Programma Messaggeri della Conoscenza
Progetto ID497: Drug Discovery
http://www.lifesci.dundee.ac.uk
http://www.unime.it/dipartimenti/scifarm
Messina 20 novembre 2014, Ore 9:30, Aula A
Dipartimento Scienze del Farmaco e dei Prodotti per la Salute
incontro pubblico
Nell’ambito del programma Messaggeri della Conoscenza promosso
congiuntamente dal Ministero dell’Istruzione, dell’Università e della Ricerca e dal
Ministero per la Coesione Territoriale, a conclusione del progetto ID 497 “Drug
Discovery”, il Dipartimento di Scienze del Farmaco e dei Prodotti per la Salute
promuove la seguente giornata di studio sul “Drug Discovery” finalizzata alla
DISSEMINAZIONE DEI RISULTATI
Introduzione ai lavori
Prof. Maria Zappalà , Direttore Dipartimento SCIFAR
Prof. Antonino Germanà, Prorettore all’Internazionalizzazione, UniME
Prof. Antonio Rapisarda, Coordinatore CLM CTF, UniME
Prof. Antonino Villari, Coordinatore CLM Farmacia, UniME
Dott.ssa Grazia De Tuzza, Responsabile Area Alta Form., Ric. Scient. e Rel. Internaz.
Presentazione del Progetto “Drug Discovery”
Prof. Antonina Saija, Dip. SCIFAR, Referente Scientifico-Didattico del Progetto ID 497
Il Drug Discovery e la ricerca su Chemical & Structural Biology of Protein-Protein
Interactions al College of Life Sciences, Univ. Dundee
Prof. Alessio Ciulli, team leader, Univ. Dundee, Scotland, UK, Docente Titolare del
Progetto ID 497 (proiezione di un filmato)
L’esperienza all’estero degli studenti del Dip. SCIFAR ed il contributo alla ricerca
su Druggability of E3 Cullin RING Ubiquitin Ligases
Sig. Martina Casale
Sig. Claudio Catalano
Sig. Federica Centorrino
Sig. Salvatore Scaffidi
Drug Discovery Computational Tools in the Scientific Community: Overview and
Successful Experiences
Prof. Stefano Alcaro, Dip. Scienze della Salute,Università “Magna Græcia” Catanzaro
Conclusioni
Messaggeri programme “Drug Discovery”:
Chemical and Structural Biology of Protein-Protein
Interactions
Alessio Ciulli
a
a
College of Life Sciences, Division of Biological Chemistry and Drug Discovery, University of
Dundee, Dundee, United Kingdom
[email protected]
The Ciulli Laboratory, based at the College of Life Sciences, University of Dundee, is
broadly interested with understanding and exploiting the ligandability of protein-protein
interactions (PPIs) and protein surfaces within complex biological systems.1 Of particular
interest are protein interfaces recognizing protein post-translational modifications (PTMs) of
amino acids. We employ a question-driven, multi-disciplinary approach that combines
chemical, biophysical, biochemical and structural techniques with the concepts of fragmentbased and structure-based drug design. Current research efforts are directed towards
targeting PPIs and PTM recognition within protein families of biological and medical
relevance within the ubiquitin and chromatin systems: 1) the multisubunit complexes Cullin
RING E3 ubiquitin ligases (CRLs); 2) multidomain proteins containing paired chromatin
reader domains. We seek to understand the chemical nature of the protein surfaces and
interfaces, and to exploit their tractability and selectivity toward small molecule modulation.
The ‘chemical probes’ we design and develop are evaluated biophysically, structurally and
inside cells as tools to address biological questions.
After delivering a series of lectures focused on Drug Discovery at the Department of
Pharmaceutical Sciences of the University of Messina, within the scope of the MIUR
exchange programme Messaggeri della Conoscenza, four amongst the top ranking
undergraduate students were selected to come to my laboratory at Dundee to carry out a
research project.2
References
1. http://www.lifesci.dundee.ac.uk/groups/alessio-ciulli/
2. see video “CIULLI LAB FILM06”
Drug Discovery Computational Tools in the
Scientific Community: Overview and Successful
Experiences
Stefano Alcaro
Laboratorio di Chimica Farmaceutica del Dipartimento di Scienze della Salute,
Università “Magna Græcia” di Catanzaro, viale Europa, 88100 Catanzaro, (Italy)
[email protected]
The development of new drugs is a challenging goal of any medicinal chemist. The integration of
computational techniques in the discovery environment is expected to speed up the process with
relatively low expenses. Today the structural biology, especially by means of the Protein Data Bank
[1] and other web bases resources, communicates directly with the medicinal chemistry, allowing to
perform the rational drug design of novel compounds in a modern fashion. In order to look for the
diffusion of such techniques in the scientific community, a detailed questionnaire has been
distributed to specialists belonging to academia and pharmaceutical industries during a meeting for
computational chemists carried out in Italy in 2011. The results of the analysis attested that among
18 different computational tools (Figure 1) three of them are ranked with high levels of potential and
real interests [2].
Data mining/meaning
Ab initio
Semi-empiricals
MTS/HTS (SAR)
Molecular Mechanics
QM/MM
Classic MD
Docking
Chemical libraries
Computational
Techniques
De novo design
2D VS
ADME prediction
3D VS
Homology Modeling
QSAR
QSPR
Protein
multiallignment
3D QSAR
Figure 1: list of the computational tools investigated by the survey.
In this communication a brief discussion about the computational most trusted methods will be
carried out and some successful stories will be presented in order to give an idea about their real
impact and advantage for the scientific community.
Finally, some considerations will be devoted to define the formative goals of high level of education
courses with the aim to enrich the competencies of experts in this field adequate to perform drug
discovery programs in academia as well as in biotech and pharmaceutical companies.
References
1. Bourne, P.E.; Westbrook, J.; Berman, H.M. The Protein Data Bank and lessons in data
management. Brief Bioinform. 2004, 5, 23-30.
2. Artese, A.; Alcaro, S.; Moraca, F.; Reina, R.; Ventura, M.; Costantino, G.; Beccari, A.R.;
Ortuso, F. State-of-the-art and diffusion of Computational Tools for Drug Design
Purposes: a Survey among Italian Academic and Industrial Institutions. Future Med
Chem, 2013, 5, 907–927.
Expression, Purification and Crystallization
of pVHL:ElonginB:ElonginC complex
a
b
Martina Casale, Morgan Gadd, and Alessio Ciulli
b
a
Dip.di Scienze del Farmaco e dei Prodotti per la Salute, Università di Messina, Italy
Division of Biological Chemistry and Drug Discovery, College of Life Sciences, Dundee, UK
b
[email protected]
Ubiquitination is a process of post-translational modification in which the ubiquitin is attached to a
substrate protein. These ubiquitination modifications of proteins regulate biological process including
cell cycle progression, DNA repair and replication and gene transcription. Ubiquitination requires
three types of enzyme: ubiquitin-activating enzymes, ubiquitin-conjugating enzymes, and ubiquitin
ligases, known as E1s, E2s, and E3s, respectively. One class of RING E3 ubiquitin ligases, called
culling-RING ligases (CRLs), consist of a cullin scaffold protein and a catalytic RING subunit (Rbx1 or
Rbx2).
In particular, the Von Hippel-Lindau (VHL) tumor suppressor is a component of a protein complex
possessing E3 ligase activity that controls the pathway of ubiquitination and protein degradation of
HIF-1 in normoxia. To explore the HIF-1 degradation we carried out the expression, purification
and crystallisation of the VCB complex that consists of VHL, elongin B and elongin C that can be
recombinantly expressed together stably in Escherichia coli. We made the purification in four
chromatography steps.
Then, for the structural studies we used the crystallization method. In order to achieve the protein
crystallization the technique of the hanging-drop vapor diffusion is one of the most popular is the one
used in the crystallization of VCB. So, a few uL of the protein solution was mixed with the same
amount of a reservoir solution containing the precipitation reagent cocktail.
A drop of this mixture was placed on a siliconized glass
slide, which covers and seals the reservoir. Since the
mixture in the drop was less concentrated than the
reservoir solution, water evaporated from the drop into
the reservoir. The drop slowly supersaturates and
subsequently nucleation and phase separation occured,
ideally resulting in crystal formation. Finally, the complex
was crystallized and these crystals were used for soaking
with fragments and the VHL:HIF-1α inhibitors
synthesised in the chemistry laboratory. Modern
synchrotrons and detectors allowed us a rapid collection
of datasets and separation of close spots in diffraction
patterns
References
1. Zimmerman ES, Schulman BA, Zheng N: Structural assembly of cullin-RING
ubiquitin ligase complexes. Structural Biology 2010, 20:714-721.
2. Min J, Yang H, Ivan M, Gertler F, Kaelin Jr. WG, Pavletich N: Structure of an HIF-1α-pVHL
Complex: Hydroxyproline Recognition in Signaling. Science 2002, vol.296 no.5574 pp.
1886-1889
Identification of new binders to the SBC complex
a
b,
Claudio Catalano, Teresa A. F. Cardote and Alessio Ciulli
b
a
Dip.di Scienze del Farmaco e dei Prodotti per la Salute, Università di Messina, Italy
Division of Biological Chemistry and Drug Discovery, College of Life Sciences, Dundee, UK
b
[email protected]
Protein-protein interactions (PPIs) are the cornerstone of the most important cellular processes. The
modulation of these interactions by small molecules offers attractive possibilities for the treatment of
human disease states. Due to their multisubunit arrangement, Cullin RING E3 ligases (CRLs) were
chosen as human model systems to probe/modulate PPIs in our project. CRLs are enzymatic
complexes that play an important role in the process of protein’s ubiquitination. There are many
different CRLs that consequently bind different substrates but they all share a common
arrangement: a scaffold protein, Cullin, an adaptor subunit, Elongin B and Elongin C, a ring finger
domain, Rbx, that binds the ubiquitin-activating enzyme E2 that, in turn, binds ubiquitin to then
transfer it to the substrate, that is bound to the substrate-binding domain. In this project the
substrate binding domain is SOCS2.
The aim of this project is to identify new binders of the SBC complex (elo-B,elo-C, SOCS2) and
SBC-GHR (which is the SBC complexed with a peptide analog of the natural substrate, the Growth
Hormone Receptor).
Searching for:
-bicyclic peptides with a chemical core (TBMB);
-bicyclic peptides obtained through air oxidation of Cys residues;
-linear 7-mer peptides.
A phage display screening of three different phage libraries of bicyclic peptides was performed.
Fig.1 Screening of
bicyclic peptides.
Image adopted from
Methods 60, 1, 2013,
46–54
References





Bullock, A. N., Debreczeni, J. E., Edwards, A. M., Sundström, M., & Knapp, S. (2006). Crystal structure of the
SOCS2-elongin C-elongin B complex defines a prototypical SOCS box ubiquitin ligase. Proceedings of the National
Academy of Sciences of the United States of America, 103(20), 7637–42.;
Chen, S., Rentero Rebollo, & Heinis, C. (2013). Bicyclic peptide ligands pulled out of cysteine-rich peptide libraries.
JACS, 135(17), 6562–9. doi:10.1021/ja400461h;
Rentero Rebollo, I., & Heinis, C. (2013). Phage selection of bicyclic peptides. Methods (San Diego, Calif.), 60(1), 46–
54;
Smith, G. P., & Petrenko, V. a. (1997). Phage Display. Chemical Reviews, 97(2), 391–410.
Wells, J. a, & McClendon, C. L. (2007). Reaching for high-hanging fruit in drug discovery at protein-protein interfaces.
Nature, 450(7172), 1001–9.
SOCS2-ElonginBC and ElonginBC protein
complexes: expression, purification and fragment
screening.
a
b
Federica Centorrino, Emil Bulatov and Alessio Ciulli
b
a
Dip.di Scienze del Farmaco e dei Prodotti per la Salute, Università di Messina, Italy
Division of Biological Chemistry and Drug Discovery, College of Life Sciences, Dundee, UK
b
[email protected]
The SOCS proteins are involved in the negative regulation of a variety of cytokine, growth factor
1
and hormone signals, particularly those mediated by the JAK/STAT signalling pathway . The
suppressor of cytokine signalling 2 (SOCS2) is a key regulator of growth hormone receptor
levels and it represents the substrate recognition subunit of an E3 ligase, in complex with
ElonginB-ElonginC as the adaptor subunit (see figure). The role of this complex is to mediate
the transfer of ubiquitin to the substrate, allowing thus the degradation process by the
2
proteasome system .
The aim of this work was to identify small molecules binding these two protein complexes trough
a screening of a fragment library of ~1150 compounds. In order to achieve this goal the first step
was to express and purify the proteins with good yields and purity, then a set of biophysical
technique (DSF, BLI and ligand based NMR) allowed to identify and increase confidence on
fragment hits and to select a small group of positive hits. Initial steps were based on the
screening of the entire library in high-throughput manner with differential scanning fluorimetry
and bio-layer interferometry, in this way we found different hits for both proteins (SOCS2ElonginB/C and ElonginB/C). Subsequently the use of NMR spectroscopy enabled to discard
same false positives and to select 17 binders to SOCS2-ElonginB/C and 9 to ElonginB/C.
(1) O'Sullivan L.A., Liongue C., Lewis R.S., Stephenson S.E., Ward A.C. «Cytokine receptor
signalling through the Jak-Stat-Socs path-way disease.» Mol. Immunol. (2007): 44:24972506.
(2) Nalepa G., Rolfe M.,Harper J.W. «Drud discovery in the ubiquitin proteasome system.»
Nature Reviews Drug Discovery (2006): 5:596-613.
Optimization of the left-hand site of small
molecules that disrupt the Von Hippel Lindau
protein and Hypoxia Inducible Factor-1α proteinprotein interaction
a
b
Salvatore Scaffidi, Carles Galdeano, Alessio Ciulli
b
a
Dip. Scienze del Farmaco e Prodotti per la Salute Università di Messina, Italy
b
College of Life Sciences, University of Dundee, Dundee, DD1 5EH, UK.
[email protected]
Protein ubiquitination occurs through a cascade of enzymatic reactions, involving an E1 ubiquitin
activating enzyme, and E2 ubiquitin conjugating enzyme and an E3 ubiquitin ligase. E3 ubiquitin
ligases confer substrate specificity to protein ubiquitination pathways, and are thus attractive drug
targets (1). However, to date, efforts to target E3 ligases using small molecules have been rewarded
with limited success, resulting in this protein family being considered as “undruggable”. In 2012
Alessio Ciulli’s group and collaborators published the first example of ligands targeting the von
Hippel-Lindau protein (VHL) (2,3,4), the substrate recognition subunit of an E3 ligase, an important
target in cancer, chronic anemia, and ischemia (5). Now, using a rational design and a molecular
recognition strategy we have very recently generated nanomolar inhibitors of the VHL:Hif-1
interaction, the natural substrate of VHL. These new inhibitors have been characterized by
Isothermal Titration calorimetry (ITC) and the binding mode was confirmed using protein X-ray
crystallography. These small molecule inhibitors of the VHL:HIF-1α interface showed huge potential
to be developed into cell-penetrant chemical probes and in the hypoxia signaling cascade.
Moreover, these ligands are perfect starting points for the development of novel drug-like PROTACS
(PROteolysis TArgeting Chimeric moleculeS) for the recruitment of target proteins to the proteasome
for degradation.
Figure 1 Crystal structure of VBC in complex with ligand
3.
REFERENCES
(1) Cohen, P.; Tcherpakov, M. Will the Ubiquitin System Furnish as Many Drug Targets as Protein Kinases? Cell. 2010, 143, 686.
(2) Buckley, D.L., et. al. Targeting the von Hippel−Lindau E3 Ubiquitin Ligase Using Small Molecules To Disrupt the VHL/HIF-1
Interaction. J. Am. Chem. Soc. 2012, 134, 4465.
(3) Van Molle, I. et al. Dissecting Fragment-Based Lead Discovery at the von Hippel-Lindau Protein:Hypoxia Inducible Factor 1α
Protein-Protein Interface. Chemistry & Biology 2012, 19, 1300.
(4) Buckley, D. L. et al. Small Molecules Inhibitors of the Interaction Between the E3 Ligase VHL and HIF-1. Angew. Chem. Int. Ed.
Engl. 2012, 51, 11463.
(5) Shen, C.; Kaelin, W.J. Jr. The VHL/HIF axis in clear renal carcinoma. Semin. Cancer Biol. 2013, 23, 18.
ALESSIO CIULLI
Email: [email protected]
Telephone: +44 (0)1382 386230
Web page: http://www.lifesci.dundee.ac.uk/people/alessio-ciulli
PRESENT APPOINTMENTS
04/2013–present
Reader in Chemical & Structural Biology, Biological Chemistry & Drug
Discovery, College of Life Sciences, University of Dundee
01/2010–present
BBSRC David Phillips Fellow
01/2010–04/2013
Group Leader, Department of Chemistry, University of Cambridge
CAREER
02–06/2009
02/2006–12/2009
EDUCATION
2002–2006
1996–2002
Visiting postdoctoral research, Yale University, USA (Craig M. Crews)
Postdoctoral research & College Research Fellow, University of
Cambridge (Chris Abell and Tom L. Blundell)
PhD Chemical Biology, University of Cambridge (Chris Abell). BBSRC
CASE Award with Astex Therapeutics (Glyn Williams)
BSc, MSc Chemistry, University of Florence, Italy (110/110 Magna
Cum Laude). Master project, CERM Laboratory (Ivano Bertini).
HONORS AND AWARDS
2012 ERC Starting Grant
2009 BBSRC David Phillips Fellowship
2008 Human Frontier Science Program Short-Term Fellowship
2006 Junior Research Fellowship, Homerton College, Cambridge
2002 Gates Cambridge Scholarship
PROFESSIONAL MEMBERSHIPS
American Chemical Society (ACS), Royal Society of Chemistry (MRSC), British Biochemical
Society, British Biophysical Society