Benedikt KesslerHWBMP/WTCHGUniversity of OxfordUKbmk@ccmp.ox.ac.ukhttp://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