Kinase Cell-Based Assays for Selectivity Assessment and Personalized Medicine

Transcription

1 Kinase Cell-Based Assays for Selectivity Assessment and Personalized Medicine Deborah J. Moshinsky, PhD January 16, 13 Cell Assay Innovations, Inc. 1

2 Outline of Presentation Focus on Cellular Assays More Physiologically Relevant Results Pim kinase drug discovery program - cellular assays for selectivity and lead optimization Personalized medicine - utilizing cellular assays to combat drug resistance - functional investigation of mutant kinases - guiding patient treatment options 2

3 Outline of Presentation Focus on Cellular Assays More Physiologically Relevant Results Pim kinase drug discovery program - cellular assays for selectivity and lead optimization Personalized medicine - utilizing cellular assays to combat drug resistance - functional investigation of mutant kinases - guiding patient treatment options 3

4 Cancer Drug Resistance eg. Imatinib Imatinib (Gleevec, Novartis) approved in 1 for Chronic Myeloid Leukemia (CML) inhibitor of Abl kinase (BCR-Abl) revolutionized treatment of CML -3% of patients develop resistance frequently caused by mutation of Abl to prevent inhibitor binding over 3 point mutations documented in patient samples T315I gatekeeper mutation common X wild type kinase drug resistant mutant kinase 4

5 A Strategy to Combat Drug Resistance Screen inhibitors against wild-type and mutant forms of kinase Successfully utilized by Ariad inhibitors against wild type Abl, [T315I], and others development of Ponatinib approved in Dec 12 (Iclusig ) inhibits wt, [T315I] and other mutant forms of Abl wild type kinase imatinib resistant mutant kinase 5

6 ClariCELL Platform for Drug-Resistant Mutants Expression Vector for Kinase Abl or Abl [T315I] or Abl [E255V] TMB Transfection HRP HRP ptyr HEK293 (or other) Compounds Lyse ELISA assay (or comparable) incubate transfer 6

7 Abl Autophosphorylation Cellular Assays Abl autophosphorylation Abl1 wild type Abl1 [T315I] Abl E255V] Negative Control P Abl wt P P Abl [T315I] Abl [E255V] kinase dead Abl Quantify autophosphorylation and measure modulation by kinase inhibitors. Screen for more effective compounds early in the drug discovery process activity against wild type and mutant targets 7

8 Abl Cellular Data - Validation Imatinib Ponatinib wt Abl -.3uM Abl [T315I] >1uM Abl [E255V] >1uM [cmpd] M 1 8 wt Abl -.1uM Abl [T315I] -.56uM Abl [E255V] -.5uM [cmpd] M Imatinib only inhibits wt form of Abl Ponatinib inhibits wt and both mutant Abl forms Correlation with clinical activity 8

9 1 Abl Cellular Data - Validation 8 wt Abl Abl [E255V] [cmpd] M 1 8 dasatinib ~.6uM imatinib -.3uM ponatinib -.1uM ref inhib 1 >6uM [cmpd] M dasatinib ~.6uM imatinib -.3uM 1 ponatinib -.1uM 8 ref inhib 1 >6uM Abl [T315I] dasatinib >1uM imatinib >1uM ponatinib -.56uM ref inhib 1 >6uM [cmpd] M dasatinib ~.1uM imatinib >1uM ponatinib -.5uM ref inhib 1 >6uM [cmpd] M 9

10 Correlation of Cellular Data with Clinical Imatinib Dasatinib Ponatinib Abl wild type IC5 (µm) Clinical Activity vs. Abl wild type Yes Yes Yes Abl [E255V] IC5 (µm) >1.1.5 Clinical Activity vs. Abl [E255V] No Yes Yes Abl [T315I] IC5 (µm) >1 >1.6 Clinical Activity vs. Abl [T315I] No No Yes General overall correlation Compounds generating IC5 in cellular assay are active in clinic. 1

11 No Cellular Activity with Reference Compound Assay Biochemical IC5 at Km ATP Biochemical IC5 at 1mM ATP Biochemical IC5 Differen<al Cellular IC5 (um) Wt Abl1 75nM 13nM 17- fold >6 Abl1 E255K 1nM 45nM 32- fold >6 Abl1 T315I 6.4nM 89nM 139- fold >6 Inhibition of Abl and mutants is sensitive to ATP concentration Cell-based potency and selectivity determinations are important! 11

12 Benefits of ClariCELL Platform Functional activity of kinase measured in a physiologically relevant setting Ser/Thr as well as Tyr kinase targets Full length kinases Human cell system Quick turnaround time for assay develoment 3-4 weeks Multiplexing capability Seed Measure Treat Lyse 12

13 Increasing Efficiency with Multiplexing traditional paradigm multiplexed paradigm stauro1 stauro2 8 8 IC5=.uM stauro2 8 8 IC5=.uM IC5=.69uM stauro stauro1 8 LOGEC5 EC LOGEC5 stauro2 EC5 stauro LOGEC staurosporine EC Result LOGEC5 EC cell-based assays IC5=.69uM LOGEC5 EC results for 3 targets in 1 assay LOGEC5 EC5 13. LOGEC5 EC LOGEC5 EC IC5 = 9.8uM Result 3 IC5=14uM 1. Result log[cmpd]um log[cmpd]um IC5=14uM 71.6 LOGEC SGI EC IC5 =.43uM LOGEC5 EC LOGEC staurosporine EC5.1 8 IC5 = 9.8uM LOGEC5 EC LOGEC5 EC5 -.5 Result 3 IC5=14uM 1. log[cmpd]um log[cmpd]um IC5=14uM 71.6 LOGEC SGI EC LOGEC5 EC5 8 IC5 =.43uM IC5=.uM. IC5=.69uM -.5 Result IC5=.uM.5. Result 1 LOGEC5 stauro2 EC5 -.5 IC5=.69uM

14 Functional Analysis of Mutant Kinases Cellular assay system useful for biological insight pathway alterations resulting from mutant kinase proteomic analysis of cell lines expressing wt vs. mutant kinases eg. RPPA analysis to map pathway alterations Working with Carna Biosciences to map mutant cell lines Characterization of known or newly discovered mutations 14

15 Guiding Physician Treatment Options Cellular system generally correlates with clinical response to inhibitors over-simplified system Utilize systems biology to model in vivo parameters in vivo drug exposure pharmacodynamics protein binding Cellular Assay Results drug influx/efflux ADME properties Model useful to guide treatment options which patients should respond best to which kinase inhibitor. 15

16 Beyond Imatinib and Abl Numerous examples of kinase inhibitor drug resistance Abl, Abl [T315I] EGFR, EGFR [T79M] Flt3, Flt3 [G697R] PDGFRa, PDGFRa [T674I] Kit, Kit [T67I] Alk, Alk [L1196M] Mutant kinases in other disease areas LRRK2, LRRK2 [G19S], others Parkinson s disease Systematically develop assays for clinically relevant mutant kinases Partners for drug discovery, functional analysis, and treatment options 16

17 Summary Mechanism-based cellular assays are important tools for selectivity assessment enabled Pim kinase project selectivity could not be predicted from biochemical results ClariCELL assays are valuable tools for personalized medicine drug discovery compounds that inhibit wt and mutant forms of kinases address cancer drug resistance functional characterization of mutations modeling to guide physician treatment options 17