Exploring Drug-Target interactions by
MS-based proteomics
Andrea Armirotti, Ph.D.
Istituto Italiano di Tecnologia, Drug Discovery and Development Department 2013
Part I
Proteomics to elucidate the
interaction mechanism
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Palmitoylethanolamide
a lipid messenger
PEA exerts anti-inflammatory and anti-nociceptive effects in animal models
by engaging the peroxisome proliferator-activated receptor-a (PPAR-a).
X
inhibition
N-acylethanolamine-hydrolyzing acid amidase
NAAA is a pharmacologically promising target
for pain and inflammation
Mazzari et al. Eur J Pharmacol. 1996
Calignano et al. Eur. J. Pharmacol. 2001
Lo Verme et al. Mol Pharmacol 2005
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NAAA Activation
Lysosomes, pH 4.5
H2N
Cys
Active
H2N
Active
NAAA
SH
Catalytic
Residue
O
R1
b-Lactones as NAAA inhibitors
O
R2
Solorzano et al. PNAS 2009
Solorzano et al. J. Med. Chem. 2010
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COOH
SPPAAPRFNVSLDSVPELRWLPVLRHYDLDLVRAAMAQVIGDRVPKWVHVLIGKVVLELERFLPQPFTGEIRG
MCDFMNLSLADCLLVNLAYESSVFCTSIVAQDSRGHIYHGRNLDYPFGNVLRKLTVDVQFLKNGQIAFTGTTFI
GYVGLWTGQSPHKFTVSGDERDKGWWWENAIAALFRRHIPVSWLIRATLSESENFEAAVGKLAKTPLIADVY
YIVGGTSPREGVVITRNRDGPADIWPLDPLNGAWFRVETNYDHWKPAPKEDDRRTSAIKALNATGQANLSLEA
LFQILSVVPVYNNFTIYTTVMSAGSPDKYMTRIRNPSRK
1) Cleaved in activation @ pH 4.5
2) Target Cysteine
CTSIVAQDSRGHIYHGRNLDYPFGNVLRKLTVDVQFLKNGQIAFTGTTFIGYVGLWTGQSPHKFTVSGDERDK
GWWWENAIAALFRRHIPVSWLIRATLSESENFEAAVGKLAKTPLIADVYYIVGGTSPREGVVITRNRDGPADIW
PLDPLNGAWFRVETNYDHWKPAPKEDDRRTSAIKALNATGQANLSLEALFQILSVVPVYNNFTIYTTVMSAGS
PDKYMTRIRNPSRK
3) Trypsin Digestion
*CTSIVAQDSR
New N-terminus peptide
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A new, potent NAAA inhibitor
ARN0077
IC50 (hNAAA)
7.3 nM
Questions
1) Covalent or not?
2) Confirmation of the target residue (N term. Cys?)
3) Molecular Mechanism of action?
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Experimental Details
•
•
•
Recombinant hNAAA purified from transfected HEK 293 cells
Activation at pH 4.5, 37°C, followed by incubation with equimolar ARN0077 (90+90 min.)
DDA (and MSe) acquisitions on Synapt G2 with nanoLC setup
FL
+077
Cont.
50 kDa
30 kDa
Full Lenght
Active hNAAA
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RS
D
Q
A
V
I
S
T
C
yMax
100
576.27
y5
262.15
y2
Native Peptide
%
256.07
114.04
274.08
173.13
86.10
I
377.18
a4
y3
387.17
676.34
788.42
y7
577.28
505.24
y4
875.45
y8
+ARN0077
adduct
876.45
677.34
578.28
359.17
789.41
458.25
877.45
1026.50 1166.23 1221.77
1353.60
0
-0
100
200
R
300
S
400
D
500
Q
600
A
700
V
800
L
900
S
1000
T
1100
1200
C
1300
(291.14)
M/z
1400
yMax
100
Native peptide
ARN077
Dmass
576.28
y5
262.15
y2
Adduct
547.23
%
377.16
y3
274.15
74.06 147.12
T
676.35
788.44
875.47
y7
y8
577.29
444.22
678.33
578.28
343.17
679.33
789.44 876.47
790.45 877.47
976.52
y9
1027.53
1079.52
y10
1283.09 1326.77
0
-0
100
200
300
400
500
600
700
800
900
1000
1100
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1200
1300
M/z
1400
Inhibition Mechanism: 3 Possible Adducts
Isomeric Species
Not
Observed
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y2
Cys side chain fragmentations
-H2O
[ARN0077]+
-CO
S-acylation diagnostic ion
is overlapping y2 isotopic
peak
Result not
conclusive
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y2
ARN0768
Cys side chain fragmentations
[ARN0768]+
-H2O
-CO
IC50 (hNAAA)
314 nM
Active analog of ARN0077
At 20.000 resolution, S-acylation
diagnostic ion is clearly
detectable
Conclusive Result
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Inhibition mechanism of b-lactones on hNAAA
1) Covalent or not?
 Covalent
2) Confirmation of the target residue (N term. Cys)
 Yes
3) Molecular Mechanism of action?
 S-acylation
References:
Armirotti et al. ACS Med. Chem. Lett. 2012
Ponzano et al J. Med. Chem. In preparation
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And what if the interaction is
NOT covalent?
(and you don’t know the target)
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MCP 2012
The trick
A non denaturing protein
chromatography conservative toward
D-T interactions
• Incubation followed by cell lysis
• Protein separation (IEX) and
fraction collection
• Drug followed by MRM
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MRM Trace
Drug
Drug in cells
Free drug
Drug + Target
IEX Chromatography
Target ID
Unboud drug
Drug bound
to target
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Part II
Drug-induced differences
in expression profiles
(test of Transomics platform)
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Drug X
inhibits
Target Y
DOWN
regulates
Protein P
Expresses
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MEF Cell Lines
WT
Group 1
Mutated
Group 2
Group 3
Group 4
Drug
(inhibitor of downregulator)
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1) Cell Lysis
2) Protein Assay
(10mg/group)
I.S. Spiking
(LYS_CHICK)
different ratios for validation
(8/10/2,5/3,3)
Reduction, Alkylation
Digestion
HDMSe
1D nLC, 2Hrs gradient
Triplicate injections
(500ng on 75mmX25cm column)
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HDMSe
ToF
MSe in the Transfer
Precursors
Fragments
Same RT and drift time
MSe using Look Up Tables
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m/z
Different slopes
Different charge states
Drift time
With LUT, the applied CE
is tailored on the precursor ion charge state
Lys_Chick
8
10
2.5
3.3
Lys_Chick Spiking Ratio: 8-10-2,5-3,3 per group
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Browsing through the Hits list...
PCK2 Protein
Mutated cells have
a better response
(unexpected result)
Drug increases
PCK2 expression
Istituto Italiano di Tecnologia, Drug Discovery and Development Department 2013
Drug X
inhibits
Target Y
DOWN
regulates
Protein PCK2
Expresses
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m/z
PCK2 Peptide
LGTPVLQALGDGDFIK
822,4507 m/z
66,8 min RT
6,55 ms DT
RT
LC-MS data maps
Full visualization options for peak picking
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Ackowledgments
Elisa Romeo
Stefano Ponzano
Benedetto Grimaldi
Claudia De Mei
Fabio Bertozzi
Gianpiero Garau
Glauco Tarozzo
Tiziano Bandiera
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