Figure 2. Alignment of phosphotransfer inactive tyrosine kinases. SRC L R L E V K L G Q G C F G E V V A I K T L E A Q V M Y V H R D L R A A N I L V DDR1 L R F K E K L G E G Q F G E V V A V K I L E V K I M F V H R D L A T R N C L V ROR1 V R F M E E L G E C A F G K I V A I K T L E A S L M F V H K D L A A R N I L I Entire human profiling panel will help to develop the specific kinase inhibitor. ROR2 V R F M E E L G E D R F G K V V A I K T L E A M L R V V H K D L A T R N V L V CCK4 L Q P I T T L G K S E F G E V V L V K S L E L E M F F V H K D L A A R N C L V Hiroshi Ishiguro, Yoshimasa Inoue, Yasuyuki Kirii, Hiroshi Ohmoto, Eiji Nishiwaki, Mamoru Matsubara, and Koichi Yokota EphA10 V T L E R S L G G G R F G E L V A V . H M E A L T L Y V H R G L A A R H V L V Carna Biosciences, Inc., Kobe Japan EphB6 I K I E E V I G T G S F G E V V A I Q A L . A A V L F V H R S L S A H S V L V 8.5 HER3 L R K L K V L G S G V F G T V V C I K V I H M L A I M V H R N L A A R N V L L kinases play critical roles in cell signaling pathways, related to cell proliferation and differentiation. Among more RYK I T L K D V L Q E G T F G R I A F V K T V E S C K L V I H K D L A A R N C V I L F H G D V A A R N I L M than 500 protein kinases from , 90 tyrosine kinases are predicted based on their sequence analysis. These TM SuRTK106 S E V L E Q I C S G S C G P I V I L K A L . I Q F H 8.0 IRESSA tyrosine kinases are classified into two types, transmembrane and cytoplasmic type. Former is known as receptor tyrosine consensus o - - - - - o g - G - o g - V o a o K - o E - - - o o o h r D o k + - N o o o subdomain 2 subdomain 6 kinase (RTK), which functions as mediator of environmental variations to intracellular signals. They are recognized as subdomain 1 subdomain 3 outstanding drug targets for aberrant cell proliferative diseases and clinically useful kinase inhibitors have been successfully 7.5 SRC C K V A D F G L A G A K F WT A P E A A D V WS F G I L L T developed. Since these inhibitors are known to have specific target among kinases, it is extremely important to clarify the DDR1 I K I A D F G M S L P I R WM A WE C I D V WA F G V T L W ROR1 V K I S D L G L S . . I R WM P P E A I D I WS F G V V L W inhibitory profile of drug candidates. This presentation is to discuss the profiling result of clinically useful tyrosine kinase 0

5 ROR2 V K I S D L G L F L P I R WM A P E A I D I WS Y G V V L W inhibitors against human tyrosine kinase profiling panel. All human tyrosine kinase were cloned and expressed by 7.0 C CCK4 V K V S A L G L S V P L R WM S P E A I D V WA F G V L M W using insect cell expression system. Most cytoplasmic tyrosine kinases have been obtained as full-length protein and I EphA10 C K I S G F G R G p G R S P WA A P E T L D V WS F G I I M W cytoplasmic regions are expressed for most RTKs. For 77 out of 90 tyrosine kinases, in vitro phosphotransferase activities have EphB6 C K V A R L G H S C L L R WA A P E V I D V WS F G I L M W HER3 V Q V A D F G V A successfully been confirmed and their assays have been developed for inhibitor profiling. Most of others such as EphB6 and 6.5 T P I K WM A L E S I D V WS Y G V T V W [consensus] RYK V K I T D N A L S TM . . . R WM A L E S L D V WA F G V T L W o: Hydrophobic CCK4, are regarded as inactive. Our entire human tyrosine kinase profiling panel (QuickScout TK Comprehensive Panel) is SuRTK106 A K L C G L G L A I P L K WL A P E R L D V WS F G I L L Y +: Hydrophobic and less bulky a powerful tool for developing kinase inhibitors with preferable specificity/selectivity. consensus o k o + D f g o + g + - - o - + p E o o D o o + o g o o o o lowercase: Moderately conserved 6.0 subdomain 8 subdomain 9 uppercase: Highly conserved 1 2 3 4 5 6 7 8 0 1 2 3 2 3 subdomain 7 2 2 a 3 1 4 1 1 2 3 4 2 4 1 2 3 4 2 L L T b T T S A S K K K S S X K N C K K K K K B C R R K C R R R K K K R K K R N R K G M I 7 T T T E A A A A A A A A K K K K X K N K B B B B R R 1 E R α R R R R R E B L O N T T T T E F L R β E Y Y E X K S A E C S E S K K R R R C R C D R O G I O M M R K I I P L L L h h h h h h h h h h h h F F F F F F Y Y Y L F A A A E E A F N R F T T Y D A A F F Y R C B B F L S R G N A C F S T U M R R F B H K A R R M S B F F F I p p p p p p p p A p p p p J J J T L T P L G G H H G G G G Y D T G T T E I M Z E E E E E E E E E E E E F F F F T D D P P Potential inactive kinases are aligned with Src, a typical active tyrosine kinase, showed in the top of the alignment (Fig. 2). Standard procedure of ELISA. Table 1. Seventy seven tyrosine kinases for the panel. 5.5 Tyrosine Kinase Only important motifs for phosphotransfer reaction are shown in this figure and residues with important functions are colored 8.5 2+ 2+ green1). Residues supposed to be responsible to lose/weaken their activities are colored red. 1) Compound solution, substrate solution containing Mg /Mn Name Structure Name Structure Name Structure with or without ATP (w/o ATP for control), and solution ABL Full Length FER Full Length LTK Kinase Domain ACK Kinase Domain FES Full Length LYN(a/b) Full Length were mixed and incubated in Streptavidin-coated 96-well plate. R In case of DDR1, although no in vitro phosphotransfer reaction has been observed, autophosphorylation of the kinase has been ALK Cytoplasmic Domain FGFR1 Cytoplasmic Domain MER Cytoplasmic Domain 8.0 GLEEVEC 2) After the incubation, the reaction was terminated by washing, ARG Full length FGFR2 Cytoplasmic Domain MET Cytoplasmic Domain suggested from overexpression experiments in 293 cells after the stimulating with collagen2). This implies the unique and before the plate was blocked. 3) Incubation with HRP- AXL Full Length FGFR3 Cytoplasmic Domain MUSK Cytoplasmic Domain BLK Full Length FGFR4 Cytoplasmic Domain P DGFRα Cytoplasmic Domain mechanism to activate the protein. On the other hand, recent report of ROR2 is clearly demonstrating tyrosine kinase activity, BMX Full Length FGR Full Length PDGFRβ Cytoplasmic Domain conjugated antibody was followed by developing with TMB 3) BRK Full Length FLT1 Cytoplasmic Domain PYK2 Full Length 7.5 in vitro with unique activation mechanism. Learning unique properties of these two kinases, investigations are now on going at solution, and then read the absorbance at 450 nm. BTK Full Length FLT3 Cytoplasmic Domain RET Cytoplasmic Domain Compound CSK Full Length FLT4 Cytoplasmic Domain RON Cytoplasmic Domain 0 Carna Biosciences. CTK Full Length FMS(CSFR) Cytoplasmic Domain ROS Cytoplasmic Domain 5

DDR2 Cytoplasmic Domain FRK Kinase Domain SRC Full Length C I Substrate, ATP (+/-), Mg2+/Mn2+ EGFR Cytoplasmic Domain FYN Full Length SRM Kinase Domain 7.0 Others in the alignment are considered inactive. For ROR1, ROR2 and CCK4, conserved Gly in subdomain 1 is replaced by Cys, Asp and Ser EphA1 Cytoplasmic Domain HCK SH3-SH2- SYK Full Length p EphA2 Cytoplasmic Domain HER2 Cytoplasmic Domain TEC Kinase Domain respectively. This mutation might weaken affinity of kinases for ATP, depending on the type of replaced residue. For EphA10 and EphB6, Enzyme EphA3 Cytoplasmic Domain HER4 Cytoplasmic Domain TIE2 Cytoplasmic Domain Cytoplasmic Domain Cytoplasmic Domain Kinase Domain highly conserved Lys at subdomain 2 is missed or replaced to Gln. The Lys is recognized as essential for maximal enzyme activity by anchoring EphA4 IGF1R TNK1 6.5 EphA5 Kinase Domain INSR Kinase Domain TRKA Cytoplasmic Domain and orienting ATP with interacting its α− and β− phosphates. EphB6 and SuRTK106 are missing Glu at subdomain 3. The Glu appears to help Wash, blocking EphA6 Cytoplasmic Domain IRR Cytoplasmic Domain TRKB Cytoplasmic Domain EphA7 Cytoplasmic Domain ITK Full Length TRKC Cytoplasmic Domain stabilizing the interaction between Lys (subdomain 2) and α− and β−phosphate of ATP. EphA10, EphB6 and HER3 have mutation on invariant EphA8 Kinase Domain JAK1 Kinase Domain TXK Kinase Domain Asp in subdomain 6. The Asp is believed to have a role as proton acceptor within phosphotransfer reaction. EphA10 and EphB6 also have Anti phospho-Y EphB1 Cytoplasmic Domain JAK2 Kinase Domain TYK2 Kinase Domain 6.0 EphB2 Cytoplasmic Domain JAK3 Kinase Domain TYRO3 Cytoplasmic Domain 2+ 1 2 3 4 5 6 7 8 0 1 2 3 2 3 2 2 a 3 1 4 1 1 2 3 4 2 4 1 2 3 4 2 L L T T b T S A S K K S K S X K N C K K K K K B C R R K C R R R K K K R N K K R R K G

M mutation at the invariant Asn in subdomain 6 which is known to chelate secondary Mg ion. CCK4, EphA10, EphB6 and SuRTK106 replace I 7 T T T E A A A A A A A A K K K K X K N K B B B B R R E 1 R α R R R R R E B L O N T T T T E F L R β E Y Y E X K S C S A E E S K K R R R C R C D R O G I O M M R K I I P L L L

Cytoplasmic Domain Cytoplasmic Domain Full Length h h h h h h h h h h h h F F F F F EphB3 KDR YES F Y Y L Y F A A A E E A F N R F T T Y D A A F F Y R C B B F L S R N G A C F S T U M F R R B H K A R R M S B F F F I p p p p p p p p A p p p p J J J T L T P L G G H H G G G G Y D T G T T E I M Z E E E E E E E E

E E E E 2+ F F F F T D Wash EphB4 Cytoplasmic Domain KIT Cytoplasmic Domain ZAP70 Full Length D Asp in subdomain 7 to Ala, Gly, Arg and Gly respectively. The Asp chelates the primary activating Mg ion to help γ-phosphate transfer. For P FAK Full Length Full Length P 5.5 RYK, a replacement of Phe-Gly in subdomain 7 to Asn-Ala shall weaken phosphotransfer activity by losing hydrogen bonding between Asp and Colorimetric development for detection Tyrosine Kinase 10.5 STAUROSPORINE Ala.

Stop 10.0 References 1) Hanks SK, Hunter T., FASEB J. 1995 May;9(8):576-96. "Protein kinases 6. The eukaryotic protein kinase superfamily: kinase (catalytic) domain structure and classification." Measure 9.5 2) Vogel W, Gish GD, et al., Mol Cell. 1997 Dec;1(1):13-23. "The discoidin domain receptor tyrosine kinases are activated by collagen." 3) Kani S, Oishi I, Yamamoto H, et al., J Biol Chem. 2004 Nov 26;279(48):50102-9. Epub 2004 Sep 15. "The Ror2 associates with and is activated by casein kinase Iε." 9.0

0 8.5 Conclusion Table 2. Inhibition Patterns of Gleevec and Iressa. 5

pIC Gleevec Iressa C

50 I 8.0 (1) Carna Biosciences has successfully collected 77 active human tyrosine kinases p Although as it is reported that Gleevec inhibits PDGFRα, PDGFRβ, KIT More than 8 PDGFRα EGFR and developed their inhibitor screening systems (QuickScoutTM TK 7.5 and ABL, Iressa inhibits EGFR, and several other less recognized targets Comprehensive Panel). are inhibited by these kinase inhibitors. KIT EphA6 (2) Although Gleevec and Iressa strongly inhibit their well known targets, their ARG, DDR2, FMS, BLK, LCK, LYN(a/b) as Gleevec targets. Between 8 to 7 7.0 PDGFRβ TM EphA4, EphA5, EphB4, PDGFRs, LYNa as Iressa targets. novel targets ("off-targets") found by QuickScout TK Comprehensive Panel. ABL HER2 6.5 (3) For the rest of our under developed tyrosine kinases, 9 out of 13 has no These results are important not only this inhibitions against novel targets ARG HER4 activities in in vitro experiment (Three of LMR family members are found as 6.0 0 1 2 3 4 5 6 7 8 1 2 3 2 3 2 2 a 3 1 4 1 1 2 3 4 2 4 1 2 3 4 2 L L T T b T S A S K K S K S X K N C K K K K K B C R R R K C R R K K K R N K K R R K G M I

may help to predict/explain side-effects of the inhibitors, but also they may 7

T T T serine/threonine kinase (data not shown)). Investigation of in vitro activity for E A A A A A A A A K K K K X N K K B B B B R R E 1 R α R R R R R E B L O N T T T T E F L R β E Y Y E X K S A E C S E S K K R R R C R C D R O G I O M M R DDR2 K I I P

EphA4 L L L h h h h h h h h h h h h F F F F F F Y Y L Y F A A A E E A F N R F T T D Y A A F F Y R C B B F L S R G N A C F S T U M F R R B H K A R R M S B F F F I p p p p p p p p A p p p p J J J T L T P L G G H H G G G G Y D T G T T E I M Z E E E E E E E E E E E E F F F F T D indicate novel therapeutic target as in the example of Gleevec, which has D

P DDR1 and ROR2 is now on going. Weak phosphotransfer activity has been FMS EphA5 5.5 P been developed and launched by Novartis AG for the treatment of Between 7 to 6 Tyrosine Kinase observed for TIE1 and suitable conditions for inhibitor screening are under Philadelphia positive chronic myeloid leukemia (CML). It's BLK EphB4 study. (Fig. 2) inhibition against Kit revealed the treatment of patients with Kit positive LCK PDGFRα Figure 1. Profiling results for well known TK inhibitors (4) Our QuickScoutTM TK Comprehensive Panel is useful to develop more advanced unresectable and/or metastatic malignant gastrointestinal stromal tumors LYNa PDGFRβ Three well known protein (tyrosine) kinase inhibitors were examined against 77 active tyrosine kinases (horizontal axis). Vertical (GIST). medicines. LYNb LYNa axis indicates pIC50 ( -log(IC50) ). The presenters wish to acknowledge K. Akita, T. Asami, H. Fukada, M. Gouda, N. Harada, M. Hatakeyama, E. Ishibushi, N. Iwata, M. Kaku, Y. Kawase, T. Myojin, R. Nakai, M. Nakamura, Y. Narumi, Y. Nishioka, C. Nukuzuma, K. Okita, and T. Tateishi for their contributions. © Carna Biosciences Inc, 2003