanno N, Wechselberger C, Seno M, Williams K, Sanicola M,
Foley S, Gullick WJ, Persico G. Endocr Relat Cancer 2000;7(4):199-226.
3. Schiffer SG, Foley S, Kaffashan A, Hronowski X, Zichittella AE, Yeo CY, Miatkowski K, Adkins HB, Damon B, Whitman M,
Salomon D, Sanicola M, Williams KP. J Biol Chem 2001;276(41):37769-37778.
4. Yan YT, Liu JJ, Luo Y, E C, Haltiwanger RS, Abate-Shen C, Shen MM. Mol Cell Biol 2002;22(13):4439-4449.
5. Adkins HB, Bianco C, Schiffer SG, Rayhorn P, Zafari M, Cheung AE, Orozco O, Olson D, De Luca A, Chen LL, Miatkowski
K, Benjamin C, Normanno N, Williams KP, Jarpe M, LePage D, Salomon D, Sanicola M. J Clin Invest 2003;112(4):575-587.
6. Schier AF. Annu Rev Cell Dev Biol 2003;19:589-621.
7. Yeo C, Whitman M. Mol Cell 2001;7(5):949-957.
8. Marasco D, Saporito A, Ponticelli S, Chambery A, De Falco S, Pedone C, Minchiotti G, Ruvo M. Proteins 2006;64(3):779788.
9. Gray PC, Harrison CA, Vale W. Proc Natl Acad Sci U S A 2003;100(9):5193-5198.
10. Risbridger GP, Schmitt JF, Robertson DM. Endocr Rev 2001;22(6):836-858.
11. Calvanese L, Saporito A, Marasco D, D'Auria G, Minchiotti G, Pedone C, Paolillo L, Falcigno L, Ruvo M. J Med Chem
2006;49(24):7054-7062.
141
AUTHOR INDEX - Eleventh Naples Workshop on Bioactive Peptides
Aaltonen Johanna
Accardo Antonella
Accettola Alessia
Adriani Walter
Afonin Sergiy
Alterio Vincenzo
Alves Isabel
Amblard Muriel
Amodeo Piero
Andreu N.
Andreu David
Antcheva Nikolinka
Arouri Ahmad
Athanasopoulos Alexandros
Attanasio Francesco
Auriemma Luigia
Auriemma Sara
Aÿ Bernhard
Baffreau Jerome
Baguinot Francesco
Bardaji Eduard
Barra Donatella
Bavoso Alfonso
Becker Jeff
Bencini Lapo
Benedetti Ettore
Benincasa Monica
Ben-Tal Nir
Berditsch Marina
Berisio Rita
Berlicki Lukasz
Bernini Andrea
Bertamino Alessia
Beyermann Michael
Bienert Michael
Bifulco Giuseppe
Biron Eric
Blayo A.L.
Blume Alfred
Bobone Sara
Bocchinfuso Gianfranco
Boettcher Jark
Boisguerin Prisca
Borissenko L.
Bracatello Angela
Bracci Luisa
Brasseur Robert
Braun Klaus
Browne Helena
Broxterman Quirinus B.
Bruckner Hans
Brunetti Jlenia
Bruno Milena
Bucci Enrico
Bunker Alex
Bürck Jochen
Burvenich C.
Cabras Tiziana
Calvanese Luisa
Campiglia Pietro
Cantel Sonia
Cantisani Marco
Capasso Dominga
Capone Stefania
Cappelli Alessandra
Carotenuto Alfonso
Cassese Angela
Castagnola Massimo
Castaldo Giuseppe
Castanho Miguel
Castiglione Mariangela
144
P37
Y5
P62
P43
L16
P54
Y11
Y6
P64, P76
L9
L9
P24
Y3
P82
P55
P61, P62
P36
P53
O5
P80
Y4, L3, P20
P15
P6
O1
P40
Y9, P9, P44, P45, P49
P60, P64, P74 P77
P11
P3
L16
P26, P72, P73, P84
P23
O6, P82
P61
Y2
Y2
P58
L11
L14
Y3
P70
P69, P71
Y3
P25, P53
L1
P6
O6, P40, P82
L10
P5
P27, P33
O4
O7, P63
O6, P40
P29
P9, P49
P46
L16
P19
P65
P87, P88
P61, P62
P85
P27, P44, P75
P38
P47
P40
P61, P62
P32
P65
P75
Y7, L3, P20, P66
P9, P49
Cataldo Sebastiano
Catania Anna
Cesarani Annalisa
Chassaing Gerard
Chatterjee Jayanta
Chau Johnny K.
Chelu Florica
Chini Maria
Chorev Michael
Ciccarelli Luciano
Cohen Leah
Coin Irene
Colombo Giorgio
Conti Stefania
Copani Agata
Correale Stefania
Correia Isabelle
Correia Ana
Cortajarena Aitziber
Cosette Pascal
Crisma Marco
Czaplewski Cezary
Czernicka Anna
D'Alessandro Cristian
D'Ambrosio Katia
D'Amico Michele
D'Andrea Luca D.
Darvaris Maria
Dathan Nina
Dathe Margitta
D'Auria Gabriella
de Boer Leonie
De Bona Paolo
De Cristofaro Andrea
De Falco S.
de la Torre B.G.
De Luca Stefania
De Lucia Maria
De Rosa Lucia
De Simone Alfonso
De Simone Giuseppina
De Simone Mariarosaria
De Spiegeleer Bart
De Vendel Jolanda
De Zotti Marta
Degenkolb Thomas
Del Gatto Annarita
Del Vecchio Silvana
Demange L.
Desmaele Enrico
Dettin Monica
Di Filippo Clara
Di Fiore Anna
Di Gaetano Sonia
Di Giacomo Daniele
Di Lello Paola
Di Marcotullio Lucia
Di Stasi Rossella
Diana Donatella
Didinger Bernd
Diederich Wibke E
DiMarchi Richard D
d'Ischia Marco
Dogne' Jean Michel
Domingues Marco
Doti Nunzianna
Dubois Carole
D'Ursi Anna Maria
Dzimbova Tatyana
Elkahoui Salem
Esposito Carla
P55
P62
P49
Y11
L11
P8
P7
P58
P85
P72
O1
Y2
O9
P65
P55
P54
Y11
Y4, P20
P86
P30
O4
O10, P12, P46
P23
P9
P57
P44
O9, P38, P86
O11
P31, P32, P45, P74
Y3
P87, P88
P8
P55
P9
P13
L9
P2, P76
P48
P86
P51, P84
P54, P57, P68
P52
P19
P6
P18, P35, P69, P71
O7, P63
O5, P2, P52
P2
L14
P54
P87
P44
P68
P54
P22
O5
P54
P38
O9, P38
P5
Y3
P85
P48
P57
Y7
Y9, P32
P77
P58, P78, P85
P28
P30
P26
New Frontiers in the Search of Bioactive Molecules: from Peptides to Drugs
P4, P51, P83
Esposito Luciana
Fabbrocino Serena
P29
Falanga Annarita
P27, P33, P60
O6, P40, P82
Falciani Chiara
Falcigno Lucia
P87, P88
Fanali Chiara
P65
Farina Biancamaria
P79, P80
Fattorusso Roberto
O9, P33, P38, P80, P81
Fehrentz Jean-Alain
L14
Feliu Lidia
P20
Ferranti Pasquale
P29
Ferre Rafael
P20
Fillat C.
L9
Finamore Emiliana
P44, P60
Flagiello Angela
P31
Foerster Guenter
Y3
Formaggio Fernando
P18, P35, P69, P71
L11
Frank Andreas
Franquelim Henri
P66
P74
Franzoso Guido
Frisch Juergen
O3
Fujioka Shio
P42
Funari Sergio S.
P3
L14
Gagne D.
Galdiero Marilena
P44, P60
Galdiero Massimiliano
P27, P33, P75
Galdiero Stefania
P27, P33, P44, P60, P64, P75
L14
Galleyrand J.C.
Gallo Pasquale
P29
Gambaretto Roberta
P87
Gennaro Renato
P11
Gierasch Lila M.
L6
Gilles Pierre
Y6
Giralt Ernesto
L15
Gispert J.D.
L9
Giuffrida Maria Laura
P55
Glowinska Agnieszka
L19
Goasdoue Nicole
Y11
Gomez V.
L9
Gomez-Monterrey Isabel
P61, P62
L16
Grage Stephan
Grande Giacinto
P69, P71
Granstrem Oleg
P43
Grieco Paolo
P61, P62
Groll Michael
L1
Grzonka Zbigniew
L8
P54
Gulino Alberto
Gunars Duburs
P46
Habdas Jan
P21
Hahm Kyung-Soo
P18, P71
P85
Halperin Jose
Haran Gilad
P86
Heine Andreas
Y3
Heras Montserrat
Y4, L3
Hilvo Mika
P68
Icriverzi Madalina
P7
P4, P84
Improta Roberto
In Yasuko
P42
Infanti Martina
P35
Innocenti Alessio
P57
Inzitari Rosanna
P65
Iommelli Francesca
P2, P52
Iozzi Sara
O6
Isernia Carla
P33, P81
P70
Jae-Il Kim
Jankowska Elzbieta
L8
Jeric Ivanka
P39
Jewginski Michal
P1, P14
Jouenne Thierry
P30
Juszczyk Paulina
L8, P12
Kafarski Pawel
P1, P14, P23
Kalvinsh Ivars
P59
Kampanaraki Aikaterini
P27, P60
Kaptein Bernard
O4
P30
Karkouch Ines
Keivish Tatiana
P59
Kessler Horst
L11
Kieltsch Iris
P47
Kiessling Volker
Y3
Kim Jin-Young
P69, P71
P18
Kim Mi-Hyun
Kirschbaum Jochen
O7
Klebe Gerhard
Y3
Kobayashi Yuji
L17
P5
Koch Mario
Koistinen Hannu
P37
Kolodziejczyk Aleksandra S.
L8
Kosson Piotr
L19
Kretzschmar Ines
P25, P53
Krezel Artur
P14
Kubiak Nina
L19
Kumaran Sowmini
O1
Kwiatkowska Anna
P50, P17
Kyung-Soo Hahm
P70
La Mendola Diego
P81
Laakkonen Pirjo
P37
Lammek Bernard
P50, P17
Landgraf Christiane
P53
O5
Langella Emma
Langlois Chantal
O5
Latajka Rafal
P1, P14
Laufer Burkhardt
L11
Lavielle Solange
Y11
Laviola Giovanni
P43
Le Chevalier-Isaad Alexandra
P85
Lecaillon Jennifer
Y6
P67
Leccia Felicia
Legault Pascale
O5
Lelli Barbara
O6, P40
Lettieri Gregory
P48
Levy Jay J
P85
Lewandrowski Peter
O3
Liepina Inta
O10, P46
Limam Ferid
P30
Linser Sebastian
P3
Lipkowski Andrzej W.
L19
Liwo Adam
O10
L9
Llop J.
Locatelli V.
L14
Loguercio Salvatore
P31
Lohner Karl
P56
Longhi Renato
P65
Loura Luis
P66
Lozzi Luisa
P40
Makker Sudesh
P6
Makowski Maciej
P1, P14
Malfi Stefania
P61, P62
Manavbasi Yasemin
P56
Manconi Barbara
P65
Mangoni Maria Luisa
P15, P22, P61
Marasco Daniela
Y9, P13, P32, P45, P74, P88
Marcelino Anna Marie C.
L6
Marcellini Hercolani Gaddi Ludovica
P22
P61, P62
Marcozzi Cristina
Marlgieri Giuseppe
P81
Y1, Y6, L14
Martinez Jean
Mas Caroline
O5
Masereel Bernard
P57
Matsui Toshiro
Y10
Mattsby-Baltzer Inger
P7
Melo Manuel
P20
Menichetti Stefano
P40
145
AUTHOR INDEX - Eleventh Naples Workshop on Bioactive Peptides
P65
Messana Irene
P32, P80
Miele Claudia
Mierke Dale
L13
Milani Alberto
P35
Millan O.
L9
Minchiotti G.
P13
Mink Christian
L16
Misicka Aleksandra
L19
Miyazawa Toshifumi
P42
P23
Mlynarz Piotr
Molnar Anca Florina
P64
Monaco Hugo L.
L5
Monfregola Luca
P76
Monti Simona Maria
Y9, P31, P32, P45, P68, P74
Mootz Henning
O8
Y10, P34
Morel Nicole
Morelli Giancarlo
P83, Y5
P40
Moretti Renato
Moretti Marta
P54
Moretto Alessandro
O4
Morgera Francesca
P24
Y5
Morisco Anna
Moulin A.
L14
Mueller Gabriele
P5
Mueller Judith
P25
Murashima Takashi
P42
Musumeci Domenica
P9, P49
Naider Fred
O1
Nakamura Shota
L17
Nakazawa Takashi
L17
Napolitano Alessandra
P48
P37
Närvänen Ale
Nasi Antonella
P29
Nguyen Leonard
P8
Niccolai Neri
O6, P82
Nikolovski Z.
L9
Novellino Ettore
P61, P62
Oliva Romina
P88
Omichinski James G.
O5
Orioni Barbara
P69, P70, P71
Ostuni Angela
P6
Pacor Sabrina
P11, P24
Pagliuca Chiara
P40
Paladino Antonella
P83
Palladino Pasquale
P77
Palleschi Antonio
P69, P71
Panico Maria Rosaria
P52
Panzella Lucia
P48
P87, P88
Paolillo Livio
Papa Salvatore
P74
Papaccioli Angela
P52
Papini Anna Maria
P85
Pappalardo Giuseppe
P55, P81
Paramelle David
Y1
Paraschiv Gabriela
L8
Park Yoonkyung
P18, P69
P68
Parkkila Seppo
Pascual J.A.
L9
Pedone Carlo
Y5, Y9, O5, O9, P2, P4,P9, P13,
P26, P27, P32, P33, P38, P45, P49,
P51, P52, P54, P67, P68, P72,P73,
P75, P80 P83, P88
Pedone Emilia
Pedone Paolo V.
Pelillo Chiara
Pensato Soccorsa
Perrissoud D.
Perry Nicole A.
Pethoukov Maxim V
Piazzon Alessandro
Pietropaolo Adriana
Pignataro Bruno
146
P26, P54, P73, P80
P67
P11
P67
L14
P8
P26
P70
P81
P55
Pileri Silvia
O6, P40
Pini Alessandro
O6, P40, P82
Pinto Marta
Y4, L3
Pinyot A.
L9
Pipkorn Ruediger
P5
Pirone Luciano
P54, P80
Planas Marta
P20
Polevaya Ludmila
P59
Polonolelli Luciano
P65
Ponticelli S.
P13
Prahl Adam
P17, P50
Przybylski Michael
L8
Ptak Tomasz
P23
Pucci Piero
P31
Ragone Raffaele
P77
Raieta Katia
P27, P60
Raiola Luca
P33, P81
Regan Lynne
P86
Reichert Johannes
L16
P67
Renda Mario
Ribeiro Marta
Y4, L3
Rispoli Giorgio
P35
Rizzarelli Enrico
P55
Rodziewicz-Motowidlo Sylwia
L8
Romanelli Alessandra
O9, P67, P86
Romankiewicz Justyna
P12
Romano Vincenza
P68
P43
Romano Emilia
Ronga Luisa
P77
Roseanu Anca
P7
Rosenfeld Yosef
P15
Rossi Filomena
P77
Rovero Paolo
P85
Roviello Giovanni
P9, P49
Royo Soledad
Y8
Ruden Serge
L16
Ruggiero Alessia
P26, P72, P73
Ruvo Menotti
Y9, P13,P32, P45,P74, P80, P88
Rzeszutek Agnieszka
P3
Sabatella Marco
Y9, P32
Sagan Sandrine
Y11
Sakarellos Constantinos
O11
Sakarellos-Daitsiotis Maria
O11, P16
Salapete Filomena
P76
Y3
Salay Luiz C.
Salvatore Marco
P2, P52
Salvatore Francesco
P75
Sandomenico Annamria
Y9, P45
Santos Nuno
Y7, P66
Saporito Angela
P88
Saviano Michele
O4, O5, P2, P52, P67
Saviello Maria Rosaria
P61, P62
P76
Scala Stefania
Scaloni Andrea
P68
Scannella Alessandra
P31
Scarabelli Guido
O9
Schmieder Peter
Y2
Schneider Joel
L12
Schroeder Grzegorz
P23
Scocchi Marco
P11
Scozzafava Andrea
P57, P68
Scrima Mario
P58, P85
Scudiero Olga
P75
Seebach Dieter
P47
Segura J.
L9
Serpe Luigi
P29
Sewald Norbert
Y8
Shai Yechiel
P15
Shcheglova Tatiana
P6
Shental-Bechor Dalit
P3
Shishkov Stoyan
P28
New Frontiers in the Search of Bioactive Molecules: from Peptides to Drugs
Singh Harpreet
Slaninova Jirina
Sleszynska Malgorzata
Smagghe Guy
Sobolewski Dariusz
Sommella Jvana
Song Yub Shin
Spiga Ottavia
Squeglia Flavia
Stachowiak Krystyna
Stankova Ivanka
Stanzione Francesca
Stavrakoudis Athanassios
Stella Lorenzo
Stenman Ulf-Håkan
Sternberg Ulrich
Strandberg Erik
Strömbeck Louise
Stueber Werner
Subra Gilles
Supuran Claudiu T.
Svergun Dmitri I
Szaniawska Bozena
Szust Justyna
Szymanska Aneta
Takahashi Ryo
Talleda Monserrat
Tamm Lukas
Tavares Isaura
Terradot Laurent
Terstappen Georg
Tesauro Diego
Theis Christoph
Togni Antonio
Toniolo Claudio
Tornatore Laura
Torsello A.
Tossi Alessandro
Tramontano Alfonso
Trif Mihaela
Tselepis Alexandros
O3
P17, P50
P17
Y10, P34
P17, P50
P52
P70
P82
P73
L8
P28
P83
O11, P16
P18, P69, P70, P71
P37
L16
L16
P7
O3
Y1, Y6
P57, P68
P26
L19
P34
L8
L17
Y4
Y3
Y4, L3
P31
L18
Y5
O7
P47
O4, P18, P35, P69, P71
Y9, P74
L14
P24
P6
P7
O11
Ulrich Anne
Vacatello Manuela
Vaccari Lisa
Van Camp John
Vedovato Natascia
Venanzi Mariano
Venditti Vincenzo
Ventura Salvador
Vercruysse Lieselot
Vergote V.
Veveris Maris
Viparelli Francesca
Vitagliano Luigi
Vitale Rosa Maria
Vitali Alberto
Vitiello Mariateresa
Vizzuso Domenica
Vogel Hans J.
Volkmer Rudolf
Wadhwani Parvesh
Waldeck Waldemar
Wheatley Mark
Wiczk Wieslaw
Wierzba Tomasz
Wiessler Manfred
Willumeit Regine
Wilmanns Matthias
Wootten Denise
Yamada Takashi
Yonath Ada
Yoonkyung Park
Yoshida Takuya
Zaat Sebastian A.J.
Zaccaro Laura
Zagari Adriana
Zannetti Antonella
Zerbe Oliver
Ziaco Barbara
Zou Chao
Zurzolo Chiara
Zweytick Dagmar
L16
P87, P88
P24
Y10, P34
P35
P69, P70, P71
P82
O10
Y10, P34
P19
P59
Y9, P32, P80
P4, P51, P54, P72, P83, P84
P57, P74, P76
P65
P27, P33, P44, P60, P75
P6
P8
P25, P53
L16
P5
O2
P12
P17
P5
P3
P73
O2
P42
L2
P71
L17
P8
O5, P2, P52, P80
P31
P2, P52
O1
O9
O1
L7
P56
147
PARTICIPANT INDEX - Eleventh Naples Workshop on Bioactive Peptides
Accardo Antonella
University of Naples Federico II
Italy
Adriani Walter
Istituto Superiore di Sanità
Italy
Akasaka Kazuhito
Kyowa Hakko Kogyo Co., Ltd.
Japan
Alterio Vincenzo
IBB-CNR
Italy
Alves Isabel
Université Pierre et Marie Curie
France
Amblard Muriel
IBMM CNRS UMR5247
France
Amodeo Pietro
Consiglio Nazionale delle Ricerche (CNR)
Italy
Andreu David
Pompeu Fabra University
Spain
Arndt Katja
University of Freiburg
Germany
Arouri Ahmad
Physical Chemistry
Germany
Auriemma Sara
Università di Napoli Federico II
Italy
Ay Bernhard
Charite
Germany
Barbagallo Alessia
CNR
Italy
Benedetti Ettore
Università di Napoli Federico II
Italy
Benincasa Monica
University of Trieste
Italy
Berisio Rita
Istituto di Biostrutture e Bioimmagini, C.N.R.
Italy
Boisguerin Prisca
Charite
Germany
Bracci Luisa
University of Siena
Italy
Brasseur Robert
Centre de Biophysique Moleculaire Numerique
Belgium
Brückner Hans
University of Giessen
Germany
Cantisani Marco
University Federico II
Capone Stefania
ETH Zurich
Italy
Switzerland
Castanho Miguel
Faculty of Medicine, Univ. Lisbon
Portugal
Castiglione Mariangela
Università Federico II
Italy
Cesarani Annalisa
Istituto di Biostrutture e Bioimmagini (CNR)
Italy
Chelu Florica
Institute of Biochemistry
Romania
Chini Maria
University of Salerno
Chorev Michael
Harvard Medical School
Italy
United States
Ciccarelli Luciano
Università di Napoli Federico II
Ciccarelli Marco
CNR
Coin Irene
Leibniz-Institute of Molecular Pharmacology
Correale Stefania
Istituto di biostrutture e Bioimmagini, C.N.R.
Crisma Marco
Institute of Biomolecular Chemistry, CNR, Padova Unit
Czaplewski Cezary
University of Gdansk
D'Ambrosio Katia
CNR
D'Andrea Luca
CNR
D'Auria Gabriella
University of Naples Federico II
D'Ursi Anna
University of Salerno
150
Italy
Italy
Germany
Italy
Italy
Poland
Italy
Italy
Italy
Italy
Dathan Nina
IBB
De Bona Paolo
Università di Catania
De Cristofaro Andrea
Istituto di Biostrutture e Bioimmagini
Degenkolb Thomas
University of Giessen
Del Gatto Annarita
IBB-CNR
De Luca Stefania
CNR
De Luca Luca
CIRPEB
De Paola Ivan
IBB-CNR
De Riccardis Francesco
Università di Salerno
De Rosa Lucia
Università Federico II
De Simone Alfonso
University of Cambridge
De Simone Giuseppina
CNR
De Simone Mariarosaria
IBB-CNR
De Spiegeleer Bart
University of Gent
De Vendel Jolanda
University of Basilicata
De Zotti Marta
Università di Padova
Diana Donatella
C.N.R.
Di Blasio Benedetto
Seconda Università di Napoli
Diederich Wibke
Philipps-University of Marburg
Di Fiore Anna
CNR
Di Giacomo Daniele
La Sapienza
Di Stasi Rossella
CNR
Domingues Marco
Instituto de Medicina Molecular
Doti Nunzianna
CNR
Dubois Carole
Universita fredirico II di napoli
Elkahoui Salem
Centre of Biotechnology of Borj-Cédria
Eberle Alex
University Hospital Basel
Esposito Carla
Università degli studi di Napoli Federico II
Esposito Luciana
CNR Istituto di Biostrutture e Bioimmagini
Falanga Annarita
University of Naples
Falcigno Lucia
University Federico II of Naples
Farina Biancamaria
CNR
Fattorusso Roberto
Seconda Università di Napoli
Fehrentz Jean-Alain
Université de Montpellier II
Ferranti Pasquale
University of Naples Federico II
Ferrari Bravo Valentina
Università di Napoli “Federico II”
Fiory Francesca
CNR
Franquelim Henri
Instituto Medicina Molecular
Italy
Italy
Italy
Germany
Italy
Italy
Italy
Italy
Italy
Italy
United Kingdom
Italy
Italy
Belgium
Italy
Italy
Italy
Italy
Germany
Italy
Italy
Italy
Portugal
Italy
Italy
Tunisia
Switzerland
Italy
Italy
Italy
Italy
Italy
Italy
France
Italy
Italy
Italy
Portugal
New Frontiers in the Search of Bioactive Molecules: from Peptides to Drugs
Galdiero Massimiliano
Second University of Naples
Italy
Galdiero Stefania
University Federico II
Italy
Gierasch Lila
University of Massachusetts
United States
Giralt Ernesto
University of Barcelona
Granstrem Oleg
Geropharm
Spain
Russian Federation
Grieco Paolo
University of Naples Federico II
Groll Michael
Adolf-Butenandt-Institut
Habdas Jan
University of Silesia
Jeric Ivanka
Rudjer Boskovic Institute
Jewginski Michal
University of Opole
Juszczyk Paulina
University of Gdansk
Kessler Horst
TU München
Kobayashi Yuji
Osaka University of Pharmaceutical Sciences
Koistinen Hannu
University of Helsinki
Kravchuk Alexander
Politecnico di Milano
Kwiatkowska Anna
Faculty of Chemistry, University of Gdansk
Latajka Rafal
Wroclaw University of Technology
Lecaillon Jennifer
Institut des Biomolécules Max Mousseron
Lelli Barbara
Università di Siena
Lettieri Gregory
University of Naples Federico II
Liepina Inta
Latvian Institute of Organic Synthesis
Linser Sebastian
GKSS Research Center
Loguercio Salvatore
University Federico II
Manavbasi Yasemin
Austrian Academy of Sciences
Mangoni Maria Luisa
La Sapienza University
Marasco Daniela
CNR
Marcellini Hercolani Gaddi Ludovica
La Sapienza University
Martinez Jean
Université de Montpellier I et II
Mascagni Paolo
Italfarmaco S.p.A
Mathieu Marc
Xigen S.A.
Melo Manuel
Faculty of Medicine - University of Lisbon
Mierke Dale
Dartmouth College
Misicka Aleksandra
University of Warsaw
Mlynarz Piotr
Wroclaw University of Technology
Moccia Maria
IBB-CNR
Molnar Anca Florina
Consiglio Nazionale delle Ricerche (CNR)
Monaco Hugo
Università di Verona
Monfregola Luca
University Federico II""
Italy
Germany
Poland
Croatia
Poland
Poland
Germany
Japan
Finland
Italy
Poland
Poland
France
Italy
Italy
Latvia
Germany
Italy
Austria
Italy
Italy
Italy
France
Italy
Switzerland
Portugal
United States
Poland
Poland
Italy
Italy
Italy
Italy
Monti Simona
CNR
Mootz Henning
Technische Universität Dortmund
Morelli Giancarlo
University of Naples Federico II
Morgera Francesca
University of Trieste
Morisco Anna
Università degli studi di Napoli Federico II
Moroder Luis
Max Planck Institute of Biochemistry
Musumeci Domenica
Bionucleon srl
Mutter Manfred
Debio RP
Nguyen Leonard
University of Calgary
Niccolai Neri
University Of Siena
Omichinski James Omichinski
Universite de Montreal
Palladino Pasquale
Università Federico II di Napoli
Panariello Simona
University of basilicata
Papaccioli Angela
CNR
Pappalardo Giuseppe
National Research Council
Paramelle David
Institut des Biomolécules Max Mousseron
Parmentier Gilles
Bachem AG
Pedone Carlo
Istituto di Biostrutture e Bioimmagini, C.N.R.
Pedone Emilia Maria
Istituto di Biostrutture e Bioimmagini, C.N.R.
Pedone Paolo Vincenzo
Seconda Università di Napoli.
Peggion Evaristo
University of Padova
Pensato Soccorsa
Università Federico II
Perretta Giuseppe
CNR-IBB
Pipkorn Ruediger
German Cancer Research Center
Pirone Luciano
Università degli studi di Napoli Federico II
Raciti Gregory
CNR
Ragone Raffaele
Second University of Naples
Raieta Katia
Facoltà di Medicina Seconda Università di Napoli
Raiola Luca
Seconda università degli studi di napoli
Ranta Tanja-Maria
University of Helsinki
Ribeiro Marta
Faculty of Medicine, Univ Lisbon
Rolka Krzysztof
University of Gdansk
Romanelli Alessandra
University of Naples
Ronga Luisa
Universita degli Studi di Napoli Federico II
Rossi Filomena
University Federico II Naples
Rothlisberger Martin
John Wiley & Sons Ltd
Roviello Giovanni
Consiglio Nazionale delle Ricerche
Royo Soledad
Bielefeld University
Italy
Germany
Italy
Italy
Italy
Germany
Italy
Switzerland
Canada
Italy
Canada
Italy
Italy
Italy
Italy
France
Switzerland
Italy
Italy
Italy
Italy
Italy
Italy
Germany
Italy
Italy
Italy
Italy
Italy
Finland
Portugal
Poland
Italy
Italy
Italy
United Kingdom
Italy
Germany
151
PARTICIPANT INDEX - Eleventh Naples Workshop on Bioactive Peptides
Ruggiero Alessia
Istituto di Biostrutture e Bioimmagini, C.N.R.
Italy
Ruvo Menotti
IBB-CNR
Italy
Rzeszutek Agnieszka
GKSS Research Centre
Sabatella Marco
Germany
CNR-IBB Istituto di Biostrutture e Bioimmagini
Salapete Filomena
University degli Studi di Napoli Federico II
Sandomenico Annamaria
Università degli Studi di Napoli Federico II
Saporito Angela
CIRPEB
Sarnataro Daniela
University of Naples Federico II
Saviano Michele
Consiglio Nazionale delle Ricerche
Scannella Alessandra
Università degli studi di Napoli Federico II
Schneider Joel
University of Delaware
Scognamiglio Liana
CNR
Scrima Mario
University of Salerno
Shishkina Anna
Moscow State University
Sleszynska Malgorzata
Faculty of Chemistry, University of Gdansk
Sommella Jvana
CNR
Stankova Ivanka
South-West University ''Neofit Rilski"
Stanzione Francesca
Università degli studi di Napoli Federico II
Stavrakoudis Athanassios
University of Ioannina
Stella Lorenzo
Università di Roma Tor Vergata
Stoineva Ivanka
Institute of Organic Chemistry Bulg.Acad. Sci
152
Italy
Italy
Italy
Italy
Italy
Italy
Italy
United States
Italy
Italy
Russian Federation
Poland
Italy
Bulgaria
Italy
Greece
Italy
Bulgaria
Stueber Werner
WS Partners Management
Germany
Subra Gilles
IBMM- CNRS UMR 5247
Søndergaard Sandra
France
Faculty of Life Sciences university of Copenhagen
Terstappen Georg
Siena Biotech SpA
Tesauro Diego
University of Naples Feerico II
Toniolo Claudio
Università di Padova
Ulrich Anne
KIT
Vercruysse Lieselot
Ghent University
Veveris Maris
Latvian Institute of Organic Synthesis
Viparelli Francesca
CNR
Vitagliano Luigi
CNR
Vitale Rosa Maria
CNR
Vitali Alberto
ICRM CNR sez Roma
Vitiello Mariateresa
Second University of Naples
Wheatley Mark
University of Birmingham
Yamada Takashi
Konan University
Yonath Ada
Weizmann Institute of Science
Zaccaro Laura
Istituto di Biostrutture e Bioimmagini-CNR
Zerbe Oliver
University of Zurich
Ziaco Barbara
Università Federico II
Zurzolo Chiara
Institut Pasteur
Denmark
Italy
Italy
Italy
Germany
Belgium
Latvia
Italy
Italy
Italy
Italy
Italy
United Kingdom
Japan
Israel
Italy
Switzerland
Italy
France