1029 A Representative Selectivity Screening Panel to Support Drug Discovery Using the ADP-Glo Kinase Activity Format

OVERVIEW RESULTS Confluence Discovery Technologies (CDT) has identified a panel of 40 that covers a CDT Selectivity Kinase Panel Heat Map of Initial Compound Selectivity Screen IC50 Analysis of Commercial Inhibitors broad spectrum of the kinome to assess # KINASE CLASS # KINASE CLASS 5Z-7 selectivity of potential drug candidates. Kinase Sutent Staurosporine Tofactinib Dastatinib Sorafenib Ibrutinib Kinase Sutent Tofactinib Dastatinib Sorafenib Ibrutinib 1 KDR TK 21 NC Oxozeaenol HER2 >1 >1 0.286 >1 0.0122 activity assays have been developed for this 2 LCK TK 22 RSK2 AGC Kinase 1uM 100nM 1uM 100nM 1uM 100nM 1uM 100nM 1uM 100nM 1uM 100nM 1uM 100nM KDR 0.0321 >1 >1 0.118 0.599 3 SRC TK 23 AKT1 AGC HER2 0 0 95 78 0 0 71 5 0 0 90 58 0 0 panel using ADP-Glo assay technology BTK >1 >1 0.107 >1 <0.005 4 BTK TK 24 PKCb AGC KDR 91 52 101 92 0 0 0 0 76 0 44 0 100 97 LCK 0.273 >1 0.0129 >1 0.0374 (Promega) and packaged into a customized BTK 0 0 96 59 0 0 99 97 0 0 100 98 0 0 5 TRKA TK 25 ROCK1 AGC SRC 0.972 >1 <0.005 >1 >1 LCK 49 0 98 87 0 0 99 96 0 0 83 62 1 0 Kinase Selectivity Profiling System for CDT. 6 SYK TK 26 AURORA A NC JAK3 >1 <0.005 >1 >1 >1 SRC 0 0 96 70 0 0 86 95 0 0 67 28 0 0 7 FGFR1 TK 27 CAMK4 CAMK SYK >1 >1 >1 >1 >1 Using these reagents, we have evaluated several JAK3 10 0 112 112 100 100 0 0 0 0 16 0 0 0 8 EPHA1 TK 28 MK2 CAMK EPHA1 >1 >1 0.0134 >1 >1 SYK 11 0 101 102 0 0 43 0 0 0 85 25 0 0 commercially available kinase inhibitors. 9 HER2 TK 29 STK33 CAMK TRKA 0.00920 >1 >1 >1 >1 EPHA1 0 0 82 22 0 0 92 91 0 0 0 0 32 0 10 JAK3 TK 30 AMPK A2 CAMK FGFR1 0.254 >1 0.769 >1 >1 Compounds were initially screened at two TRKA 98 95 100 99 50 12 69 16 26 0 20 3 97 74 11 MLK2 TKL 31 MARK1 CAMK Aurora 1 >1 0.848 >1 >1 >1 FGFR1 76 6 99 95 30 2 80 21 34 0 48 3 24 2 concentrations and “hits” were then evaluated in >1 >1 >1 >1 >1 12 NEK2 TKL 32 CHK1 CAMK Aurora 1 14 0 94 68 43 10 35 9 0 36 4 0 10 7 CK2a1 IKKb >1 >1 >1 >1 >1 a dose -response. In selected cases, inhibitor 13 IRAK4 TKL 33 PIM1 CAMK CK2a1 0 0 17 7 11 18 37 16 0 0 2 0 15 11 NEK2 >1 >1 >1 >1 >1 potency measurements were made with and 14 ALK4 TKL 34 DAPK1 CAMK IKKb 9 0 77 41 23 19 32 12 0 0 4 2 17 16 PLK1 >1 >1 >1 >1 >1 15 ASK1 STE 35 IKKB NC NEK2 28 19 49 18 18 16 44 12 0 15 7 10 8 11 without a one-hour preincubation with to 16 HPK1 STE 36 CDK1 CMGC PLK1 23 0 38 8 13 0 29 1 1 18 0 0 0 0 CK1g1 >1 >1 >1 >1 >1 identify time-dependent inhibition. Here, we 17 PAK3 STE 37 ERK2 CMGC CK1g1 52 65 11 -10 12 1 22 9 23 56 0 0 0 0 CHK1 0.0650 >1 >1 >1 >1 18 MST1 STE 38 GSK3b CMGC CHK1 82 30 95 96 25 23 5 0 0 0 7 0 15 8 MAPKAPK2 >1 >1 >1 >1 >1 report the results of our in-house selectivity 19 MINK1 STE 39 CK2a1 CMGC MAPKAPK2 0 0 80 30 13 4 0 0 0 0 18 0 31 9 AMPK A2/B1/G1 0.0309 >1 >1 >1 >1 AMPK A2/B1/G1 93 63 101 100 37 4 12 0 0 0 26 3 53 17 MARK1 0.267 0.961 >1 >1 >1 assessment and compare them to those 20 CK1g1 CK1 40 CLK3 CMGC MARK1 53 0 101 99 47 12 26 2 0 0 20 0 24 4 PIM1 >1 >1 >1 >1 >1 published by Zarrinkar, et al (Nature Table 1: CDT custom kinase panel and the PIM1 6 0 100 96 23 0 2 0 0 0 13 0 30 0 CAMK4 0.0629 >1 >1 >1 >1 CAMK4 84 37 97 88 34 16 18 5 0 0 32 0 33 0 Biotechnology, Volume 26, 127-132 (2008). respective kinase classification DAPK1 >1 >1 >1 >1 >1 DAPK1 43 35 108 59 31 10 STK33 0.0411 >1 >1 >1 >1 STK33 95 24 90 55 31 10 22 0 5 0 9 0 31 1 ATK1 >1 >1 >1 >1 >1 AKT1 0 0 99 89 23 37 14 5 0 0 0 0 4 12 Kinome Representation by PKCb II >1 >1 >1 >1 >1 PKCb II 0 0 100 95 42 0 0 0 0 0 0 0 0 0 ROCK1 >1 >1 >1 >1 >1 ROCK1 21 2 101 94 20 7 20 6 4 0 14 8 10 14 CDT Selectivity Panel RSK2 0.667 >1 >1 >1 >1 RSK2 64 20 101 100 28 28 30 20 16 0 16 13 22 25 CDK1/ A2 >1 >1 >1 >1 >1 CDK1/Cyclin A2 8 0 103 100 23 24 26 19 7 1 17 19 28 17 INTRODUCTION ERK2 >1 >1 >1 >1 >1 ERK2 0 0 43 26 28 38 35 22 0 0 2 13 51 26 GSK3b >1 >1 >1 >1 >1 GSK3b 27 8 108 104 43 35 36 15 40 0 46 25 38 18 Protein kinases are a major family of in CLK3 >1 >1 >1 >1 >1 CLK3 33 0 43 21 34 18 25 11 0 0 4 0 25 10 ASK1 >1 >1 >1 >1 >1 the human proteome with over 500 members and ASK1 46 7 93 43 9 0 32 22 0 0 0 0 16 7 MINK1 0.214 >1 >1 >1 >1 MINK1 89 39 101 99 31 8 20 21 4 0 33 0 32 0 regulate a variety of cellular processes through MST1 >1 >1 >1 >1 >1 MST1 0 0 98 92 19 2 16 26 0 0 13 0 17 0 PAK3 >1 >1 >1 >1 >1 their roles in pathways. PAK3 92 76 98 97 20 7 60 29 0 0 9 5 34 16 >1 >1 0.792 >1 >1 HPK1 4 3 87 37 25 18 96 63 0 0 20 16 74 19 HPK1 Dysregulated kinase activities have been >1 >1 0.0682 >1 >1 ALK4 20 0 99 93 0 96 87 0 0 ALK4 implicated in a number of diseases including MLK2 75 30 100 93 11 23 20 11 0 0 21 10 47 0 MLK2 >1 >1 >1 >1 >1 0.205 >1 >1 >1 >1 inflammation, cancer, metabolic and immune IRAK4 18 20 0 3 8 7 13 8 25 19 7 6 7 9 IRAK4 disorders. Thus, these enzymes represent Table 2: Percent inhibition analysis of commercially available inhibitors in CDT’s Table 3: IC50 analysis of commercially available attractive targets for therapeutic intervention and selectivity kinase panel. Compounds were run at two concentrations, 100nM and inhibitors in CDT’s selectivity kinase panel. several small molecule kinase inhibitors are now 1uM, with 10uM ATP in duplicate and values were averaged. Data is displayed as a Compounds were run in duplicate in a 10 point approved as drugs. One of the major challenges heat map where the color green reflects a % inhibition of less than 30%, the color dose-response curve using three fold dilutions. in the development of kinase inhibitors is yellow indicates a % inhibition of between 30% and 70% and the color red indicates a Duplicates were averaged and data was fit using a selective inhibition of the target kinase, as most % inhibition of greater than 70%. 4 parameter fit. Also, superimposed on the table inhibitors described to date act at the highly is the heat map of the % inhibition results of the conserved ATP . To evaluate the Figure 1: Human Kinome tree with CDT selectivity compounds screened at 1uM as seen in Table 2 . selectivity of kinase inhibitors, several vendors panel kinases indicated by yellow circles. have established kinase selectivity profiling services that include large numbers of kinases. Identification of Time-Dependent Inhibition Validation of CDT’s Selectivity Kinase Panel

However, for efficient SAR cycles in drug 10 0.6

A. Sutent B. discovery, a large number of compounds need to

A. B. ) 1 Dasatinib be assessed for selectivity in a quick and cost- 100 100 Sorafenib 0.4 ( uM effective manner. 0.1 80 IC50 = 0.0387 uM 80 Kd IC50 =0.789 uM n 60 60 0.01

o IC50=0.599 uM 0.2 i

Confluence Discovery Technologies (CDT) has t i Ambit Ambit n

b 40 i 40 io developed an internal kinase panel of 40 kinases t 0.001

nh I 20 Score Selectivity Ambit with a wide coverage of the kinome which yields 20 0 % IC50 = 5.03 uM nhibi 0.0001 I 0 0.0001 0.001 0.01 0.1 1 10 0 0.2 0.4 0.6 representative kinase selectivity information at a 0 % fraction of the cost and time of the larger -20 CDT IC50 (uM) CDT Selectivity Score -20 commercially available kinase panels. The CDT -40 Figure 3: Panel A shows scatter plot comparing IC50 values obtained from CDT selectivity panel utilizes the ADP-Glo kinase activity assay kinase panel and Kd values previously reported by Ambit (Zarrinkar, et al.). The solid line format (Promega), allowing for a single assay 0.001 0.01 0.1 1 10 0.001 0.01 0.1 1 10 100 represents a 1:1 correlation and the dotted lines represent a 3-fold difference. Panel B shows technology to evaluate kinases with different Ibrutinib (uM) Ibrutinib (uM) a scatter plot comparing selectivity scores (# of kinases with Kd or IC50 < 3uM or 100nM / # substrate specificities. The customized Kinase Figure 2: Time dependence assay of Ibrutinib against the kinases JAK3 (Panel A) and KDR (Panel B). of kinases tested) of CDT’s selectivity kinase panel against Ambit (Zarrinkar, et al.). Solid Selectivity Profiling System kits were purchased Inhibitor potency was measured with (red) and without (blue) a 60 minute preincubation of compound shapes represent 3uM data and open shapes represent 100nM data. Circles are from Promega and consisted of paired enzyme- and enzyme prior to initiation of assay with substrate/ATP mix. representative of Tofactinib data, squares of Dasatinib data, upside down triangles of Sutent substrate strips, permitting rapid assay data, and regular triangles of Sorafinib data. The line represents a 1:1 correlation. preparation for large numbers of kinases.

The scheme used to perform the assay, as adapted from the Promega procedure, is as CONCLUSIONS REFERENCES follows: • CDT has developed a selectivity kinase panel, using Promega’s Kinase Selectivity Profiling System, that

can be used to predict the selectivity profile of compounds against a larger selectivity panel 1) Zarrinkar, P., et al. A Quantitative Analysis of Kinase Inhibitor Selectivity.

Nature Biotechnology, 2008; 26, 127-132 • Percent inhibition results suggest a good correlation between the two inhibitor concentrations tested • Potency (IC50) measurements correlated well with initial % Inhibition values at 1uM 2) Zegzouti, H. Flexible and targeted inhibitor profiling using the Luminescent • 3% false positive rate observed ADP-Glo Kinase Assay Platform. Promega Corporation. • 1.5% negative hit rate observed

2.5 μL 2.5 μL • Demonstrated that using this technology time dependence of compounds can be determined 3) Manning G, et al. The Complement of the Human Genome.

Science, 2002 Dec; 298 (5600): 1912-1934 50nL Compound Plate • Demonstrated a good correlation in IC50 values obtained at CDT when compared to data previously reported by Ambit (Zarrinkar, et al.) and is representative of a large kinase selectivity panel

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