© 2011 Nature America, Inc. All rights reserved. should should be addressed to J.R.P. ([email protected]). 1 novel identify to were goals Our duplicate. in 300 of panel a against inhibitors known 178 of screen parallel scale screens target-centric by conventional identify to target primary single a toward than rather targets kinase specific of number small a toward is focused activity inhibitory whose inhibitors, multitargeted reveal to and interest of kinases for inhibitors predicted new unexpected is identify approach parallel This optimized. chemically then and identified are patterns selectivity desired showing Compounds of compound each selectivity the to reveal binant kinases protein recom of panel comprehensive a against manner target-blind a in compounds of libraries screening involves approach, alternative An are then tested for against selectivity a panel of representative kinases. and a of of kinase compounds molecules The interest. resulting small centric manner involving high-throughput screening of large numbers unknown. important an remains thus inhibition functional predict to assays these of ability The activity. of catalytic inhibition functional than rather to kinases, cases, however, these methods measure the binding of small molecules against of sizable fractions the 518 human protein kinases selectivity target kinase to profile of methods to development led the have advances technological of Recent part kinome. the a substantial However, of inhibitors the kinase is across selectivity seldom assessed settings. and clinical in research of both inhibitors the effects preting of kinase inhibitors for their targets is critical for predicting and inter is inhibition tive a kinase major challenge selec highly However, achieving compounds. chemical by synthetic ATP-binding that of can ence be pocket exploited conserved a highly targets because of their central roles in cellular and signaling the pres of therapeutic classes important most the are among kinases Protein elucidate kinase function and for interpreting the results of experiments involving kinase inhibitors. specific, diverse kinases. The results have implications for drug development and provide a resource for selecting compounds to interactions occur with seemingly unrelated kinases, revealing how large-scale profiling can identify inhibitors multitargeted of unexpected interactions between protein kinases and kinase inhibitors, with a wide spectrum of promiscuity. Many off-target available kinase inhibitors against a panel of 300 recombinant protein kinases. Quantitative analysis revealed complex and often kinase comprehensive selectivity of most inhibitors. Here we used functional assays to profile the activity of 178 commercially inhibit multiple kinases. of Interpretation experiments that use these compounds is confounded by a lack of data on the agents. Owing to evolutionary conservation of the ATP-, most kinase inhibitors that target this site promiscuously protein Small-molecule kinase inhibitors are widely used to elucidate cellular signaling pathways and are promising therapeutic Theonie Anastassiadis reveals features of kinase inhibitor selectivity C nature biotechnology nature Received 1 September; accepted 23 September; published online 30 October 2011; Cancer Cancer Biology Program, Fox Chase Cancer Center, USA. Pennsylvania, Philadelphia, e sd hg-hogpt nyai asy o odc a large- a conduct to assay enzymatic high-throughput a used We target- a in discovered been have inhibitors kinase Traditionally, omprehensive assay omprehensive of kinase catalytic activity 11 , 1 2 . Indeed, multitargeted inhibitors are challenging challenging are inhibitors multitargeted Indeed, .

1 advance online publication online advance , Sean , W Sean Deacon 1– 6 . Knowing the selectivity . Knowing the selectivity 1 2 3 , , Karthik Devarajan . 7 , 8 . . In many

9 , 1 0 - - - - . protein kinase families ( families kinase protein Table 1 Supplementary from all inhibit major kinases ( protein subfamilies kinase to known compounds 178 comprised library The tools. research as and used testing, compounds in compounds primarily drugs, clinical Administration–approved Drug and Food US included inhibitors compared are inhibition kinase for assays indirect more which against standard the is method This well-validated substrate. specific a toward activity catalytic kinase measures directly which assays, filter-binding conventional on based assay ric radiomet a We HotSpot, used kinases. protein human recombinant 300 of panel a using assays kinase low-volume conducted we itors, To directly test the kinase selectivity of a large number of kinase inhib map interaction A kinase-inhibitor RESULTS points for the development of multitargeted kinase inhibitors. for orphan kinases for which few inhibitors currently exist and starting commonly used kinase inhibitors. In addition, we report potential leads,small commonly inhibited by many compounds, kinases that are resistant to Systematic, quantitative analysis of the results revealed kinases that are measuring pairwise inhibition of a single by a single compound.resultsprises generated from>100,000independent functionalassays ­knowledgelargestavailablethepublic itstype ofthedomain, in com our to set, data resulting The families. kinase major the of all geting tar agents clinical and compounds research used widely represent specificities of a large panel of kinase inhibitors. The compounds tested inhibitor chemotypes for specific kinase targets and to reveal the target constructs constructs and substrates used is provided in kinase the of listing complete A tested. compounds the of 87.6% of d 2 o Reaction Reaction Biology Corporation, Malvern, USA. Pennsylvania, Correspondence i The kinase panel tested includes members of all major human human major all of members includes tested panel kinase The : 1 1 0 - , , Haiching Ma . molecule inhibition, and unexpected off-target activities of many 1 0 3 8 / n b t . 2 0 1 7 2 & Jeffrey R Peterson ). Fig. 1 Fig. b ) and includes the intended targets targets intended the includes and ) 7 . Our collection of kinase kinase of collection Our . Supplementary Supplementary Table 2 e c r u o s e r 1 Fig. Fig. 1 a and  - - - - .

© 2011 Nature America, Inc. All rights reserved. been reported and structural studies have revealed the structural structural the revealed have have studies kinases Aurora structural of and reported inhibitors been isoform-specific with finding, Consistent this molecules. small by targeted differentially be can 2 Fig. ( kinases family and FGFR PDGFR Aurora, relevant pat similar by clinically of the members included inhibited Exceptions were of compounds. terns and clustered often were identity by sequence related activity closely kinases Similarly, their together. grouped ally of gener were compounds related similarity the structurally on expected, As patterns. both based cluster to inhibitors and performed kinases was clustering hierarchical Two-way down be ( to window set browser a within data analyzed or the of loaded queries specific kinase or compound allows that website internet an database, (KIR) Resource Inhibitor in in in map form heat lution a as presented is pair tor observed. was inhibition kinase 20% in at pairs which least for kinase-inhibitor all replicate, second the versus replicate one in activity kinase remaining the as plotted pair kinase-inhibitor one represents point each which in plot scatter a as set data resulting Figure 1 analysis the from (Online Methods set) and data the of (0.18% and identified eliminated disparate replicates We activity). kinase remaining as to referred (henceforth reactions control solvent of age percent a as results phosphorylation strate sub average the expressed and duplicate in inhibitor combination (kinase-inhibitor pair) activity. inhibitory off-target weaker capture 0.5 use to chose we nM, 66 of targets primary their toward compounds these for (IC concentration inhibitory maximum 10 0.5 of concentration a at tested were compounds all simplicity, For ( tool online (KIR) Resource Inhibitor Kinase the in table data in presented is map heat this of version high-resolution labeled, activity. A fully kinase of map a heat as presented map interaction kinase-inhibitor entire the of analysis clustering hierarchical ( observed. was activity kinase of inhibition >20% which for pair kinase-inhibitor each 2 for replicate 1 versus replicate in ( 33). ref. on Technology based Signaling Cell of courtesy reproduced is and adapted was illustration (kinome kinome human the representing a on dendrogram dots blue by represented is panel screening the in kinases of distribution family. ( library, kinase by inhibitor the of ( analysis. 1 Figure e c r u o s e r  inhibition isoform-specific for basis h

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C Atypical Lipi CAMK kinase-inhibitor pairs with low affinity (weaker than 1 the of 13.1% Conversely,only (>50%). inhibition functional showed kinase-inhibitor interactions with high affinity (strongeractivitymeasured oursingle-dosein study ( than 100 nM kinase expected the withagreement good generallyshowed affinities dred fifty-four kinase-inhibitor pairs overlapped with ouraffinities studyfor a andlarge numbertheir of kinase-inhibitor interactions to kinases. Two recent studies used a competitive binding assaydata towith previousderive large-scale studies of the binding of small molecules ity remains uncertain. To assess this, we compared our kinase inhibitionextent to which these binding assays predict inhibition of catalytic activtion of kinase catalytic activity. Although convenient for screening, the detect kinase-compound interactions without directly measuring A inhibivariety of high-throughput screening approaches platforms assay have multiple across data of been Comparison devised to pound binding pound com by denaturation thermal from kinases protecting involves ing >50% inhibition, as expected. d An alternative approach to monitoring kinase-compound bind kinase-compound monitoring to approach alternative An CMGC CAM advance online publication online advance b K TK Kinases screened 3 . To assess this approach to predict kinase inhibition, . Toinhibition, kinase approach to this predict assess AG CK C Compounds STE TKL 1

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© 2011 Nature America, Inc. All rights reserved. tor set. The untargeted kinases, including COT1, NEK6/7 and p38 and NEK6/7 COT1, including kinases, untargeted The inhibi set. tor this by kinome the of coverage good demonstrating inset), ( tested by compounds any of the inhibited not were panel by ( >50% kinase each of activity catalytic the inhibited that tested compounds (S score selectivity a to respect with the kinases ranked we this, do To inhibition. small-molecule to sensitivity be inhibited by a given compound or whether kinases differed in their to likely equally was panel the in kinase each We whether asked next druggability kinase of Analysis correlated. significantly are inhibition and binding ity, although activ catalytic inhibit that functionally compounds to ability predict appreciable false-positive and false-negative rates with respect to their exhibit assays binding kinase-inhibitor that suggest together, taken comparisons, these from findings The quadrant). dashed left lower exhibiting without activity kinase of tion 117 out inhibi showed >50% pairs of inhibitor 3,926 rant). Likewise, by activity kinase >50% ( inhibiting showed compounds of number significant a However, predicted. as activity, catalytic of inhibition showed also temperature melting kinase the ( pair kinase-inhibitor ( temperature melting reported in change of the function a as assay functional our in activity kinase remaining the plotted we control. Ctrl, activity. catalytic of inhibition >50% show yet stability thermal affect marginally only which compounds contains quadrant left lower the whereas activity kinase inhibiting without stabilization thermal significant showed that compounds includes quadrant right upper resulting The activity. catalytic of inhibition for threshold 50% the highlights line horizontal dashed The the denotes line vertical dashed The pairs. kinase-inhibitor 3,926 for binding compound by caused control, solvent to In affinity. given the of inhibitor an for activity kinase remaining expected for line) (dotted curve a theoretical with compared be can and line) (solid curve dose-response a sigmoidal to fit were data resulting ( affinity binding compound In inhibitors individual of presence the in kinases by denaturation assay binding inhibitor kinase- a quantitative by pairs kinase-inhibitor overlapping of interactions ( studies. profiling interaction kinase-inhibitor previous with study 2 Figure nature biotechnology nature scaffolds may be less successful. By contrast, a subset of kinases kinases of inhibited subset broadly were a HGK/MAP4K4 and TRKC contrast, FLT3, By including successful. less be may scaffolds ATP-mimetic traditional using screens for list a which target suggest a , b b a , remaining kinase activity is plotted as a function of kinase- of a function as plotted is activity kinase , remaining ) Scatter plots compare our results with studies that examined examined that studies with results our compare plots ) Scatter remaining kinase activity is plotted against the change in in change the against plotted is activity kinase remaining Selectivity score, S(50%) 0. 0. 0. 0.

T 0 1 2 3 4 Comparison of functional inhibition data generated in this this in generated data inhibition functional of Comparison m Fig. Fig. changes >4 °C, the hit threshold used previously 3 and COT1/MAP3K8 P38 MAPKAPK3 CTK MATK DYRK4 Supplementary Table 4 Supplementary δ HIPK1 WNK3 GRK3 GRK2 VRK1 NEK7 NEK6 1 JNK3 P38 /MAPK13 , Fig. Fig. 2 2 ( γ a K ), or an assay measuring resistance to thermal thermal to resistance measuring assay an or ),

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shift threshold used in ref. 3. ref. in used threshold shift (this study) –1 0 0 15 50 e c r u o s e r 0 0 T m shift 1 4 ). For inhibi For ). ( ° C) x 2 0 ) has been been has ) 0  - - - - © 2011 Nature America, Inc. All rights reserved. a multitargeted inhibitor, highly selective for ErbB family members, a limited number of other tyrosine kinase targets and the serine/threonine kinase kinase serine/threonine the and targets kinase tyrosine other of number a limited members, family ErbB for selective highly inhibitor, a multitargeted benzyloxyanilino)-6,7-dimethoxyquinazoline, Technology. ( Signaling Cell of courtesy reproduced is and adapted was dendrogram kinome The activity. 25–50% lightest, activity; 10–25% lighter, activity; remaining 0–10% darkest, potency: similarity sequence acid amino on based kinases human all of a dendrogram on presented are compounds representative three of selectivity in presented is table complete The compounds. scoring highest five the inset, right the and scores Gini lowest the with compounds five the highlights inset Left inhibition). (selective kinase one only of inhibition 1 indicates of a score whereas inhibition) (promiscuous kinases all of inhibition equal indicates 0 of score A Gini selectivity. inhibitor score Gini by sorted ( 4 Figure single any with correlation linear consistent a observe not did but with to respect properties either the Gini score or score the selectivity promiscuity fied correlations and compound properties between physicochemical identi have studies profiling kinase-inhibitor previous promiscuity, spots). (darker targets its in activity kinase residual lower producing by a score Gini achieves higher masitinib dendrograms), right and (middle scores Gini were kinases targeted by the compounds with the median and highest of number comparable a Although panels. bottom the in shown are scores Gini highest and median lowest, the with compounds three the of spectra target The kinases. family ErbB of inhibitors distinct ( most compounds the Among selective spectrum. target broad known their with sistent ( scores Gini lowest the exhibited analogs Table 5 ( selective to most the cuous of results to analysis this rank the from compounds the most promis Weof 0). score (a Gini the used kinases tested all across equally uted distrib is or 1) of score Gini (a target single a only toward directed is kinases) for all of sum as inhibition the (calculated of a compound activity inhibitory aggregate the a which to on degree the 1, to 0 of reflects, scale score Gini The concentration. screening compound the by influenced strongly is it although threshold, hit arbitrary an Gini the on coefficient based selectivity inhibitor previously kinase a for metric calculated described therefore We scored. any be for not threshold could hit kinase the meet not did that compounds addition, compounds of orders rank different produce can thresholds hit scores generated from the selectivity same data set but using different Indeed, shown). (not threshold this below just kinases other of tion inhibi of deal great a despite kinases, of number limited a with old criterion, hit met they the hit favorably scored compounds because thresh the several as inhibition percent arbitrary an using data our on ( hit an threshold arbitrary tested (with an greater than affinity e c r u o s e r  kinase. individual an for activity remaining percent the to corresponds bar Each CHK2. a

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© 2011 Nature America, Inc. All rights reserved. tor, a 4,6-dianilinopyrimidine EGFR inhibitor (CAS no. 879127-07-8) inhibi uni-specific most the In fact, (EGFR). receptor factor growth kinases ­specific are several inhibitors intended to target the epidermal any in ( panel the kinase other than potently more 20% least compounds at target Nineteen primary their kinases. inhibited related differ closely of achieving of inhibition challenge ential the highlights finding This panel). in bars (short targets secondary and primary their between differences potency slight only showed these of most and targets. these against potency absolute the indicates bar two most sensitive kinase targets, and the left-right positioning of this these of inhibition of potency differential the denotes bar, therefore, ( target inhibited potently most of second the activity kinase remaining the indicates edge most right the and kinase inhibited potently most the for activity kinase remaining the denotes bar the of edge leftmost the which in graph bar horizontal a as results the Weplotted least. to score) numerical kinase. Compounds were then ranked from most uni-specific (highest potently most the inhibited of most potently next the from activity the of kinase inhibited activity kinase remaining the subtracting by compound each for calculated was score uni-specificity A panel. screen ing the in homologs close without targets kinase for bias a has it kinases addition, In subfamily. same those the from if isoforms related even closely are potency, similar with kinase one than more control. Ctrl, panel. test our in ATM included **, not was 202190. SB kinase inhibitor kinase MAP p38 the for compound control a is negative 202474 SB *, plot. a in ranked shown is panel the in kinase each on compounds individual the of effect the panel, gray.in right shown the In are target intended the not is target sensitive most the which for compounds Six targets. sensitive most their and targets, intended potency, their differential 20% least at showed that compounds the identifies table central The shown. are 5% least at of potency a differential with compounds Only targets. inhibited potently most two its against inhibitor corresponding the of activity differential the reflects bar each of length horizontal the Thus, 5%). of bins in shown is activity kinase remaining (% inhibited is kinase sensitive most next the which with potency the reflects boundary right the and target sensitive most its inhibits compound the which with potency the depicts bar horizontal each of boundary left The tested. kinase other any than potently more kinase single 5 Figure nature biotechnology nature IC reported a with 1 5 Few compounds in the panel showed any degree of uni-specificity of uni-specificity any showed degree panel in the Few compounds 1 0 2 5

Uni-specific kinase inhibitors. The left panel presents a graphical table of compounds ranked based on the compound’s ability to inhibit a inhibit to ability compound’s the on based ranked compounds of table a graphical presents panel left The inhibitors. kinase Uni-specific 3 0 25 Kinase activityin5%bins 3 0 4 5 40 50 of 21 nM for EGFR for nM 21 of 5 50 55 Fig. Fig. Fig. Fig.

60 advance online publication online advance 65 5 5 , middle). Among these 19 uni- most Among these , middle). , leftmost panel). The length of each each of length The panel). leftmost , 70 75 80 2 2 , inhibited EGFR catalytic catalytic EGFR inhibited , GSK-3b inhibitor p38 MAPkinase JNK inhibitorVlll JNK inhibitorlX Chk2 inhibitorll EGFR inhibito Akt inhibitor SB 202474* ATM kinase GTP-14564 PD 158780 PD 174265 Tandutinib SC-68376 inhibitor ll AG 1478 AG 1024 Gefitinib inhibitor BPIQ-l Name DMBI Vll l Fig. Fig. l X r 5 , leftmost leftmost , 387867-13-2 587871-26-9 184475-35-2 172747-50-1 894804-07-0 175178-82-2 581098-48-8 925681-41-0 318480-82-9 487021-52-3 171179-06-9 174709-30-9 216163-53-0 312917-14-9 516480-79-8 879127-07-8 34823-86-4 65678-07-1 5812-07-7 CAS no.

- - - - FLT3/ c-KIT p38 MAPK INACTIVE KIT/ FLT3 c-FMS/ c- Intended PDGFR IGF-1R PDGFR GSK-3 gis te oe tres ee ofre ( confirmed were targets novel the against greater potency the intended and novel their cases targets.In both all the dose-response relationship for five uni-specific compounds against sensitivity of different to kinases the same compound, we determined identified. compounds specific one uni- of most the AG1024, inhibitor kinase tyrosine of IGF1R the target sensitive more much a as RIPK2 kinase serine/threonine the identified we example, For target. intended the of subfamily kinase the of outside falls hit off-target potent most the DMBI, compound the of that case, one but all in Remarkably, compounds. these of gets tar kinase unknown hitherto represent hits off-target potent more these cases In the all panel. ATM of screening not because a the was part in included not was kinases inhibitor all kinase ATM The against plot. sorted a compounds as 6 panel these of 5 of activity the ( target to intended were they kinases the than potently more kinases target. therapeutic important this of inhibitors of development the to devoted attention unequal or, likely,the more EGFR of features unique reflect could here tified matic kinases family ErbB related closely the among inhibition this compound the also ability highlights to achieve isoform-selective MRCK by ­activity >94% but its target, inhibited next most inhibited potently ATM** EGFR EGFR EGFR EGFR EGFR EGFR CHK2 target p38 AKT JNK JNK Fig. Fig. To validate the use of our single-dose screening data to rank the the rank to data screening single-dose our of use the Tovalidate other inhibited compounds uni-specific 17 top 6 of the Strikingly, α 5 selectivity of this and other uni-specific EGFR inhibitors iden inhibitors EGFR of and this uni-specific other selectivity , center, gray rows). The rightmost panel of of panel rightmost The rows). gray center, , α PDGFR Primary Haspin GSK-3 RIPK2 EGFR EGFR EFGR EGFR EGFR EGFR CHK2 JNK3 JNK3 CK1 PIM FLT3 FLT3 PIM p38 , by only 22%. In contrast to other EGFR inhibitors tested, tested, inhibitors EGFR other to contrast In 22%. only by , hit α 3 3 δ α Kinase activity(%ctrl) 100 100 100 100 100 10 10 10 10 10 1 1 1 1 1 Individual kinases 10 10 10 10 10 upeetr Fg 5 Fig. Supplementary e c r u o s e r Figure Figure 100 100 100 100 100 2 2 . The . The 5 shows shows dra 300 300 300 300 300 ). ).  - - -

© 2011 Nature America, Inc. All rights reserved. warranted if attempting to extrapolate these these extrapolate to attempting if warranted is caution robust, and rapid economical, is activity kinase measure compounds. these for targets unknown previously represent compounds these cases, In all targets. intended their than potently more kinases other inhibit that were compounds found tion of Six new targets. uni-specific inhibitor kinase single a characteriza the We only is to target inhibited. prioritize metric this used which within window dosing widest the vide pro to expected are uni-specificity of degree greatest the exhibiting Compounds and targets. most its sensitive kinase next most sensitive its toward inhibitor an of activity differential the assessing titatively subfamilies. kinase for particular selective inhibitors of new data set in guiding both tool compound present selection the and of the developmentutility the illustrate findings These subfamilies. other from subfamily this of kinases, divergence AGC sequence greater to inhibit due perhaps selectively to employed be can chemotypes distinct of variety a that demonstrating subfamily, kinase AGC the of members other all almost are compounds these by shared targets no clear structural similarity in the compounds. In fact, the secondary ( together Y-27632 and IV Inhibitor Kinase Rho Rockout, clustered spectrum target on based hierarchical clustering Strikingly, ROCK. than KHS and PRKX of inhibition other kinase (data any not than shown). potently By more contrast, fasudil significantly compound showed II and more I this potent ROCK both indeed, inhibited and, (0.738) glycyl-H-1152 for selectivity (HA-1077) fasudil agent clinical the and Y-27632 IV), Inhibitor Kinase (Rho glycyl-H-1152 Rockout, kinase): (Rho-associated ROCK kinase AGC the subfamily example, of inhibitors For four contains well-established collection the compound function. kinase investigating for most inhibitor the choosing selective for basis powerful a provide should interest of kinase specific a targeting inhibitors multiple across scores Gini of methods by previous used thresholds hit arbitrary for a measure of inhibitor kinase selectivity to elucidate kinase biology. We therefore applied the Gini coefficient as agents of therapeutic efficacy for essential may be not selectivity kinase strong Although reported. are sets data profiling kinase ever-larger as important increasingly is supported by our data. Quantitative assessment of inhibitor selectivity of the importance of broad profiling kinase compounds and these are inhibitors kinase characterized unex an number pected of interactions with even off-target kinases, for highly revealed have studies profiling inhibitor kinase Previous DISCUSSION multitargeted kinase inhibition. inhibitors for their novel targets and in some starting pointscases, for compounds the here uni-specific described provide new and selective anti-cancer target, though few inhibitors have been reported contributes to chromosomal organization and has been suggested as an ( Haspin kinase protein nuclear cell–specific germ haploid the kinase, inhibitor SB202190 kinase (ref. MAP p38 the of analog inactive an SB202474, Additionally, TrkC. and FLT3 of inhibitor potent highly a be to DMBI inhibitor, receptor factor growth platelet-derived weak the revealed example, results the For compounds. these for targets new inhibited potently These findings confirm the accuracy of our single-dose data and reveal e c r u o s e r  out in the was carried our First, screen efficacy. of cellular prediction Fig. Fig.

Although the high-throughput assay used here to systematically systematically to here used assay high-throughput the Although of quan a way as of uni-specificity concept the We introduce also 5 ). This atypical family kinase phosphorylates histone H3 and H3 histone phosphorylates kinase family atypical This ). 23), showed significant inhibition of only one 28 , 2 9 2 . Gini score analysis revealed greatest greatest revealed analysis score Gini . 7 , it is critical , for it used compounds tool is critical 1 , 2 . These findings have emphasized emphasized have findings These . Supplementary Fig. 2 Fig. Supplementary 1 5 , , thus avoiding the necessity in vitro in 2 results to the the to results . Comparison . Comparison 24– ), despite despite ), 2 6 . Thus, - - - -

HTML HTML version of the paper at http://www.nature.com/nbt/index.html. The authors declare competing interests:financial details accompany the full-text written by J.R.P. with input from the other authors. by T.A. and J.R.P. with input from S.W.D. and H.M., and the manuscript was bycollected S.W.D., statistical analysis was performed by K.D., data were analyzed The study was conceived by J.R.P., S.W.D. and H.M., data experimental was R44 CA114995 to H.M.). supported partially by the US National Institutes of Health (RO1 HG003818 and CA006927 to Fox Chase Cancer Center. HotSpot developmenttechnology was by US National Institutes of Health awards RO1 GM083025 to J.R.P. and P30 Keystone Program in Head and Neck Cancer of Fox Chase Cancer Center and This work was supported by a W.W. Smith Foundation Award, funding from the Corp. for developing the Kinase Inhibitor Resource (KIR) web application tool. laboratory for comments on the manuscript and R. Hartman of Biology Reaction We gratefully acknowledge B. Turk, A. Andrews and members of the Peterson provides one example of how large-scale kinase profiling can iden can profiling kinase large-scale how of example one provides their function. Our identification of a inhibitor uni-specific of Haspin of elucidation facilitate greatly would kinases understood poorly ing subset of the kinome efficacy cellular with correlates kinase- generally of interactions inhibitor analysis biochemical and interactions, concerning kinase-inhibitor information of resource rich a provide here presented data the Nevertheless, expected. reasonably be can kinases untested not included in the panel. Thus, additional off-target are activities against kinases of minority a activity, catalytic kinase of measurements kinase panel tested here is among the largest available for biochemical and only one such state was assayed for each kinase. Third, though the states conformational multiple adopt can kinases many addition, In or milieu. in kinase the cellular in the context of differ the full-length could compound a with interactions whose constructs truncated by represented are panel the in kinases many the Second, in context. differ cellular may here determined kinases inhibited of order rank ATP of concentration cellular the and binding ATP for constant Michaelis-Menten the by also but kinase, the for inhibitor the of affinity intrinsic the by only not dictated is context for ATP. Potency of in ATP-competitive the inhibitors cellular kinase presence of 10 Note: Supplementary information is available on the online the in version of the paper at http://www.nature.com/naturebiotechnology/.available are references associated any and Methods M models. cell in functions kinase elucidate to tool a powerful be will kinases, protein of human majority the against ity here, with activ characterized that we the collection inhibitor expect Finally, this. as such maps interaction kinase-inhibitor robust using developed ( tion inhibi kinase multitargeted be for can opportunities set new reveal data to mined present the how illustrate we such addition, In support to studies. kinase, per nine average on candidates, of library determination structure for crystallographic kinases to stabilize can be used inhibitors kinase kinases of unliganded crystals diffracting obtain plasticity, to can which make conformational it difficult considerable studies may also benefit from the present study. Protein kinases exhibit new tool tify compounds to stimulate new Crystallographic research. CO AUTH Ac ethods Protein kinase research has been predominantly on focused a small k MP no Fig. 4 Fig. O E TI w R R advance online publication online advance CON NG le 1 3 d b to facilitate analysis of potential drug polypharmacology to of analysis facilitate drug polypharmacology potential FI ). Indeed, new statistical methods have been recently recently been have methods statistical new Indeed, ). g TRIBUTI µ N m M M ATP regardless of the of affinity kinases individual A en NC ts 3 IA 1 . The identification of selective inhibitors target ON L I 3 N S Te aa e peetd ee rvds a provides here presented set data The . T E R E STS nature biotechnology nature N a t u r e 3

B 3 0 2 . Thus, the relative relative the Thus, . i o . . ATP-competitive t e c h n o l o g y website. 3 0 . - - - -

© 2011 Nature America, Inc. All rights reserved. 16. 15. 14. 13. 12. 11. 10. 9. 8. 7. 6. 5. 4. 3. 2. 1. reprints/index.html. R Published online at http://www.nature.com/nbt/index.html. nature biotechnology nature eprints and permissions information is available online at http://www.nature.com/ at online available is information permissions and eprints

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© 2011 Nature America, Inc. All rights reserved. on the estimated mean and s.d. of this distribution. The red vertical lines in in lines vertical red The distribution. this of s.d. and mean estimated the on region enclosed by these vertical lines contains 75.6% of the observations based for D, plot density exponential double the in lines vertical gray the by determined D (as retained of differences distribution of the we mean of the 1 s.d. within observations measurements, assay the in noise inherent the for account To in D is versus displayed methods likelihood maximum using estimated were 1a Fig. ( distributed exponentially double be to determined was D difference the plots, quantile-quantile and estimation density kernel Using pair. kinase-inhibitor each for computed were observations duplicate from (D) difference the and (CV) from variation of removed coefficient the were and analysis duplicates further across values identical or miss either observations with ing pairs kinase-inhibitor and zero to truncated were values negative All in duplicate. pair kinase-inhibitor for each activity of compound methods. Statistical r Kinome tree representations were prepared using Kinome Mapper ( tions. IC reac sulfoxide) (dimethyl vehicle to compared samples test in activity kinase remainingpercent the as wereexpressed data activity kinase enzyme, inactive acid. After subtraction of background phosphoric derived 0.75% from in control filters of reactions washing containing extensive by removed was phosphate ting of the reactions onto P81 ion exchange filter paper (Whatman). Unbound 10 of of a mixture of ATP (Sigma) and Compounds were delivered into the reaction, followed ~20 min later by addition details of individual kinase reaction components see Brij35, 0.02 mg/ml BSA, 0.1 mM Na MgCl mM 10 7.5, pH Hepes mM 20 buffer; reaction in preparedwere cofactors required with along pairs kinase/substrate specific Briefly, platform. “HotSpot”assay the using Corporation Biology Reaction at Kinase assays. in provided Table 2 is Supplementary used kinases recombinant of description complete A of >98%. purity average an with Laboratories LC or Biosciences EMD from either Materials. METHODS ONLINE nature biotechnology nature e a c t i o µ Supplementary Fig. 1a Supplementary n M. Reactions were carried out at 25 °C for 120 min, followed by spot by followed min, 120 for °C 25 at out carried were Reactions M. , b b 50 ). Its location and scale parameters (and hence the mean and s.d.) s.d.) and mean the hence (and parameters scale and location Its ). i values and curve fits were obtained using Prism (GraphPad Software). o iae niios ( inhibitors Kinase l o g

y In vitro . c o m /

a Outlier Outlier detection profiling of the 300 member kinase panel was performed p p Supplementary Figure 1e Figure Supplementary . s / k i n ) for further analyses of ) analyses compound activity. for The further o m 33 e upeetr Tbe 1 Table Supplementary P ATP (PerkinElmer) to a final concentration / m 3 VO a . . Raw data were as measured percentage p p 4 e , , 2 mM DTT, 1% DMSO (for specific r / L a u n c h for all pairs of data points. of points. data pairs for all K Supplementary Supplementary Table 2 i n 3 2 4 o , 1 mM EGTA, 0.02% 0.02% EGTA, mM 1 , . A scatter plot of CV CV of plot scatter A . m e . h ) were obtained obtained were ) t Supplementary Supplementary m ). h t t p : / / w w w ). ). - - - . tering based on 1 – Spearman rank correlation as the distance metric and and metric data. the to distance applied was No scaling the linkage. average as correlation rank Spearman – 1 on based tering heat map of compound was activity obtained using clus two-way hierarchical reordered A value. that at truncated were >100 values and zero to truncated clustering Hierarchical in set data complete of the context the within are shown points) data (black outliers The analysis. further from excluded and observations outlying as identified were by blue circles, represented band, this of and 0.5 outside CV cut-off the above located were that observations the of remainder the approach, twofold this on Based ~0.5. of cut-off CV a to corresponds and threshold this represents in line horizontal pink The expected. are vations computingby the thresholdoutliers beyondare which a certain prespecifiedobservations number ofextreme obser whether determine to performed then log-normal distribution and its parameters were robustly estimated. A test was for the plot positions the top and bottom 1%) were fit to the quantile-quantile these data ( for earlier D described distribution of CV was determined and its parameters estimated using methods the First, points. of data set reduced on this based CV to the applied then was pairs. kinase-inhibitor remaining the for recomputed CV the and data of set current the from excluded were tions observa These observations. these represent region this within circles black 1e Figure Supplementary 36. 35. 34. kinases. all a and binding of 1 for the coefficient a Hill assumes tion (IC + (100/(1 − µ (100 = activity equation: the to according calculated Kinase analysis. activity extremevalues. and VGAM libraries using ment M))) and the Cheng-Prusoff equation Cheng-Prusoff the and M))) The distribution-based outlier detection method outlined by outlined method van der detection outlier Loo The distribution-based Computations were carried out in the R statistical language and environ and language statistical R the in out carried were Computations

a dr o, ... itiuinbsd ule dtcin o uiait data. (K constant inhibition univariate the between Relationship W.H. for Prusoff, & Y. detection Cheng, outlier Distribution-based M.P.J. Loo, der van models. linear generalized vector Reduced-rank T.J. Hastie, & T.W. Yee, h cnetain f niio wih ass 0 e cn ihbto (I inhibition cent per 50 causes which inhibitor of concentration the enzymatic reaction. enzymatic 2010). Hague, The Netherlands, (Statistics 10003 paper Discussion Modelling Supplementary Figure 1f Figure Supplementary Supplementary Fig. 1c

3 , 15–41 (2003). 15–41 , Biochem. Pharmacol. Biochem. 3 . Negative values for remaining kinase activity were were activity kinase remaining for values Negative . 4 The theoretical kinase activity curve in curve activity kinase The theoretical . The log-normal distribution . provided distribution The the best log-normal fit for also represent these limits whereas the green and and green the whereas limits these represent also , d ). For outlier detection, the data . 3

6 22 relating relating , 3099–3108 (1973). 3099–3108 , Supplementary Figure Supplementary K i and IC and doi:10.1038/nbt.2017 K m,ATP 50 . This calcula This . Figure 2 Figure of 10 of 10 ( excluding 50 µ o an of ) 1 50 M M for a and ) was Stat. /0.5 /0.5

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