High Throughput Kinase Discovery, Inhibitor Screening and Specificity Analysis using Multi-Array Technology Nisar Pampori, Pankaj Oberoi, Ryan Swenerton, Ilia Davydov, Stefanie Nelson, Hans A. Biebuyck, John H. Kenten, and Jacob N. Wohlstadter TM TM A division of Meso Scale DiagnosticsTM, LLC. High Throughput Kinase Discovery, Inhibitor Screening and Specificity Analysis using Multi-Array Technology Abstract We developed a high-throughput system for biological discovery and compound screening. A systematic and deterministic approach allowed us to categorize and quantify functional aspects of our library of 22,000 mammalian proteins (based on Unigene) coded by 50,000 individual full- length cDNA clones. Our methodology combined MSD's proprietary Multi-ArrayTM detection technology with high-throughput cell-free production, labeling, immobilization and purification of proteins. Here we report on several approaches we used to identify and characterize proteins with tyrosine kinase activity. First, we screened a set of 20,000 clones (14,000 unique Unigenes) for proteins with tyrosine kinase activity using a screening assay that simultaneously measured auto- phosphorylation and phosphorylation of a consensus tyrosine kinase substrate. The screen recovered known tyrosine kinases, previously unidentified kinases and, under low stringency conditions, proteins that recruited kinases. Second, we selected a redacted set of clones on the basis of homology to known kinases and demonstrated that we could identify ~ 70% of the expected tyrosine kinases in this redacted set. Third, we used MSD Multi-Array technology to test, in parallel, the specificity of 21 of these well-characterized kinases for a panel of known kinase inhibitors. Finally, we carried out a focused screen of 2,000 compounds for inhibitors of two of the identified kinases. The combination of high-throughput protein expression and Multi-Array technology allows for a unified approach to the discovery of biological targets and the identification of small molecules that regulate their activity. TM TM A division of Meso Scale DiagnosticsTM, LLC. High Throughput Kinase Discovery, Inhibitor Screening and Specificity Analysis using Multi-Array Technology High Throughput In-Vitro Expression Screening for Protein Tyrosine Kinase Activity Multi-Array Label TM α−Phosphotyrosine (MSD TAG ) Antibody (α−pY) Multi-Array Label Phosphate α−Phosphotyrosine (MSD TAGTM) Antibody (α−pY) ATP Substrate Transfer to second plate for assay ATP Biotinylated Expressed Autophosphorylation α−pY protein (assay in same plate) Streptavidin Capture protein Multi-Array Plate Kinase activity assay In-situ protein expression Autophosphorylation Assay Kinase activity was determined by cDNA clones The extent of auto-phosphorylation capturing the phosphorylated of the immobilized tyrosine kinase substrate with an anti-phosphotyrosine Clones expressed and labeled in was detected using MSD Multi-Array (α-pY) antibody and detection using 96-well format technology. MSD Multi-Array technology. Expressed protein immobilized on plate and purified Kinase phosphorylates consensus substrate or itself (autophosphorylation) TM TM A division of Meso Scale DiagnosticsTM, LLC. High Throughput Kinase Discovery, Inhibitor Screening and Specificity Analysis using Multi-Array Technology Screening of 20,000 cDNA Clones 10000000 Validation Evenly Spaced Controls through 1 week of 1000000 screening (10,000 clones) 100000 SYK BTK FERT2 Neg. Controls Kinase Binding Kinases Protein (SCAP2) 10000 1000 Batch 1 Batch 2 Batch 3 Batch 4 Batch 5 Batch 6 Results SYK 400 HCK 50,000 cDNA clones l a n Known Tyrosine Kinases g i 300 S Other Hits 20,000 clones expressed and labeled d e z i in a 96-well format. d r a d n a 200 t Immobilized on plate S TM 100 Tyrosine Kinase assay on Sector HTS Reader HCK BTK FGFR1 SCAP2 FRK Lyn FGR MATK Identified Kinases and Kinase binding protein 0 Entire Process <1 month 25 50 75 100 125 150 175 200 225 250 275 300 20,000 Clones TM TM A division of Meso Scale DiagnosticsTM, LLC. High Throughput Kinase Discovery, Inhibitor Screening and Specificity Analysis using Multi-Array Technology Kinase Activity from Our Library Demonstration of tyrosine kinase specificity of our assay across the kinase genome 0 g o l o m o 100 H Positive kinases are e s a shown in yellow. n i K e n i n o 200 e r h T - e n i r e S Genome 300 MSD Library Hits 400 300 200 100 0 Tyrosine Kinase Homolog Kinase Kinases in the genome and kinases in our library by activity gene similarity score. The overlap in the two distributions demonstrates the high degree of coverage of our library. Putative kinases scored as having high homology to known tyrosine kinases and confirmed as such in our activity-based assays. Ser/Thr Kinase Tyr Kinase Homology Homology TM TM A division of Meso Scale DiagnosticsTM, LLC. High Throughput Kinase Discovery, Inhibitor Screening and Specificity Analysis using Multi-Array Technology Protein Tyrosine Kinases Active from High Throughput Expressed Clones Consensus substrate Autophosphorylation hemopoieticcell kinase Gardner-Rasheedfeline sarcoma viral (v-fgr) oncogenehomolog protein tyrosine kinase2 beta c-src tyrosine kinase Eph receptor A7 fibroblast growth factor receptor 1 Tyrosine Kinase Family Tree fer (fms/fps related) protein kinase Bruton agammaglobulinemia tyrosine kinase Yamaguchi sarcoma viral (v-yes-1) oncogenehomolog FYN oncogenerelated to SRC EPHB2 endothelial-specific receptor tyrosine kinase EPHB1 kit oncogene EPHA3 EPHB3 Roussarcoma oncogene EPHA4 kinaseinsert domain protein receptor EPHA5 EPHA7 EPHB4 Eph receptor B4 EPHA8 fibroblast growth factor receptor 4 megakaryocyte-associated tyrosine kinase EPHA1 LMR2 EPHB6 EPHA2 INSR LMR1 B lymphoid tyrosine kinase IGF1R spleen tyrosine kinase TNK1 neurotrophic tyrosine kinase HCK LYN INSRR ACK1 TYK2JAK1 PTK2 B-cell src-homology tyrosine kinase JAK3 PTK2B fibroblast growth factor receptor 2 LCK JAK2 ZAP70 ERBB4 FGR BLK SYK EGFR fyn-related kinase FYN ERBB2 lymphocyte-protein tyrosine kinase ERBB3 SRC MATK CDC-like kinase2 YES1 CSK Yamaguchi sarcoma viral (v-yes) oncogenehomolog FRK DDR1 BMX non-receptor tyrosine kinase ABL2 DDR2 AXL Januskinase 1 (a protein tyrosine kinase) ABL1 MERTK EphB1 ROS1 TYR03 Eph receptor B3 BMX RYK anaplastic lymphoma kinase (Ki-1) TXK MET BTK MST1R hemopoieticcell kinase FER TEK LTK NTRK1 TEC FES TIE FYN oncogenerelated to SRC ITK RET ALK NTRK3 NTRK2 CDC-Like Kinase1 CDC-like kinase3 ROR1/2 FLT3 PTK7 fibroblast growth factor receptor 1 (fms-related tyrosine kinase 2) MUSK CDC like kinase 4 FLT1 FGFR4 TTK protein kinase KDR FLT4 FGFR3 wee 1 homolog (S. pombe) KIT KIN - Kinases from Screens lymphocyte-specific protein tyrosine kinase CSF1R FGFR2 FGFR1 PDGFRB - Kinases used in specificity study control PDGFRA vector Negatives(n=10) TK substrate TK binding protein 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 0 0 0 0 0 0 , , , , , , , , , 1 1 0 0 0 0 0 0 0 1 1 0 0 0 0 0 1 1 0 0 0 , , , 1 1 0 1 Signal on Sector HTS TM TM A division of Meso Scale DiagnosticsTM, LLC. High Throughput Kinase Discovery, Inhibitor Screening and Specificity Analysis using Multi-Array Technology Multi-Array Tyrosine Kinase Specificity of Inhibitors Towards In-Vitro Tyrosine Kinase Activity (Autophosphorylation and Substrate) Hierarchical cluster analysis of inhibitors and kinases IC50 Compound Known Effect (µM) (10X IC50) Damnacanthal LCK 0.62 AG-1433 PDGFR 5 VEGFR2 Inhibitor KDR (Vegfr2) 0.07 WHI-P131 Jak3 7.8 VEGFR Inhibitor KDR (Vegfr2) 2 PP1 FYN 0.6 Oxindole 1 Flk1 0.39 AG-1024 IGF1R 18 AG-957 Abl 0.75 LFM-A13 BTK 17.2 Erbstatin analog EGFR 0.78 Lavendustin A EGFR 0.011 AG879 NTRK1 10 AG-17 PDGFR 0.5 Genistein EGFR 2.6 AG-1296 PDGFR 1 AG-1478 EGFR 0.003 PP2 LCK 0.004 Compound-56 EGFR 6-pM SU-1498 Flk1 0.7 AG-490 EGFR 0.1 AG-538 IGF1R 0.06 t t r r c c k k K K K K K K X X K K K K K K 7 7 4 4 2 2 R R 3 3 4 4 2 2 B B i i 1 1 1 1 r r t t d d - - S S e e Y Y T T C C C C a a b b K K R R R R 2 2 K K G G S S M M k k r r R R r r T T S S K K C C L L f f F F B B h h F F h h R R H H K K F F e e F F B B n n g g T T p p T T p p F F G G T T G G F F E E E E P P F F N N F F 0 1 2 Tyrosine Kinases Relative Kinase Activity Autophosphorylation Consensus substrate A survey of known inhibitors reveals patterns of their actions on a set of tyrosine kinases, allowing classification of these kinases into groups reflecting their lineage. The individual drug profiles are useful supplements to general studies of the physical organic basis of chemical phenotypes. TM TM A division of Meso Scale DiagnosticsTM, LLC. High Throughput Kinase Discovery, Inhibitor Screening and Specificity Analysis using Multi-Array Technology From Biological Discovery to Compound Discovery Rapid progression to titratable inhibitors for chemical genomic and other types of study. The biological screening format used previously is also employed here to screen compounds. 50,000 cDNA clones 140 ) K C 120 H ( n 100 o y i t t Identified Kinases i a v l i t y r c 80 o A h p % s o 60 h p o t u 40 A Z > 0.5 Express and label 20 0 Sample Number Immobilized on plate Screen Kinase for Inhibitor 160 125 A 140 100 IC50~ 600nM 120 ) K C H y ( t 75 i 100 v i n t o y c i t t A i a v l i t y 80 % r c 50 o A h p % s 60 o h p o 25 t u 40 A 20 0 0.01 0.1 1 10 100 0 A Concentration (µM) 0 20 40 60 80 100 120 140 160 % Activity Autophosphorylation (FGFR1) TM TM A division of Meso Scale DiagnosticsTM, LLC.