A Representative Kinase Selectivity Screening Panel to Support Drug Discovery Using the

ADP-Glo Kinase Activity Format

1029

INTRODUCTION

RESULTS

CONCLUSIONS

Protein kinases are a major family of enzymes in the human proteome with over 500 members and regulate a variety of cellular processes through their roles in signal transduction pathways. Dysregulated kinase activities have been implicated in a number of diseases including inflammation, cancer, metabolic and immune disorders. Thus, these enzymes represent attractive targets for therapeutic intervention and several small molecule kinase inhibitors are now approved as drugs. One of the major challenges in the development of kinase inhibitors is selective inhibition of the target kinase, as most inhibitors described to date act at the highly conserved ATP active site. To evaluate the selectivity of kinase inhibitors, several vendors have established kinase selectivity profiling services that include large numbers of kinases. However, for efficient SAR cycles in drug discovery, a large number of compounds need to be assessed for selectivity in a quick and cost-effective manner. Confluence Discovery Technologies (CDT) has developed an internal kinase panel of 40 kinases with a wide coverage of the kinome which yields representative kinase selectivity information at a fraction of the cost and time of the larger commercially available kinase panels. The CDT panel utilizes the ADP-Glo kinase activity assay format (Promega), allowing for a single assay technology to evaluate kinases with different substrate specificities. The customized Kinase Selectivity Profiling System kits were purchased from Promega and consisted of paired enzyme-substrate strips, permitting rapid assay preparation for large numbers of kinases. The scheme used to perform the assay, as adapted from the Promega procedure, is as 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

• Percent inhibition results suggest a good correlation between the two inhibitor concentrations tested • Potency (IC50) measurements correlated well with initial % Inhibition values at 1uM

• 3% false positive rate observed • 1.5% negative hit rate observed

• Demonstrated that using this technology time dependence of compounds can be determined • 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

OVERVIEW

Confluence Discovery Technologies (CDT) has identified a panel of 40 kinases that covers a broad spectrum of the kinome to assess selectivity of potential drug candidates. Kinase activity assays have been developed for this panel using ADP-Glo assay technology (Promega) and packaged into a customized Kinase Selectivity Profiling System for CDT. Using these reagents, we have evaluated several commercially available kinase inhibitors. Compounds were initially screened at two concentrations and “hits” were then evaluated in a dose-response. In selected cases, inhibitor potency measurements were made with and without a one-hour preincubation with enzyme to identify time-dependent inhibition. Here, we report the results of our in-house selectivity assessment and compare them to those published by Zarrinkar, et al (Nature Biotechnology, Volume 26, 127-132 (2008).

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REFERENCES

50nL Compound Plate

2.5 μL 2.5 μL

Heat Map of Initial Compound Selectivity Screen

Table 2: Percent inhibition analysis of commercially available inhibitors in CDT’s selectivity kinase panel. Compounds were run at two concentrations, 100nM and 1uM, with 10uM ATP in duplicate and values were averaged. Data is displayed as a heat map where the color green reflects a % inhibition of less than 30%, the color yellow indicates a % inhibition of between 30% and 70% and the color red indicates a % inhibition of greater than 70%.

IC50 Analysis of Commercial Inhibitors

Table 3: IC50 analysis of commercially available inhibitors in CDT’s selectivity kinase panel. Compounds were run in duplicate in a 10 point dose-response curve using three fold dilutions. Duplicates were averaged and data was fit using a 4 parameter fit. Also, superimposed on the table is the heat map of the % inhibition results of the compounds screened at 1uM as seen in Table 2 .

Kinome Representation by CDT Selectivity Panel

Figure 2: Time dependence assay of Ibrutinib against the kinases JAK3 (Panel A) and KDR (Panel B). Inhibitor potency was measured with (red) and without (blue) a 60 minute preincubation of compound and enzyme prior to initiation of assay with substrate/ATP mix.

Identification of Time-Dependent Inhibition

A. B.

Validation of CDT’s Selectivity Kinase Panel

Figure 3: Panel A shows scatter plot comparing IC50 values obtained from CDT selectivity kinase panel and Kd values previously reported by Ambit (Zarrinkar, et al.). The solid line represents a 1:1 correlation and the dotted lines represent a 3-fold difference. Panel B shows a scatter plot comparing selectivity scores (# of kinases with Kd or IC50 < 3uM or 100nM / # of kinases tested) of CDT’s selectivity kinase panel against Ambit (Zarrinkar, et al.). Solid shapes represent 3uM data and open shapes represent 100nM data. Circles are representative of Tofactinib data, squares of Dasatinib data, upside down triangles of Sutent data, and regular triangles of Sorafinib data. The line represents a 1:1 correlation.

1) Zarrinkar, P., et al. A Quantitative Analysis of Kinase Inhibitor Selectivity. Nature Biotechnology, 2008; 26, 127-132

2) Zegzouti, H. Flexible and targeted inhibitor profiling using the Luminescent ADP-Glo Kinase Assay Platform. Promega Corporation.

3) Manning G, et al. The Protein Kinase Complement of the Human Genome. Science, 2002 Dec; 298 (5600): 1912-1934

CDT Selectivity Kinase Panel

Table 1: CDT custom kinase panel and the respective kinase classification

B. A.

Figure 1: Human Kinome tree with CDT selectivity panel kinases indicated by yellow circles.

Sutent Staurosporine Tofactinib Dastatinib Sorafenib Ibrutinib 5Z-7 Oxozeaenol

Kinase 1uM 100nM 1uM 100nM 1uM 100nM 1uM 100nM 1uM 100nM 1uM 100nM 1uM 100nM HER2 0 0 95 78 0 0 71 5 0 0 90 58 0 0

KDR 91 52 101 92 0 0 0 0 76 0 44 0 100 97

BTK 0 0 96 59 0 0 99 97 0 0 100 98 0 0

LCK 49 0 98 87 0 0 99 96 0 0 83 62 1 0

SRC 0 0 96 70 0 0 86 95 0 0 67 28 0 0

JAK3 10 0 112 112 100 100 0 0 0 0 16 0 0 0

SYK 11 0 101 102 0 0 43 0 0 0 85 25 0 0

EPHA1 0 0 82 22 0 0 92 91 0 0 0 0 32 0

TRKA 98 95 100 99 50 12 69 16 26 0 20 3 97 74

FGFR1 76 6 99 95 30 2 80 21 34 0 48 3 24 2

Aurora 1 14 0 94 68 43 10 35 9 0 36 4 0 10 7

CK2a1 0 0 17 7 11 18 37 16 0 0 2 0 15 11

IKKb 9 0 77 41 23 19 32 12 0 0 4 2 17 16

NEK2 28 19 49 18 18 16 44 12 0 15 7 10 8 11

PLK1 23 0 38 8 13 0 29 1 1 18 0 0 0 0

CK1g1 52 65 11 -10 12 1 22 9 23 56 0 0 0 0

CHK1 82 30 95 96 25 23 5 0 0 0 7 0 15 8

MAPKAPK2 0 0 80 30 13 4 0 0 0 0 18 0 31 9

AMPK A2/B1/G1 93 63 101 100 37 4 12 0 0 0 26 3 53 17

MARK1 53 0 101 99 47 12 26 2 0 0 20 0 24 4

PIM1 6 0 100 96 23 0 2 0 0 0 13 0 30 0

CAMK4 84 37 97 88 34 16 18 5 0 0 32 0 33 0

DAPK1 43 35 108 59 31 10

STK33 95 24 90 55 31 10 22 0 5 0 9 0 31 1

AKT1 0 0 99 89 23 37 14 5 0 0 0 0 4 12

PKCb II 0 0 100 95 42 0 0 0 0 0 0 0 0 0

ROCK1 21 2 101 94 20 7 20 6 4 0 14 8 10 14

RSK2 64 20 101 100 28 28 30 20 16 0 16 13 22 25

CDK1/Cyclin A2 8 0 103 100 23 24 26 19 7 1 17 19 28 17

ERK2 0 0 43 26 28 38 35 22 0 0 2 13 51 26

GSK3b 27 8 108 104 43 35 36 15 40 0 46 25 38 18

CLK3 33 0 43 21 34 18 25 11 0 0 4 0 25 10

ASK1 46 7 93 43 9 0 32 22 0 0 0 0 16 7

MINK1 89 39 101 99 31 8 20 21 4 0 33 0 32 0

MST1 0 0 98 92 19 2 16 26 0 0 13 0 17 0

PAK3 92 76 98 97 20 7 60 29 0 0 9 5 34 16

HPK1 4 3 87 37 25 18 96 63 0 0 20 16 74 19

ALK4 20 0 99 93 0 96 87 0 0

MLK2 75 30 100 93 11 23 20 11 0 0 21 10 47 0

IRAK4 18 20 0 3 8 7 13 8 25 19 7 6 7 9

# KINASE CLASS # KINASE CLASS

1 KDR TK 21 PLK1 NC 2 LCK TK 22 RSK2 AGC 3 SRC TK 23 AKT1 AGC 4 BTK TK 24 PKCb AGC 5 TRKA TK 25 ROCK1 AGC 6 SYK TK 26 AURORA A NC 7 FGFR1 TK 27 CAMK4 CAMK 8 EPHA1 TK 28 MK2 CAMK 9 HER2 TK 29 STK33 CAMK

10 JAK3 TK 30 AMPK A2 CAMK 11 MLK2 TKL 31 MARK1 CAMK 12 NEK2 TKL 32 CHK1 CAMK 13 IRAK4 TKL 33 PIM1 CAMK 14 ALK4 TKL 34 DAPK1 CAMK 15 ASK1 STE 35 IKKB NC 16 HPK1 STE 36 CDK1 CMGC 17 PAK3 STE 37 ERK2 CMGC 18 MST1 STE 38 GSK3b CMGC 19 MINK1 STE 39 CK2a1 CMGC 20 CK1g1 CK1 40 CLK3 CMGC

Ambi

t Kd

(uM

)

10

1

0.1

0.01

0.001

0.0001

CDT IC50 (uM) 0.0001 0.001 0.01 0.1 1 10

Sutent

Dasatinib

Sorafenib

0.6

0.4

0.2

0

Ambi

t Sel

ectiv

ity S

core

CDT Selectivity Score 0 0.2 0.4 0.6

Ibrutinib (uM)0.001 0.01 0.1 1 10

%In

hibi

tion

-20

0

20

40

60

80

100

IC50 = 5.03 uM

IC50 = 0.0387 uM

Ibrutinib (uM)0.001 0.01 0.1 1 10 100

-40

-20

0

20

40

60

80

100

IC50=0.599 uMIC50 =0.789 uM

% In

hibi

tion

Kinase Sutent Tofactinib Dastatinib Sorafenib Ibrutinib HER2 >1 >1 0.286 >1 0.0122 KDR 0.0321 >1 >1 0.118 0.599 BTK >1 >1 0.107 >1 <0.005 LCK 0.273 >1 0.0129 >1 0.0374 SRC 0.972 >1 <0.005 >1 >1 JAK3 >1 <0.005 >1 >1 >1 SYK >1 >1 >1 >1 >1

EPHA1 >1 >1 0.0134 >1 >1 TRKA 0.00920 >1 >1 >1 >1 FGFR1 0.254 >1 0.769 >1 >1

Aurora 1 >1 0.848 >1 >1 >1 CK2a1 >1 >1 >1 >1 >1 IKKb >1 >1 >1 >1 >1 NEK2 >1 >1 >1 >1 >1 PLK1 >1 >1 >1 >1 >1

CK1g1 >1 >1 >1 >1 >1 CHK1 0.0650 >1 >1 >1 >1

MAPKAPK2 >1 >1 >1 >1 >1 AMPK A2/B1/G1 0.0309 >1 >1 >1 >1

MARK1 0.267 0.961 >1 >1 >1 PIM1 >1 >1 >1 >1 >1

CAMK4 0.0629 >1 >1 >1 >1 DAPK1 >1 >1 >1 >1 >1 STK33 0.0411 >1 >1 >1 >1 ATK1 >1 >1 >1 >1 >1

PKCb II >1 >1 >1 >1 >1 ROCK1 >1 >1 >1 >1 >1 RSK2 0.667 >1 >1 >1 >1

CDK1/Cyclin A2 >1 >1 >1 >1 >1 ERK2 >1 >1 >1 >1 >1

GSK3b >1 >1 >1 >1 >1 CLK3 >1 >1 >1 >1 >1 ASK1 >1 >1 >1 >1 >1 MINK1 0.214 >1 >1 >1 >1 MST1 >1 >1 >1 >1 >1 PAK3 >1 >1 >1 >1 >1 HPK1 >1 >1 0.792 >1 >1 ALK4 >1 >1 0.0682 >1 >1 MLK2 >1 >1 >1 >1 >1 IRAK4 0.205 >1 >1 >1 >1