Future of Novel Drugs: DUBs Drug Discovery Platform LifeSensors Inc.

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1 Future of Novel Drugs: DUBs Drug Discovery Platform LifeSensors Inc. 271 Great Valley Parkway Malvern PA Phone: Fax:

2 LifeSensors Capabilities ~35 active DUBs in LifeSensors Library ~25 DUB assays developed Confirmation and counter screening to eliminate nuisance compounds Assays for determining compound action in the cell Ability to screen ~500,000 compounds

3 Business Model Help customer succeed in identifying true DUBs inhibitors or binders Establish HTS platforms in house or screen customer libraries at LifeSensors All IP and data belong to the customer Work performed under CDA and Master Service Agreement Custom assay development Fee for service model, initiate work upon signing the agreement

4 Ubiquitylation Ubiquitylation is one of the most important post-translation modifications (PTMs) regulating the stability and activity of proteins. E1: Ubiquitin activating enzyme E2: Ubiquitin conjugation enzyme E3: Ubiquitin ligase E1 C E2 C C E3 K substrate ATP AMP

5 Deubiquitylating Enzymes (DUBs) DUBs are a large group of proteases that remove ubiquitin from proteins substrate substrate DUB DUB

6 DUBs functions Save ubiquitin tagged proteins from being degraded Involve in more subtle editing of the polyubiquitin signal Cleave ubiquitin precursors Recycle ubiquitin monomers It is now understood that a tight balance between ubiquitylation and deubiquitylation is required for cellular survival underlying the equal importance of E3s and DUBs.

7 Mammalian DUB families Family name Number Members ubiquitin C-terminal hydrolases 4 BAP1, UCHL1, UCHL3, UCHL5 (UCHs) ubiquitin-specific proteases (USPs) 56 USP1, USP2, USP3, USP4, USP5, USP6, USP7, USP8, USP9X, USP9Y, USP10, USP11, USP12, USP13, USP14, USP15, USP16, USP18, USP19, USP20, USP21, USP22, USP24, USP25, USP26, USP27X, USP28, USP29, USP30, USP31, USP32, USP33, USP34, USP35, USP36, USP37, USP38, USP39, USP40, USP41, USP42, USP43, USP44, USP45, USP46, USP47, USP48, USP49, USP50, USP51, USP52, USP53, USP54 Machado Joseph disease proteases (MJD, Josephins) 4 Ataxin-3, Ataxin-3-Like, JosD1, JosD2 ovarian tumour proteases (OTUs) JAB1/MPN/Mov34 Metalloenzymes (JAMMs) 16 A20, Cezanne, Cezanne2, TRABID, VCPIP1, OTUB1, OTUB2, OTUD1, OTUD3, OTUD4, OTUD5,OTUD6A,OTUB6B,OTU1,YOD1, Otulin 11 BRACC36, CSN5, CSN6, PSMD7, PSMD14, AMSH, AMSH-LP, EIF3H, MPND, MYSM1, PRPF8 Ristic, G., Tsou, W. L., & Todi, S. V. (2014). An optimal ubiquitin-proteasome pathway in the nervous system: the role of deubiquitinating enzymes.frontiers in molecular neuroscience, 7, 72.

8 DUB Drug Targets and Disease DUBs Role/Disease USP14 USP15 USP30 USP7 A20 CEZANNE AMSH Neurodegenerative conditions, e.g., Alzheimer s and Parkinson s Negative regulator of T cell activation Antagonizes parkin ligase activity, Parkinson s disease Cancer therapy Ubiquitin chain assembly and disassembly in NF-kB pathway, cancer NF-kB activation and expression of inflammatory genes Endosome-associated DUB for K63 chains Ataxin3 Machado-Joseph disease (MJD) / spinocerebellar ataxia type 3

9 Targeting DUBs for Diseases Ubiquitylation and deubiquitylation regulate cellular homeostasis, dysfunction in ubiquitin pathway result in diseases. Deubiquitylation of histones, transcription factors and their co-factors plays a major role in regulating epigenetics by DUBs. As a result DUBs are valuable therapeutic targets.

10 Examples of DUB Inhibitors DUB Pan-inhibitor USP1 Compounds PR-619 Pimozide, ML323, SJB2-043 and SJB3-019A, GW7647 USP7 HBX 19,818 and HBX 28,258, P5091 and P22077, Pimozide USP10 USP14 USP46 Spautin-1 b-ap15, AC17 Pimozide

11 Unique Assays Discover Novel Compounds Customizable o Studies (right figure) indicated that each DUB prefer different assay/substrate. LifeSenors offers variety of assays and will optimize the right assay for your DUB. Efficient o LifeSensors has years of experience assaying every family of DUBs. Comprehensive o Our team is able to profile candidate compounds, perform HTS and determine selectivity. 13 >50% FP assay out of 33 (39%) 25 >50% CHOP assay out of 59 hits (42% Confirm) 22 >50% Ub-Rho assay out of 73 hits (30% Confirm) 10K compounds were screened with three different assays, which yielded different hits.

12 DUB Drug Screening Tools High Throughput Screening (HTS) assays CHOP reporter assay Diubiquitin internally quenched fluorescence (IQF) assay Ubiquitin fluorophore assays Confirm the hits through cellular assays UbiQuant S assay (ELISA / AlphaLISA) UbiTest (Immunoblot based assay)

13 Cellular Drug Target Confirmation UbiQuant S ELISA Relative quantification of substrate ubiquitylation levels in a cellular context. Poly-Ubiquitin capture reagent Apply cell lysates Chemiluminescence Capture poly- Ub proteins Poly-Ubiquitn capture reagent Protein of interest (POI) Apply POI antibodies Other proteins Poly-ubiquitin Antibody

14 Cellular Drug Target Confirmation UbiQuant S AlphaLISA The AlphaLISA format is a homogenous assay, which could efficiently determine substrate ubiquitylation status. Emission 615 nm 1 O 2 Excitation 680 nm Acceptor Beads Target T Ub U Ub B Ub E Ub Donor Beads

15 UbiTest Cellular Drug Target Confirmation Detection of transfected and endogenous substrate ubiquitylation. Problems of traditional IP/IB method for detection of target ubiquitylation: Cumbersome Signal dilution Antibodies do not detect target with Ub Hard to qualify Principle of UbiTest. A more definitive method for demonstrating protein ubiquitylation is to couple immunoprecipitation with digestion by a broad spectrum deubiquitylase prior to immunoblot analysis. An increased signal for the protein of interest (POI) after DUB treatment is a clear indication that the protein was ubiquitylated even if there was no clear reactivity in the untreated sample.

16 Confirm hits through cellular assay UbiTest - Example Target 1 Target 2 Target 3 Determine endogenous target proteins ubiquitylation using UbiTest. Jurkat cells were treated with indicated compounds and lysed in RIPA buffer. Anti-Ub TUBE1 agarose resin was added for pull down total polyubiquitylated proteins and then elution was incubated with DUB, USP2core. Immunoblot (IB) of the assay is shown on the left, increased signal of targets treatment indicates they are poly-ub modified.

17 Contact We are your partner for drug discovery in ubiquitylation! Michelle Gordon x 339 LifeSensors Inc. 271 Great Valley Parkway Malvern PA