Novel tools for DUB inhibitor specificity profiling in cancer · Novel tools for DUB inhibitor...
Transcript of Novel tools for DUB inhibitor specificity profiling in cancer · Novel tools for DUB inhibitor...
Benedikt KesslerHWBMP/WTCHGUniversity of [email protected]://www.ccmp.ox.ac.uk/kessler-group
Novel tools for DUB inhibitor specificity profiling in cancer
Novartis April 2013
Chemical Biology & Mass Spectrometry
Group and Interests
MHC presentation
Immune recognition(T‐cells)
Ubiquitin‐Proteasome System
Neurodegenerative disease
Inflammation
Cancer
Bacterial and Viral Infections
Ubiquitin System in Human Diseases
Ub linkage type adds to biological complexity
Cancer Target discovery & “Learn” how to use themNeurodegeneration Identification for disease interventionPathogens
DUB enzyme Biology Human DiseaseUCH-L1 AKT – -Catenin – NF-B Parkinson’s disease (mutation/OE)
B-cell malignancies, pancreas, colorectal, breast (OE)USP6 (Tre2) OncogeneUSP8 RTK, Wnt and HH pathways OncogeneUSP14 26S editing Ataxia (mouse)CYLD Wnt, NF-B Cylindromatosis (mutations)VDU1, 2 bind VHL Renal carcinoma
DUBs in Disease (1)
USP2 Mdm2/4, FASN, NF-B, c-Myc Prostate / breast cancer (OE)USP7 / 7S* Mdm2/4, PTEN, FOXO4 (p53) Diverse cancers (OE)USP15 TGF--R1, -Catenin, SMADS Glioblastoma, breast & ovarian (OE)Cezanne 1 EGRF turnover Breast cancer (amplification, OE)OTUB1 UBC13/RNF 168, p53, RhoA DNA damage, prostate cancerUSP1/UAF1 Chk1 & ID1-3 (CSC) Melanoma, colon, lung, osteosarcome (OE, activation)USP9X Mcl-1, -catenin, TGF- Colorectal, breast, lung, lymphoma (OE)USP10 p53, AR, autophagy Melanoma (OE)USP13 MITF oncogene 10-20% of melanomasUSP22 p53, MYC Aggressive cancers (OE)USP4 TGF--R1, -CateninUSP17 (Dub3) Cdc25A turnover – GTPases Breast, lung, colon hematopoietic cancers (OE)USP33 Met signaling - apoptosis Breast cancer
*Khoronenkova Mol.Cell. 2012
Problems, Challenges & Opportunities:
• How to discover & choose the right target(s) relevant for disease ?• Best way to manipulate these targets for effective intervention ?
• Good knowledge about molecular target and pathway• Substrate identity and function are unknown for most DUBs
• Understand how your target functions• Inhibitor development and substrate ID in vitro• Need to explore them in a cellular environment
DUBs in infection: DUB-like enzyme PathogenUL36USP HSVCoV PLpro SARSL protein Hemorrhagic fever virusAvp AdenovirusYopJ YersiniaChlaDub1, ChlaDub2 ChlamydiaPFDub1 Plasmodium Falciparum
DUBs in Disease (2)
Know How Your DUB Works: Structural Information for Inhibitor targeting & design
OTUB2‐UbBr2yOTU1‐UbBr3
OTUB2‐UbBr2vOTU‐Ub
OTUB2‐UbBr2OTUB1‐Ubal‐UBC13‐Ub
OTUB2‐UbBr2DEN1‐NEDD8
Altun M, 2013 Otubain‐2 catalytic centre‐ Specific features of DUB cysteine proteases:‐ Unusual triade‐ Often in an “unproductive conformation”in apo form
USP Inhibitors
UCH Inhibitors
Others
DUB Inhibitor Target/Disease association/Therapeutic potential
USP7: prostate cancer, non‐small cell lung adenocarcinoma,
USP8: Sensitivity to glioblastoma
USP14: Neurodegeneration, ataxia
UCH‐L1: Parkinson’s disease
Plpro: SARS corona virus
Small molecular DUB inhibitors in the UPSNovel Therapeutics and Research Tools
Edelmann MJ, Nicholson B, Kessler BM. Exp. Rev. Mol. Med. 2011
N
N
OCl
N
N
HBX41,108USP7
N
O
NH2
H
GRL0617Plpro
Isatin O-acyl oximesUCH-L1
NCl
Cl
Br
NO
O
O
UCH-L3
O
N
NO
OH
O
Isatin derivative
N
N
N O
N
N
HBX 90,397USP8
P022077USP7
PR-619Broad specificity DUB inhibitor
UI1
15d-PGJ2
NNF
O
USP14
N
SSNN
NH2 NH2
SF
F
S
O
O
O
O
COOH
(1)
(3) (7)
(2)
(5) (6)(4)
(9)(8)
UCH-L3UCH-L1
Altun M et.al.Chem&Biol 2011
Structures of DUB inhibitors PR-619 and P22077 and in vitro DUB inhibition profiles
p53 p53
MDM2
USP7
Cell survivalCell death
Nicholson B et.al.Cell Biochem Biophys 2011
visualizationretrieval specificity trap
125Ibiotinfluorescentgroup
peptidenon-peptidylmoiety
epoxidevinyl sulfoneflourophosphonateacyloxyketone
-SH -S-+active enzyme
inactive enzyme
+
detected
not detected
ChemoproteomicsLooking at DUBs in Cells: Activity-Based Probes
HAUb-derived probes
NH
CNHAUb
NH
SO O
HAUb
NH
O
OHAUb
BrNH
HAUb
C lNH
HAUb
NH
BrHAUb
NH
O
HA Ub75
Michael acceptors Alkyl halides
NH
SO O
HAUb
reactivegroup
Borodovsky A, Ovaa H, Nagamallesawari K, Tudeviin G‐E, Wilkinson KD, Ploegh HL and Kessler BM. Chemistry & Biology. 2002, 9 (10), 1149‐1159
Biochemical Validation of DUB Inhibition in Cells
Altun M et.al.Chem&Biol 2011
VME
O
UbiquitinH2N
Enzyme
Activesite+
NH
VME
O
UbiquitinH2N
Enzyme
HATag
HATag
NH
DUB inhibitorsPR‐619P22077
Altun et al., BBA 2012
DUB Inhibitor Profiling in Cells using a Mass Spectrometry Approach
1. Label‐Free Quantitation ‐ UPLC‐MSE
2. Label‐Free Quantitation / SILAC – UPLC‐Orbitrap Velos –LC‐Progenesis / MaxQuant
1. Normalisation based on Ubiquitin derived peptides
Label-Free / SILAC Quantitation MS
Nano‐UPLC25cm column1.7M particle size~7000psi
HA‐IPLabelled extractsuntreated
HA‐IPLabelled extractsInhibitor treated
In‐sol digestionUPLC‐MS/MS
In‐sol digestionUPLC‐MS/MS
Label‐free quantitation:Comparison of DUB derived peptide peakabundances
Time
Intensity
Activity-Based Proteomics for DUB Inhibitor Profiling in Living CellsActivity-Based Proteomics for DUB Inhibitor Profiling in Cells
49 DUBs covered
Harper Lab
Altun M et.al.Chem&Biol 2011
Expanding to NEDD8, SUMO, etc...
(out of 71 known human Cysteine protease DUBs = 69%)
Intensity
based
absolute qu
antification (iB
AQ)
DUBs in HEKs
DUB Activity Versus Abundance
Correlation betweenAbundance and labelling (activity)
+ ‐
Adapted from Geiger T Mol Cell P roteomics2012 Kessler BM Curr. Opin Chem Biol 2013
Altun Chem&Biol 2011
P5091 is a USP7 Selective Inhibitor
Chauhan D. et.al.Mol Cell 2012
USP7 : A Therapeutic Target in Multiple Myeloma (MM)
Combination of P5091 and Lenalidomide, SAHA, or Dex Trigger Synergistic Anti‐MM ActivityP5091 Overcomes Bortezomib‐Resistance
USP7 ExpressionAnd PrognosticRelevance inMM Cells
Chauhan D. et.al.Mol Cell 2012
O
HA Ub NH
O
OMe1
O
HA Ub NH
2
A
Br
O
HA Ub NH
O
HN
3O
NH
HA Ub OO O
O
OH
NNN
O
HN
4
Development of Fluorescent Ub probes
McGouran et al., OBC 2012
N3‐CH2‐CH2‐CH2‐Cy3“Click‐Chemistry”
Active DUB profiles in cells
Blue: DAPI; green: lyso‐tracker; red/yellow: DUBs
Patent P38458GB
Active DUB Profiles in Cells
CTRL (dye alone) HA‐Ub‐VA‐Cy3 HA‐Ub‐VA‐Cy5
McGouran, 2012
“Chariot” protein transfection reagentHEK293T cells
Information about localized DUB activity & inhibitionin cells
Patent P38458GB
“Branched”Ub Probes to Profile DUB Linkage Specificity
Iphöfer, ChemBiochem 2012Information about DUB inhibition & Ub linkage specificity
in a cellular environment
Differential profiling by IB Mass Spec
Novel di-Ubiquitin Probes Coupled by „Click“Chemistry
Joanna McGouran et al., 2013. Submitted
Purification and Characterisation of di-Ubiquitin active site probes
Joanna McGouran et al., 2013. Submitted
Challenges for future UPS / DUB drug development
• Obtaining specificity in targeting DUBs – demonstrated with selective USP7 inhibitors
• Chemoproteomics for DUB (inhibition) mechanism of action in cells – DUBs as drug targets
• USP7 inhibition has anti‐tumour activity in vivo – synergistic with other drugs
• Novel di‐Ub probes begin to address DUB Ub‐linkage specificity in cells
• Opportunities for •Defining DUB subsets for different Ub‐linkages•DUB Ub‐linkage inhibitor screening•Deconvoluting DUB function: DUB – substrate probes to capture DUB(s) for a given substrate in cells
Conclusions & Perspectives
Mikael Altun (Swe)Holger KramerLianne WillemsSelina Gaertner Joanna McGouran Mukram MackeenRoman FischerRebecca KonietznyEdward KoganNicola Ternette
Benedikt Kessler HWBMPOxford, UK
Ben NicholsonCraig LeachTauseef ButtProgenra Inc., Malvern, PA, USAAlex IphoeferLothar JentschRaimo FamkeHelmholtz‐Zentrum fuer Infektionsforschung Braunschweig, Germany
Thank You !
Mikael Altun
Joanna McGouran
Holger Kramer
Shoumo BhattacharyaAyman Al Haj ZenCVD, Oxford, UK
Selina Gaertner
Dharminder ChauhanKenneth AndersonDana Farber Cancer InstituteBoston, USA