Compound-Target Engagement Cell-Based Assays. Assays, Validation, and Publications

Transcription

1 Compound-Target Engagement Cell-Based Assays Assays, Validation, and Publications 1

2 Agenda What is Compound-Target Engagement? Cell-Based Target Engagement Assays Customer needs Products and services Assay benefits How the assays work Applications and Validation Data Case Study, Publications, and Posters Conclusions 2

3 What is Compound-Target Engagement? Cellular target engagement is the binding of a small molecule to its protein target in a living, cell-based system Small Molecule (Compound) Intracellular Protein Target (Unbound State) Compound-Protein Target Engagement (Compound-Bound State) 3

4 Cellular Target Engagement Data Drive Drug Discovery Importance of cellular target engagement data for multiple drug discovery paradigms Target-specific approach Target-Specific Biochemical Screen Target-Specific Cellular Assays Cellular & Animal Disease Models Target Validation Phenotypic screening approach Phenotypic Screen Target Deconvolution Studies Proof of Cellular Target Engagement Target Validation 4

5 Customer Assay Needs Cell-based assays for compound-target engagement Monitoring compound cell entry and target engagement enables assessment of compound potency and confirmation of mode of action Customers need a cell-based binding assay to answer: Is my compound cell-penetrant? Does my compound bind to its intended or off-targets in the native cellular environment? How potent is the compound-target interaction? Assay requirements Quantitative, sensitive, HTS-compatible, easy-to-use, and low cost Broadly applicable to a wide range of cellular targets (selective) Provides accurate inhibitor rank order potencies 5

6 InCELL Compound-Target Engagement Assays DiscoverX offers 2 InCELL target engagement assay platforms InCELL Hunter platform offers researchers cell lines, ready-to-use kits, and profiling services for specific targets InCELL Pulse platform offers researchers ready-to-use kits for specific targets and a do-it-yourself kit perfect for creating your own compound-target engagement assay Create your own assay Products 35+ Stable and rigorously validated InCELL Hunter cell lines 65+ InCELL Hunter and InCELL Pulse ready-to-use kits with cells and reagents InCELL Pulse starter kit to make your own cellbased target engagement assays Profiling and Custom Services InCELL Hunter high throughput screening, lead optimization, and selectivity testing Multiple target classes Custom assay development Pools or clones Flexible cell types Custom constructs 6 Find your target and solution at discoverx.com/incell

7 InCELL Compound-Target Engagement Assay Benefits Universal cell-based binding assays Ideal assays for determining compound cell entry and target engagement in the native cellular environment Easily measure binding with a chemiluminescent readout Requires no custom chemical tracer or antibody reagents Provides a convenient solution when functional assays are difficult or unavailable Rapidly make your own target engagement cell-based assays after cloning in <2 weeks Generate biologically-relevant data for more confident drug discovery decisions 7

8 Agenda Cell-Based Target Engagement Assays Customer needs Products and services Assay benefits How the assays work Applications and Validation Data Case Study, Publications, and Posters Conclusions 8

9 Enzyme Fragment Complementation (EFC) InCELL assays are based on the proprietary DiscoverX EFC Technology β-galactosidase Enzyme Donor (ED) β-galactosidase Enzyme Acceptor (EA) EFC is a patented detection technology based on two recombinant β-galactosidase fragments Small peptide fragment (enzyme donor, ED) Large protein fragment (enzyme acceptor, EA) Separately, the fragments are inactive, but when combined, they form an active enzyme that hydrolyzes substrate to produce a chemiluminescent signal 9

10 How InCELL Target Engagement Assays Work InCELL Hunter and InCELL Pulse assays allow for monitoring of compound cell entry and target interaction Compound binding stabilizes the target protein that is fused with epl (the enzyme donor) Complementation between the epl tagged target and the added larger enzyme acceptor (EA) fragment forms an active β-galactosidase This increases the chemiluminescent signal upon substrate addition in a dose dependent manner 1 The enzyme donor, enhanced ProLabel (epl), is a dual-purpose peptide tag ideally suited for InCELL assays

11 How InCELL Hunter Assays Work Relies on proteins in a steady-state (dynamic) Platform based on protein steady-state stability in the presence of a compound Proteins are synthesized and degraded as part of normal biology This is the protein-specific steady-state that remains the same if no compound is present There are 2 InCELL Hunter assay formats Both assay measure changes in protein levels in a dose dependent manner Stabilized Compound-Protein Complex Assay Format* Destabilized Partner Protein Assay Format Compound binding increases protein stability (less protein degradation) Greater EFC signal corresponds to greater compound-target engagement Compound binding destabilizes protein-protein interaction Greater EFC signal corresponds to less compound-target engagement 11 Protein steady-state is dynamic and is defined as the rate of change between protein synthesis and degradation. * Most common assay format for the majority of InCELL Hunter assays.

12 How InCELL Pulse Assays Work Highly versatile platform beyond InCELL Hunter Platform based on protein thermal stability in the presence of a compound Heat Denaturation Customizable parameter Proteins are sensitive to heat denaturation Compound binding protects the protein target from thermal denaturation (a customizable parameter) allowing for EFC (and higher assay signal) When compound binding is absent, the target protein forms denatured aggregates with poor EFC (lower signal) 12

13 InCELL Pulse Target Engagement Starter Kit DIY Kit Make your own cellular compound-target engagement assays Kit provides reagents and detailed protocols for designing and developing InCELL Pulse assays Measure compound cellular potency against user-defined protein targets of interest by detecting changes in protein thermal stability Cloning & Transfection Sections XX Assay Development Protocol Overview Target gene cloning into picp expression vectors Transfection of mammalian host cells (precedes all phases of assay development) Phase I Assay Development Optimize EFC-based detection of the epl-tagged target protein Create your own target engagement cell-based assays after cloning in <2 weeks Ultimate control over assay development Test multiple constructs in parallel - Full length proteins or compound binding domains, binding domain mutants, Test binding in cell types of choice Transiently screen for compound binding without investing in lengthy and costly stable cell line development Assay Development Sections XX Stable Cell Line Generation Supplemental Information Phase II Assay Development Thermal shift assay Phase III Assay Development Isothermal inhibitor dose-response curves Stable Cell line Generation Optional 13 Catalog No Series; DIY = Do-it-yourself

14 Agenda Cell-Based Target Engagement Assays Customer needs Products and services Assay benefits How the assays work Applications and Validation Data Case Study, Publications, and Posters Conclusions 14

15 Applications for Compound-Target Engagement Including validation data using InCELL Hunter and InCELL Pulse Both Platforms are Valid for All Applications Determine compound cell permeability and target engagement Characterize different types of inhibitor classes Rank order compounds and compare to literature Confirm cellular potencies correlate with multiple methods Analyze challenging targets Characterize inhibitor mechanism of action Compare compound binding potencies Measure partner protein destabilization Access More Targets with 2 Platforms (Coverage Represents Targets) InCELL Pulse Example Protein Targets Kinases Epigenetic Targets Bromodomains Methyltransferases Hydrolases InCELL Hunter 15

16 Determine Compound Cell Entry and Target Binding in the Native Cellular Environment Example using InCELL Hunter G9a Methyltransferase Assay Published biochemical IC 5 s indicate compounds bind with defined affinities outside the cell InCELL Hunter assay indicates compounds with similar biochemical IC 5 do not have the same binding affinity to the target inside a cell This data suggests UNC321 is not as cell permeable as UNC638 or not as good at engaging the target in an intracellular environment 1 8 G9a Methyltransferase UNC638 BIX-1294 UNC Compound [M] UNC638 BIX1294 UNC321 Published Biochemical IC 5 15 nm 18 nm 15 nm Published Cellular IC 5 81 nm 5 nm N/A InCELL Hunter Cellular IC nm 95 nm 1183 nm 16 Nature Chemical Biology. 211; 7(8):

17 Characterize Different Types of Inhibitor Classes Example using InCELL Pulse ABL1 Tyrosine Kinase Assay Obtain accurate cellular potency measurements for type I-, II-, and allosteric-abl1 inhibitors Type I and II ABL1 Inhibitors Allosteric ABL1 Inhibitor 17 Inhibitor Inhibitor Type EC 5 (nm) Assay Window Hill Slope Dasatinib I Imatinib II GNF2 Allosteric 2, PI13 Lipid Kinase Inhibitor >1, n/a n/a

18 Rank Compound Cellular Potency and Compare with Literature Example using InCELL Hunter HCK Tyrosine Kinase and MEK1 Assays Rank order compounds tested and correlate with published data InCELL Hunter HCK Assay InCELL Hunter MEK1 Assay Dasatinib SKI-66 LCK Inhibitor K, 252a Inhibitor [M] Rank order: Dasatinib > Lck Inhibitor > SKI-66 > K-252A Comparison data pulled from existing binding and activity assay publications Davis et al. 211, Nat Biotech; Anastassiadis et al. 211 Nat Biotech; and Fabian et al. 25 Nat Biotech Compound InCELL Hunter Cellular IC 5 [nm] IC 5 [nm] Reference PD Henderson et al. Mol Cancer Ther. 21 U Granas et al. JBS 26 MEK Inhibitor I Wilkinson et al. Assay D&D 28 PD Solit et al. Nature 26 18

19 Confirm Cellular Steady-State Levels Correlate with WB Analysis Example using InCELL Hunter BRD4(1) Bromodomain Assay Bromodomains, such as BRD4(1), are epigenetic proteins that have no enzymatic activity and limited number of assays available This analysis focuses on steady-state levels of BRD4(1) and correlates results between InCELL Hunter assay and WB using JQ1 (BRD4 inhibitor, ~5 nm IC 5 ) Dose response curve data shows an increase in signal suggesting an increase of protein levels, and the observation is supported with the WB data InCELL Hunter BRD4(1) Assay BRD4(1) Western Blot Confirmation JQ1+ 1 nm JQ1 1 nm JQ1 BRD4(1) Ligand [M]

20 Analyze Challenging Targets Example using InCELL Pulse MTH1 Hydrolase and G9a Methyltransferase Assays Hydrolases and epigenetic targets (e.g. methyltransferases) are challenging targets to address using traditional cell-based assay approaches InCELL Pulse assays offers a simple and robust solution Rank order potencies in agreement with published data InCELL Pulse MTH1 Hydrolase Assay InCELL Pulse G9a Methyltransferase Assay 2 Inhibitor EC 5 (nm) Assay Window Hill Slope TH S-Crizotinib SCH , PI13 >1, n/a n/a Inhibitor EC 5 (nm) Assay Window Hill Slope UNC UNC BIX PI13 >1, n/a n/a

21 Characterize Inhibitor Mechanism of Action Example using InCELL Hunter PRMT3 Methyltransferase Assay The novel allosteric inhibitor, SGC77, binds to the methyltransferase domain of protein arginine methyltransferase 3 (PRMT3) SGC77 engages and stabilizes PRMT3 in both HEK 293 and A549 cell types InCELL Hunter PRMT3 Assay (HEK 293 cell type) (A459 cell type) 21 Kaniskan et al, Angew Chem Int Ed Engl. 215, 45(17) HEK 293 = transformed human embryonic kidney cells. A549 = adenocarcinoma of human alveolar basal epithelial cells.

22 Identify Novel Inhibitors by Targeting Different Domains Example using InCELL Hunter BLK and MEK1 Kinase Assays Identify ATP-competitive inhibitors Assay target construct includes ATP-binding site Dasatinib, known ATP-competitive inhibitor to BLK kinase target, is able to bind with a cellular IC 5 of 45.5 nm ATP-Competitive BLK Inhibitor Identify allosteric inhibitors Assay target construct includes ATPbinding and allosteric sites U126 binds at the MEK1 orthosteric site (natural ligand binding site; ATPsite), while the PD32591 (more potent) binds to the allosteric site Allosteric MEK1 Inhibitors 22

23 Compare Compound Binding Potencies Example using InCELL Hunter BLK and MEK1 Kinase Assays Confirm target specificity using two different kinase targets and analyzing target engagement using the same compounds in each assay BLK-specific compound (Dasatinib) does not affect MEK MEK-specific compounds (PD32591 and U126) do not affect BLK InCELL Hunter MEK1 Assay CHO-MEK Dasatinib U126 PD InCELL Hunter BLK Assay U2OS-BLK PD32591 U126 Dasatinib Compound [M] Compound [M] 23

24 Measure Partner Protein Destabilization Example of the InCELL Hunter Bcl2-Bim Assay Compound binding destabilizes protein-protein interaction Compound (ABT-737) binds to untagged target protein (BCL2) and disrupts the targetpartner protein interaction. The epl tagged partner protein becomes destabilized. BCL2 (B-cell CLL/lymphoma 2) BIM (BCL2-like 11) Greater EFC signal corresponds to less compound-target engagement Destabilized Partner Protein Assay Format Bcl2-Bim BCL [A BT-737], M BIM EC 5 S/B 1.4 nm

25 Select Kinase Cell-Based Target Engagement Assays InCELL Hunter focused (cell lines and Assay Ready express kits) RIPK2 R IP K 2 AMPK Inhibitor Compound C C D K 1/2 Inhibitor III Vandetanib XL-288 CSF1R CSF1R 8 D asatinib XL Sunitinib ABT Dasatanib SKI LCK Inhibitor K-252a HCK D asatinib Lck Inhibitor XL-88 FGR Compound [M] Compound [M] Compound [M] Inhibitor [M] SIK1 S D asatinib PD TAE-684 SKI-66 SRC SRC Dasatinib SKI Vandetanib LCK Inhibitor PIM1P 1 SGI-1778 P IM 1 Inhibitor III Bisindolylmaleimide Compound [M] Inhibitor [M] Compound [M] PCI CGI-1746 LCK Inhibitor BTK BTK CLK2 CLK2 JAK3 Inhibitor VI A GSK A TAE-684 EXEL-288 Sunitinib PYK2 PTK2B CSNK1D SB 2225 SB 2358 PD CSNK1D BLK BLK D asatinib Lck Inhibitor Src Inhibitor I Inhibitor [M] Inhibitor [M] Inhibitor [M] Inhibitor [M] Inhibitor [M] 25

26 Select Kinase Cell-Based Target Engagement Assays InCELL Pulse focused Quantitative EC 5 data for cellular target engagement for inhibitors of diverse kinases, including under-studied and well-studied kinases and common off-target kinases identified in biochemical inhibitor selectivity screens Validated Kinase Assays* for 7 of the 8 Phylogenetic Groups Balanced Kinase Representation Across Human Kinome AAK1 Ser/Thr Kinase AURKA Ser/Thr Kinase Kinase Target ABL1 ABL1(T315I) ACVR1 BTK AAK1 AKT1 AURKA BRAF BUB1 CAMK2A CSNK2A2 ERK1 VPS34 FAK IGF1R JAK2(JH1) KIT GAK HASPIN MEK1 p38-alpha PAK4 PKAC-alpha PLK1 SRPK1 Class Tyr Kinase Ser/Thr Kinase Lipid Kinase BTK Tyr Kinase VPS34 Lipid Kinase * Expression vectors available upon request 26 Refer to InCELL Pulse Target Engagement Kinase Reference Set pdf for a complete set of kinases and details (available at

27 Select InCELL Hunter Bromodomain Assays Available 6 BRD2(1) JQ 1 (+ ) BRD4(1) 8 E12348 TAF1L(2) I-B E T E PF1 BD1A (+)-JQ 1 [M ] Com pound [M ] Compound [M] BRDT(1) BRDT(1,2) JQ 1(+) PF-1 BRD3(1,2) 7 JQ 1(+) 6 PF-1 E BD1A BD1A BRD3(1) JQ 1(+) PF-1 E12348 I-B et (+)-JQ 1 [M ] Compound [M] Compound [M] Compound [M] 27

28 Agenda Cell-Based Target Engagement Assays Customer needs Products and services Assay benefits How the assays work Applications and Validation Data Case Study, Publications, and Posters Conclusions 28

29 Case Study: SGC-DiscoverX Kinase Collaboration SGC s comprehensive kinase chemical probe development initiative Develop potent and selective chemical probes for full kinome Use probes to identify new targets for diverse disease indications Proof of cellular target engagement a key pillar of the strategy Biochemical Potency and Selectivity (KINOMEscan ) Cellular Target Engagement Potency (InCELL Pulse ) Phenotypic Screens in Diverse Cell Assays Target ID for New Disease Indications InCELL Pulse used to evaluate SGC inhibitors of understudied kinases GAK and HASPIN kinases Potency rank orders established 29 SGC-University of N. Carolina: David Drewry, Bill Zuercher, Christopher Asquith

30 InCELL Pulse GAK and HASPIN Data Establish Cellular Potency Rank Order for SGC Inhibitors GAK_1 GAK_2 GAK_3 GAK_4 GAK_5 GAK EC 5 45 nm EC 5 21 nm EC 5 38 nm EC 5 8,3 nm EC 5 >1, nm HASPIN More Potent HASPIN_1 HASPIN_2 HASPIN_3 HASPIN_4 HASPIN_5 Less Potent EC 5 92 nm EC 5 76 nm EC 5 1,4 nm EC 5 5,1 nm EC 5 >3, nm 3 SGC-University of N. Carolina: David Drewry, Bill Zuercher, Christopher Asquith

31 Select InCELL Publications and Posters InCELL Pulse A High-Throughput Dose-Response Cellular Thermal Shift Assay for Rapid Screening of Drug Target Engagement in Living Cells, Exemplified Using SMYD3 and IDO1 (McNulty, et al.. M. Holbert, 217) Related to GSK SLAS poster 216 (link) InCELL Pulse: A Novel Cellular Target Engagement Assay Platform for Drug Discovery InCELL Hunter DOT 216 & SLAS 217 conf. DiscoverX posters (link) Development of Three Orthogonal Assays Suitable for the Identification and Qualification of PIKfyve Inhibitors (Fogarty, et al., 217) Small-Molecule Target Engagement in Cells (minireview; Schurmann, et al., 216) Examining Ligand-Based Stabilization of Proteins in Cells with MEK1 Kinase Inhibitors (Auld DS, et al., 215) Cell-Based Protein Stabilization Assays for the Detection of Interactions between Small-Molecule Inhibitors and BRD4 (Schulze et al., 215) A Potent, Selective and Cell-Active Allosteric Inhibitor of Protein Arginine Methyltransferase 3 (PRMT3) (Kaniskan et al., 215) Evaluation of InCELL Hunter Technology for use with Bromodomain targets (Gatfield and Craggs, SLAS poster, 215) 31 (Epigenetics and Kinase tabs) (posters)

32 DOT 216 & SLAS 217 InCELL Pulse Poster A Novel Cellular Target Engagement Assay Platform for Drug Discovery InCELL Pulse platform for cellular compound-target engagement applications Rapid and simple protocol Validated assays for diverse targets Difficult targets hydrolases and methyltransferases Under-studied, common off-target and well-studied kinases Kinase Target ABL1 FAK ABL1(T315I) IGF1R ACVR1 JAK2(JH1) BTK KIT AAK1 GAK AKT1 HASPIN AURKA MEK1 BRAF p38-alpha BUB1 PAK4 CAMK2A PKAC-alpha CSNK2A2 PLK1 ERK1 SRPK1 VPS34 Class Tyr Kinase Ser/Thr Kinase Lipid Kinase Balanced Kinase Assay Representation 32 Treiber et al. poster and talk at DOT 216; Poster at SLAS (posters) * Expression vectors available upon request

33 InCELL Compound-Target Engagement Assays Conclusions Products and services to confirm compound permeability and target binding in the native cellular environment Starter kit to create your own target engagement cell-based assays Easy-to-use protocol for a target of your choice Generate biologically relevant data for more confident drug discovery decisions 33 For additional information, visit discoverx.com/incell.