《细胞生物学》课程教学资源（文献资料）Protein kinases - the major drug targets of the twenty-first century

PERSPECTIVESseveral protein kinases, such as the RHO-TIMELINEdependent protein kinase ROCK,but it isunclear whether its clinicalefficacy resultsfrom inhibition of this or other proteinProtein kinases the major drugkinases,or whetheritisduetoa non-kinaseeffect.ROCKcanconstrictbloodvesselsbytargets of the twenty-first century?inhibitingsmooth-musclemyosinphosphatase,and Y27632amorespecificROCK inhibitor - might exert its anti-Philip Cohenhypertensive effects in this waylo.It was the discovery that staurosporineProteinphosphorylation regulates mostHidaka. Naphthalene sulphonamides, such(FIG.n),an antifungal agent that is producedaspectsofcellife,whereasabnormalas N-(6-amino-hexyl)-5-chloro-1-naph-bybacteria ofthegenus Streptomyces,was ananomolar inhibitor of PKCu that reallyphosphorylation is acauseorthalenesulphonamide (W7),which hadconsequenceofdisease.Agrowingalready been developed as antagonists of themade the pharmaceutical industry sit up andinterestin developing orallyactive proteincalcium-binding protein calmodulin, weretake notice. This discovery led several compa-kinase inhibitors has recently culminated inalso found (at higher concentrations)tonies to make and test many derivatives of thistheapprovalofthefirstofthesedrugsfoinhibit several proteinkinases.However,bisindolyl maleimide.Two such compounds.clinical use. Protein kinases have nowwhenthe naphthalene ring wasreplaced bywhich were developed by Roche (Ro31-become the secondmost importantgroupisoquinoline, Hidaka observed that the8220)12 and Goedke (Go6850; also calledGF109203X)", were used subsequently inof drugtargets,afterG-protein-coupledderivatives were no longer calmodulinreceptors.Here,Igiveapersonalviewofantagonists,but retained the ability tocell-based assays by hundreds of laboratoriessomeofthemostimportantadvancesthatinhibitproteinkinases.Oneofthesecom-to invoke a myriad of rolesfor PKC.However,have shaped this field.pounds - termed H8 (FIG 1)- was a muchthese and several other bisindolyl maleimidesstronger inhibitor of cyclic-AMP- andwerelater shown to lack specificity,and inhib-Protein phosphorylation was originally identi-cGMP-dependent protein kinases than fourited several other protein kinases in vitro%14fied as a regulatory mechanism for the controlNevertheless, several bisindolyl malei-otherprotein kinases tested,whereasofglycogenmetabolism,and ittookmanyanother (H7)inhibited cyclic-nucleotide-mideshaveprogressed tohuman clinicalmore years before its general significance camedependent protein kinases and PKC withtrials, although it is unclear whether theirto be appreciated'.Two observations that weresimilarpotency.These compoundswereefficacystemsfromtheinhibitionofPKCmade morethan 20 years ago,and did muchATPcompetitiveand, importantly,were cellanother protein kinase or even the com-tostimulateawarenessofthe importanceofpermeable, which indicated their potentialbined inhibition of several proteinkinasesprotein phosphorylation,also provided theuseinvivosThecompound 7-hydroxystaurosporinefirst connectionsbetween abnormal proteinAlthoughtheseand manyotherisoquino-(UCN-01; TABLE 1; FIG.2)also blocks cell-cyclephosphorylation and disease (see TIMELINE).linesulphonamides that were developed sub-progression and might act by inhibiting thesequentlyare of relativelylow potencyandcell-cycle checkpoint-control kinase CHK1In1978,RayEriksonfoundthatthetrans-formingfactoroftheRoussarcomavirusinhibitseveral proteinkinases,oneofthese(REE15).N-benzoylstaurosporine(PKC412)which is being developed by Novartis,has(v-Src)wasaproteinkinase’,and-duringacompounds(HA1077,alsoknownasAT877short visit to Yasutomi Nishizuka's laboratoryorfasudil hydrochloride;TABLEI; FIG.2)pro-entered humanclinicaltrialsforthetreatin 1981-—Monique Castagna discovered thatgressed to human clinical trials in the earlyment of advancedcancers (TABLE 1).Italsotumour-promoting phorbol esters were1990s.ItwasapprovedinJapanin1995forinhibits retinal neovascularizationandlaserpotentactivatorsofproteinkinaseC(PKC)3thetreatmentof cerebral vasospasmafterinduced choroidal neovascularization inthatprobablymimicked activation bythesurgeryfor subarachnoid haemorrhage,andmouse models,and is nowinPhaseI clinicalsecond messenger diacylglycerol.associated cerebral ischaemic symptoms.trials for thetreatment ofischaemic retino-HA1077hadnomarkedsideeffectsoverapathyl6,Another bisindolyl maleimide,The first protein-kinase inhibitorsLY333531, which inhibits the PKC-β isoformtwo-week period when given by intravenousThe first protein-kinase inhibitors weremore potently than other PKC isoforms,isinjection(see,for example,REFs6,7).Atdevelopedintheearly1980sbyHiroyoshimicromolarconcentrations,HA1077inhibitsreported to normalize the elevated levels ofNATUREREVIEWSDRUGDISCOVERYVOLUME1APRIL20023092002NaturePublishingGroup

© 2002 Nature Publishing Group PERSPECTIVES Hidaka. Naphthalene sulphonamides, such as N-(6-amino-hexyl)-5-chloro-1-naph￾thalenesulphonamide (W7), which had already been developed as antagonists of the calcium-binding protein calmodulin, were also found (at higher concentrations) to inhibit several protein kinases. However, when the naphthalene ring was replaced by isoquinoline, Hidaka observed that the derivatives were no longer calmodulin antagonists, but retained the ability to inhibit protein kinases. One of these com￾pounds — termed H8 (FIG. 1) — was a much stronger inhibitor of cyclic-AMP- and cGMP-dependent protein kinases than four other protein kinases tested, whereas another (H7) inhibited cyclic-nucleotide￾dependent protein kinases and PKC with similar potency. These compounds were ATP competitive and, importantly, were cell permeable, which indicated their potential use in vivo 5 . Although these and many other isoquino￾linesulphonamides that were developed sub￾sequently are of relatively low potency and inhibit several protein kinases, one of these compounds (HA1077, also known as AT877 or fasudil hydrochloride; TABLE 1; FIG. 2) pro￾gressed to human clinical trials in the early 1990s. It was approved in Japan in 1995 for the treatment of cerebral vasospasm after surgery for subarachnoid haemorrhage, and associated cerebral ischaemic symptoms. HA1077 had no marked side effects over a two-week period when given by intravenous injection (see, for example, REFS 6,7). At micromolar concentrations, HA1077 inhibits several protein kinases, such as the RHO￾dependent protein kinase ROCK8 , but it is unclear whether its clinical efficacy results from inhibition of this or other protein kinases, or whether it is due to a non-kinase effect. ROCK can constrict blood vessels by inhibiting smooth-muscle myosin phos￾phatase9 , and Y27632 — a more specific ROCK inhibitor — might exert its anti￾hypertensive effects in this way10. It was the discovery that staurosporine (FIG. 1), an antifungal agent that is produced by bacteria of the genus Streptomyces, was a nanomolar inhibitor of PKC11 that really made the pharmaceutical industry sit up and take notice. This discovery led several compa￾nies to make and test many derivatives of this bisindolyl maleimide. Two such compounds, which were developed by Roche (Ro31- 8220)12 and Goedke (Go6850; also called GF109203X)13, were used subsequently in cell-based assays by hundreds of laboratories to invoke a myriad of roles for PKC. However, these and several other bisindolyl maleimides were later shown to lack specificity, and inhib￾ited several other protein kinases in vitro8,14. Nevertheless, several bisindolyl malei￾mides have progressed to human clinical trials, although it is unclear whether their efficacy stems from the inhibition of PKC, another protein kinase or even the com￾bined inhibition of several protein kinases. The compound 7-hydroxystaurosporine (UCN-01; TABLE 1; FIG. 2) also blocks cell-cycle progression and might act by inhibiting the cell-cycle checkpoint-control kinase CHK1 (REF. 15). N-benzoyl staurosporine (PKC412), which is being developed by Novartis, has entered human clinical trials for the treat￾ment of advanced cancers (TABLE 1). It also inhibits retinal neovascularization and laser￾induced choroidal neovascularization in mouse models, and is now in Phase I clinical trials for the treatment of ischaemic retino￾pathy16. Another bisindolyl maleimide, LY333531, which inhibits the PKC-β isoform more potently than other PKC isoforms, is reported to normalize the elevated levels of Protein phosphorylation regulates most aspects of cell life, whereas abnormal phosphorylation is a cause or consequence of disease. A growing interest in developing orally active protein￾kinase inhibitors has recently culminated in the approval of the first of these drugs for clinical use. Protein kinases have now become the second most important group of drug targets, after G-protein-coupled receptors. Here, I give a personal view of some of the most important advances that have shaped this field. Protein phosphorylation was originally identi￾fied as a regulatory mechanism for the control of glycogen metabolism, and it took many more years before its general significance came to be appreciated1 . Two observations that were made more than 20 years ago, and did much to stimulate awareness of the importance of protein phosphorylation, also provided the first connections between abnormal protein phosphorylation and disease (see TIMELINE). In 1978, Ray Erikson found that the trans￾forming factor of the Rous sarcoma virus (v-Src) was a protein kinase 2 , and — during a short visit to Yasutomi Nishizuka’s laboratory in 1981 — Monique Castagna discovered that tumour-promoting phorbol esters were potent activators of protein kinase C (PKC)3 that probably mimicked activation by the second messenger diacylglycerol4 . The first protein-kinase inhibitors The first protein-kinase inhibitors were developed in the early 1980s by Hiroyoshi NATURE REVIEWS | DRUG DISCOVERY VOLUME 1 | APRIL 2002 | 309 Protein kinases — the major drug targets of the twenty-first century? Philip Cohen TIMELINE

PERSPECTIVESTimeline|Keyeventsinthedevelopmentofprotein-kinaseinhibitorsPD98059 is shown to reverse thephenotypeofRAS-transfomed cellines bypreventngtheactiationfMKK1byRAFThe first nanomolarinhibitors of receptorPhamaceuticalprotein tyrosine kinases are developedThe frst oncogenesoquinolnecompanies start toGleevec (CGP57148, STGleevec is approvedsulphonamides are shownis shown to be adevelop inibitors ofOytokine-synthesis ant-nfammatory diugs571) enters human clinicalfor clinical use in theprotein kinase.to inhibit protein Kinasesprotein tyrosine kinaseareshowntoinhibitp38MAPKtrialsforthetreatmentof CMUnited States.1984Late1980s199119961999197819811986199419952001Tumour-promotingStairosporihelis shownThvee-dirmensional structure ofthe frstThefrst proteinnehitoRapamycin is approvedphorbol esters areto be ananomolar(fasudi, HA1077)is approvedfordinical use as anprotein kinase is determined (PKA)immunosuporessart inshown to activate PKC.Inhibitor ofPKC.nJapanforthetreatmentofThe immunosuppressant cyclosporin isthe Urited StatesCerebral vasospasm.shown to inhibit a proteinphosphatase(PP2B)The immunosuppressant rapamycin isshown to irhibit a protein knase (Tor).CML_chronic myelogenousleukaemia:EGF,epidermalgrowthfactor;MAPKmitogen-activatedproteinkinase;MKK1,mitogen-activatedproteinkinasekinase1PKA,proteinkinaseA;PKC,proteinkinaseC,Tor,targetofrapamycinPKC activity in the retina and kidneys ofsoil sample), was approved for clinical use inimmunosuppressive side effects.It is onlydiabetic rats7. It is in Phase III clinical trials1983, and has revolutionized organ transplan-more recently that the potential of rapamycinfor thetreatmentof diabeticmicrovasculartation byits abilityto prevent graft rejection.as an immunosuppressant was explored, anddisease (TABLE 1).The staurosporine ana-However, the mechanism by which it inhib-it was approved for clinical use in 1999. Sincelogue CEP-1347 (FIG.2) is reported to preventited T-cell activation remained unknown forthat time, rapamycin has rapidly become thethe activation of the c-JUN amino-terminalmany years. Schreiber and his colleaguesimmunosuppressantof choicetopreventkinase (INK) by inhibiting the mixed-lineageshowed that the complex that is formedrejection after kidney transplantation,kinase (MLK),which is anupstream' compo-between cyclosporin and cyclophilin (itsbecause its side effects areless severethannent ofthis signalling pathwayls. It has enteredintracellular receptor protein)was a potentthose of cyclosporin. It also prevents the rejec-human clinical trials for the prevention ofand specific inhibitor of calcineurin0, ation of pancreatic islets, thetransplantation ofCazcalmodulin-dependent protein phos-neuronal apoptosis and neurodegeneration.which is a potential way to treat insulin-phatase that had been identified ten years pre-dependent diabetes mellitus (IDDM)withoutviously in a completely different context?l.Immunosuppressant drugsthe need for daily injections of insulinBy the end of the 1980s, no protein-kinaseFK506isproduced by StreptomycesThe anticancer properties of rapamycin,inhibitors had entered human clinical trials.tsukubaensis, a soil bacterium that originateswhich was the first cytostatic agent to be iden-Moreover, virtually all ofthe protein-kinasefrom the north of Japan.The complex that istified, were first noted in the mid-1970s, andinhibitors that had been developed were ATPformedbetweenFK506andtheFK-bindingthe compound was sent to the Nationalcompetitive, and the difficulties that wereprotein (FKBP) also inhibits calcineurin,Cancer Institute (NCI)intheUnited Statesinvolved in developing compounds withwhich indicates a similar mechanism offor testing by Suren Sehgal, a scientist atsufficient potency to compete with theATPaction to that ofcyclosporin",The inhibitionAyerst Research Laboratories in Montreal.concentrationsthat arepresent in theintra-ofcalcineurinpreventsthedephosphoryla-Theimpressiveactivityofrapamycinagainstcellularmillieu (2-10mM)werebecomingtion of transcriptionfactorsoftheNFATsolidtumours,whenused in combinationapparent. Furthermore, with the determina-(nuclearfactorofactivatedTcells)familyinwith chemotherapy,led the NCI to designatetion of thefirst three-dimensional structureT cells, which inhibits their entry into theit a'priority drug. However, when Ayerstof a protein kinase (protein kinaseA;PKA)19,nucleus.Thisstopstheproductionofinter-closeddowntheMontreal laboratoriesinithad become apparent that the residues thatleukin-2 (IL-2),and hence T-cell prolifera-1982,the rapamycin programme was aban-doned.It was only when the two subsidiarieswere involved inbindingATPwere conservedtion.So,an important drug that is alreadyinfromkinasetokinase.Amyththereforebeganclinical useexerted its effectsbymodulatingofAmericanHomeProductsWyethandto permeate thefield that it was impossible'tothe phosphorylation of one or more intracel-Ayerst-- merged in 1988 that Sehgal manageddevelop protein-kinase inhibitors with thelular proteins.togettheprojectresurrected.CC1779,a closerequisitepotencyand specificityRapamycin(FIG.1)isproduced byanalogueof rapamycinthathasimprovedA turning point came in 1991, whenStreptomyceshygroscopicus,asoilbacteriumpharmacological properties,hasshownactiv-Stuart Schreiber identified cellular targets forthat originates from Easter Island (Rapa Nuiity against a wide range of cancers2, andcyclosporin (FIG 1) and FK506. Cyclosporin A,is the native namefor Easter Island)and wasPhase II trials are underway. Anotherwhich is a naturally occurring secondaryidentified more than 30 years ago.It was orig-rapamycin analogue, RAD001,which is beingmetabolite that is produced by Tolypocladiuminally purified as an antifungal agent,but wasdeveloped by Novartis, has also enteredinfilatum(a fungus isolated from a Norwegianinitiallydiscardedbecause of itsundesirablehuman clinical trials (TABLE 1).310APRIL2002VOLUME1www.nature.com/reviews/drugdisc2002Nature Publishing Group

© 2002 Nature Publishing Group 310 | APRIL 2002 | VOLUME 1 www.nature.com/reviews/drugdisc PERSPECTIVES immunosuppressive side effects22. It is only more recently that the potential of rapamycin as an immunosuppressant was explored, and it was approved for clinical use in 1999. Since that time, rapamycin has rapidly become the immunosuppressant of choice to prevent rejection after kidney transplantation, because its side effects are less severe than those of cyclosporin. It also prevents the rejec￾tion of pancreatic islets, the transplantation of which is a potential way to treat insulin￾dependent diabetes mellitus (IDDM) without the need for daily injections of insulin. The anticancer properties of rapamycin, which was the first cytostatic agent to be iden￾tified, were first noted in the mid-1970s, and the compound was sent to the National Cancer Institute (NCI) in the United States for testing by Suren Sehgal, a scientist at Ayerst Research Laboratories in Montreal. The impressive activity of rapamycin against solid tumours, when used in combination with chemotherapy, led the NCI to designate it a ‘priority drug’. However, when Ayerst closed down the Montreal laboratories in 1982, the rapamycin programme was aban￾doned. It was only when the two subsidiaries of American Home Products — Wyeth and Ayerst — merged in 1988 that Sehgal managed to get the project resurrected. CCI779, a close analogue of rapamycin that has improved pharmacological properties, has shown activ￾ity against a wide range of cancers23, and Phase II trials are underway. Another rapamycin analogue, RAD001, which is being developed by Novartis, has also entered human clinical trials (TABLE 1). soil sample), was approved for clinical use in 1983, and has revolutionized organ transplan￾tation by its ability to prevent graft rejection. However, the mechanism by which it inhib￾ited T-cell activation remained unknown for many years. Schreiber and his colleagues showed that the complex that is formed between cyclosporin and cyclophilin (its intracellular receptor protein) was a potent and specific inhibitor of calcineurin20, a Ca2+–calmodulin-dependent protein phos￾phatase that had been identified ten years pre￾viously in a completely different context21. FK506 is produced by Streptomyces tsukubaensis, a soil bacterium that originates from the north of Japan. The complex that is formed between FK506 and the FK-binding protein (FKBP) also inhibits calcineurin, which indicates a similar mechanism of action to that of cyclosporin20. The inhibition of calcineurin prevents the dephosphoryla￾tion of transcription factors of the NFAT (nuclear factor of activated T cells) family in T cells, which inhibits their entry into the nucleus. This stops the production of inter￾leukin-2 (IL-2), and hence T-cell prolifera￾tion. So, an important drug that is already in clinical use exerted its effects by modulating the phosphorylation of one or more intracel￾lular proteins. Rapamycin (FIG. 1) is produced by Streptomyces hygroscopicus, a soil bacterium that originates from Easter Island (Rapa Nui is the native name for Easter Island) and was identified more than 30 years ago. It was orig￾inally purified as an antifungal agent, but was initially discarded because of its undesirable PKC activity in the retina and kidneys of diabetic rats17. It is in Phase III clinical trials for the treatment of diabetic microvascular disease (TABLE 1). The staurosporine ana￾logue CEP-1347 (FIG. 2) is reported to prevent the activation of the c-JUN amino-terminal kinase (JNK) by inhibiting the mixed-lineage kinase (MLK), which is an ‘upstream’ compo￾nent of this signalling pathway18. It has entered human clinical trials for the prevention of neuronal apoptosis and neurodegeneration. Immunosuppressant drugs By the end of the 1980s, no protein-kinase inhibitors had entered human clinical trials. Moreover, virtually all of the protein-kinase inhibitors that had been developed were ATP competitive, and the difficulties that were involved in developing compounds with sufficient potency to compete with the ATP concentrations that are present in the intra￾cellular millieu (2–10 mM) were becoming apparent. Furthermore, with the determina￾tion of the first three-dimensional structure of a protein kinase (protein kinase A; PKA)19, it had become apparent that the residues that were involved in binding ATP were conserved from kinase to kinase. A myth therefore began to permeate the field that it was ‘impossible’ to develop protein-kinase inhibitors with the requisite potency and specificity. A turning point came in 1991, when Stuart Schreiber identified cellular targets for cyclosporin (FIG. 1) and FK506. Cyclosporin A, which is a naturally occurring secondary metabolite that is produced by Tolypocladium inflatum (a fungus isolated from a Norwegian The first oncogene is shown to be a protein kinase. Tumour-promoting phorbol esters are shown to activate PKC. Staurosporine is shown to be a nanomolar inhibitor of PKC. Three-dimensional structure of the first protein kinase is determined (PKA). The immunosuppressant cyclosporin is shown to inhibit a protein phosphatase (PP2B). The immunosuppressant rapamycin is shown to inhibit a protein kinase (Tor). The first nanomolar inhibitors of receptor protein tyrosine kinases are developed. Cytokine-synthesis anti-inflammatory drugs are shown to inhibit p38 MAPK. Gleevec is approved for clinical use in the United States. Gleevec (CGP57148, STI- 571) enters human clinical trials for the treatment of CML. 1978 1981 1984 1986 Late 1980s 1991 1994 1995 1996 1999 2001 The first protein-kinase inhibitor (fasudil, HA1077) is approved in Japan for the treatment of cerebral vasospasm. PD98059 is shown to reverse the phenotype of RAS-transformed cell lines by preventing the activation of MKK1 by RAF. Timeline | Key events in the development of protein-kinase inhibitors CML, chronic myelogenous leukaemia; EGF, epidermal growth factor; MAPK, mitogen-activated protein kinase; MKK1, mitogen-activated protein kinase kinase 1; PKA, protein kinase A; PKC, protein kinase C; Tor, target of rapamycin. Rapamycin is approved for clinical use as an immunosuppressant in the United States. Isoquinoline sulphonamides are shown to inhibit protein kinases. Pharmaceutical companies start to develop inhibitors of protein tyrosine kinases

PERSPECTIVESRemarkably, the intracellular receptor for工rapamycin is also FKBP,but in contrast to the0FK506-FKBP complex,rapamycin-FKBPIZdoes not inhibit calcineurin. Michael HallHNCHaidentified the molecular target of0=S=0NHrapamycin-FKBP in yeast as being a proteinOCHkinase, which he termed the ‘target ofOCHrapamycin (Tor)24.Themammalian homo-HC-NHlogue, which is called mTOR, or theFKBP-rapamycin-associated protein (FRAP),StaurosporineHBPD98059was identified subsequently.Rapamycin wasDthefirst drugto be approvedfor clinical usethat seems to inhibit one protein kinasespecifically,0=HNHOmTOR is involved in a phosphatidylino-sitol (PtdIns)-3-kinase-dependent signallingnpathway that has a crucial role in cell-cycleHNprogression from G1-to-S phase,as well asaRin IL-2-stimulated T-cell proliferation; theseOHare activities that underlie its anticancer andOlomoucine:R,=H2R2=2TyrphostinSB203580immunosuppressiveproperties.ConsistentRoscovitine:R,=C2Hs,R2=CH3withitsrequirementfortheGi-to-Strans-ition,the cytostatic effects of CCI779 aremostmarked in tumours in which the level ofCHaCHaCH.10NPtdins-3,4,5-trisphosphate (the product of theNOCHOHclass 1 Ptdins 3-kinases) is elevated, such asOCHaOHOoccurs in tumours that are deficient in theACHphosphatase-and-tensin homologue (PTEN),nCHAwhich is the phosphatase that convertsPtdIns(3,4,5)P, to PtdIns(4,5)P,(REFS 25,26).AOCHCSAIDSCyclosporinRapamycinDuring bacterial infection, lipopolysaccharide(LPS)--a component of the cell wall ofFigure1|Chemical structures of some compoundsthat havebeen importantfor stimulatinggram-negative bacteria-triggers cells of theinterestinthedevelopmentofprotein-kinase inhibitors.immune system to produce proinflammatorycytokines, such as tumour-necrosis factorpotent pyridinyl-imidazole inhibitors of TNFthree-dimensional structure of p38 MAPK in(TNF) and IL-1, which are released into thecirculation, where they help to mount theproduction,such as SB202190,were shown tocomplex with closely related compounds.These studies showed that only a portion ofimmune responses thatfight-and eventuallypreventtranslationofthemessengerRNAkill-the invading bacteria.However,thisthe drug interacts with the residues that bindthat encodesTNEAprotein thatboundthesedefence mechanism is a double-edged sword,pyridinyl imidazoles specifically was purifiedATP, whereas the 4-fluorophenyl moietybecause the uncontrolled production offrom the cell extracts, which led to its identifibinds in a small hydrophobic groove that isproinflammatorycytokines can bea causeofcation as a mitogen-activated proteinkinaseadjacent to the ATP-binding site* (FIG.3). Thischronic inflammatory diseases, such as(MAPK)family member that was termed thegroove is created bythe presence ofa smallCSAID-binding protein (CSBP)9. This pro-rheumatoidarthritis,inflammatorybowelside chain (threonine)at position 106,and itsdisease and septic shock.For this reason,tein kinase was identified independently bymutationtoa bulkyhydrophobicresidue,several other laboratories30-2,and is nowmany companies have sought to developwhich is found at this position in most othercompounds that suppress TNF production.usually called p38 MAPK.At least threeproteinkinases,makesp38MAPK insensitiveMore than 20 years ago, SmithKline andinhibitors of p38MAPKhavenow enteredtoSB203580(REFs35.36).ThefindingthatFrench developed a new class of pyridinylhuman clinical trials for the treatment ofspecificityis conferred largelybyinteractionimidazoles-exemplifiedbySKF86002-rheumatoidarthritisorpsoriasis(TABLE1)with residues that lie near,but out with, thethat showed efficacy in animal models ofand several other companies have potentATP-binding pocket also explains the highinhibitors of this enzyme at a preclinical stage.chronicinflammatory disease.Theyweredegreeof specificity of several otherATPAlthough SB203580 (FIG. 1) and the closelytherefore called cytokine-synthesis anticompetitiveinhibitorsofdifferentproteininflammatorydrugs(CSAIDs seeREE.27forarelated SB202190 areATP competitivekinasesthatwere developed subsequentlyreview of early work in this area).inhibitors, they are relatively specificfor p38Examples include SU5402,an inhibitor of theIn the late 1980s, John Lee and colleaguesMAPK in vitro and do not inhibit manyfibroblast-growth-factor receptor (FGFR)otherproteinkinasesthat havebeentestedfound that SKF86002 suppressed the LPS-tyrosinekinase7,and two cyclin-dependentinduced production of IL-1 and TNF inThe basis for this high degree of specificityprotein-kinase inhibitorsroscovitinehuman monocytes Subsequently,morewas shownbythedeterminationof the(REE 38; FIG 1) and purvalanoP’ (FIG 3).NATUREREVIEWSDRUGDISCOVERYVOLUME1APRIL20023112002NaturePublishingGroup

© 2002 Nature Publishing Group PERSPECTIVES three-dimensional structure of p38 MAPK in complex with closely related compounds. These studies showed that only a portion of the drug interacts with the residues that bind ATP, whereas the 4-fluorophenyl moiety binds in a small hydrophobic groove that is adjacent to the ATP-binding site34 (FIG. 3). This groove is created by the presence of a small side chain (threonine) at position 106, and its mutation to a bulky hydrophobic residue, which is found at this position in most other protein kinases, makes p38 MAPK insensitive to SB203580 (REFS 35,36). The finding that specificity is conferred largely by interaction with residues that lie near, but out with, the ATP-binding pocket also explains the high degree of specificity of several other ATP competitive inhibitors of different protein kinases that were developed subsequently. Examples include SU5402, an inhibitor of the fibroblast-growth-factor receptor (FGFR) tyrosine kinase37, and two cyclin-dependent protein-kinase inhibitors — roscovitine (REF. 38; FIG. 1) and purvalanol39 (FIG. 3). Remarkably, the intracellular receptor for rapamycin is also FKBP, but in contrast to the FK506–FKBP complex, rapamycin–FKBP does not inhibit calcineurin. Michael Hall identified the molecular target of rapamycin–FKBP in yeast as being a protein kinase, which he termed the ‘target of rapamycin’ (Tor)24. The mammalian homo￾logue, which is called mTOR, or the FKBP–rapamycin-associated protein (FRAP), was identified subsequently. Rapamycin was the first drug to be approved for clinical use that seems to inhibit one protein kinase specifically 8 . mTOR is involved in a phosphatidylino￾sitol (PtdIns)-3-kinase-dependent signalling pathway that has a crucial role in cell-cycle progression from G1-to-S phase, as well as in IL-2-stimulated T-cell proliferation; these are activities that underlie its anticancer and immunosuppressive properties. Consistent with its requirement for the G1-to-S trans￾ition, the cytostatic effects of CCI779 are most marked in tumours in which the level of PtdIns-3,4,5-trisphosphate (the product of the class 1 PtdIns 3-kinases) is elevated, such as occurs in tumours that are deficient in the phosphatase-and-tensin homologue (PTEN), which is the phosphatase that converts PtdIns(3,4,5)P3 to PtdIns(4,5)P2 (REFS 25,26). CSAIDs During bacterial infection, lipopolysaccharide (LPS) — a component of the cell wall of gram-negative bacteria — triggers cells of the immune system to produce proinflammatory cytokines, such as tumour-necrosis factor (TNF) and IL-1, which are released into the circulation, where they help to mount the immune responses that fight — and eventually kill — the invading bacteria. However, this defence mechanism is a double-edged sword, because the uncontrolled production of proinflammatory cytokines can be a cause of chronic inflammatory diseases, such as rheumatoid arthritis, inflammatory bowel disease and septic shock. For this reason, many companies have sought to develop compounds that suppress TNF production. More than 20 years ago, SmithKline and French developed a new class of pyridinyl imidazoles — exemplified by SKF86002 — that showed efficacy in animal models of chronic inflammatory disease. They were therefore called cytokine-synthesis anti￾inflammatory drugs (CSAIDs; see REF. 27 for a review of early work in this area). In the late 1980s, John Lee and colleagues found that SKF86002 suppressed the LPS￾induced production of IL-1 and TNF in human monocytes28. Subsequently, more potent pyridinyl-imidazole inhibitors of TNF production, such as SB202190, were shown to prevent translation of the messenger RNA that encodes TNF. A protein that bound these pyridinyl imidazoles specifically was purified from the cell extracts, which led to its identifi￾cation as a mitogen-activated protein kinase (MAPK) family member that was termed the CSAID-binding protein (CSBP)29. This pro￾tein kinase was identified independently by several other laboratories30–32, and is now usually called p38 MAPK. At least three inhibitors of p38 MAPK have now entered human clinical trials for the treatment of rheumatoid arthritis or psoriasis (TABLE 1), and several other companies have potent inhibitors of this enzyme at a preclinical stage. Although SB203580 (FIG. 1) and the closely related SB202190 are ATP competitive inhibitors, they are relatively specific for p38 MAPK in vitro and do not inhibit many other protein kinases that have been tested8,33. The basis for this high degree of specificity was shown by the determination of the NATURE REVIEWS | DRUG DISCOVERY VOLUME 1 | APRIL 2002 | 311 N O N N H N N H N N N N N N HN S F HN R1 R2 R2 S HN H N CH3 N H N N H N O O O O N H N N H N O O O N O N N O O O CH3 CH3 CH3 CH3 CH3 CH3 CH3 OH O OH OCH3 O OCH3 OH O N O O O OH OCH3 O H H H O O O O NH OCH3 H3C O O NH2 OCH3 OH H8 Staurosporine PD98059 Olomoucine: R1 = H2, R2 = H2 Roscovitine: R1 = C2H5, R2 = CH3 Tyrphostin SB203580 Cyclosporin Rapamycin N H O HO HO Figure 1 | Chemical structures of some compounds that have been important for stimulating interest in the development of protein-kinase inhibitors

PERSPECTIVESTable 1Some small-molecule inhibitors of protein kinases that are undergoing human clinical trialsKinase targetedInhibitorDiseaseStatusCompanyTyrosine kinasesCancerABL (KITR, PDGFR)Gleevec (Glivec,STI-571)NovartisFDAapproval, May2001EGFRZD1839(ressa)CancerPhasellIAstraZenecaOSI774OSIPharmaceuticals/RochelCancerPhase IlIGenentechCI1033PfizerPhaselEGFR,ERB2RCancerEKB569CancerPhaselWyeth-AyerstGW2016GlaxoSmithKlineCancerPhaselPKI166CancerPhaselNovartisVEGFR (PDGFR, FGFR)SU6668Pharmacia CoporationCancerPhaselVEGFRPTK787/ZK222584Novartis/Schering-PloughCancerPhasellVEGFR (EGFR)ZD6474CancerPhaselAstraZenecaCEP2583CancerPhasellNGFRCephalonSerine/threoninekinasesPKC412NovartisPhase!PKC.KITR,PDGFR?Cancer,retinopathyCDKs?AventisCancerPhasellFlavopiridolCDK2CYC202CyclacelCancerPhaselMKK1PD184352PfizerCancerPhaselRAF?CancerPhaselBAY43-906OnyxPhamaceuticals/BayerUCN-01PhaselCHK1,PKC,others?Kyowa HakkoCancermTORCCI779Wyeth-AyerstCancerPhasellRAD001NovartisCancerPhaselRapamycin (Sirolimus)Wyeth-AyerstFDAapproval,1999ImmunosuppressionROCK?HA1077 (AT877, fasudi)Asahi Chemical IndustryApproved in 1995 (Japan)CerebralvasospasmEnLilyPKCβLY333531PhasellIDiabetic retinopathyp38/SAPK2aSB281832GlaxoSmithKlineRheumatoid arthritisPhaselBIRB0796Boehringer IngelheimRheumatoid arthritisPhasellRo320-1195RocheRheumatoid arthritisPhaselMLKCEP-1347PhaselCephalonNeurodegenerationABL_Abelson tyrosine kinase; CDK,cyclin-dependent kinase; CHK1,checkpoint kinase 1:EGFR,epidermal-growth-factor receptor,ERB2R,ERB2 receptor;FDA,US Food and Drug Administration; FGFR,fibroblast-growth-factor receptor,KITR, c-KIT receptor; MKK1, mitogen-activated protein kinase kinase1MLK,mixed-lineage proteinkinase;mTOR,targetof rapamycin (mammalian):NGFR,nerve-growth-factor receptor:p3B.p3Bmitogen-activated proteinkinase;PDGFR,platelet-derived-growth-factor receptor; PKC,protein kinase C; ROCK,RHO-dependent proteinkinase; SAPK,stress-activated proteinkinaseVEGFR,vascular-endothelial-growth-factor receptor.Anticanceragentsinhibit protein tyrosine kinases (reviewed incancers that target particular protein kinasesCyclosporin and rapamycin were the firstREFS 4042)in this pathway.compounds to be approved as drugs thatGrowth factors, such as epidermal growthSeveral antibodies that bind to the extra-exert theireffectsbyinhibitinga particularfactor (EGF), activatethe classical RAS-cellular domain of the EGFR are undergoingprotein phosphatase or protein kinase.dependentMAPKcascade,whichis requiredclinical trials,including C225 (also calledHowever,these compounds-as well as thefor the proliferation of some cells and theCetuximab), which was developed byCSAIDs that inhibit p38 MAPK-were notdifferentiation ofothers. However,the uncon-Imclone Systems and is in Phase IlI clinicaldeveloped as a result of thisknowledge,andtrolled activation of this pathway is nowtrialsforthetreatment of several cancers.their clinical efficacy was known beforeknownto cause cancer.This can occurasaC225 has to be administered intravenously;theirmechanism of action was elucidated.Itresult of the overexpression of particularhowever, several promising orally activeis in the field of cancer in which much of thegrowth-factor-receptor tyrosinekinases ordrugs that are potent and relatively specificeffortto develop drugs thattarget specifictheir mutation to constitutively active forms.inhibitorsoftheEGFRtyrosinekinaseareprotein kinases has been concentrated. AtFor example,theEGF receptor (EGFR)isnow well advanced in human clinical trialspresent,someofthemostpromisingdrugsoverexpressedinmanycancersofepithelial(TABLE1).TheAstraZeneca compoundin development as anticancer agents areorigin,suchaslungandbreastcancers.TheZD1839,whichisnowcalledIressa,hasinhibitors of protein tyrosine kinases.mutation of RAS itselfto constitutivelyshown marked efficacy against severalactive,oncogenicforms occurs in 25% ofProgress in this area has beengreatlyinflu-cancersin human clinical trials (reviewedenced by seminal studies that were carriedhuman cancers, whereas the protein kinasein REE.43),and will probablybe approvedout 10-15 years ago, which identifiedRAF was discovered to bea viral oncogene.for clinical use in 2002 (FIG.2).Anotherimportantcompound classes,suchasTherehasthereforebeenconsiderableinterestcompound,OSI774,isalsoinPhaseI1Iquinazolines and tyrphostins, that potentlyin the development of drugs to treat differentclinical trials43.312APRIL2002VOLUME1www.nature.com/reviews/drugdisc2002NaturePublishingGroup

© 2002 Nature Publishing Group 312 | APRIL 2002 | VOLUME 1 www.nature.com/reviews/drugdisc PERSPECTIVES cancers that target particular protein kinases in this pathway. Several antibodies that bind to the extra￾cellular domain of the EGFR are undergoing clinical trials, including C225 (also called Cetuximab), which was developed by Imclone Systems and is in Phase III clinical trials for the treatment of several cancers. C225 has to be administered intravenously; however, several promising orally active drugs that are potent and relatively specific inhibitors of the EGFR tyrosine kinase are now well advanced in human clinical trials (TABLE 1). The AstraZeneca compound ZD1839, which is now called Iressa, has shown marked efficacy against several cancers in human clinical trials (reviewed in REF. 43), and will probably be approved for clinical use in 2002 (FIG. 2). Another compound, OSI774, is also in Phase III clinical trials43. inhibit protein tyrosine kinases (reviewed in REFS 40–42). Growth factors, such as epidermal growth factor (EGF), activate the classical RAS￾dependent MAPK cascade, which is required for the proliferation of some cells and the differentiation of others. However, the uncon￾trolled activation of this pathway is now known to cause cancer. This can occur as a result of the overexpression of particular growth-factor-receptor tyrosine kinases or their mutation to constitutively active forms. For example, the EGF receptor (EGFR) is overexpressed in many cancers of epithelial origin, such as lung and breast cancers. The mutation of RAS itself to constitutively active, oncogenic forms occurs in 25% of human cancers, whereas the protein kinase RAF was discovered to be a viral oncogene. There has therefore been considerable interest in the development of drugs to treat different Anticancer agents Cyclosporin and rapamycin were the first compounds to be approved as drugs that exert their effects by inhibiting a particular protein phosphatase or protein kinase. However, these compounds — as well as the CSAIDs that inhibit p38 MAPK — were not developed as a result of this knowledge, and their clinical efficacy was known before their mechanism of action was elucidated. It is in the field of cancer in which much of the effort to develop drugs that target specific protein kinases has been concentrated. At present, some of the most promising drugs in development as anticancer agents are inhibitors of protein tyrosine kinases. Progress in this area has been greatly influ￾enced by seminal studies that were carried out 10–15 years ago, which identified important compound classes, such as quinazolines and tyrphostins, that potently Table 1 | Some small-molecule inhibitors of protein kinases that are undergoing human clinical trials Kinase targeted Inhibitor Company Disease Status Tyrosine kinases ABL (KITR, PDGFR) Gleevec (Glivec, STI-571) Novartis Cancer FDA approval, May 2001 EGFR ZD1839 (Iressa) AstraZeneca Cancer Phase III OSI774 OSI Pharmaceuticals/Roche/ Cancer Phase III Genentech EGFR, ERB2R CI1033 Pfizer Cancer Phase I EKB569 Wyeth-Ayerst Cancer Phase I GW2016 GlaxoSmithKline Cancer Phase I PKI166 Novartis Cancer Phase I VEGFR (PDGFR, FGFR) SU6668 Pharmacia Corporation Cancer Phase I VEGFR PTK787/ZK222584 Novartis/Schering-Plough Cancer Phase II VEGFR (EGFR) ZD6474 AstraZeneca Cancer Phase I NGFR CEP2583 Cephalon Cancer Phase II Serine/threonine kinases PKC, KITR, PDGFR? PKC412 Novartis Cancer, retinopathy Phase I CDKs? Flavopiridol Aventis Cancer Phase II CDK2 CYC202 Cyclacel Cancer Phase I MKK1 PD184352 Pfizer Cancer Phase I RAF? BAY43-906 Onyx Pharmaceuticals/Bayer Cancer Phase I CHK1, PKC, others? UCN-01 Kyowa Hakko Cancer Phase I mTOR CCI779 Wyeth-Ayerst Cancer Phase II RAD001 Novartis Cancer Phase I Rapamycin (Sirolimus) Wyeth-Ayerst Immunosuppression FDA approval, 1999 ROCK? HA1077 (AT877, fasudil) Asahi Chemical Industry Cerebral vasospasm Approved in 1995 (Japan) PKCβ LY333531 Eli Lilly Diabetic retinopathy Phase III p38/SAPK2a SB281832 GlaxoSmithKline Rheumatoid arthritis Phase I BIRB0796 Boehringer Ingelheim Rheumatoid arthritis Phase II Ro320-1195 Roche Rheumatoid arthritis Phase I MLK CEP-1347 Cephalon Neurodegeneration Phase I ABL, Abelson tyrosine kinase; CDK, cyclin-dependent kinase; CHK1, checkpoint kinase 1; EGFR, epidermal-growth-factor receptor; ERB2R, ERB2 receptor; FDA, US Food and Drug Administration; FGFR, fibroblast-growth-factor receptor; KITR, c-KIT receptor; MKK1, mitogen-activated protein kinase kinase 1; MLK, mixed-lineage protein kinase; mTOR, target of rapamycin (mammalian); NGFR, nerve-growth-factor receptor; p38, p38, mitogen-activated protein kinase; PDGFR, platelet-derived-growth-factor receptor; PKC, protein kinase C; ROCK, RHO-dependent protein kinase; SAPK, stress-activated protein kinase; VEGFR, vascular-endothelial-growth-factor receptor

PERSPECTIVESAnother promising approach to the devel-opment of anticancer drugs with broadOHDhoyefficacy is based on Judah Folkman's 25-year-old idea of destroying theblood supply to atumour.Angiogenesis depends on growthO=S=0factors,such as platelet-derived growth factor(PDGF), FGF and vascular endothelialOCHagrowth factor (VEGF).Antibodies haveHic-NHobeen raised that bind to the extracellularFasudilUCN-01CEP-1347domain of theVEGF receptor,and onethathasbeendevelopedbyGenentechisnowinPhase IlI clinical trials as an anti-angiogenic2Nagent.However, several promising orallyNactive inhibitors of the VEGF receptorO(VEGFR)protein tyrosinekinase-—includingSU6668,PTK787andZD6474(TABLE1)are undergoing human clinical trialsGleevecressa(reviewedinREE.43)Cyclin-dependent protein kinases (CDKs)CHaNhave essential roles in cell proliferation,which has stimulated considerableinterest inthe development of inhibitors of theseHCenzymes to suppress tumour growth.TwoCDK inhibitors that are undergoing humanTclinical trials are shown in TABLE I,the first ofOHOwhich was Flavopiridol.More potent andSU6668FlavopiridolPD184352specific CDK inhibitors have been developedFigure 2|Chemical structures of some small-molecule inhibitors of protein kinases that haveby many other companies, but whether theybeen approved or are undergoing human clinical trials.Further information is given in the TIMELINE.have entered human clinical trials has not yetbeen disclosed.Interestingly, certain indirubin dyes,entered human clinical trials (CGP57148B;conformation.The compound BIRBo796which are the active ingredients in anancient Chinese herbal remedythat has beenlatercalledSTI-571andthenGleevec/(TABLE 1) has recently been shown to interactGlivec) was discovered around 1992 (FIG.2)46,with p38 MAPK in an analogous way (S. Jakes,used for centuries to treat diseases such ascancer, are potent CDK inhibitors4s. So,but itsdevelopment wasnot initially givenapersonal communication).Although Gleevec is a relatively specificwithout anyone realising it, CDK inhibitorshigh priority because of the relatively lowmight actually have been in clinical use forinhibitor of ABL, it also inhibits the c-KITincidenceofCMLSupportforthedevelop-mentofCGP57148Bcame asa result ofaand PDGF-receptor tyrosine kinases withrather a long time!pivotal study that was carried out by Briansimilar potency.It has been undergoingGleevecDruker, which profiled this compound inclinical trials for the treatment of gastro-A landmark event occurred in May 2001exvivocolony-formingassaysusing cellsintestinal stromaltumoursand other cancerswhen Gleevec-thefirst importantdrugtofromCMLandcontrolcells47.Aftertheini-in which c-KIT or PDGF-receptor signallingbe developed by targeting a protein kinasetiation ofa clinical trial, spectacular efficacyis deregulateds,and was recentlyapprovedspecifically (the Abelson tyrosine kinase,and minimal side effects were shown.Thisfor the treatment of gastrointestinal stromalABL)wasapprovedforclinicaluse.ABLledtoitsextremelyrapidapprovalforclini-tumours.becomesfused to the breakpoint clustercal use,and has created greatexcitement inregion (BCR) protein as a result of a chromo-the pharmaceutical industry (reviewed inPreventingkinase activationsome rearrangement in nearly all cases ofREFS 43,49).Alan Saltiel and his colleagues at Parke-Davischronic myelogenous leukaemia (CML).ThisAlthough Gleevec is an ATP competitivedeveloped the compound PD98059 (FIG.1)creates akinase with enhanced activity thatinhibitor, the determination of the three-during a screen toidentifyinhibitors of amight cause CML by eliciting uncontrolled'constitutively'active mutant of MAPKdimensional structureofABLin complexactivationoftheMAPKcascadewithGleevecshowedthatthedrugextendskinase-1 (MKK1),whichcanbeproducedbyA programme to develop an inhibitor ofmuchfurther into the catalytic sites.In par-mutating the residues that are phosphoryl-ABL was initiated by Nick Lydon atatedbyRAFto glutamicacid(MKK1-EE)53ticular, it straddles thehighly conservedNovartis in 1986.However,itwasonlyamino-terminal region oftheactivationloopHowever,wewere surprised to find thatwhenbaculovirusexpressionsystemsforthatcontrolsthecatalyticactivityofmanyPD98059doesnotinhibitMKK1thathadthe large-scale production of several pro-proteinkinases by switching them from anbeen activated by RAF.Because MKK1-EEtein tyrosine kinases were developed byinactive dephosphorylated form to an activehas<1% of the activity of phosphorylatedphosphorylated state. Intriguingly, the bind-MKK1, this indicates that the screen selectedTom Robertsand NickLydon in thelate1980s that the programmereally started toingofGleevec induces a structural transitionfor compounds that bind much moretake off. The compound that subsequentlythat causesthekinasetoadopttheinactivestrongly to the inactive form of MKK1.NATUREREVIEWSDRUGDISCOVERYVOLUME1APRIL20023132002NaturePublishingGroup

© 2002 Nature Publishing Group PERSPECTIVES conformation. The compound BIRB0796 (TABLE 1) has recently been shown to interact with p38 MAPK in an analogous way (S. Jakes, personal communication). Although Gleevec is a relatively specific inhibitor of ABL, it also inhibits the c-KIT and PDGF-receptor tyrosine kinases with similar potency51. It has been undergoing clinical trials for the treatment of gastro￾intestinal stromal tumours and other cancers in which c-KIT or PDGF-receptor signalling is deregulated52, and was recently approved for the treatment of gastrointestinal stromal tumours. Preventing kinase activation Alan Saltiel and his colleagues at Parke-Davis developed the compound PD98059 (FIG. 1) during a screen to identify inhibitors of a ‘constitutively’ active mutant of MAPK kinase-1 (MKK1), which can be produced by mutating the residues that are phosphoryl￾ated by RAF to glutamic acid (MKK1-EE)53. However, we were surprised to find that PD98059 does not inhibit MKK1 that had been activated by RAF. Because MKK1-EE has < 1% of the activity of phosphorylated MKK1, this indicates that the screen selected for compounds that bind much more strongly to the inactive form of MKK1. Another promising approach to the devel￾opment of anticancer drugs with broad efficacy is based on Judah Folkman’s 25-year￾old idea of destroying the blood supply to a tumour. Angiogenesis depends on growth factors, such as platelet-derived growth factor (PDGF), FGF and vascular endothelial growth factor (VEGF). Antibodies have been raised that bind to the extracellular domain of the VEGF receptor, and one that has been developed by Genentech is now in Phase III clinical trials as an anti-angiogenic agent. However, several promising orally active inhibitors of the VEGF receptor (VEGFR) protein tyrosine kinase — including SU6668, PTK787 and ZD6474 (TABLE 1) — are undergoing human clinical trials (reviewed in REF. 43) . Cyclin-dependent protein kinases (CDKs) have essential roles in cell proliferation, which has stimulated considerable interest in the development of inhibitors of these enzymes to suppress tumour growth. Two CDK inhibitors that are undergoing human clinical trials are shown in TABLE 1, the first of which was Flavopiridol44. More potent and specific CDK inhibitors have been developed by many other companies, but whether they have entered human clinical trials has not yet been disclosed. Interestingly, certain indirubin dyes, which are the active ingredients in an ancient Chinese herbal remedy that has been used for centuries to treat diseases such as cancer, are potent CDK inhibitors45. So, without anyone realising it, CDK inhibitors might actually have been in clinical use for rather a long time! Gleevec A landmark event occurred in May 2001 when Gleevec — the first important drug to be developed by targeting a protein kinase specifically (the Abelson tyrosine kinase, ABL) — was approved for clinical use. ABL becomes fused to the breakpoint cluster region (BCR) protein as a result of a chromo￾some rearrangement in nearly all cases of chronic myelogenous leukaemia (CML). This creates a kinase with enhanced activity that might cause CML by eliciting uncontrolled activation of the MAPK cascade. A programme to develop an inhibitor of ABL was initiated by Nick Lydon at Novartis in 1986. However, it was only when baculovirus expression systems for the large-scale production of several pro￾tein tyrosine kinases were developed by Tom Roberts and Nick Lydon in the late 1980s that the programme really started to take off. The compound that subsequently entered human clinical trials (CGP57148B; later called STI-571 and then Gleevec/ Glivec) was discovered around 1992 (FIG. 2)46, but its development was not initially given a high priority because of the relatively low incidence of CML. Support for the develop￾ment of CGP57148B came as a result of a pivotal study that was carried out by Brian Druker, which profiled this compound in ex vivo colony-forming assays using cells from CML and control cells47. After the ini￾tiation of a clinical trial, spectacular efficacy and minimal side effects were shown48. This led to its extremely rapid approval for clini￾cal use, and has created great excitement in the pharmaceutical industry (reviewed in REFS 43,49). Although Gleevec is an ATP competitive inhibitor, the determination of the three￾dimensional structure of ABL in complex with Gleevec showed that the drug extends much further into the catalytic site50. In par￾ticular, it straddles the highly conserved amino-terminal region of the ‘activation loop’ that controls the catalytic activity of many protein kinases by switching them from an inactive dephosphorylated form to an active phosphorylated state. Intriguingly, the bind￾ing of Gleevec induces a structural transition that causes the kinase to adopt the inactive NATURE REVIEWS | DRUG DISCOVERY VOLUME 1 | APRIL 2002 | 313 N N H N O NH OCH3 H3C O CEP-1347 UCN-01 OH S N O O HN Fasudil N N N H N H N N N O Gleevec Iressa O OH O Cl N HO CH3 OH Flavopiridol H N Cl I H N F F O O PD184352 SU6668 H3CO N N HN F Cl N O O N H O OH O N N H N O O HO O OCH3 S S Figure 2 | Chemical structures of some small-molecule inhibitors of protein kinases that have been approved or are undergoing human clinical trials. Further information is given in the TIMELINE

PERSPECTIVESrelapseinpatientswhohavetheadvancedstage of CMLs.However,although suchresistancepresentsnewtherapeuticchal-lenges, resistance to Gleevec has been seenonly in the most advanced forms of CML, inwhich extensive genomic instability occurs,which leads to many additional geneticchanges.Most protein-kinase inhibitors that areidentified by high-throughput screening areATP competitiveand,althoughinhibitorsofhigh specificitycan clearlybeobtainedbytargeting the ATP-binding site, the develop-Figure 3|Basis for the specificity of two ATP-competitive protein-kinase inhibitors.a|ComparisonofthebindingofSB203580(blue)andATP(vellow)top38MAPK34.b|Comparisonofthebindingofpurval-mentof sufficientpotencytocompetewithanol (blue)and ATP (yellow) to CDK239,Drug specificity is largely determined by the partof the molecule thattheATP concentrations that arepresent indoes not interact with residues that bind ATP, but which occupies a hydrophobic binding pocket near thevivo is still a problem, especially for theATP-binding site. CDK2, cyclin-dependent kinase 2: p38 MAPK, p38 mitogen-activated protein kinase.many protein kinases that have2Michaelis-Mentenconstant(K)forATPof10 μM or less. Indeed, it might be no coinci-This led us to discover that PD98059 prevents23 protein-kinase inhibitors areknown to bedencethataplethora of potent inhibitorsthe activation of MKK1 by RAF$4. So,undergoing human clinical trials (TABLE1)thattargetthep38MAPKhavebeendevel-PD98059is notakinaseinhibitor,butbindsHowever,manyothershaveentered clinicaloped, because its K for ATP is above 0.1 mM.to the inactive, dephosphorylated form oftrialswithouttheir structuresbeing dis-It is surprising that virtually no compoundsMKKI,preventing its activation by RAF bothclosed, and a great many more are stillat thethat competefor binding with the proteinin vitro and in cell-based assays.The moresubstrate have been identified. Unlike ATPpreclinical stage,As a result, protein kinasespotentcompounds U0126 (REE55)andare already the second largest group of drugcompetitiveinhibitors,such compoundsPD184352 (REE56;TABLE1:FIG.2),whichweretargets afterG-protein-coupledreceptors,mighthave thepotentialto preventa proteindeveloped subsequently,also seem to preventand they account for 20-30% of the drug dis-kinase from phosphorylating some sub-the activation of MKKlmuch more potentlycovery programmes of many companies.strates but not otherso,and their develop-than they inhibit MKK1 activity.There is clearly no shortage of potential tar-mentis clearly an important challengeforPD98059isoneofthefewsyntheticpro-gets, as protein kinases comprise the largestthe future. A much greater emphasis ontein-kinase inhibitors thatis not ATP com-enzyme family, with ~500 being encoded bydeveloping compounds that bind preferen-petitive. The way in which it was identified isthe human genome.tially to the inactive forms of proteintherefore instructive, and raises the possibil-In thefield of cancer, in which much of thekinases, or which prevent oneprotein kinaseity that completely different compoundseffort so far has been directed, protein-kinasefrom activating another, might well pay div-mightbe obtainedbyscreening witheitherinhibitors are proving to be well toleratedidends.Smarterwaystoscreenfororhigh- or low-activity forms of particular pro-compared with conventional chemothera-design-inhibitors of the activityand acti-tein kinases.PD98059 reverses the phenotypepeutic treatments. This is encouraging,vation of particular proteinkinases on theof RAS-transformed cell linesss,andbecause several protein-kinase inhibitorsbasis of a detailed understanding of theirPD184352 was reported to inhibit the growththathaveenteredhumanclinical trialsarecatalytic and regulatory properties is likelyof human colontumours that werenotveryspecific.Thishasbecomeobvioustobecomemoreandmoreimportant.implanted intomicewithoutcausing obvi-only since the introduction of much largerFinally,it should be mentioned thatous adverse sideeffects overseveralmonthspanels of protein kinases for profiling speci-protein-kinase inhibitors are important notoftreatment.For this reason,PD184352 hasficity More extensive use of even largerjustforthetreatmentof disease,butalso asnow entered Phase I clinical trials. Thepanelswill aidthefuturedevelopmentofreagents to help us understand more aboutabsence of side effects that have beenmorespecificprotein-kinaseinhibitors,andthe physiological roles of protein kinases.reportedsofar has come as a surprise to thealsoallowtheseenzymestobereclassifiedThe number of papers that have been refer-cell-signalling community,because theclassi-according to the similarities of their ATP-enced in this article that have been cited morecal MAPK cascade has been implicated in sobinding sitesratherthantheiramino-acidthan1,000times (forexample,REFs5,11,13,many cellular processes. However,perhaps itssequences28,53,54)isanindicationoftheacuteneedforIt willbecome more important to showessential roles inproliferationand differen-such compounds bythecell-signalling com-tiation are required only during embryo-that a drug is exerting its effect through inhi-munity-Many specific protein-kinasegenesis, and the pathway might be far lessbition of a particular protein kinase, byinhibitors that cannot be used as drugs forcrucial for normal function in adults thanshowingthat the effect ofthe drugdisappearsreasonsoftoxicity,pharmacologyorsolubil-previously supposedwhen a drug-resistant mutant is over-ity,suchasPD98059andSB203580,couldbeexpressed orreplacesthewild-typeenzyme.extremelyuseful research reagents.Pharma-The futureHowever, this has been shown in onlyafewceutical companies have muchto gain fromThree protein-kinase inhibitors (fasudil,cases (for example, REFs57,58).Themutationthe discoveries that will be made by exploit-rapamycin and Gleevec)and one protein-ing such compounds, and it is to behopedof a singleamino acid can makeakinasephosphatase inhibitor (cyclosporin) have sodrug resistant7sand mutations in ABL thatthat more will be released for general use infar been approved for clinical use,and a furthermakeitresistanttoGleevecarethecauseofthefuture.314APRIL2002VOLUME1www.nature.com/reviews/drugdisc2002Nature Publishing Group

© 2002 Nature Publishing Group 314 | APRIL 2002 | VOLUME 1 www.nature.com/reviews/drugdisc PERSPECTIVES relapse in patients who have the advanced stage of CML59. However, although such resistance presents new therapeutic chal￾lenges, resistance to Gleevec has been seen only in the most advanced forms of CML, in which extensive genomic instability occurs, which leads to many additional genetic changes. Most protein-kinase inhibitors that are identified by high-throughput screening are ATP competitive and, although inhibitors of high specificity can clearly be obtained by targeting the ATP-binding site, the develop￾ment of sufficient potency to compete with the ATP concentrations that are present in vivo is still a problem, especially for the many protein kinases that have a Michaelis–Menten constant (Km) for ATP of 10 µM or less. Indeed, it might be no coinci￾dence that a plethora of potent inhibitors that target the p38 MAPK have been devel￾oped, because its Km for ATP is above 0.1 mM. It is surprising that virtually no compounds that compete for binding with the protein substrate have been identified. Unlike ATP competitive inhibitors, such compounds might have the potential to prevent a protein kinase from phosphorylating some sub￾strates but not others60, and their develop￾ment is clearly an important challenge for the future. A much greater emphasis on developing compounds that bind preferen￾tially to the inactive forms of protein kinases, or which prevent one protein kinase from activating another, might well pay div￾idends. Smarter ways to screen for — or design — inhibitors of the activity and acti￾vation of particular protein kinases on the basis of a detailed understanding of their catalytic and regulatory properties is likely to become more and more important. Finally, it should be mentioned that protein-kinase inhibitors are important not just for the treatment of disease, but also as reagents to help us understand more about the physiological roles of protein kinases. The number of papers that have been refer￾enced in this article that have been cited more than 1,000 times (for example, REFS 5,11,13, 28,53,54) is an indication of the acute need for such compounds by the cell-signalling com￾munity. Many specific protein-kinase inhibitors that cannot be used as drugs for reasons of toxicity, pharmacology or solubil￾ity, such as PD98059 and SB203580, could be extremely useful research reagents. Pharma￾ceutical companies have much to gain from the discoveries that will be made by exploit￾ing such compounds, and it is to be hoped that more will be released for general use in the future. 23 protein-kinase inhibitors are known to be undergoing human clinical trials (TABLE 1). However, many others have entered clinical trials without their structures being dis￾closed, and a great many more are still at the preclinical stage. As a result, protein kinases are already the second largest group of drug targets after G-protein-coupled receptors, and they account for 20–30% of the drug dis￾covery programmes of many companies. There is clearly no shortage of potential tar￾gets, as protein kinases comprise the largest enzyme family, with ~500 being encoded by the human genome. In the field of cancer, in which much of the effort so far has been directed, protein-kinase inhibitors are proving to be well tolerated compared with conventional chemothera￾peutic treatments. This is encouraging, because several protein-kinase inhibitors that have entered human clinical trials are not very specific. This has become obvious only since the introduction of much larger panels of protein kinases for profiling speci￾ficity 8 . More extensive use of even larger panels will aid the future development of more specific protein-kinase inhibitors, and also allow these enzymes to be reclassified according to the similarities of their ATP￾binding sites rather than their amino-acid sequences. It will become more important to show that a drug is exerting its effect through inhi￾bition of a particular protein kinase, by showing that the effect of the drug disappears when a drug-resistant mutant is over￾expressed or replaces the wild-type enzyme. However, this has been shown in only a few cases (for example, REFS 57,58). The mutation of a single amino acid can make a kinase drug resistant57,58, and mutations in ABL that make it resistant to Gleevec are the cause of This led us to discover that PD98059 prevents the activation of MKK1 by RAF54. So, PD98059 is not a kinase inhibitor, but binds to the inactive, dephosphorylated form of MKK1, preventing its activation by RAF both in vitro and in cell-based assays. The more potent compounds U0126 (REF. 55) and PD184352 (REF. 56; TABLE 1; FIG. 2), which were developed subsequently, also seem to prevent the activation of MKK1 much more potently than they inhibit MKK1 activity8 . PD98059 is one of the few synthetic pro￾tein-kinase inhibitors that is not ATP com￾petitive. The way in which it was identified is therefore instructive, and raises the possibil￾ity that completely different compounds might be obtained by screening with either high- or low-activity forms of particular pro￾tein kinases. PD98059 reverses the phenotype of RAS-transformed cell lines53, and PD184352 was reported to inhibit the growth of human colon tumours that were implanted into mice without causing obvi￾ous adverse side effects over several months of treatment56. For this reason, PD184352 has now entered Phase I clinical trials. The absence of side effects that have been reported so far has come as a surprise to the cell-signalling community, because the classi￾cal MAPK cascade has been implicated in so many cellular processes. However, perhaps its essential roles in proliferation and differen￾tiation are required only during embryo￾genesis, and the pathway might be far less crucial for normal function in adults than previously supposed. The future Three protein-kinase inhibitors (fasudil, rapamycin and Gleevec) and one protein￾phosphatase inhibitor (cyclosporin) have so far been approved for clinical use, and a further a b Figure 3 | Basis for the specificity of two ATP-competitive protein-kinase inhibitors. a | Comparison of the binding of SB203580 (blue) and ATP (yellow) to p38 MAPK34. b | Comparison of the binding of purval￾anol (blue) and ATP (yellow) to CDK239. Drug specificity is largely determined by the part of the molecule that does not interact with residues that bind ATP, but which occupies a hydrophobic binding pocket near the ATP-binding site. CDK2, cyclin-dependent kinase 2; p38 MAPK, p38 mitogen-activated protein kinase

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J.efal Eflicacyand safetyofaspeclicprotein kinasestheeflect ofCCI-779,anmTORinhibitor,inprecinicalinhibitor af the BCR-ABLtyrosine kinase inchronicNational Cancer Institute:models of breast cancer.Endocr. Relat. Cancer 8,myeloid leukaemia. N. Engl. J. Med. 344, 10311037http://www.ncnih.gow249258 (2001).(2001),Access to this interactive links box is free online.NATUREREVIEWSDRUGDISCOVERYVOLUME1APRIL20023152002NaturePublishingGroup

© 2002 Nature Publishing Group PERSPECTIVES 49. Druker, B. J. & Lydon, N. Lessons learned from the development of an Abl tyrosine kinase inhibitor for chronic myelogenous leukaemia. J. Clin. Invest. 105, 3–7 (2000). 50. Schindler, T. et al. Structural mechanism of STI-571 inhibition of Abelson tyrosine kinase. Science 289, 1938–1942 (2000). 51. Buchdunger, E. et al. Abl protein-tyrosine kinase inhibitor, STI-571, inhibits in vitro signal transduction mediated by c-Kit and PDGF receptors. J. Pharmacol. Exp. Ther. 295, 139–145 (2000). 52. Joensuu, H. et al. Effect of the tyrosine kinase inhibitor STI571 in a patient with a metastatic gastrointestinal stromal tumor. N. Engl. J. Med. 344, 1052–1056 (2001). 53. Dudley, D. T., Pang, L., Decker, S. J., Bridges, A. J. & Saltiel, A. R. A synthetic inhibitor of the mitogen-activated protein kinase cascade. Proc. Natl Acad. Sci. USA 92, 7686–7689 (1995). 54. Alessi, D. R., Cuenda, A., Cohen, P., Dudley, D. T. & Saltiel, A. R. 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A common phosphate binding site explains the unique substrate specificity of GSK3 and its inactivation by phosphorylation. Mol. Cell 7, 1321–1327 (2001). Acknowledgements I thank J. Adams, S. Cartlidge, R. Ford, S. Jakes, B. Machin and N. Lydon for helpful discussions. I apologise to the many scientists whose important discoveries could not be included or referenced in this article because of space restrictions. The work carried out in my laboratory is supported by the UK Medical Research Council, The Royal Society of London, Diabetes UK, The Louis Jeantet Foundation, AstraZeneca, Boehringer Ingelheim, GlaxoSmithKline, NovoNordisk and Pfizer. Online links DATABASES The following terms in this article are linked online to: Cancer.gov: http://www.cancer.gov/cancer_information/ breast cancer | chronic myelogenous leukaemia | colon tumour | gastrointestinal stromal cancer | lung cancer LocusLink: http://www.ncbi.nlm.nih.gov/LocusLink/ ABL | BCR | calcineurin | calmodulin | CDK2 | CHK1 | cyclophilin | EGF | EGFR | FGFR | FKBP | IL-1 | IL-2 | JNK | c-KIT | MKK1 | MLK | p38 MAPK | mTOR | PDGF | PDGF receptor | PKA | PKC | PKC-β | PtdIns 3-kinase | PTEN | RAF | ROCK | TNF | VEGF | VEGFR Medscape DrugInfo: http://promini.medscape.com/drugdb/search.asp Cyclosporin | Gleevec OMIM: http://www.ncbi.nlm.nih.gov/Omim/ chronic myelogenous leukaemia | insulin-dependent diabetes mellitus | inflammatory bowel disease | rheumatoid arthritis Saccharomyces Genome Database: http://genome-www.stanford.edu/Saccharomyces/ Tor FURTHER INFORMATION Encyclopedia of Life Sciences: http://www.els.net/ protein kinases National Cancer Institute: http://www.nci.nih.gov/ Access to this interactive links box is free online. 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