Kinase Inhibitors

7/27/2019 Kinase Inhibitors

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Kiass hav bcom o of th most itsivlprsd classs of drg targt ith approximatl 30distict kias targts big dvlopd to th lvl of aPhas I cliical trial. Th vast maorit of ths targts arbig ivstigatd for th tratmt of cacr. Hovr,drglatio of kias fctio has b implicatd iothr disordrs, icldig immological, rologi-cal, mtabolic ad ifctios disas. This has gr-atd cosidrabl itrst i th dvlopmt of smallmolcl kias ihibitors for th tratmt of thsdisordrs1. Th crrt poplarit of kiass as drg tar-gts is driv b th covrgc of svral factors. First,ith approximatl 518 kiass codd i th hmagom, virtall vr sigal trasdctio procss isird throgh a phosphotrasfr cascad, sggstigthat ihibitio of kias activit ca licit a ral phsi-ological rspos. Accordigl, ihibitio of th trosikias activit of th ocogic BCR(brakpoit clstrrgio)ABL1 fsio proti, formatio of hich is acasativ trasformig vt i chroic mloid lka-

mia (CML), is a validatd mas for thraptic itr-vtio2,3. Scod, dspit a high dgr of cosrvatioi th ATP bidig sit, highl slctiv small molclsith favorabl pharmactical proprtis ca b dvl-opd4. Third, srprisigl, ihibitio of kias activit iormal clls ca oft b tolratd, prstig a thra-ptic ido for th slctiv killig of tmor clls.For xampl, dasatiib, a drg rctl approvd for thtratmt ofimatiib-rsistat CML, pottl ihib-its all i mmbrs of th Src famil of kiass (admros othr trosi kiass), t xhibits a morfavorabl sid ffct profil tha covtioal ctotoxicchmothrap5,6. Forth, drgs sch as imatiib hav had

high-profil sccss, xhibitig p to 80% rspos ratsi chroic-phas CML patits7. Hovr, dspit thsmotivatig factors, th fild facs sigificat challgsfrom drg rsistac, lack of ihibitor slctivit, lackof ihibitor fficac ad difficlt i drg targt valida-tio for particlar disas sttigs. I this Rvi, xami th approachs crrtl i s to dsig ad

validat th xt gratio of kias ihibitors.

Kinases in cancer

Ocolog drg discovr has bfitd sigificatl fromprogrss i drstadig ho to targt kiass ithsmall molcls rlativ to othr disas idicatios. Oraso for this is that ma kiass hav b fod tob itimatl ivolvd i th procsss ladig to tmorcll prolifratio ad srvival. First, thr ar kias tar-gts that hav bcom imprvios to ormal rglatormchaisms folloig gtic mtatio or traslocatio(Spplmtar iformatio S1 (tabl) lists kiass thathav b itsivl ivstigatd). Ths kiass hav

trasformig capacit ad ar thrfor cosidrd tob ocogic. Th costittiv activit of this class ofkias targt maks thm sstial for srvival ad/orprolifratio of th cacr cll. This so-calld ocogaddictio8,9 rdrs th cacr cll xcptioall sscp-tibl to th appropriat kias ihibitor. Th sccss ofmtatioall markd kiass as drg targts has moti-

vatd a itsiv ffort to srv th kiom across abroad rag of tmor tps for mtatios. Ths std-is hav covrd a larg mbr of kiass barigmtatios, hich ar crrtl i th procss of bigfctioall charactrizd10,11. Prhaps th most otablsccss from ths fforts as th discovr thatPI3KCA

*DanaFarber Cancer

Institute, Department of

Cancer Biology, HarvardMedical School, Department

of Biological Chemistry and

Molecular Pharmacology,

250 Longwood Avenue,

Boston, Massachusetts

02115, USA.Harvard Medical School,

Department of Microbiology

and Molecular Genetics, 200

Longwood Avenue, Boston,

Massachusetts 02115, USA.

Correspondence to N.S.G.

e-mail: Nathanael_Gray@

dfci.harvard.edu

doi:10.1038/nrc2559

Targeting cancer with small moleculekinase inhibitors

Jianming Zhang*, Priscilla L. Yang and Nathanael S. Gray*

Abstract | Deregulation of kinase activity has emerged as a major mechanism by which

cancer cells evade normal physiological constraints on growth and survival. To date, 11

kinase inhibitors have received US Food and Drug Administration approval as cancer

treatments, and there are considerable efforts to develop selective small molecule inhibitors

for a host of other kinases that are implicated in cancer and other diseases. Herein we discussthe current challenges in the field, such as designing selective inhibitors and developing

strategies to overcome resistance mutations. This Review provides a broad overview of some

of the approaches currently used to discover and characterize new kinase inhibitors.

R E V I E W S

28 | jAnuARy 2009 | VOLuMe 9 www.au.cm/ws/cac

2009 Macmillan Publishers Limited. All rights reserved
http://www.uniprot.org/uniprot/P11274http://www.uniprot.org/uniprot/P00519http://www.cancer.gov/Templates/drugdictionary.aspx?CdrID=315885http://www.cancer.gov/Templates/drugdictionary.aspx?CdrID=37862http://www.nature.com/nrc/journal/v9/n1/suppinfo/nrc2559.htmlhttp://www.ncbi.nlm.nih.gov/sites/entrez?Db=gene&Cmd=ShowDetailView&TermToSearch=5290http://www.ncbi.nlm.nih.gov/sites/entrez?Db=gene&Cmd=ShowDetailView&TermToSearch=5290mailto:[email protected]:[email protected]:[email protected]:[email protected]://www.ncbi.nlm.nih.gov/sites/entrez?Db=gene&Cmd=ShowDetailView&TermToSearch=5290http://www.nature.com/nrc/journal/v9/n1/suppinfo/nrc2559.htmlhttp://www.cancer.gov/Templates/drugdictionary.aspx?CdrID=37862http://www.cancer.gov/Templates/drugdictionary.aspx?CdrID=315885http://www.uniprot.org/uniprot/P00519http://www.uniprot.org/uniprot/P11274


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Synthetic lethal

Two gn hav a

ynthtic-lthal phnotyp if

th individual gn dltion

ar not lthal but th

combind mutation i. A

ynthtic-lthal intraction i

proof of a gntic intraction.

is frqtl mtatd i mros cacrs ad that thsmtatios impart trasformig pottial in vitro as llas in vivo1214. Similarl, th V600e mtatio, locatdi th activatio loop ofBRAF, has b implicatd idvlopmt of carciomas of th ski, ovar, throid,colo ad pacras15,16. Th V617F activatig mtatioi th ato-ihibitor psdokias domai ofjAK2 isfrqtl fod i polcthamia vra, sstial throm-bocthamia, ad idiopathic mlofibrosis, ad hasstimlatd th rapid progrssio of svral jAK2 ihibi-tors ito Phas I stdis17. Rctl, both somatic adgrmli mtatios that activat aaplastic lmphomakias (ALK) hav b discovrd i roblastoma,a dvastatig childhood tmor for hich targtdthrapis ar rgtl dd18.

For a scod class of kias targt, ihibitio of thkias rslts i a ynthtic-lthal photp h pairdith aothr o-lthal mtatio i th particlarpatholog of th tmor cll19. Althogh ot ocogicad rarl mtatd i cacr, ths kiass ar prfr-tiall rqird for th srvival ad/or prolifratio ofcacr clls ad ma b locatd i k sigallig path-as dostram of trasformig ocogs. examplsicld MeK1 ad MeK2 (also ko as MAP2K1 adMAP2K2), hich ar locatd i th critical MAPK path-a20, mTOR (also ko as FRAP1), hich is locatdi th PI3KAkt sigallig sstm21, ad th ribosomal

S6 kias (RSK), hich is dostram of th fibroblastgroth factor (FGF) i th cotxt of FGF rcptor(FGFR)-dpdt prolifratio22. Othr xampls ofthis class of targts icld kiass that ar rqird tosstai rapid prolifratio ad/or srvival i th prs-c of abormal ploid, sch as th ccli-dpdtkiass (CDKs), hich rglat cll ccl trasitios; thArora kiass, hich ar sstial for accratl divid-ig chromosoms to ach daghtr cll; ad th Polo-likkiass, hich ar importat drig both mitosis adctokisis23. It shold b otd that th mchaistic basisfor slctiv ctotoxicit of ma ihibitors i this scodclass is poorl drstood.

A third class of kias targts ar xprssd i thtmor or i srrodig tisss ad ar rqird fordiffrt stags of tmor formatio ad maitaci th hma host. For xampl, th rotrophicgroth factor rcptor (nTRK2) is sstial for allo-ig som clls to srviv dtachmt ad ma alsob rqird for tmor cll mtastasis24. Additioalxampls icld th vasclar dothlial groth fac-tor rcptor (VeGFR) ad th FGFR kiass, hich arimportat i dvlopig ad sstaiig tmor bloodsppl25, as ll as th M2 splic isoform of prvatkias, hich is rqird for th tmorigic sitch toarobic glcolsis that occrs i cacr clls26.

For svral rasos, ihibitig mtatioall acti-vatd kiass has rsltd i th most dramatic clii-cal rsposs. As ths kiass ar markd b mtatioth ar thrfor th most radil idtifid b DnAsqcig 10,27. Frthrmor, a sbst of ths mtatdkiass ar thmslvs trasformig, makig it mchasir to gir mchaisticall rlvat clllar assasthat ca b sd drig th cors of ihibitor discov-

r ad optimizatio. Kias targts of th scod adthird classs ar ot dirctl trasformig bt istad arrqird for th srvival, prolifratio ad/or tmorig-sis of cacr clls. As sch, th ca b highl cotxtdpdt ad mch mor difficlt to ivstigat i cllcltr sstms. Th cotxt-dpdt rol of kiassi tmorigsis provids opportitis ad challgsfor thraptic targt slctio. A rct xampl is thatPTen-dficit cacrs dpd o PI3K to sstaiactivatio of th PI3K patha, hras PI3K kiasactivit appars to b rqird to sstai th prolifratioof stablishd tmors28,29 Tpicall, kiass that ca btargtd throgh a sthtic-lthal itractio spcific tocacr clls mst b ivstigatd ad validatd b a larglmpirical procss. Likis, kias targts rqird fortmorigsis ar sall valatd i aimal tmormodls that ar oft ot accrat rflctios of th dis-as voltio i hmas, so thr is a d for gtictmor modls that ca mor accratl rcapitlat thdiffrt stags of tmor progrssio ad ca b sd toivstigat th impact of slctiv kias ihibitors.

Kinase inhibitor binding sites

Proti kiass ar dfid b thir abilit to catals thtrasfr of th trmial phosphat of ATP to sbstratsthat sall cotai a sri, throi or trosi rsid.Th tpicall shar a cosrvd arragmt of scodar

strctr lmts that ar arragd ito 12 sbdomaisthat fold ito a bi-lobd cataltic cor strctr ith ATPbidig i a dp clft locatd bt th lobs 3032.ATP bids i th clft ith th adi rig formighdrog bods ith th kias hig th sgmtthat cocts th amio- ad carbox-trmial kiasdomais. Th ribos ad triphosphat grops of ATPbid i a hdrophilic chal xtdig to th sbstratbidig sit that fatrs cosrvd rsids that arsstial to catalsis. All kiass hav a cosrvd activa-tio loop, hich is importat i rglatig kias activ-it ad is markd b cosrvd DFG ad APe motifs(hich rfr to o-lttr amio acid abbrviatios) at

A a gac

Small-moleculekinaseinhibitorsarebeingintensivelypursuedasnewanticancer

therapeutics.Todate,approximately80inhibitorshavebeenadvancedtosomestage

ofclinicalevaluation.

Understandingthestructuralbasisofkinaseinhibitorselectivityiscrucialtothe

ultimategoalofdevelopingselectiveinhibitorstotargeteverymemberofthekinome.

MostcurrentlyknownkinaseinhibitorstargettheATPbindingsitewiththekinase

activationloopintheactive(type1)orinactive(type2)conformation.

Newkinaseinhibitorsareprimarilydevelopedwithacombinationofmethods,

includinghigh-throughputscreeningusingbiochemicalorcellularassays,analogue

synthesis,structure-guideddesignandfragment-basedassemblystrategies.

Therepertoireofkinasestargetedbyagiveninhibitorcanbedeterminedbyprofiling

itsactivityinbindingandenzymaticassaysagainstextensivepanelsofrecombinant

kinases,byprofilingactivityincellularassaysandbyaffinityapproachesintegrated

withdetectionbymassspectrometry.

Kinaseinhibitorresistanceresultingfromselectionformutantallelesorupregulation

ofalternativesignallingpathwaysisarecurrentthemeintheclinic.Strategiesfor

developingmultipleinhibitorstargetingdifferentkinasesitesandfordiscovering

synergisticinhibitorcombinationsareurgentlyneeded.

R E V I E W S

nATuRe ReVIewS |CAnCer VOLuMe 9 | jAnuARy 2009 |29

http://www.uniprot.org/uniprot/P15056http://www.uniprot.org/uniprot/O60674http://www.ncbi.nlm.nih.gov/entrez/dispomim.cgi?id=263300http://www.ncbi.nlm.nih.gov/entrez/dispomim.cgi?id=187950http://www.ncbi.nlm.nih.gov/entrez/dispomim.cgi?id=187950http://www.ncbi.nlm.nih.gov/entrez/dispomim.cgi?id=254450http://www.uniprot.org/uniprot/Q9UM73http://www.ncbi.nlm.nih.gov/entrez/dispomim.cgi?id=256700http://www.uniprot.org/uniprot/P42345http://www.uniprot.org/uniprot/Q16620http://www.uniprot.org/uniprot/P14618http://www.uniprot.org/uniprot/P14618http://www.uniprot.org/uniprot/P60484http://www.uniprot.org/uniprot/P42338http://www.uniprot.org/uniprot/P42338http://www.uniprot.org/uniprot/P42338http://www.uniprot.org/uniprot/P60484http://www.uniprot.org/uniprot/P14618http://www.uniprot.org/uniprot/P14618http://www.uniprot.org/uniprot/Q16620http://www.uniprot.org/uniprot/P42345http://www.ncbi.nlm.nih.gov/entrez/dispomim.cgi?id=256700http://www.uniprot.org/uniprot/Q9UM73http://www.ncbi.nlm.nih.gov/entrez/dispomim.cgi?id=254450http://www.ncbi.nlm.nih.gov/entrez/dispomim.cgi?id=187950http://www.ncbi.nlm.nih.gov/entrez/dispomim.cgi?id=187950http://www.ncbi.nlm.nih.gov/entrez/dispomim.cgi?id=263300http://www.uniprot.org/uniprot/O60674http://www.uniprot.org/uniprot/P15056


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|

N

N N O

CH3

N

Cl

Cl

S

H3C

N

O

H

H

O HN

O

NH

ON

HO

Hydrophobicpocket I

Hydrophobicpocket I

Adenine site

Hydrophobicpocket II

Hydrophobicpocket II

Hinge

Hinge

Hinge

Hinge

T315 gatekeeper

DFG motif

DFGmotif

DFG motif

Activationloop

Activationloop

Allosteric site

a

b

N

N

N

HN

N

O

H

O HN

O

NH

O

HO

N

O

H

N

N

CH3

O O

H3C N

O

O

NH

H

O

Allosteric

site

F381

D380

H

N

H

Heterocycle

An organic compound with a

ring tructur containing

non-carbon atom uch a

ulphur, oxygn or nitrogn a

part of th ring.

Pharmacophore

A molcular framwork that

carri th ntial fatur

rponibl for th biological

activity of a drug.

th start ad d of th loop, rspctivl. Th activa-tio loop ca assm a larg mbr of coformatiosith th xtrms big a coformr that is catalticallcomptt ad sall phosphorlatd, ad a iac-tiv coformr i hich th activatio loop blocks thsbstrat bidig sit. Most kias ihibitors discov-rd to dat ar ATP comptitiv ad prst o tothr hdrog bods to th amio acids locatd i thhig rgio of th targt kias, thrb mimickig thhdrog bods that ar ormall formd b th adirig of ATP32,33(FIG. 1). Th maorit of kias ihibi-tors do ot xploit th ribos bidig sit (a xcptiobig AZD0530, a ovl Src ad Abl dal famil kiasihibitor34) or th triphosphat bidig sit of ATP.

Type 1 inhibitors. This tp of ihibitor costitts thmaorit of ATP-comptitiv ihibitors ad rcogizsth so-calld activ coformatio of th kias (FIG. 1a), acoformatio othris codciv to phosphotrasfr33.Th prpodrac of tp 1 ihibitors ma b a co-sqc of ma compods havig b discovrd

sig zmatic assas i hich kiass r itrodcdi thir activ coformatio ad bcas ma kiasihibitors hav b sthsizd to mimic ATP (ad achothr). Tp 1 ihibitors tpicall cosist of a htrocyclicrig sstm that occpis th pri bidig sit, hr itsrvs as a scaffold for sid chais that occp th adacthdrophobic rgios I ad II (FIG. 2).

Type 2 inhibitors. B cotrast, tp 2 kias ihibi-tors rcogiz th iactiv coformatio of th kias(FIG. 1b). Th coformatio that is rcogizd b tp 2ihibitors is somtims rfrrd to as DFG-ot oig toth rarragmt of this motif. Movmt of th acti-

vatio loop to th DFG-ot coformatio xposs aadditioal hdrophobic bidig sit dirctl adact toth ATP bidig sit. Th origial discovr that ihibi-tors sch as imatiib ad sorafib bid i th tp 2coformatio as srdipitos, bt sbsqt aalsisof mltipl tp 2 kias ihibitor co-crstal strctrshas rvald that all shar a similar pharmacophor adxploit a cosrvd st of hdrog bods (FIG. 1b).

Figure 1 | Kas b bdg mds. Kinase inhibitorprotein interactions are depicted by ribbon structures (left)

and chemical structures (right). The chemical structures depict hydrophobic regions I and II of ABL1 (shaded beige and

yellow respectively) and hydrogen bonds between the kinase inhibitor (inhibitor atoms engaged in hydrogen bonds to

hinge are highlighted in green or to allosteric site in red) and ABL1 are indicated by dashed lines. The DFG motif (pink),

hinge and the activation loop of ABL1 are indicated in the ribbon representations. The kinase inhibitors are shown in light

blue. a |ABL1 in complex with the type 1 ATP-competitive inhibitor PD166326 (Protein Data Bank (PDB) ID 1OPK)104.

Shown here is the DFG-in conformation of the activation loop (dark blue). b |The DFG-out conformation of the activation

loop of ABL1 (dark blue) with the type 2 inhibitor imatinib (PDB ID 1IEP)105. The allosteric pocket exposed in the DFG-out

conformation is indicated by the blue shaded area (right).

R E V I E W S


http://www.cancer.gov/Templates/drugdictionary.aspx?CdrID=453588http://www.cancer.gov/Templates/drugdictionary.aspx?CdrID=299013http://www.rcsb.org/pdb/explore.do?structureId=1OPKhttp://www.rcsb.org/pdb/explore/explore.do?structureId=1IEPhttp://www.rcsb.org/pdb/explore/explore.do?structureId=1IEPhttp://www.rcsb.org/pdb/explore.do?structureId=1OPKhttp://www.cancer.gov/Templates/drugdictionary.aspx?CdrID=299013http://www.cancer.gov/Templates/drugdictionary.aspx?CdrID=453588


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http://www.rcsb.org/pdb/explore/explore.do?structureId=1O6Lhttp://www.uniprot.org/uniprot/P48736http://www.cancer.gov/Templates/drugdictionary.aspx?CdrID=435988http://www.uniprot.org/uniprot/P10721


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http://www.rcsb.org/pdb/explore/explore.do?structureId=1S9Jhttp://www.uniprot.org/uniprot/P04626http://www.uniprot.org/uniprot/P00533http://www.uniprot.org/uniprot/P62942http://www.cancer.gov/Templates/drugdictionary.aspx?CdrID=42555


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http://www.rcsb.org/pdb/explore/explore.do?structureId=1SM2http://www.nature.com/nrc/journal/v9/n1/suppinfo/nrc2559.htmlhttp://www.uniprot.org/uniprot/Q06187http://www.uniprot.org/uniprot/P35968http://www.nature.com/nrc/journal/v9/n1/suppinfo/nrc2559.html


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Polyketide

scondary mtabolit from

bactria, fungi, plant and

animal with divr biological

activiti and pharmacological

proprti.

Isostere

A chmical group with imilar

iz and lctronic proprti.

Homology model

A cla of mthod for

contructing a

thr-dimnional tructur of

a targt protin for which only

th qunc i availabl,

providd at lat on mpirical

thr-dimnional tmplat

tructur with >30% qunc

idntity i availabl.

IC50

Inhibitor concntration for

half-maximum rpon.

EC50

effctor concntration for

half-maximum rpon.

Enrichment factorTh ratio of th numbr of

biologically activ compound

dicovrd uing

computationally bad

lction to th numbr found

by random crning.

Chemical space

Th pac pannd by all

poibl (that i, nrgtically

tabl) toichiomtrical

combination of lctron and

atomic nucli and topologi

(iomr) in molcul and alo

compound in gnral.

sd to prdict a bidig mod of th ihibitor to a thr-dimsioal homolog modl of th kias. Aalogsar th prpard to modlat potc, slctivit adpharmacological proprtis. Isostric rplacmts arsd to improv ths proprtis hil prsrvig thstrolctroic cotacts rsposibl for bidig toth kias. For xampl, mros mdicial chmis-tr fforts hav spad scod-gratio ihibitorsfrom th progitor compod PD180970 (ReF. 59), aprido[2,3-d]primidi class compod that pottlihibits a varit of trosi kiass. Ths icldPD173074, a slctiv ihibitor ofFGFR1, FGFR3 adFGFR4 (ReF. 60); BI-2356, a slctiv ihibitor of th Polo-lik kiass61; ad BI-D1870, a ihibitor of RSK62. Allthr compods icorporat th prido[2,3-d]primi-di cor motif(BOX 2) bt driv slctivit from sidchai sbstittios. Drig th cors of compod opti-mizatio, it is tpicall importat to moitor strctractivit rlatioships i both biochmical ad clllarkias assas bcas th biochmical kias assa illfrqtl ot prdict hich compods xhibit optimalclllar activit. Althogh cll prmabilit ad itracl-llar accmlatio ca somtims accot for ths dif-frcs, i othr cass it appars that th phsiologicallrlvat form of th kias is ot accratl rflctd bth biochmical kias assa, spciall h ol thkias domai is sd. For xampl, th isli-lik

groth factor rcptor 1 (IGF1R) ihibitor Aew541 dis-plas almost idtical potc for ihibitio of IGF1R(IC

50= 150 M) ad isli rcptor (IC

50= 140 M) i

biochmical kias assas; hovr, i clllar assas thcompod is 25-fold mor slctiv for IGF1R vrssth isli rcptor (eC

50, IGFR= 86 M)63.

Structure-informed design. I additio to gidig aa-log sthsis, strctr-iformd dsig is sd idivrs as to facilitat ihibitor discovr ad opti-mizatio. Prhaps th most frqt s is to dtrmith k itractios formd bt th ihibitor ad thactiv sit ad to lar hr fctioalit ma

b itrodcd to modlat potc or slctivit.Altrativl, crstal strctrs ca b sd to dsighbrid compods i hich a portio of o ihibitor isgraftd oto a portio of aothr, gidd b sprimpo-sitio of th bod strctrs. This ca b a spciallffctiv a to crat tp 2 ihibitors b grat-ig molclar amalgams bt ko tp 1 ad 2ihibitors33,64. Hbrid dsig ca also ivolv graftig aslctivit dtrmiat from o ihibitor sris otoaothr. Crstal strctrs ar also sd for virtal ligadscrig fforts, i hich ligads ar discovrd sigcomptatioal approachs. Hovr, th nrichmntfactor that ar drivd from ths fforts ar ot sfficitto mak this approach grall applicabl65.

Fragment-based inhibitor discovery. Fragmt-basdihibitor discovr ivolvs idtificatio of moitisthat bid to diffrt portios of th activ sit of th tar-gt kias follod b covalt likag of ths moitisto crat a ihibitor tmplat66. Fragmts that bidto th activ sit of itrst grall origiat i a librar

ad ca b slctd comptatioall or xprimtallsig nMR or crstallograph. Tpical fragmts ar oflo molclar mass (


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|

N

N N

N

HN

O

CH3

O

HN O

N

CH3

H3C

N

N N

N

HN

O

CH3

OH

F F

N

N N NHHN

OHN

O

O

N

CH3

H3C

N

N N OHN

CH3

Cl

Cl

F

PD180970

Pan-tyrosinekinase inhibitor PD173074

FGFR1, FGFR3and FGFR4

BI-2356

PLK1, PLK2and PLK3

BI-D1870

RSK1, RSK2, RSK3and RSK4

Bisindolylmaleimide

Two fud indol ring bridgd

by a malimid that can act a

an ATP-comptitiv kina

inhibitor.

stdis agaist slctd kiass of itrst. Ths, dspitth availabilit of ths larg pals, ol limitd slc-tivit data ar availabl for ma commol sd kiasihibitors68. Hovr, a sris of paprs xamiig thkias spcificit profils of ma cliical ad prcliicalstag kias ihibitors hav rctl b pblishd70,71.For xampl, bidig assas r sd to dmostratthat VX-680 (ReF. 72), a primidi-drivd Arorakias ihibitor, also bids to th cliicall impor-tat T315I mtat of BCRABL1, hich is rsistatto crrtl approvd ihibitors70. Charactrizatioof th clllar activit agaist T315I BCRABL1 adco-crstallograph ith a ABL1 mtat73 spportdthis fidig ad hav motivatd sbsqt cliicalivstigatio.

A scod approach cosists of masrig th abilitof a tst compod to altr th mltig tmpratr (T

m)

of a kias74. Th largr th shift i Tm, th tightr thbidig affiit of th ihibitor is to th kias, as mai-fstd b icrasd stabilizatio of kias trtiar strc-tr. This approach as rctl sd to discovr thatLy333531, a ptativl highl slctiv biindolylmalim-id proti kias C ihibitor that is big dvlopdfor diabtic rtiopath, also possssd pott ihibitoractivit agaist Pim famil kiass, hich ar pottiallkamia targts74.

Clllar slctivit of kias ihibitors ca b val-atd ithr sig pals of cll lis that ar girdto rport o ihibitio of a particlar kias or sigbiasd protomics approachs. Ma sch cll lis

hav b dvlopd from th mri Ba/F3 cll li75.This pro-B-cll li ormall rqirs th ctokiitrlki 3 (IL-3) to prolifrat. Trasformatio b aocogic kias, hovr, lads to IL-3-idpdtprolifratio ad srvival sch that th rat of cllgroth ad prolifratio ca b sd as a rad-ot ofitraclllar kias ihibitio. Ctotoxicit that rsltsfrom spcific kias ihibitio ca b discrimiatd fromo-spcific ctotoxicit b rsc throgh th itro-dctio of IL-3. Pals of kias-trasformd Ba/F3 cllshav b cratd b sig ithr atrall occrrigocogic fsio protis (for xampl, BCRABL1 adnPMALK) or b cratig artificial fsios btkias domais ad th proti mltimrizatio domaiTl. Th Tl domai as sd bcas it is fod i sv-ral atrall occrrig fsio protis ad it xhibits astrog abilit to mltimriz fsd protis76. Othr cll

lis hav b gratd throgh th s of rportrg assas. ubiasd protomics-basd approachs forcharactrizig kias ihibitor slctivit ivolv ithrtraditioal affiit chromatograph77 or comptitivdisplacmt assas. For xampl, a comptitiv dis-placmt assa as stablishd i hich a affiitbad rsi barig sv diffrt ATP-comptitiv lig-ads that displad littl slctivit as dmostratdto b abl to captr hdrds of diffrt kiass fromHLa cll xtracts78. Th iclsio i th cll lsat of atst ihibitor of itrst that spcificall bids its targtrdcs th amot of fr targt availabl for captro th rsi. This tcholog as rctl sd to mak

Box 2 | Methods to create selective kinase inhibitors

Syss aagus ad ssc acm

Pyrido[2,3-d]pyrimidinessuchasPD180970(seethefigure)wereoriginally

identifiedasinhibitorsoftheSrcfamilykinasesbutweresubsequently

determinedtobehighlypromiscuoustyrosinekinaseinhibitors.Crystallizationof

PD180970withSRCrevealedthatthephenylaminopyrimidinemotifbindstothe

hingeandthedichlorophenylringissituatedinhydrophobicmotifII.Focused

medicinalchemistryeffortstovarytheanilineanddichlorophenylsubstitutentsresultedintheidentificationoffunctionalitythatmaintainedpotencyagainst

fibroblastgrowthfactorreceptors(FGFRs)yetimpartedahighdegreeofkinase

selectivity95.SubsequentcrystallographyofPD173074withFGFR1in

conjunctionwithexaminationofstructureactivityrelationshipsrevealedthat

the3,5-dispositionofthemethoxygroupsappearstobeakeyselectivity

determinantthatisuniquelytoleratedbyFGFR 60.Ahighlyselectiveinhibitorof

thePLKs,BI-2356(ReF. 61),wascreatedbyreplacingthepyrimidoneringof

PD180970withasaturatedsix-memberedringcontainingacyclizedaminoacid.

Herethekeyselectivitydeterminants,asdeterminedbycrystallographyand

analoguesynthesis,aredistributedthroughoutthestructureandincludethe

orthomethoxygroup,theN-methylgroupandtheethyl-substitutedchiral

centre96.AninhibitorofRSKs,BI-D1870,wasindependentlyderivedfromthe

samescaffoldasBI-2356byvaryingtheside-chainfunctionalgroups 62.

Sucu-md dsg

Thelargenumberofavailableinhibitorkinaseco-crystalstructuresmakesitpossibletograftstructuralfeaturesthatarepresentinonestructureonto

another.Forexample,newtype2inhibitorshavebeencreatedbyintroducinga

3-trifluoromethylbenzamidegrouptooccupytheallostericbindingpocket

(FIG. 1).Thisapproachallowedthecreationofanewtype2inhibitorwitha

completelydifferentselectivityprofile64.Asecondexampleisthedevelopment

ofapotentPI3KorPI3Kinhibitor,PIK-294,throughintroductionofa

3-hydroxyphenylgroupthatoccupieshydrophobicregionII(theso-called

affinitypocketforPI3Ks)37.

R E V I E W S

nATuRe ReVIewS |CAnCer VOLuMe 9 | jAnuARy 2009 |35

http://www.cancer.gov/Templates/drugdictionary.aspx?CdrID=485305http://www.uniprot.org/uniprot/P08700http://www.uniprot.org/uniprot/P06748http://www.uniprot.org/uniprot/P06748http://www.uniprot.org/uniprot/P08700http://www.cancer.gov/Templates/drugdictionary.aspx?CdrID=485305


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Steric clash

Atom contain an lctron hll

with a dfind radiu that

prvnt atom not ngagd

in a covalnt bond from

occupying th am volum.

A tric clah occur whn two

atom ar placd clor than

th um of thir atomic radii.

Farnesyltransferase activity

Pot-tranlational modification

of protin that conit of

attaching an ioprnyl group to

a cytin ridu.

th imprssiv discovr that imatiib, prhaps o of thmost xtsivl ivstigatd kias ihibitors, had a pr-

viosl sspctd additioal rcptor trosi kiastargt: th discoidi domai rcptor DDR1(ReFs 78,79).

evalatio of kias ihibitor slctivit o aorgaismal lvl rmais a sigificat rsarch chal-lg. Crrt fforts ar sall limitd to dtrmi-ig drg distribtio to varios orga compartmtsad moitorig a pharmacodamic markr, sch as aparticlar phosphorlatio sit. w ko from stdissig isolatd cll lis that diffrt lisca xhibitdramaticall diffrt rsposs to a giv ihibitor, soit is clar that d mthods to globall moitorth chags i phosphorlatio that rslt from kiasihibitio i th cotxt of a livig orgaism. This illallo mor accrat valatio of o- ad off-targtffcts of th ihibitor that ca b valatd i rlatioto fficac ad toxicit.

Kinase inhibitor resistance mechanisms

As ma kias ihibitors xrt thir ctotoxic ffcts

primaril b ihibitig a spcific kias, thr is a strogslctiv prssr for clls to acqir rsistac throghmtatios i th kias g that abrogat drg bid-ig. Additioal o-mtatio kias ihibitor rsistacmchaisms hav b docmtd, icldig targtamplificatio i th cas of BCRABL1 i CML patits80ad prglatio of altrativ kias pathas sch ashpatoct groth factor rcptor i th acqisitioof rsistac to eGFR kias ihibitors that has bobsrvd i lg cacr81. Oig to th rapid prolifra-tio of cacr clls, th acqisitio of mtatios cofr-rig drg rsistac has bcom a rcrrig thm ith cliic. Idd, rsistac as a rslt of kias mta-tios has b docmtd for ihibitors of BCRABL1(Spplmtar Iformatio S4 (figr)), eGFR, FLT3,KIT ad PDGFR50,8286. To dat th most xtsiv clii-cal ad laborator charactrizatio of rsistac-casigmtatios has b prformd for BCRABL1 i thcotxt of imatiib ad scod-gratio ihibitors.Additioall, it has b sho i svral hamatologi-cal tmors that qisct stm clls ar rfractor totrosi kias ihibitors, ad ths cll poplatios arprobabl also ivolvd i rsistac mchaisms87,88.

Ihibitor rsistac cofrrd b mtatio at th gat-kpr rsid so calld bcas th siz of th amioacid sid chai at this positio dtrmis th rlativaccssibilit of a hdrophobic pockt locatd adact to

th ATP bidig sit (hdrophobic pockt II, FIG. 1) appars to b a commo thm for a varit of kiass.Accss to this pockt is importat to ma kiasihibitors bcas hdrophobic itractios i this sitar crcial for th bidig affiit of th ihibitor. Forxampl, th most rcalcitrat of th BCRABL1 mtatsis T315I, hich harbors a mtatio i th gatkprrsid. Althogh th gatkpr rsid oft comsi clos cotact ith tp 1 ad tp 2 ATP bidigsit ihibitors, it tpicall dos ot itract ith ATP 33.Cosqtl, mtatio of th gatkpr rsid gr-all cass littl or o chag i kias activit bt has thpottial to cofr ihibitor rsistac throgh a varit

of biochmical mchaisms. Th T315I gatkpr mta-tio i BCRABL1 impds drg bidig throgh loss ofa crcial hdrog bodig itractio to th ihibitorad itrodctio of a tric clah. Mtatio of th eGFRT790 rsid to mthioi idcs rsistac to thqiazoli-basd ihibitors gfitiibadrlotiib50,89 bicrasig th affiit for ATP, hich ffctivl aksth affiit of ATP-comptitiv ihibitors.

Gatkpr mtatios hav b fod to cofrihibitor rsistac i a mbr of additioal cass. Thgatkpr mtatio G697R i FLT3 idcs rsistacto th tp 1 starospori drivativPKC412(ReF. 86). Asstmatic ivstigatio of gird gatkpr mta-tios i ABL1, PDGFR, SRC ad FGFR1 dmostratdthat rsistac ca b achivd agaist a varit of slc-tiv ad o-slctiv ihibitors of ths kiass90. Forxampl, th T341M mtatio of SRC rslts i rsist-ac to th tp I prido[2,3-d]pridio PP58, mta-tio of T681I of PDGFR cofrs rsistac to bothPP58 ad imatiib, ad th V561M mtatio of FGFR1cofrs rsistac to PP58 (ReF. 90). Thr is crrtl

dbat as to hthr th gatkpr mtatio is pr-xistig or acqird folloig ihibitor tratmt. Thgatkpr mtats of BCRABL1 (T315I), PDGFR(T674I), eGFR (T790M) ad KIT (T670I) ar th mostfrqtl rportd mtats for ach of ths kiass.This sggsts that mtatio of th gatkpr amioacid is likl to b a commo occrrc i cliicalkias ihibitor rsistac ad that gral mthods todsig ihibitors that ca ovrcom this mtatio oldb sfl.

Svral stratgis ar big ivstigatd to ovrcomkias ihibitor rsistac mtats. A first approach isto dvlop ihibitors that ca tolrat divrs amioacids at th gatkpr positio as discssd for T315IBCRABL1 ihibitors i BOX 3. A scod approach is totargt th kias ith ihibitors that bid at altrativbidig sits. For xampl, th ABL1 sbstrat bidigsit has b targtd b On012380, a vil slpho-cotaiig ihibitor91,92, ad th mristat bidig sithas b targtd b th GnF2 class of ihibitors 39. Athird approach ivolvs targtig othr pathas thatma b rqird for BCRABL1-mdiatd trasforma-tio, sch as th chapro fctio of HSP90 (ReF. 93) orfarnyltranfra activity94 Ths approachs hav bdmostratd to ork i cll cltr ad fforts arcrrtl dra to appl thm cliicall.

PerspectivesKias ihibitor drg discovr has progrssd dramati-call i th past dcad. I additio to th cliical approvalof a mbr of drgs, fficit approachs for thdvlopmt of pott ad slctiv ihibitors ith dsir-abl proprtis hav bcom stablishd. Kias ihibitordsig has bfitd immsl from crstallograph ith lcidatio of bidig mods ad xpctd ihib-itor-idcd coformatioal rarragmts. Idd,mch of crrt kias ihibitor discovr taks placthrogh ratioal drg dsig rathr tha throgh high-throghpt scrig ad mpirical optimizatio o thbasis of strctractivit rlatioships. Sophisticatd

R E V I E W S


http://www.uniprot.org/uniprot/Q08345http://www.uniprot.org/uniprot/P08581http://www.nature.com/nrc/journal/v9/n1/suppinfo/nrc2559.htmlhttp://www.uniprot.org/uniprot/P36888http://www.cancer.gov/Templates/drugdictionary.aspx?CdrID=43649http://www.cancer.gov/Templates/drugdictionary.aspx?CdrID=38428http://www.cancer.gov/Templates/drugdictionary.aspx?CdrID=462346http://www.uniprot.org/uniprot/P09619http://www.uniprot.org/uniprot/P09619http://www.uniprot.org/uniprot/P12931http://www.uniprot.org/uniprot/P16234http://www.uniprot.org/uniprot/P16234http://www.uniprot.org/uniprot/P16234http://www.uniprot.org/uniprot/P12931http://www.uniprot.org/uniprot/P09619http://www.cancer.gov/Templates/drugdictionary.aspx?CdrID=462346http://www.cancer.gov/Templates/drugdictionary.aspx?CdrID=38428http://www.cancer.gov/Templates/drugdictionary.aspx?CdrID=43649http://www.uniprot.org/uniprot/P36888http://www.nature.com/nrc/journal/v9/n1/suppinfo/nrc2559.htmlhttp://www.uniprot.org/uniprot/P08581http://www.uniprot.org/uniprot/Q08345


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protomic approachs hav b dvlopd i coc-tio ith pals of zm assas to allo for a mor thor-ogh aotatio of kias ihibitor slctivit. It is fairto sa that th chmical challg of makig pott adslctiv ihibitors ith good pharmacological propr-tis ca b ovrcom for most kiass of itrst sigcrrtl availabl tchologis. Sigificat advacs havb mad to discovr ad validat th kias targts thatar k ocogic drivrs ad thrfor most appropriatto targt ith ihibitors. Hovr, dspit ths advacsa mbr of sigificat challgs rmai. First, mtatihibitor rsistat kiass sm to volv qickl, adappropriat mlti-targtd ihibitors or combiatiosd to b plad i advac of cliical applicatio.

Scod, th mchaistic basis of xpctd toxici-tis obsrvd drig th prcliical ad cliical stagsof ihibitor dvlopmt d to b stdid mor rig-orosl. Improvd docmtatio of kias ihibitorspcificitis, mtabolits ad obsrvd toxicitis oldprovid a valabl databas for drstadig hthrthr ar particlar kiass of hich ihibitio sholdb avoidd or spcific sbstrctrs that rslt i prob-lmatic mtabolits. Giv th prohibitivl high costsof dvlopig drgs, a collctiv ffort i this arafrom all orgaizatios gagig i hma cliical trialsith kias ihibitors old b stifid to avoid rpat-ig mistaks. Sch a databas old b a trmdosasst to th rsarch commit oig to th prc-dtd siz of th proti kias famil i drg discov-r ad th fact that lss tha a qartr of th kiom hasb pharmacologicall itrrogatd to dat.

Third, thr is a d for mor prdictiv tmor mod-ls ad bttr as to moitor targt ihibitio i hmasi a miimall ivasiv fashio. As ol a small fractio ofth tmorigsis that occrs i hmas ca b rcapit-

latd i cll cltr ad aimal modls, d a bttrabilit to moitor th dvlopmt of cacr i hmas.This old icld miimall ivasiv mthods to dis-covr ad moitor biomarkrs that ma srv as lad-marks for th voltio of a tmor i hmas. O caimagi th implmtatio of imagig modalitisi coctio ith protomic tchologis to moitorchags i sigallig protis ad mtabolits. Th dvl-opmt of arl dtctio tchologis ma allo s todiscovr ad radicat tmors bfor th hav acqirdth fll rag of srvival capabilitis that mak stablishdtmors ad mtastass so rsistat to thrap.

Forth, as kias sigallig cascads ivolv aitricat arra of itrcoctd circits d todvlop mor sophisticatd modllig of ho th arrprogrammd i rspos to ocogic vts ad ith prsc of ihibitors. This iformatio ill b vitalto mor ratioal applicatio of combiatios of ihibi-tors. Sccssfll mtig ths challgs ill rqir ahighl itrdiscipliar collaboratio bt chmis-tr, biolog, comptatio, proti crstallograph, phar-macolog ad cliical ivstigatio. Ths, th ftrpromiss icrmtal advacs agaist a disas thativitabl ill affct s all.

Box 3 |Development of mutant-targeting BCRABL1 inhibitors

Thediscoveryofimatinibresistancemutationshasencouragedthesuccessful

developmentofnewATP-competitiveinhibitorssuchasnilotinib(AMN107)97and

dasatinib(BMS-354825)98.Nilotinibisaphenylaminopyrimidinethathasapproximately

20-foldhighercellularactivitythanimatinibandinhibitsthemajorityofBCR

(breakpointclusterregion)ABL1mutants,withtheexceptionofT315I.Dasatinibisa

thiazole-derivedcompoundthathasanapproximately50-foldlowercellularEC50than

imatinibandinhibitsvirtuallyallknownBCRABL1mutants,butagainwiththeexceptionoftheT315Imutant.Whereasnilotinibhasaselectivityprofilesimilarto

imatinib(BCRABL1,PDGFRandKIT),dasatinibhasapan-tyrosinekinaseinhibitor

profileincludingtheSrcfamily(SRC,YES,FYNandLYN),BCRABL1,KIT,

platelet-derivedgrowthfactorreceptor(PDGFR),BMX,Ephfamily,vascular

endothelialgrowthfactorreceptor2andTIE2(ReF. 99).Nilotinibbindstotheinactive

conformation(type2,DFGout)whereasdasatinibbindstotheactiveconformationof

theABL1kinasedomain(type1,DFGin)100.Bothinhibitorsmakeahydrogen-bonding

interactiontotheside-chainhydroxylgroupofT315.Thedevelopmentofthese

inhibitorsdemonstratesthatbyincreasingaffinitytotheATPbindingsitethroughmore

extensiveandcomplementaryhydrogen-bondingandhydrophobicinteractions,itis

possibletoovercomebothasubsetoftheactivesiteanddistalmutationsinBCRABL1.

Owingtothedirectstericintrusionoftheisobutylsidechainandlossofahydrogen-

bondinginteractioninthemiddleoftheATPcleft,however,theT315Imutationrenders

completeresistancetobothcompounds101

TwonewinhibitorsthatarecapableofinhibitingT315IBCRABL1haverecentlybeenreportedinconjunctionwithco-crystalstructureswithT315IBCR-ABL1:PPY-A101and

PHA-739358(ReF. 102).Bothinhibitorsrecognizetheactiveconformationofthekinase

(type1,DFGin).TheselectivityprofileofPPY-AhasnotbeenreportedbutPHA-739358

isreportedtohaveabroadselectivityprofile(IC50


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Competing interests statement.The authors declare competing financial interests: see web

version for details.

DATABASESEntrez Gene:http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=gene

PI3KCA

National Cancer Institute Drug Dictionary:http://www.

cancer.gov/drugdictionary/

AT7519 | dasatinib | erlotinib | gefitinib|imatinib|nilotinib|

PKC412 | rapamycin|sorafenib| VX-680

Protein Data Bank:http://www.rcsb.org/pdb/home/home.do

1IEP | 1O6L | 1OPK | 1S9J|1SM2

OMIM:http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=OMIM

essential thrombocythemia | myelofibrosis| neuroblastoma |

polycythaemia vera

UniProtKB:http://www.uniprot.org

ABL1 | ALK| BCR | BRAF|BTK|DDR1|EGFR| FGFR1|FKBP1A|

FRAP1|hepatocyte growth factor receptor|IGF1R | IL-3 |

insulin receptor|JAK2 | KIT| M2 splice isoform of pyruvate

kinase | NPM| NTRK2 | PDGFR | PDGFR | PI3K|PI3K|

PI3K | PTEN |RET | RSK1|RSK2| RSK4 | VEGFR2

FURTHER INFORMATIONN. S. Grays homepage:http://research4.dfci.harvard.edu/

gray_lab/home.htm

Catalogueof somatic mutations in cancer:http://www.

sanger.ac.uk/genetics/CGP/cosmic/

Mutations of kinases in cancer: http://strubiol.icr.ac.uk/

extra/mokca/protein.php

PyMOL molecular viewer: http://www.pymol.org/

SUPPLEMENTARY INFORMATION

See online article:S1 (table) | S2(figure) |S3 (box) | S4(figure)

All linKS Are ACtive in the online pDf

R E V I E W S

nATuRe ReVIewS | CAnCer VOLuMe 9 | jAnuARy 2009 | 39
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