Staurosporine) to Validate the Assay, IC50 for Non-Specific Kinase Inhibitor Staurosporine Was Determined

Comprehensive Protein Kinase Profiling Panels for Inhibitor Selectivity Screening

Yusuke Kawase, Hiroshi Ohmoto, Yasuyuki Kirii, Yoshimasa Inoue, and Hiroshi Ishiguro, Carna Biosciences, Inc., Kobe, Japan

Abstract

It is well known that protein kinases are key elements in intracellular signaling pathways that control many physiological processes. Further, it has been demonstrated that the activity of protein kinases are altered in several human diseases such as cancer and autoimmune disorders. Therefore, molecules that modulate kinase functions are expected to be promising new drugs. To avoid predictable side effects of protein kinase inhibitors, determining their specificity is an important issue. Recently, Carna Biosciences have succeeded in extracting and cloning the entire (88) tyrosine kinase genes and is developing a complete tyrosine kinase assay panel for kinase inhibitor specificity profiling. A description of this panel system, as well as progress made with developing a profiling system for serine/threonine kinases will be presented. Each kinase assay will be available in homogeneous platforms using IMAPTM technology, TR-FRET and AlphaScreenTM, all of which are suitable for HTS assays. IMAPTM technology and AlphaScreenTM are registered trademarks of Molecular Devices Corporation and PerkinElmer. Tyrosine Kinases in Carna Biosciences

AXL EGFR Protein Available PDGFR TYRO3 EGFR HER2/ErbB2 PDGFRα MER Available Soon PDGFRβ AXL FMS HER4/ErbB4 KIT RON FLT3 RYK VEGFR MET HER3/ErbB3 JAKA KDR FLT4 FLT1 TIE1 TIE2 FGFR1 RET JAK1TYK2 FGFR JAK3 FGFR4 ACK TNK1 FGFR3 JAK2 INSR FGFR2 LMR1 FES LMR2 LMR IGF1R IRR FER INSR ROS LMR3 LTK ALK BTK BMX ITK EphA10 SuRTK106 TXK CSK CCK4 EphB6 TEC TEC CTK PYK2 FAK EphA1 SYKZAP70 SRM ARG ABL ROR2 BRK ROR1 MUSK FRK EphA2 DDR1 DDR2 TRKA EphA8

BLK DDR TRKC TRK FGR EphB4 TRKB

FYN HCK LCK EphA6 SRC LYN EphB1 YES SRC EphA7 EphB3 EphB2

EphA4 EphA3 EphA5 Fig. 1 TK kinome. Carna Biosciences suceeded in collecting all TK genes. TK described in the GREEN and BLUE letters are available in Carna Biosceinces as the protein. BLUE kinases EPH are also available for profiling study. GRAY kinases will be available 1Q/2005.

Serine/Threonine Kinases in Carna Biosciences AGC CAMK STE AKT2 PKC-ε CaMK4 PHKg1 MAP2K3 BARK1 PKC-γ CaMK2-α PIM1 MAP2K7 Protein Available θ BARK2 PKC- CHK1 PKD2 MAP3K5 Available Soon CRIK ROCK1 CHK2 TRIO MAP3K3 PDK1 ROCK2 DAPK1 α Fig.2 STKs collecting in Carna Biosciences. We are also trying to clone entire PKA-c RSK2 MAPKAPK2 serine / threonine kinase genes. The kinases described in the GREEN and PKC-α smMLCK BLUE letters are available in Carna Biosceinces as the protein. BLUE kinases are also available for profiling study. GRAY kinases will be available in 2005.

Carna Biosciences is developing entire TK profiling panel firstly with 96-well format ELISA platform to exclude potential compound interference.

Procedure Km for ATP Carna Biosciences's standard condition to evaluate inhibitory activity of compounds is performed with ATP at the concen- Compound trarions around Km. The most of known kinase inhibitors so far are the ATP competitor. Using too much ATP may miss the positive signal.

Substrate / ATP / Divalent Metal Ion EphA7 FER ITK 0.05 0.07 0.05 0.06 0.04 0.04 Enzyme 0.05 0.03 0.03 ] ] ] 0.04 V V V [ [ [ 0.02 0.03 0.02 Wash, Blocking 0.02 0.01 µ µ 0.01 Km = K3m0 µ=M 30 M 0.01 Km = 7 M Km = 40 µM Conjugation 0.00 0 0 0 200 400 600 800 1000 0 100 200 300 0 200 400 600 800 1000 [ S ] [ S ] [ S ]

Wash JAK1 MER PDGFRβ 0.06 0.05 0.14 0.12 0.05 0.04 0.10 Enzyme Reaction for Detection 0.04 0.03 ] ] ] 0.08 V V 0.03 V [ [ [ 0.02 0.06 Stop 0.02 0.04 µ 0.01 µ 0.01 Km = 15 M Km = 40 µM 0.02 Km = 15 M Measure 0 0 0.00 0 200 400 600 800 1000 0 200 400 600 800 1000 0 100 200 300 [ S ] [ S ] [ S ] Fig. 3 Standard scheme of ELISA. Streptavidine coated- plate, biotinylated peptide substrates (which contains Fig.4 Representative data of Km for ATP. The Michaelis-Menten plot calculated from ATP concentrations and initial velocity 1 tyrosine in their sequences) are used in the kinase values were shown. Initial velocity was determined by a linear least-squares fit from the data obtained with 5 to 30 min reaction and HRP-conjugated anti-phospho antibody reaction. ATP concentrations were varied from 0.3 to 1000 nM by 3 in common ratio. The Km values for EphA7, FER, ITK, β µ µ and TMB were used for detection. Absorbance at OD JAK1, MER and PDGFR were 30, 7, 40, 15, 40 and 15 M, respectively. The values were 1 to 100 M for the most of tested 450 nm was measured. (62) TKs.

IC50 Determination (Staurosporine) To validate the assay, IC50 for non-specific kinase inhibitor Staurosporine was determined. HCK FER EGFR EphA7 100 100 100 100 IC50 = 3000 nM 80 80 80 80 n o n n n o t i o o 60 i t i 60 t i i t i b

i 60

i 60 i b i b b h i i h n h h n I I

n 40 I

I 40

40 40 % % % %

20 20 20 20 IC50 = 100 nM IC50 = 1 nM IC50 = 3 nM 0 0 0 0 0.1 1 10 100 1000 10000 0.1 1 10 100 1000 10000 0.01 0.1 1 10 100 0.01 0.1 1 10 100 1000 Staurosporine (nM) Staurosporine (nM) Staurosporine (nM) Staurosporine (nM)

PDGFRβ ITK JAK1 MER 100 100 100 100

80 80 80 80 n n n n o o o o t i

t i 60 t i

60 60 i t i i i

i 60 b b b i b i i i h h h h n n n I n I I

40 40 40 40 % % % %

20 20 20 20 IC50 = 50 nM IC50 = 1 nM IC50 = 2 nM IC50 = 0.05 nM 0 0 0 0 0.1 1 10 100 1000 10000 0.01 0.1 1 10 100 0.01 0.1 1 10 100 1000 0.001 0.01 0.1 1 10 Staurosporine (nM) Staurosporine (nM) Staurosporine (nM) Staurosporine (nM)

Fig.5 Representative data of IC50 values for Staurosporine. The ATP were used at the concentrations around Km for each kinase. The concentrations of each kinases were lower than 250 ng/mL. The IC50 values for EGFR, EphA7, FER, HCK, ITK, JAK1, MER and PDGFRβ were 3000, 100, 1, 3, 50, 1, 2 and 0.05 nM, respectively.

Tyrosine Kinase Profile (Staurosporine)

100%

80% n o t i

i 60% b i h n I

40% %

20%

0% 2 4 L K G K X K K S 1 2 R 1 2 3 4 7 8 1 2 4 K 1 2 3 4 R 1 2 1 4 K R R K 1 2 3 IT K K α β 2 S C M 3 0 B L R XL L M R T M SK R R F A A A A A A B B B A ER ES R R R R G K K T T C 1 SR IR IT K K K K C YN ER ET S R R ET O EK O A A A A B B B B F C D D h h h h h h h h h F F F F F F F F L L L L H ER ER F IN A A A L L M M U F F R R SR SR SYK T YES P7 D D EG G G G G F F F F H H G J J J G G PYK YR A Ep Ep Ep Ep Ep Ep Ep Ep Ep F F F F I M T Z PD PD

Fig.6 Inhibition study with 62 TKs were conducted with Staurosporine at 1 mM. The ATP were used at the concentrations around Km for each kinase. Staurosporine showed more than 50 % inhibition on the most of tested TKs.

The IMAPTM fluorescence polarization (FP) assay platform is a generic, homogeneous system applicable to a variety of enzymes, including protein kinases. IMAPTM is based on the high affinity binding of phosphate to immobilized trivalent metals. It has been applied to a wide variety of kinases spanning the whole kinome. Principle IMAP-able TKs Low FP To apply the TKs to HTS assay, TK titrations were performed with IMAPTM platform. EGFR / AXL Family FGFR Family / TIE2 KINASE 500 500 PO4 ATP Phosphopeptide Fluorescent 400 400 Peptide Substrate Product

300 300 P )

IMAP P ) ( m + III Binding ( m P

M P

Reagent F 200 EGFR F 200 HER4 FGFR1 FGFR2 AXL FGFR4 100 MER 100 RET TYRO3 TIE2 III M PO4 0 0 0.01 0.1 1 10 100 0.01 0.1 1 10 100 µ High FP Kinase ( g/mL) Kinase (µg/mL) PDGFR / VEGFR Family SRC / TEC Family Fig.7 The IMAPTM technology is based on the high affinity binding of III phosphate by immobilized metal (M ) coordination complexes on 500 500 nanoparticles. This IMAPTM "binding reagent" complexes with phosphate groups on phosphopeptides generated in a kinase reaction. Such binding 400 400 causes a change in the rate of the molecular motion of the peptide, and results in an increase in the FP value observed for the fluorescent label 300 300 P ) P ) ( m attached at the end of the peptide. This assay, unlike antibody-based ( m P P F kinase assays, is applicable to a wide variety of kinases without regard to F 200 200 TM FMS BRK the substrate peptide sequences. IMAP can be also used to analyze FLT3 FGR FLT4 HCK phosphatase activity, simply by starting out with fluorescent phospho- 100 100 FLT1 BMX peptide. This figure and the legend are by courtesy of Molecular Devices KDR BTK Corporation. 0 0 0.001 0.01 0.1 1 10 100 0.001 0.01 0.1 1 10 100 Kinase (µg/mL) Kinase (µg/mL)

Serine /Threonine Kinase IMAP Eph / MET/ TRK Family Others Carna Biosciences has been developing serine / threonine kinase IMAPTM 500 500 assay. Currently, six serine / threonine kinases are available and the number is increasing rapidly. 400 400 Staurosporine

P ) 300 300 γ P ) A PKC- ( m ( m

100 P P F 200 F EphA1 200 80 ALK EphB2 FER n n o o MET FES t i t i i i 60 100 b 100 JAK2 b i RON i h h ROS n TRKB n I I

40 % % 0 0 20 IC50 = 0.8 nM 0.001 0.01 0.1 1 10 100 1000 0.001 0.01 0.1 1 10 100 Kinase (µg/mL) Kinase (µg/mL) 0 0.1 1 10 100 Fig.8 Representative data of TK IMAPTM assay. All the TKs currently available in Carna Biosciences were Staurosporine (nM) tested with IMAPTM. Using 6 different substrates, 42 TKs were implied as IMAPTM applicable, the EC50 B PKC-ε values were under 5 µg/mL. HCK showed lowest EC50 value at 7 ng/mL. This experiment was conducted 100 in collaboration with Dr. Liz Gaudet, Molecular Devices Corporation.

80 n o t i i 60 b i h n I 40 Summary % 20 IC50 = 0.5 nM 0 Carna Biosciences has been creating entire TK profiling panel. 0.01 0.1 1 10 100 Staurosporine (nM) Currently 62 kinds of TK profiling panel has been completed. Y-27632 Validation with Staurosporine proved the reliability of the assay. TM C ROCK2 These TKs were potentially applicable to IMAP homogeneous assay. 100 The rest of TKs for profiling and STK profiling will be available soon. 80 n o t i

i 60 b i h n I

40 % 20 IC50 = 1 nM 0 0.01 0.1 1 10 100 Y-27632 (nM) D CRIK 100 Fig.9 Representative data of IC50 values by 80 Staurosporine A), B), or n o

t i Y-27632 C), D). ATP was

i 60 b i h used at the concentrations Please contact us to; n I 40 around Km for each kinase. % γ [email protected] 20 he IC50 values for PKC- , Carna is a goddess in Greek IC50 = 4 nM ε PKC- , ROCK2 and CRIK & mythology who protects human 0 were 0.8, 0.5, 1 and 4 nM, Visit web site; health. This image is the symbol 0.1 1 10 100 1000 respectively. Y-27632 (nM) www.carnabio.com of Carna Biosciences. © Carna Biosciences Inc., 2004