2010 Targeted covalent drugs of the kinase family

Available online at Targeted covalent drugs of the kinase familyJuswinder Singh,Russell C Petter and Arthur F KlugeIn the past decade tremendous progress has been made toward a new class of therapeutics termed ‘targeted covalent drugs’,in which structure-based approaches are employed to create small molecules that inactivate their protein target through targeted covalent attachment to a specific cysteine.In the kinase field,this approach is demonstrating promise in overcoming the potency,selectivity,and efficacy challenges currently faced by reversible kinase inhibitors,with several advancing into late stage clinical testing.This design paradigm has been successfully applied to making drug candidates for epidermal growth factor receptor (EGFR),Her2,and Bruton’s tyrosine kinase (Btk).Here we review recent pre-clinical and clinical advances with targeted covalent kinase inhibitors,and the potential for broader application of the approach.AddressAvila Therapeutics,100Beaver Street,Waltham,MA,United States Corresponding author:Singh,Juswinder (jsingh@ )Current Opinion in Chemical Biology 2010,14:475–480This review comes from a themed issue on Next Generation TherapeuticsEdited by Michael Gelb and Adrian Whitty Available online 6th July 20101367-5931/$–see front matter#2010Elsevier Ltd.All rights reserved.DOI 10.1016/j.cbpa.2010.06.168IntroductionRecent successes in targeted cancer therapy have made kinase inhibition the preeminent point of intervention in developing novel anti-cancer agents.The persistent chal-lenges in kinase inhibitor drug discovery have been poor selectivity across the family of kinases and the high intracellular concentrations of the endogenous competi-tive substrate,ATP.The principal mechanism to address these challenges has been the design and development of reversible,ATP-competitive inhibitors.Highly targeted covalent,irreversible inhibitors afford an alternative mechanism to overcome these challenges,and inhibitors in this class exhibit distinct pharmacology:remarkable selectivity,prolonged pharmacodynamics relative to exposure,and the potential to overcome resistance mutations by virtue of time-dependent inactivation.Historically,irreversible inhibition has been a proven approach for many classes of drug targets [1,2 ],though most of these covalent drugs were discovered throughserendipity.Despite the ubiquity of drugs that operate by irreversible mechanisms,concerns about potential modi-fication of off-target proteins have led pharma to avoid this approach [2 ].A new approach to the discovery of targeted irreversible inhibitors using structure-based design has been exemplified in the epidermal growth factor receptor (EGFR)family of kinases.This approach,reviewed by Fry in 2000[3],offers an attractive route to a new therapeutic class of kinase inhibitors with potentially superior profiles and exceptional selectivity.Over the past decade,the unique benefits of targeted,covalent kinase inhibitors have been proven.Specifically,covalent inhibitors have demonstrated exceptional potency in overcoming endogenous ATP competition and selectivity that is largely orthogonal to that afforded by conventional,non-covalent inhibitors.In addition,sustained potency in the face of resistance mutations has emerged as an important attribute of this mechanism.To date,five irreversible kinase inhibitors have been introduced into clinical study (Figure 1),though taken together the kinases targeted by irreversible inhibitors represent only a small fraction of all kinases targeted by therapeutic agents (Figure 2).This review focuses on their design,progress pre-clinically and clinically,and some thoughts on the path forward.Informatics as an organizing tool for initiating inhibitor designThis review highlights progress in the design of drugs based on targeting a rare cysteine found in the ATP binding site of EGFR and a few related protein kinases.In the 1990s it was recognized that the potential reactivity of the thiol sidechain of cysteine residues allowed the kinase family of protein drug targets to be organized into subgroups that could be selectively targeted for irrevers-ible inhibition by covalent modification [4].A seminal proof-of-concept experiment showed that thioadenosine covalently inactivated EGFR by forming a disulfide bond with Cys797.This study demonstrated that a particular kinase suitable for targeted covalent inhibition could be identified by informatics,and that a specific,covalent inhibitor could be designed by modeling an electrophilic group based upon the binding mode of an existing revers-ible inhibitor.Subsequent alignment of all the kinases in the human genome [5]and systematic matching of their structural domains led to the identification of 10kinases that share a cysteine that structurally corresponds to Cys797in EGFR (Figure 3).In the intervening years since the original proof-of-concept finding,there has been explosive growth in the number of x-ray structures for kinases,including EGFR,and the small molecules thatCurrent Opinion in Chemical Biology 2010,14:475–480reversibly bind to them [6],thus greatly expanding the opportunities for design of targeted covalent inhibitors.Mechanism-based and structure-based design of ErbB family kinase inhibitorsThe key design process is to identify that portion of a high-affinity ligand that is proximal to the targeted cysteine and install electrophilic functionality at that position with geometry that is compatible with the for-mation of the critical bond.In a refinement of this approach,a general base is included at an adjacent site on the inhibitor to accelerate bond formation by activat-ing the reacting Cys [7](Figure 4).Refinements in the linker that position the warhead near the cysteine thiol,and modifications in the original scaffold itself,can also play a key role in optimizing selectivity and target inac-tivation rate.Irreversible inhibitors that emerge from such an optimization program are rapid inactivators of the targeted kinase,but exhibit remarkable selectivity,both with respect to other kinases and other proteins more generally.Several 4-anilinoquinazolines,a common scaffold in reversible EGFR inhibitors,have been transformed into irreversible inhibitors by substitution with acrylamide as the electrophilic warhead.These compounds exhibit low reactivity with DTT in enzyme assays and with gluta-thione in cellular assays,showing that their reactivity toward nonspecific thiols is low.Successful modification of the target kinase required proper positioning of the warhead relative to the target cysteine,in that congeners bearing an acrylamide in a less-than-optimal geometry476Next Generation TherapeuticsFigure1Cys-targeted covalent kinase inactivators in clinical development.Current Opinion in Chemical Biology 2010,14:475–480were poor inactivators.Most compelling was the demon-stration that these irreversible inhibitors outperformed their reversible counterparts in tumor growth inhibition in vitro and in animal models [8–10].This was a con-sequence of the covalent drug achieving more complete inhibition of EGFR signaling and more durable inhibition of the target once silenced since return of activity was dependent upon protein resynthesis.The generalizability of this approach toward EGFR and Her2was demon-strated using an irreversible inhibitor based on a thieno-pyrimidine scaffold with a tunable acetylenic warhead that demonstrated superior efficacy in a xenograft model compared to the reversible inhibitor,lapatinib [11].Several heterocyclic scaffold –warhead combinations were explored by teams within Parke-Davis,Wyeth,Boehrin-ger-Ingelheim,and others,in the search for drug-like leads of the ErbB family of kinases as potential candidates for cancer therapy [3].Ultimately,in each case,an acryl-amide or a substituted acrylamide proved to be the war-head of choice for imparting potency against the enzyme and in cells,and in obtaining good in vivo activity.Fiveclinical candidates have emerged from optimization of leads generated from the application of a modular design paradigm:the 4-anilinoquinazoline series yielded CI-1033[12]and BIBW2992[13]and the 4-anilino-3-cyano-quinoline series yielded neratinib and pelitinib [14].The structure of PF-00299804,also an irreversible ErbB inhibitor,has not been disclosed.Clinical progress for ErbB-targeted covalent drugsA first generation of ErbB family targeted drugs,in-cluding the reversible inhibitors (gefitinib,erlotinib,and lapatinib)and monoclonal antibodies (e.g.,cetux-imab and trastuzumab)has been shown to produce therapeutic benefit against a variety of cancers.These drugs address the overexpression of EGFR and Her2,as well as activating somatic mutations that drive increased signaling by ErbB family proteins in many cancers.However,their effectiveness has been blunted by issues such as a significant number of non-responding patients and a decreasing response-rate during drug treatment [15,16].Targeted covalent drugs of the kinase family Petter,Kluge and Singh 477Figure2A schematic representation of the human kinome family members:(a)Those family members currently being targeted in clinical trials with reversible,small-molecule inhibitors (data taken from /chembl ).(b)The three targets currently being pursued clinically using irreversible covalent inhibitors,which represents a small fraction of the total drug target space.Current Opinion in Chemical Biology 2010,14:475–480With three irreversible inhibitors in late-stage clinical testing there is optimism about the first approval for a targeted covalent drug [17 ].Neratinib is an irreversible,dual EGFR/Her2inhibitor that,like the approved revers-ible drug lapatinib,targets the inactive conformation of the kinase.Recently,results from a Phase 2trial com-posed of breast cancer patients with tumors positive for ErbB-2,who either had or had not undergone previous trastuzumab therapy,showed neratinib had substantial clinical activity and was well tolerated.The objective response rates were 24%among patients with prior tras-tuzumab treatment and 56%in the trastuzumab-naı¨ve cohort.Pfizer has recently announced advancement of neratinib to Phase 3trials for metastatic breast cancer [18–20].Two irreversible inhibitors,BIBW2992and PF-00299804,which are dual inhibitors of EGFR and Her2,are in pivotal Phase 3trials for non-small cell lung cancer (NSCLC)[13,21].A major challenge for the treatment of NSCLC patients,some of whom achieve dramatic responses to the reversible EGFR inhibitors erlotinib/gefitinib,is the de-velopment of drug resistance.In approximately 50%of patients who relapse there is evidence of resistance due to mutation at T790M in the kinase binding site [15,16].Initially,it had been assumed that T790M resistance was due to loss of affinity of the drug to the kinase binding site,however x-ray crystallography studies show that the drugs can adopt similar binding modes and have relatively modest differences in affinity to the T790M form of the protein and to WT [22].It appears that the basis for resistance in cells is due to an increased affinity for ATP to the T790M mutant form of the enzyme making it more difficult for a reversible drug to compete with the mM-levels of intracellular ATP [23 ].The irreversible inhibitors BIBW2992and PF-00299804retain some activity against the T790M mutant of EGFR.This is probably a consequence of time-dependent inac-tivation,wherein covalency overcomes high competing concentrations of ATP,enabling silencing of the drug target given sufficient residence time on the mutant protein [24].Detailed cellular profiling in 98cell lines of 11irreversible inhibitors,including BIBW2922,along with 10reversible inhibitors showed relatively modest activity against the T790M mutation [25 ].It remains to be seen if effective inhibition of T790M mutant protein with the inhibitors BIBW2992and PF-00299804can be achieved at clinical doses that do not lead to unaccep-table toxicities mediated by inhibition of wild-type EGFR.Recently,a novel chemotype,an acrylamide-substituted 2,4-dianilino-5-chloropyrimidine,has been reported that shows potent inhibition of T790M mutant EGFR [26 ].Interestingly,this lead has only modest activity against wild-type EGFR.This mutant-selective profile may turn out to be useful in a clinical setting to enable a greater therapeutic window in inhibiting mutant EGFR while minimizing toxicities mediated by the inhibition of wt-EGFR [27,28].478Next Generation TherapeuticsFigure3Ten kinases unified by sequence alignment of cysteine in EGFR.Figure4Selective formation of the key covalent bond with Cys797on EGFR.Current Opinion in Chemical Biology 2010,14:475–480Btk as a drug target for targeted covalent inhibitionTargeted covalent inhibition has also been applied to Bruton’s tyrosine kinase(Btk),an enzyme that infor-matics has identified to contain a Cys at a structurally equivalent position to that seen in EGFR.Exciting pre-clinical and emerging clinical data with an irreversible small-molecule inhibitor support the covalent approach.Btk is expressed in B-cells and is critically involved in their proliferation,development,differentiation,and sur-vival.Btk represents an exciting target for discovery of drugs modulating the B-cell receptor(BCR)pathway [29,30].Its role in the autoimmune disease X-linked agammaglobulinemia(XLA)has been traced to a mutation in the human Btk.The prominent role of Btk in the BCR signaling pathway and its highly restricted expression pattern makes Btk an attractive drug target for B-cell lymphomas and leukemias.Btk has been reported to be highly phosphorylated in follicular lymphoma patient cells compared to non-malignant B-cells from patients[31].The activated B-cell(ABC)subset of dif-fuse large B-cell lymphoma(DLBCL),which is associ-ated with the lowest5-year survival rate(30%),expresses genes normally induced in human peripheral B-cells after BCR stimulation and has a gene expression profile suggesting these cells are in a chronically activated BCR state[32].Recently,the importance of Btk for survival of the activated B-cell-like subset of DLBCL has been reported[33 ].Historically it has been difficult to design selective inhibi-tors of Btk;however,recently there has been substantial pre-clinical and clinical progress in defining selective compounds that irreversibly inhibit Btk.The covalent inhibitor PCI-32765(Figure1)has recently entered clinical development and was identified through a struc-ture-based design program similar to that used for inhibi-tors of the ErbB family targets.Beginning with a reversible1-cyclopentyl-4-aminopyrazolo[3,4-d]pyrimi-dine core structure with a diphenyl ether that imparted good activity against Btk and the Src-family kinases,the cyclopentyl was replaced by3-piperidyl,which was then substituted with an acrylamide warhead for bonding to the target cysteine in Btk[34].In addition,a series of2,4-dianilinopyrimidine-based irreversible inhibitors have been developed that exhibit potent activity against the Btk enzyme[35].Enzyme and cellular data for two of these,AVL-101and AVL-291,were presented at the Annual Meeting of the AACR in April,2009. Preliminary evidence of clinical activity was recently reported for PCI-32765in selected patients with relapsed or refractory B-cell non-Hodgkin’s lymphoma in a Phase 1b study(51st meeting of the American Society of Hema-tology,2009).A‘covalent probe’was used to quantify occupancy of the target,Btk,and thereby correlate phar-macodynamics(PD)of Btk inhibition with administered dose.The covalent probe assay uses afluorescently tagged probe derived from PCI-32765that can covalently bond to any Btk in the sample not already bonded to a molecule of the drug candidate.This probe was added to peripheral blood mononuclear cell(PBMC)lysates from clinical trial subjects,ex vivo.PD studies showed the average drug occupancy of Btk in patient PBMCs was 89%at4h post-dose and77%at24h post-dose.Ex vivo stimulation assays showed near complete functional inhi-bition of the Btk responsive Fc e receptor signaling path-way in basophils and substantial inhibition of the BCR pathway.The ability to correlate target occupancy with dosing in humans represents a powerful aspect to the targeted covalent inhibitor approach and should enable a rational approach to defining a suitable dose in patients. ConclusionsThe current clinical progress with targeted covalent kinase inhibitors underlines their promise as novel thera-pies.We expect that ongoing clinical success with the current targeted covalent inhibitors will stimulate interest in broader application of the approach.Despite the major effort on kinase inhibitor drug discovery in the pharma-ceutical industry,the primary focus has been on revers-ible non-covalent inhibition.Given the challenges in achieving potency and selectivity across the family,the adoption of alternative mechanisms of action for inhibi-tors is warranted and the data provided in this review support the benefits of targeted covalent inhibition.To date,approximately10%of the human kinome has been targeted with inhibitors in clinical development,almost all of which are reversible inhibitors.An analysis of protein kinases suggests that approximately20%may be amenable to a Cys-based targeting approach[36]. We expect this to be an active and growing area of drug discovery and development.References and recommended readingPapers of particular interest,published within the period of review, have been highlighted as:of special interestof outstanding interest1.Robertson JG:Mechanistic basis of enzyme-targeted drugs.Biochemistry2005,44:5561-5571.2.Potashman MH,Duggan ME:Covalent modifiers:an orthogonal approach to drug design.J Med Chem2009,52:1231-1246. 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