kinase inhibitors
TRANSCRIPT
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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
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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
30 | jAnuARy 2009 | VOLuMe 9 www.au.cm/ws/cac
2009 Macmillan Publishers Limited. All rights reserved
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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.
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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
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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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17. Morgan, K. J. & Gilliland, D. G. A role for JAK2
mutations in myeloproliferative diseases.Annu. Rev.
Med.59, 213222 (2008).
18. Mosse, Y. P. et al. Identification ofALKas a major
familial neuroblastoma predisposition gene. Nature
455, 930935 (2008).
19. Kaelin, W. G. Jr. The concept of synthetic lethality in
the context of anticancer therapy. Nature Rev. Cancer
5, 689698 (2005).20. Wang, J. Y., Wilcoxen, K. M., Nomoto, K. & Wu, S.
Recent advances of MEK inhibitors and their clinical
progress. Curr. Top. Med. Chem.7, 13641378(2007).
21. Faivre, S., Kroemer, G. & Raymond, E. Current
development of mTOR inhibitors as anticancer agents.
Nature Rev. Drug Discov.5, 671688 (2006).22. Hu, Y. et al. 90-kDa ribosomal S6 kinase is a direct
target for the nuclear fibroblast growth factor receptor
1 (FGFR1): role in FGFR1 signaling.J. Biol. Chem.
279, 2932529335 (2004).
23. Malumbres, M. & Barbacid, M. Cell cycle kinases in
cancer. Curr. Opin. Genet. Dev.17, 6065 (2007).
A comprehensive review of the cell cycle kinases
currently being developed as targets for novel
inhibitors.
24. Geiger, T. R. & Peeper, D. S. Critical role for TrkB
kinase function in anoikis suppression,
tumorigenesis, and metastasis. Cancer Res.67,
62216229 (2007).
25. Kerbel, R. S. Tumor angiogenesis. N. Engl. J. Med.
358, 20392049 (2008).
26. Christofk, H. R. et al. The M2 splice isoform of
pyruvate kinase is important for cancer metabolism
and tumour growth. Nature452, 230233 (2008).
27. Greenman, C. et al. Patterns of somatic mutation in
human cancer genomes. Nature446, 153158
(2007).
28. Jia, S. et al. Essential roles of PI(3)K-p110 in cell
growth, metabolism and tumorigenesis. Nature454,
776779 (2008).29. Wee, S. et al. PTEN-deficient cancers depend on
PIK3CB. Proc. Natl Acad. Sci. USA105,
1305713062 (2008).
30. Johnson, L. N., Lowe, E. D., Noble, M. E. & Owen, D. J.
The Eleventh Datta Lecture. The structural basis for
substrate recognition and control by protein kinases.
FEBS Lett.430, 111 (1998).31. Manning, G., Whyte, D. B., Martinez, R., Hunter, T. &
Sudarsanam, S. The protein kinase complement of
the human genome. Science298, 19121934
(2002).
32. Traxler, P. & Furet, P. Strategies toward the design of
novel and selective protein tyrosine kinase inhibitors.Pharmacol. Ther.82, 195206 (1999).
33. Liu, Y. & Gray, N. S. Rational design of inhibitors that
bind to inactive kinase conformations. Nature Chem.
Biol.2, 358364 (2006).
34. Hennequin, L. F. et al. N.-(5-chloro-1,3-benzodioxol-4-
yl)-7-[2-(4-methylpiperazin-1-yl)ethoxy]-5-(tetrahydro-
2H-pyran-4-yloxy)quinazolin-4-amine, a novel, highly
selective, orally available, dual-specific c-Src/Abl
kinase inhibitor.J. Med. Chem.49, 64656488
(2006).
35. Manley, P. W., Cowan-Jacob, S. W. & Mestan, J.
Advances in the structural biology, design and clinical
development of Bcr-Abl kinase inhibitors for the
treatment of chronic myeloid leukaemia. Biochim.
Biophys. Acta1754, 313 (2005).
36. Wan, P. T. et al. Mechanism of activation of the
RAFERK signaling pathway by oncogenic mutations
of B-RAF. Cell116, 855867 (2004).37. Knight, Z. A. et al. A pharmacological map of the
PI3-K family defines a role for p110alpha in insulinsignaling. Cell125, 733747 (2006).
An impressive illustration of the integration of
chemical and biological approaches to discover
and characterize isoform-selective PI3K inhibitors.
38. Ohren, J. F. et al. Structures of human MAP kinase
kinase 1 (MEK1) and MEK2 describe novel
noncompetitive kinase inhibition. Nature Struct. Mol.
Biol.11, 11921197 (2004).
39. Adrian, F. J. et al. Allosteric inhibitors of Bcr-abl-
dependent cell proliferation. Nature Chem. Biol.2,
95102 (2006).
40. Barnett, S. F. et al. Identification and characterization
of pleckstrin-homology-domain-dependent and
isoenzyme-specific Akt inhibitors. Biochem. J.385,
399408 (2005).
41. Lindsley, C. W. et al. Allosteric Akt (PKB) inhibitors:
discovery and SAR of isozyme selective inhibitors.
Bioorg. Med. Chem. Lett.15, 761764 (2005).
42. McIntyre, K. W. et al. A highly selective inhibitor of
IB kinase, BMS-345541, blocks both joint
inflammation and destruction in collagen-induced
arthritis in mice.Arthritis Rheum.48, 26522659
(2003).
43. Grimsby, J. et al. Allosteric activators of glucokinase:
potential role in diabetes therapy. Science301,
370373 (2003).
44. Guertin, K. R. & Grimsby, J. Small molecule
glucokinase activators as glucose lowering agents: a
new paradigm for diabetes therapy. Curr. Med. Chem.
13, 18391843 (2006).45. Sullivan, J. E. et al. Inhibition of lipolysis and
lipogenesis in isolated rat adipocytes with AICAR, a
cell-permeable activator of AMP-activated protein
kinase. FEBS Lett.353, 3336 (1994).46. Sanders, M. J. et al. Defining the mechanism of
activation of AMP-activated protein kinase by the
small molecule A-769662, a member of the
thienopyridone family.J. Biol. Chem.282,
3253932548 (2007).
47. Cohen, M. S., Zhang, C., Shokat, K. M. & Taunton, J.
Structural bioinformatics-based design of selective,
irreversible kinase inhibitors. Science308, 13181321
(2005).
This study is an instructive example of how a
selective irreversible inhibitor of RSK was designed.48. Kwak, E. L. et al. Irreversible inhibitors of the EGF
receptor may circumvent acquired resistance to
gefitinib. Proc. Natl Acad. Sci. USA102, 76657670
(2005).
49. Rabindran, S. K. et al. Antitumor activity of HKI-272,
an orally active, irreversible inhibitor of the HER-2
tyrosine kinase. Cancer Res.64, 39583965
(2004).
50. Kobayashi, S. et al. An alternative inhibitor overcomes
resistance caused by a mutation of the epidermal
growth factor receptor. Cancer Res.65, 70967101
(2005).
51. Fry, D. W. et al. A specific inhibitor of the epidermal
growth factor receptor tyrosine kinase. Science265,
10931095 (1994).
52. Heymach, J. V., Nilsson, M., Blumenschein, G.,
Papadimitrakopoulou, V. & Herbst, R. Epidermal
growth factor receptor inhibitors in development for
the treatment of non-small cell lung cancer. Clin.
Cancer Res.12, 4441s4445s (2006).53. Felip, E., Santarpia, M. & Rosell, R. Emerging drugs
for non-small-cell lung cancer. Expert Opin. Emerg.
Drugs12, 449460 (2007).
54. Wissner, A. et al. Dual irreversible kinase inhibitors:
quinazoline-based inhibitors incorporating two
independent reactive centers with each targetingdifferent cysteine residues in the kinase domains of
EGFR and VEGFR-2. Bioorg. Med. Chem.15,
36353648 (2007).
55. Pan, Z. et al. Discovery of selective irreversible
inhibitors for Brutons tyrosine kinase. ChemMedChem
2, 5861 (2007).
56. Cohen, M. S., Hadjivassiliou, H. & Taunton, J. A
clickable inhibitor reveals context-dependent
autoactivation of p90 RSK. Nature Chem. Biol.3,
156160 (2007).
57. Schirmer, A., Kennedy, J., Murli, S., Reid, R. &
Santi, D. V. Targeted covalent inactivation of protein
kinases by resorcylic acid lactone polyketides.
Proc. Natl Acad. Sci. USA103, 42344239 (2006).
58. Li, B., Liu, Y., Uno, T. & Gray, N. Creating chemical
diversity to target protein kinases. Comb. Chem. High
Throughput Screen7, 453472 (2004).
59. Kraker, A. J. et al. Biochemical and cellular effects of
c-Src kinase-selective pyrido[2,3-d]pyrimidine tyrosine
kinase inhibitors. Biochem. Pharmacol.60, 885898(2000).
60. Mohammadi, M. et al. Crystal structure of an
angiogenesis inhibitor bound to the FGF receptor
tyrosine kinase domain. EMBO J.17, 58965904
(1998).61. Lenart, P. et al. The small-molecule inhibitor BI 2536
reveals novel insights into mitotic roles of polo-like
kinase 1. Curr. Biol.17, 304315 (2007).
62. Sapkota, G. P. et al. BI-D1870 is a specific inhibitor of
the p90 RSK (ribosomal S6 kinase) isoforms in vitro
and in vivo. Biochem. J.401, 2938 (2007).
63. Scotlandi, K. et al. Antitumor activity of the insulin-like
growth factor-I receptor kinase inhibitor NVP-AEW541
in musculoskeletal tumors. Cancer Res.65,
38683876 (2005).64. Okram, B. et al. A general strategy for creating
inactive-conformation abl inhibitors. Chem. Biol.13,
779786 (2006).
65. Dubinina, G. G. et al.In silico design of protein kinase
inhibitors: successes and failures.Anticancer Agents
Med. Chem.7, 171188 (2007).
66. Gill, A. New lead generation strategies for protein
kinase inhibitors fragment based screening
approaches. Mini Rev. Med. Chem.4, 301311
(2004).
67. Wyatt, P. G. et al. Identification ofN-(4-piperidinyl)-4-
(2,6-dichlorobenzoylamino)-1H-pyrazole-3-
carboxamide (AT7519), a novel cyclin dependent
kinase inhibitor using fragment-based X-ray
crystallography and structure based drug design.J. Med. Chem.51, 49864999 (2008).
68. Bain, J. et al. The selectivity of protein kinase
inhibitors: a further update. Biochem. J.408,
297315 (2007).69. Venter, J. C. et al. The sequence of the human genome.
Science291, 13041351 (2001).
70. Carter, T. A. et al. Inhibition of drug-resistant mutants
of, ABL, KIT, and EGF receptor kinases. Proc. Natl
Acad. Sci. USA102, 1101111016 (2005).
71. Fabian, M. A. et al. A small molecule-kinase
interaction map for clinical kinase inhibitors. Nature
Biotechnol.23, 329336 (2005).
72. Harrington, E. A. et al. VX-680, a potent and selective
small-molecule inhibitor of the Aurora kinases,
suppresses tumor growth in vivo. Nature Med.10,
262267 (2004).
73. Young, M. A.et al. Structure of the kinase domain of
an imatinib-resistant Abl mutant in complex with the
Aurora kinase inhibitor VX-680. Cancer Res.66,
10071014 (2006).
74. Fedorov, O. et al. A systematic interaction map of
validated kinase inhibitors with Ser/Thr kinases.
Proc. Natl Acad. Sci. USA104, 2052320528
(2007).
75. Warmuth, M., Kim, S., Gu, X. J., Xia, G. & Adrian, F.
Ba/F3 cells and their use in kinase drug discovery.
Curr. Opin. Oncol.19, 5560 (2007).
76. Melnick, J. S. et al. An efficient rapid system for
profiling the cellular activities of molecular libraries.
Proc. Natl Acad. Sci. USA103, 31533158 (2006).
77. Godl, K. et al. An efficient proteomics method to
identify the cellular targets of protein kinase inhibitors.
Proc. Natl Acad. Sci. USA100, 1543415439
(2003).
78. Bantscheff, M. et al. Quantitative chemical proteomics
reveals mechanisms of action of clinical ABL kinase
inhibitors. Nature Biotechnol.25, 10351044
(2007).
79. Peters, E. C. & Gray, N. S. Chemical proteomics
identifies unanticipated targets of clinical kinase
inhibitors.ACS Chem. Biol.2, 661664 (2007).80. le Coutre, P. et al. Induction of resistance to the
Abelson inhibitor STI571 in human leukemic cells
through gene amplification. Blood95, 17581766
(2000).81. Engelman, J. A. et al. MET amplification leads to
gefitinib resistance in lung cancer by activating ERBB3
signaling. Science316, 10391043 (2007).
This article describes upregulation of an alternative
pathway as a mechanism of resistance to kinase
inhibitors.
82. Chu, S. et al. Detection of BCRABL kinase mutations
in CD34+ cells from chronic myelogenous leukemia
patients in complete cytogenetic remission on
imatinib mesylate treatment. Blood105, 2093
2098 (2005).
83. Hughes, T. & Branford, S. Molecular monitoring of
BCRABL as a guide to clinical management in
chronic myeloid leukaemia. Blood Rev.20, 2941
(2006).
84. Roumiantsev, S . et al. Clinical resistance to thekinase inhibitor STI-571 in chronic myeloid leukemia
by mutation of Tyr-253 in the Abl kinase domain
P-loop. Proc. Natl Acad. Sci. USA99, 1070010705
(2002).
85. Fletcher, J. A. & Rubin, B. P. KIT mutations in GIST.
Curr. Opin. Genet. Dev.17, 37 (2007).
86. Cools, J. et al. Prediction of resistance to small
molecule FLT3 inhibitors: implications for molecularly
targeted therapy of acute leukemia. Cancer Res.64,
63856389 (2004).
87. Graham, S. M. et al. Primitive, quiescent, Philadelphia-
positive stem cells from patients with chronic myeloid
leukemia are insensitive to STI571 in vitro. Blood99,
319325 (2002).
88. Copland, M. et al. Dasatinib (BMS-354825) targets
an earlier progenitor population than imatinib in
primary CML but does not eliminate the quiescent
fraction. Blood107, 45324539 (2006).
R E V I E W S
38 | jAnuARy 2009 | VOLuMe 9 www.au.cm/ws/cac
2009 Macmillan Publishers Limited. All rights reserved
-
7/27/2019 Kinase Inhibitors
12/12
89. Pao, W. et al. Acquired resistance of lung
adenocarcinomas to gefitinib or erlotinib is associated
with a second mutation in the EGFR kinase domain.
PLoS Med.2, e73 (2005).
90. Blencke, S. et al. Characterization of a conserved
structural determinant controlling protein kinase
sensitivity to selective inhibitors. Chem. Biol.11,
691701 (2004).
This article provides an impressive illustration of
the general importance of the gatekeeper position.
91. Gumireddy, K. et al. A non-ATP-competitive inhibitor
of BCRABL overrides imatinib resistance. Proc. NatlAcad. Sci. USA102, 19921997 (2005).
92. Gumireddy, K. et al. ON01910, a non-ATP-competitive
small molecule inhibitor of Plk1, is a potent anticancer
agent. Cancer Cell7, 275286 (2005).93. Gorre, M. E., Ellwood-Yen, K., Chiosis, G., Rosen, N. &
Sawyers, C. L. BCRABL point mutants isolated from
patients with imatinib mesylate-resistant chronic
myeloid leukemia remain sensitive to inhibitors of the
BCRABL chaperone heat shock protein 90. Blood
100, 30413044 (2002).
94. Copland, M. et al. BMS-214662 potently induces
apoptosis of chronic myeloid leukemia stem and
progenitor cells and synergises with tyrosine kinase
inhibitors. Blood111, 28432853 (2008).
95. Hamby, J. M. et al. Structure-activity relationships for
a novel series of pyrido[2,3-d]pyrimidine tyrosine
kinase inhibitors.J. Med. Chem.40, 22962303
(1997).
96. Kothe, M. et al. Selectivity-determining residues in
Plk1. Chem. Biol. Drug Des.70, 540546 (2007).
97. Weisberg, E. et al. Characterization of AMN107, a
selective inhibitor of native and mutant BcrAbl.
Cancer Cell7, 129141 (2005).
98. Shah, N. P. et al. Dasatinib (BMS-354825) inhibits
KITD816V, an imatinib-resistant activating mutation
that triggers neoplastic growth in most patients with
systemic mastocytosis. Blood108, 286291
(2006).
99. Karaman, M. W. et al. A quantitative analysis of kinase
inhibitor selectivity. Nature Biotechnol.26, 127132
(2008).
This article features an impressive profiling of
inhibitors against a large panel of kinases.
100.Vajpai, N.et al. Solution conformations and dynamics
of ABL kinase-inhibitor complexes determined byNMR substantiate the different binding modes of
imatinib/nilotinib and dasatinib.J. Biol. Chem.283,
1829218302 (2008).
101. Zhou, T. et al. Crystal structure of the T315I mutant of
AbI kinase. Chem. Biol. Drug Des.70, 171181
(2007).
102. Modugno, M. et al. Crystal structure of the T315I Abl
mutant in complex with the aurora kinases inhibitor
PHA-739358. Cancer Res.67, 79877990 (2007).
103. Carpinelli, P. et al. PHA-739358, a potent inhibitor of
Aurora kinases with a selective target inhibition profile
relevant to cancer. Mol. Cancer Ther.6, 31583168
(2007).
104. Nagar, B. et al. Structural basis for the autoinhibition
of c-Abl tyrosine kinase. Cell112, 859871 (2003).
105. Nagar, B. et al. Crystal structures of the kinase domain
of c-Abl in complex with the small molecule inhibitors
PD173955 and imatinib (STI-571). Cancer Res.62,
42364243 (2002).
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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