Vol 462 24/31 Deemer 29 doi:1.138/nture8622 ovel mutnt-seletive kinse inhiitors ginst T79M Wenjun Zhou 1,2 *, Dli Ern 3,4 *, Ling Chen 3,4 *, Ci-Hong Yun 1,2 *, Dnn Li 3,4, Mrzi Cpelletti 3,4, Alexis B. Cortot 3,4, Luin Chirie 5, Roxn E. Io 6,7, Roert Pder 5, John R. Engen 6,7, Kwok-Kin Wong 3,4,8,9, Mihel J. Ek 1,2, thnel S. Gry 1,2 & Psi A. Jänne 3,4,8 The linil effiy of epiderml growth ftor reeptor () kinse inhiitors in -mutnt non-smll-ell lung ner (SCLC) is limited y the development of drug-resistne muttions, inluding the gtekeeper T79M muttion 1 3. Strtegies trgeting T79M with irreversile inhiitors hve hd limited suess nd re ssoited with toxiity due to onurrent inhiition of wild-type 4,5. All urrent inhiitors possess struturlly relted quinzoline-sed ore sffold nd were identified s ATP-ompetitive inhiitors of wild-type. Here we identify ovlent pyrimidine inhiitor y sreening n irreversile kinse inhiitor lirry speifilly ginst T79M. These gents re 3- to 1-fold more potent ginst T79M, nd up to 1-fold less potent ginst wildtype, thn quinzoline-sed inhiitors in vitro. They re lso effetive in murine models of lung ner driven y T79M. Co-rystlliztion studies revel struturl sis for the inresed poteny nd mutnt seletivity of these gents. These mutnt-seletive irreversile kinse inhiitors my e linilly more effetive nd etter tolerted thn quinzoline-sed inhiitors. Our findings demonstrte tht funtionl phrmologil sreens ginst linilly importnt mutnt kinses represent powerful strtegy to identify new lsses of mutnt-seletive kinse inhiitors. kinse inhiitors, gefitini nd erlotini, re effetive linil therpies for SCLCs tht hrour tivting muttions in the kinse domin 1,6. The most ommon muttions, L858R nd dele746_a75, imprt oth n inresed ffinity for gefitini or erlotini nd deresed ffinity for ATP reltive to wild-type (WT) 7,8. The linil effiy of gefitini or erlotini is, however, ultimtely limited y the development of quired drug resistne suh s y muttion of the gtekeeper T79 residue (T79M), whih is deteted in 5% of linilly resistnt ptients 2,3. Unlike the nlogous T315I muttion in ABL, whih introdues steri impediment for imtini inding, T79M only modestly ffets gefitini inding. However, more importntly, it restores the ffinity for ATP, similr to tht of WT 9. Most inhiitors re sed on 4-nilinoquinzoline ore sffold nd were initilly identified s ATP-ompetitive inhiitors of WT. They inlude irreversile inhiitors tht, unlike gefitini, ontin n eletrophili funtionlity tht undergoes Mihel ddition retion with onserved ysteine residue present in (Cys 797). The ovlent nture of these ompounds llows them to hieve greter oupny of the ATP site reltive to reversile inhiitors, thus providing the ility to inhiit T79M in prelinil models, despite the inresed ATP ffinity onferred y this seondry muttion 4,1,11. However, ll urrent irreversile inhiitors re less potent in ell-line models hrouring T79M thn those with n -tivting muttion lone (Supplementry Fig. 1) nd, t linilly hievle onentrtions, these gents do not inhiit T79M in vitro 1 13. Beuse the ATP ffinity of T79M is similr to WT, the onentrtion of quinzoline-sed inhiitors required to inhiit T79M will lso effetively inhiit WT. In ptients, this onurrent inhiition of WT results in skin rsh nd dirrhoe, nd limits the ility to hieve plsm onentrtions suffiient to inhiit T79M. Consequently, the linil effiy of the irreversile inhiitors CI-133, HKI-272 nd PF29984 hs een limited, espeilly in ptients with gefitinior erlotini-resistnt SCLC, nd the dose-limiting toxiity hs een dirrhoe nd skin rsh 5,14,15. We hypothesized tht the nilinoquinzoline sffold my not e the most potent or speifi for inhiiting T79M euse it relies on the smll size nd hydrogen onding intertions with the gtekeeper threonine of WT. We prepred foused lirry of ommon kinse inhiitor ore sffolds where one of the side hins ws modified with n rylmide group t position tht moleulr modelling predited to ret with Cys 797. This lirry ws sreened for ompounds tht ould inhiit the growth of oth gefitini-resistnt (PC9GR; dele746_a75/t79m) nd -sensitive (PC9; dele746_a75) ell lines ut were not toxi up to 1 mm ginst A549 (KRAS mutnt) or H3122 (EML4-ALK) ells. We ompred our findings with oth reversile (gefitini) nd irreversile inhiitors (CL-387,785 nd HKI-272). Three losely relted pyrimidines,, WZ42 nd WZ84, were identified from the sreen tht possessed up to 3-fold lower hlf-mximum inhiitory onentrtion (IC 5 ) ginst the PC9GR ells ompred with linil-stge inhiitors suh s HKI-272 (Fig. 1, nd Supplementry Tle 1). We oserved similr inresed poteny of the WZ ompounds in the H1975 (L858R/ T79M) ell line nd in B/F3 ells hrouring T79M (Fig. 1 nd Supplementry Tles 1 nd 2). The inresed ellulr poteny orrelted with inhiition of, nd ERK1/2 phosphoryltion in SCLC ell lines (Fig. 1 nd Supplementry Fig. 2) nd with the more potent inhiition of phosphoryltion y WZ42 in IH-3T3 ells expressing different T79M mutnt lleles (Fig. 1d nd Supplementry Fig. 3). The profile ginst ERBB2 ws mrkedly different: the WZ ompounds were less potent thn CL-387,785 or HKI-272 (Supplementry Tles 1 nd 2) nd did not inhiit ERBB2 1 Deprtment of Cner Biology, 2 Deprtment of Biologil Chemistry nd Moleulr Phrmology, 3 Lowe Center for Thori Onology, 4 Deprtment of Medil Onology, Dn- Frer Cner Institute, 44 Binney Street, Boston, Msshusetts 2115, USA. 5 Deprtment of Pthology, Brighm nd Women s Hospitl, Boston, Msshusetts 2115, USA. 6 The Brnett Institute of Chemil & Biologil Anlysis, 7 Deprtment of Chemistry nd Chemil Biology, orthestern University, Boston, Msshusetts 2115, USA. 8 Deprtment of Mediine, Brighm nd Women s Hospitl nd Hrvrd Medil Shool, Boston, Msshusetts 2115, USA. 9 Ludwig Center t Dn-Frer/Hrvrd Cner Center, Boston, Msshusetts 2115, USA. *These uthors ontriuted eqully to this work. 17 29 Mmilln Pulishers Limited. All rights reserved
ATURE Vol 462 24/31 Deemer 29 Y Conentrtion (µm) H X Cl X=O, Y=H WZ42 X=O, Y=OMe WZ84 X=S, Y=H p perk 1/2 ERK 1/2.1.1 1. H O WZ42 CL-387,785.1.1 1..1.1 1. IC 5 (nm) IC 5 (nm) d 1 8 6 4 2 1 8 6 4 2 Conentrtion (nm) EGF (1 ng ml 1 ) 1 8 6 4 2 4, 3, 2, 1, 1 8 6 4 4, 3, 2, 1, 1 8 6 4 2 2 H3255 PC9 H1975 PC9 GR 2 18 16 14 12 1 4, 3, 2, 1, 4, 3, 2, 1, 1 8 6 4 2 1 8 6 4 2 1 8 6 4 2 L858R E746_A75 L858R/T79M E746_A75/T79M Gefitini CL-387,785 HKI-272 WZ42 WZ84 WZ42 CL-387,785 Gefitini HKI-272 1 1 1, 1 1 1, 1 1 1, 1 1 1, + + + + + + + + + + + + + + + + Figure 1 WZ42, nd WZ84 re novel inhiitors, suppress the growth of -T79M-ontining ell lines nd inhiit phosphoryltion., Chemil strutures of the WZ ompounds., IC 5 vlues for SCLC ell lines (top) nd B/F3 ells (ottom), with genotypes orresponding to the SCLC ell lines, treted with indited drugs. Growth ws ssessed using the MTS (3-(4,5-dimethylthizol-2-yl)-5-(3- roxymethoxyphenyl)-2-(4-sulfophenyl)-2h-tetrzolium) survivl ssy., Comprison of, WZ42 nd CL-387,785 on signlling in phosphoryltion in 3T3 ells expressing the ERBB2 gtekeeper (T798I) muttion (dt not shown). Anlysis of reominnt T79M kinse inuted with y eletrospry mss spetrometry reveled stoihiometri ddition of one inhiitor moleule to the PC9 GR ells. The ells were treted with the indited onentrtions of eh drug for 16 h. Cell extrts were immunolotted to detet the indited proteins. d, Comprison of inhiitors on phosphoryltion in 3T3 ells expressing del E746_A75/T9M. The ells were treted with indited onentrtions of eh drug for 16 h nd stimulted with EGF (1 ng ml 21 ) 15 min efore lysis. Cell extrts were immunolotted to detet the indited proteins. protein. Anlysis of pepsin digest of the modified protein y tndem mss spetrometry identified Cys 797 s the site of modifition, thus verifying ovlent ond formtion etween nd (Supplementry Fig. 4). Perentge ontrol 125 1 75 5 25 CL-387,785 HKI-272 WZ42 WZ84 WZ42 CL-387,785 Gefitini HKI-272 1 1 1, Conentrtion (nm) EGF (1 ng ml 1 ) ++++ ++++ ++++ ++++ 1 1 1, 1 1 1, 1 1 1, 1 3 1 2 1 1 1 1 1 Drug onentrtion (µm) Erlotini WZ42 d 1 P <.1 P >.5 positive (%) 8 6 4 2 Figure 2 WZ42 is less potent thn quinzoline inhiitors ginst WT in vitro nd in vivo., viii B/F3 ells treted with WZ or quinzoline inhiitors. The men (n 5 6) nd stndrd devition is plotted for eh drug nd onentrtion., Comprison of inhiitors on phosphoryltion in 3T3 ells expressing WT. The ells were treted with indited onentrtions of eh drug for 16 h nd stimulted with EGF (1 ng ml 21 ) 15 min efore lysis. Cell extrts were 29 Mmilln Pulishers Limited. All rights reserved Erlotini WZ42 immunolotted to detet the indited proteins., Immunohistohemil nlysis of hir ul from erlotini- or WZ42-treted mie using nd py1173. Only tretment with erlotini results in signifint inhiition of phosphoryltion. Sle r, 5 mm. d, Quntifition of frequeny of phospho- stining from vehile- (n 5 3), erlotini- (n 5 3) nd WZ42- (n 5 2) treted mie. The mens nd stndrd devitions re plotted for drug tretment. 171
ATURE Vol 462 24/31 Deemer 29 We profiled nd WZ42 ginst pnel of 4 kinses using the Amit kinome sreening pltform (Supplementry Tle 3 nd Supplementry Fig. 5). For WZ42, kinses tht exhiited greter thn 95% inhiition reltive to the dimethylsulphoxide ontrol (Amit sore,5) t 1 mm were seleted for mesurement of their dissoition onstnts (Supplementry Tle 3). In ddition to, we oserved potent inhiition of severl of the ten kinses tht possess ysteine t the sme position s, inluding suset of the TECfmily kinses (Supplementry Fig. 6). Cross-retivity with BMX hs een reported for irreversile quinzoline-derived inhiitors 16. To onfirm whether the oserved inding tivity trnslted into ellulr inhiition, WZ42 nd were profiled ginst B/F3 ells trnsformed with TEL fusions of BMX, BLK, JAK2 nd JAK3. WZ42, whih possesses n ortho-methoxy group t the C2-niline sustituent, is more seletive for thn (Supplementry Tle 4). We next determined whether the inresed poteny of the WZ ompounds ginst mutnt lso pplied to WT. We used WT H11 ells 17 nd B/F3 ells hrouring the viii muttion, whih ontins WT kinse domin (Fig. 2 nd Supplementry Tle 2). These ompounds were 3- to 1-fold less potent, with WZ42 eing lest potent, thn CL-387,785 nd HKI-272 t inhiiting the growth of the WT ells. Furthermore, WZ42 ws 1-fold less effetive t inhiiting phosphoryltion of WT thn the quinzoline inhiitors (Fig. 2). Similrly, WZ42 inhiited kinse tivity of reominnt L858R/T79M protein more potently thn tht of WT, wheres the opposite ws oserved with HKI-272 nd gefitini (Supplementry Fig. 7, ). To understnd etter the poteny nd reltive seletivity for T79M over WT, we determined the rystl struture of WZ42 in omplex with T79M (Fig. 3,, Supplementry Fig. 8 nd Supplementry Tle 5). The ompound inds within the ATP-inding left of the enzyme, forming the expeted ovlent ond with Cys 797. As expeted sed upon o-strutures of relted pyrimidine-derived inhiitors with CDK2 (ref. 18), JK1 (ref. 19) nd FAK 2, the nilinopyrimidine ore of WZ42 forms identte hydrogen onding intertion with the hinge residue Met 793 (Fig. 3). The hlorine sustituent on the pyrimidine ring ontts the mutnt gtekeeper residue, Met 79. The hydrophoiity onferred y this muttion likely ontriutes to the poteny of these ompounds ginst the T79M mutnt. The niline ring forms hydrophoi intertion with the -ron of Gly 796 nd its methoxy sustituent extends towrds Leu 792 nd Pro 794 in the hinge region. The greter seletivity of the WZ42 ompound likely derives from the ft tht oth JAK3 nd TEC-fmily kinses hve ulkier residue (tyrosine in JAK3, phenyllnine in TEC-fmily kinses) in the position of Leu 792, whih would e expeted to interfere sterilly with the methoxy group in WZ42. The retive rylmide moeity nd the linking phenyl ring omprise the other rm of the inhiitor. The linker phenyl ring lies roughly perpendiulr to the pyrimidine ore; this orienttion juxtposes the rylmide with the thiol of Cys 797 for ovlent ond formtion (Fig. 3). We further determined whether WZ42 is effetive in vivo y using mouse lung ner models hrouring either L858R/T79M or Del E746_A75/T79M. We hose WZ42 for the in vivo studies euse in vitro it ws lest potent ginst WT nd other ysteine-ontining kinses (Supplementry Tles 2 nd 4) ut ws effetive ginst T79M. A phrmokineti study ws performed to determine the hievle plsm onentrtion (429 ng ml 21 ), hlflife (2.5 h) nd the orl iovilility (24%) of WZ42 (Supplementry Tles 6 8). In phrmodynmi study, WZ42 effetively inhiited, nd ERK1/2 phosphoryltion (Fig. 4), whih ws ssoited with signifint inrese in TdT-medited dutp nik-end lelling (TUEL)-positive nd signifint derese in Ki67-positive ells ompred with mie treted y vehile lone (Fig. 4, ). To evlute whether WZ42 imprted differentil effet on WT in vivo, we evluted phosphoryltion in the hir ul from mouse skin fter tretment with either erlotini or WZ42 172 Aniline Leu 792 Met 793 Pro 794 WZ84 29 Mmilln Pulishers Limited. All rights reserved Gly 796 Pyrimidine Methoxy Met 79 Methylpiperzine Arylmide Leu 718 WZ42 Vl 726 Cys 797 WZ42 Figure 3 Crystl struture of WZ42 ound to T79M., Chemil strutures of WZ84 nd WZ42 re shown shemtilly in mnner resemling the onformtion dopted in omplex with the kinse., Crystl struture of WZ42 in omplex with T79M mutnt (PDB ID 3IKA). WZ42 inds the tive onformtion of the kinse, with oth the regultory C-helix nd the DFG segment of the tivtion loop in their inwrd, tive positions. The kinse is shown in rion representtion (lue) with the ound inhiitor in yellow. Side-hin nd min-hin toms re shown for seleted residues tht ontt the ompound. Expeted hydrogen onds to the kone mide nd ronyl toms of Met 793 re indited y dshed lines. ote lso the ovlent ond with Cys 797. The struture ws refined to rystllogrphi R vlue of 21.3% (R free 5 25.4%) with dt extending to 2.9-Å resolution (see Methods for further rystllogrphi detils). (Fig. 2, d). Only erlotini signifintly inhiited phosphoryltion in the hir ul. In 2-week effiy study, WZ42 tretment resulted in signifint tumour regressions ompred with vehile lone in oth T79M-ontining murine models (Fig. 4d, e nd Supplementry Fig. 9). Histologil evlution of the lungs fter tretment onfirmed signifint resolution of the tumour nodules, with only few smll residul nodules nd nodule remnnts tht hd evidene of tretment effet with deresed ellulrity nd inresed firosis onsistent with remodelling/srring (Fig. 4f). There were no signs of overt toxiity ompred with mie treted y vehile lone during the study s ssessed y hnges in weight (dt not shown), serum retine nd totl white lood ell ount (Supplementry Fig. 1). Our studies identify novel struturl lss of kinse inhiitors tht re effetive in vitro nd in in vivo models hrouring the T79M muttion. Given the mrked tivity in models with estlished T79M, we determined whether WZ42 tretment ould lso prevent the development of T79M using in vitro models hrouring -tivting muttions. Unlike with gefitini or HKI-272, whih when used t their hievle plsm onentrtions led to development of T79M in vitro 13,21,22,
ATURE Vol 462 24/31 Deemer 29 p perk1/2 ERK1/2 Del E746_A75/T79M WZ42 umer of positive ells 2.5 mg kg 1 p perk1/2 ERK1/2 L858R/T79M TUEL 4 35 3 25 2 15 1 5 12 * 1 8 6 * 4 2 WZ42 WZ42 TUEL Ki67 Figure 4 WZ42 inhiits phosphoryltion nd indues signifint tumour regression in murine models of T79M., Two doses seprted y 16 h of WZ42 (2.5 mg kg 21 or 25 mg kg 21 ) or vehile were dministered to dele746_a75/t79m or L858R/T79M mie with MRI-onfirmed tumours. The mie were killed, the lungs isolted, grossly disseted nd sujeted to ell lysis. Cell extrts were immunolotted to detet the indited proteins., Immunohistohemil nlyses of tumours from dele746_a75/t79m mie from using indited ntiodies. Sle r, 5 mm., Quntifition of TUEL- nd Ki67-positive ells from tumour nodules (n 5 4) from vehile- nd WZ42-treted mie. The mens Ki67 d e Reltive tumour volume (%) 25 2 15 1 5 L858R/T79M w w P =.1 Del19/T79M 2w WZ42 2w P =.18 WZ42 WZ42 L858R/T79M f Del19/T79M L858R/T79M Del19/T79M w w 2w WZ42 2w WZ42 nd stndrd devitions re plotted. *, P,.5. d, MRI imges of vehile- or WZ42-treted mie t seline ( weeks: w) nd fter 2 weeks (2w) of tretment. e, Quntifition of the reltive tumour volume from MRI imges from vehile-treted mie (E746_A75/T79M (n 5 3); L858R/ T79M (n 5 4)), nd WZ42-treted L858R/T79M (n 5 3) nd E746_A75/T79M (n 5 3) mie. The mens nd stndrd devitions re plotted. f, Tumours from vehile- nd WZ42-treted mie stined with hemtoxylin nd eosin. Low-power view (inset) demonstrtes neromplete resolution of tumours in the WZ42-treted mie. Sle r, 1 mm. we were unle to isolte ny -T79M-ontining lones from WZ42-treted B/F3 or PC9 SCLC ells (Supplementry Tle 9). These findings suggest tht WZ42 ould lso e used s initil therpy for ptients with -mutnt SCLC nd my ultimtely led to longer time to disese progression thn urrently hieved with gefitini 1. Our rystllogrphi studies provide insight into why WZ42 is so muh more effetive ginst L858R/T79M thn HKI-272. Although oth shre the irreversile omponent, the nilinopyrimidine sffold of WZ42 is n intrinsilly etter fit for the mutnt gtekeeper methionine (Supplementry Fig. 11). To test this hypothesis further, we prepred WZ43, reversile nlogue of WZ42 tht is non-retive towrds Cys 797. WZ43 inds to the L858R/T79M mutnt 1-fold more tightly thn it does to the WT (Supplementry Tle 1), onfirming tht the sffold per se is indeed speifi for the mutnt kinse. Importntly, the WZ ompounds rely on ovlent ond formtion for potent ellulr inhiition, s evidened y the 1-fold inrese in the IC 5 of WZ42 ginst the C797S mutnts nd y the signifintly redued ellulr IC 5 of WZ43 ginst T79M ontining B/F3 ells (Supplementry Tle 2). These oservtions highlight the importne of using lirry of irreversile kinse inhiitors, s WZ43 would not hve een identified in the initil ellulr sreen. Muttions, inluding those t the gtekeeper residue, re ommon mehnism of drug resistne to kinse inhiitors. The urrent pproh, using ellulr sreen expressing the mutnt kinse of interest, n e pplied to identify novel gents speifilly ginst drug resistne or onogeni muttions implited in humn ners. Suh gents my truly e ner seletive, linilly more potent nd less toxi thn those tht lso onurrently inhiit the WT kinse. The gents desried here re unique in tht they inhiit oth the drug sensitizing nd resistne muttions ut re seletive ginst WT 29 Mmilln Pulishers Limited. All rights reserved 23 25. Further studies re needed to determine whether this lss of inhiitors will e linilly effetive in ptients with mutnt ners hrouring -T79M-medited quired drug resistne. METHODS SUMMARY Kinse inhiitors. Gefitini, CL-387,785 nd HKI-272 were otined from ommeril soures. The WZ ompounds were synthesized using four-step hemil synthesis tht is desried in detil in the Supplementry Methods. The finl produts were verified y 1 H nuler mgneti resonne nd liquid hromtogrphy mss spetrometry. Cell lines. wild-type nd mutnt SCLC, B/F3 ells nd IH-3T3 ells were ultured s previously desried 1. The PC9GR4 ells were generted s previously desried nd verified to ontin dele746_a75/t79m y diret sequening 21. Cell prolifertion nd growth ssys were performed using olorimetri ssy s previously desried 26. Site-direted mutgenesis ws performed using the Quik Chnge Site-Direted Mutgenesis kit (Strtgene) ording to the mnufturer s instrutions. kinse ssys. In vitro inhiitory enzyme kineti ssys using reominnt L858R/T79M nd WT protein nd were performed using the ATP/ ADH oupled ssy system in 96-well formt s previously desried 7. Crystl struture determintion nd refinement. The struture of WZ42 in omplex with T79M ws determined s previously desried 9. Mouse studies. All studies involving mie were pproved y the Dn-Frer Cner Institute Animl Cre nd Use Committee. -TL (T79M/L858R) mie were generted s previously desried 4. exon 19 deletion-t79m (TD)-induile itrnsgeni mie were similrly generted nd hrterized. Mie were treted either with vehile (1% 1-methyl-2-pyrrolidinone:9% PEG-3) lone or WZ42 t 25 mg kg 21 gvge dily. This ws followed y mgneti resonne imging (MRI) snning s previously desried 4,27. Histology, immunohistohemistry nd immunolotting nlyses were performed ording to stndrd protools, s previously desried 4,1. Genertion of drug-resistnt ells. -ethyl--nitrosoure mutgenesis ws performed using L858R nd DelE746_A75 B/F3 ells s previously desried 28. Treted ells were expnded in 1 nm WZ42, 1 mm WZ42, 173
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Aknowledgements This study is supported y grnts from the tionl Institutes of Helth RO1CA11446 (P.A.J.), R1CA135257 (P.A.J.), R1CA8942 (M.J.E.), R1CA13876-2 (.S.G.), R1CA1162 (M.J.E.), R1AG2441 (K.-K.W.), R1 CA122794 (K.-K.W.), R1GM759 (J.R.E.), tionl Cner Institute Lung SPORE P5CA9578 (P.A.J. nd K.-K.W.), the Ceily nd Roert Hrris Foundtion (K.-K.W.), Uniting Aginst Lung Cner (K.-K.W.), the Flight Attendnt Medil Reserh Institute (K.-K.W.)., the Hzel nd Smuel Bellin Reserh Fund (P.A.J.) nd the Dmon Runyon Foundtion Cner Innovtion Awrd (.S.G.). This work is ontriution 948 from the Brnett Institute. The uthors thnk Si Advntium Phrm for performing the phrmokineti studies. Author Contriutions W.Z., D.E., Li.C., C.-H.Y., D.L., M.C. A.B.C. designed experiments, onduted studies nd nlysed dt. R.E.I. nd J.R.E. performed nd nlysed the mss spetrometry studies. Lu.C. nd R.P. performed the histologil nd immunohistohemistry nlyses. M.J.E., K.-K.W.,.S.G. nd P.A.J. designed the experiments, nlysed dt nd wrote the mnusript. The Wong, Ek, Gry nd Jänne lortories ontriuted eqully to this work. Author Informtion Struturl dt re deposited in Protein Dt Bnk under ession numer 3IKA. Reprints nd permissions informtion is ville t www.nture.om/reprints. The uthors delre ompeting finnil interests: detils ompny the full-text HTML version of the pper t www.nture.om/ nture. Correspondene nd requests for mterils should e ddressed to.s.g. (thnel_gry@dfi.hrvrd.edu) or P.A.J. (pjnne@prtners.org). 174 29 Mmilln Pulishers Limited. All rights reserved