Discovery of mobocertinib, a potent, oral inhibitor of EGFR exon 20 insertion mutations in non-small cell lung cancer.

In the treatment of non-small cell lung cancer (NSCLC), patients harboring exon 20 insertion mutations in the epidermal growth factor receptor (EGFR) gene (EGFR) have few effective therapies because this subset of mutants is generally resistant to most currently approved EGFR inhibitors. This report describes the structure-guided design of a novel series of potent, irreversible inhibitors of EGFR exon 20 insertion mutations, including the V769_D770insASV and D770_N771insSVD mutants. Extensive structure-activity relationship (SAR) studies led to the discovery of mobocertinib (compound 21c), which inhibited growth of Ba/F3 cells expressing the ASV insertion with a half-maximal inhibitory concentration of 11 nM and with selectivity over wild-type EGFR. Daily oral administration of mobocertinib induced tumor regression in a Ba/F3 ASV xenograft mouse model at well-tolerated doses. Mobocertinib was approved in September 2021 for the treatment of adult patients with advanced NSCLC with EGFR exon 20 insertion mutations with progression on or after platinum-based chemotherapy.

Therapeutic strategies to overcome crizotinib resistance in non-small cell lung cancers harboring the fusion oncogene EML4-ALK.

The echinoderm microtubule-associated protein-like 4 (EML4)-anaplastic lymphoma kinase (ALK) fusion oncogene represents a molecular target in a small subset of non-small cell lung cancers (NSCLCs). This fusion leads to constitutive ALK activation with potent transforming activity. In a pivotal phase 1 clinical trial, the ALK tyrosine kinase inhibitor (TKI) crizotinib (PF-02341066) demonstrated impressive antitumor activity in the majority of patients with NSCLC harboring ALK fusions. However, despite these remarkable initial responses, cancers eventually develop resistance to crizotinib, usually within 1 y, thereby limiting the potential clinical benefit. To determine how cancers acquire resistance to ALK inhibitors, we established a model of acquired resistance to crizotinib by exposing a highly sensitive EML4-ALK-positive NSCLC cell line to increasing doses of crizotinib until resistance emerged. We found that cells resistant to intermediate doses of crizotinib developed amplification of the EML4-ALK gene. Cells resistant to higher doses (1 µM) also developed a gatekeeper mutation, L1196M, within the kinase domain, rendering EML4-ALK insensitive to crizotinib. This gatekeeper mutation was readily detected using a unique and highly sensitive allele-specific PCR assay. Although crizotinib was ineffectual against EML4-ALK harboring the gatekeeper mutation, we observed that two structurally different ALK inhibitors, NVP-TAE684 and AP26113, were highly active against the resistant cancer cells in vitro and in vivo. Furthermore, these resistant cells remained highly sensitive to the Hsp90 inhibitor 17-AAG. Thus, we have developed a model of acquired resistance to ALK inhibitors and have shown that second-generation ALK TKIs or Hsp90 inhibitors are effective in treating crizotinib-resistant tumors harboring secondary gatekeeper mutations.

Discovery of 5-(arenethynyl) hetero-monocyclic derivatives as potent inhibitors of BCR-ABL including the T315I gatekeeper mutant.

Ponatinib (AP24534) was previously identified as a pan-BCR-ABL inhibitor that potently inhibits the T315I gatekeeper mutant, and has advanced into clinical development for the treatment of refractory or resistant CML. In this study, we explored a novel series of five and six membered monocycles as alternate hinge-binding templates to replace the 6,5-fused imidazopyridazine core of ponatinib. Like ponatinib, these monocycles are tethered to pendant toluanilides via an ethynyl linker. Several compounds in this series displayed excellent in vitro potency against both native BCR-ABL and the T315I mutant. Notably, a subset of inhibitors exhibited desirable PK and were orally active in a mouse model of T315I-driven CML.

Mobocertinib (TAK-788): A Targeted Inhibitor of EGFR Exon 20 Insertion Mutants in Non-Small Cell Lung Cancer.

Most EGFR exon 20 insertion (EGFRex20ins) driver mutations in non-small cell lung cancer (NSCLC) are insensitive to approved EGFR tyrosine kinase inhibitors (TKI). To address the limitations of existing therapies targeting EGFR-mutated NSCLC, mobocertinib (TAK-788), a novel irreversible EGFR TKI, was specifically designed to potently inhibit oncogenic variants containing activating EGFRex20ins mutations with selectivity over wild-type EGFR. The in vitro and in vivo activity of mobocertinib was evaluated in engineered and patient-derived models harboring diverse EGFRex20ins mutations. Mobocertinib inhibited viability of various EGFRex20ins-driven cell lines more potently than approved EGFR TKIs and demonstrated in vivo antitumor efficacy in patient-derived xenografts and murine orthotopic models. These findings support the ongoing clinical development of mobocertinib for the treatment of EGFRex20ins-mutated NSCLC. SIGNIFICANCE: No oral EGFR-targeted therapies are approved for EGFR exon 20 insertion (EGFRex20ins) mutation-driven NSCLC. Mobocertinib is a novel small-molecule EGFR inhibitor specifically designed to target EGFRex20ins mutants. Preclinical data reported here support the clinical development of mobocertinib in patients with NSCLC with EGFR exon 20 insertion mutations.See related commentary by Pacheco, p. 1617.This article is highlighted in the In This Issue feature, p. 1601.

Airway management in patients who develop neck hematomas after carotid endarterectomy.

BACKGROUND: Progressive airway compromise from neck hematoma and edema is a feared complication of carotid endarterectomy (CEA). Despite this, the relationship of airway management technique to patient outcome has not been systematically studied in this population. We report the rate of successful airway management using various techniques in post-CEA patients. METHODS: A 10-year retrospective analysis was conducted to identify patients requiring airway management for neck exploration within 72 hours after CEA at Mayo Clinic, Rochester, MN. RESULTS: Three thousand two hundred twenty-five patients underwent CEA over a 10-year period at our institution. Forty-four (1.4%) required neck exploration for hematoma, and 42 of these required airway management immediately before neck exploration surgery. (The tracheal tube had not been removed after CEA in the remaining 2 patients.) The average interval between the completion of CEA and return to the operating room for hematoma evacuation was 6.0 +/- 6.0 hours (mean +/- SD; range, <1-32 hours). Fiberoptic airway management, performed before the induction of anesthesia, was successful in 15 of 20 patients (75%) and, in patients in whom fiberoptic tracheal intubation failed, direct laryngoscopy (DL) was successful in all 5 (3 before and 2 after the induction of general anesthesia). In the remaining 22 patients, DL was used as the initial management technique without a trial of fiberoptic intubation. DL was successful in 5 of 7 patients (71%) when performed before induction of general anesthesia and was successful in 13 of 15 patients (87%) when performed after induction of general anesthesia. Hematoma decompression facilitated DL in 3 of 4 failures of DL; tracheostomy was performed in the remaining patient. An arterial site of bleeding was subsequently identified in 36% of patients in whom no difficulty was encountered during laryngoscopy for hematoma evacuation versus 6% in whom difficulty was noted (P = 0.03). In 36 of 44 patients (82%), the tracheal tube was removed within 24 hours of surgery for neck exploration. No adverse events related to airway management were noted. There were no deaths at 2 weeks after hematoma evacuation. CONCLUSIONS: Multiple techniques resulted in successful airway control both before and after the induction of general anesthesia. Tracheal intubation was accomplished with both fiberoptic visualization and DL. In instances of poor direct visualization of the glottis, decompression of the airway by opening of the surgical incision may facilitate intubation of the trachea.

Novel N9-arenethenyl purines as potent dual Src/Abl tyrosine kinase inhibitors.

Novel N(9)-arenethenyl purines, optimized potent dual Src/Abl tyrosine kinase inhibitors, are described. The key structural feature is a trans vinyl linkage at N(9) on the purine core which projects hydrophobic substituents into the selectivity pocket at the rear of the ATP site. Their synthesis was achieved through a Horner-Wadsworth-Emmons reaction of N(9)-phosphorylmethylpurines and substituted benzaldehydes or Heck reactions between 9-vinyl purines and aryl halides. Most compounds are potent inhibitors of both Src and Abl kinase, and several possess good oral bioavailability.

SRC inhibitors in metastatic bone disease.

Src tyrosine kinase was the first gene product shown to have an essential function in bone using recombinant DNA technology after its expression was knocked out in mice approximately 15 years ago. Since then, our understanding of the regulation of bone catabolism has advanced significantly with the identification of other key enzymes that regulate osteoclast formation, activation, and survival after their knockout in mice or recognition of mutations in them in humans. This led to the discovery or development of specific inhibitors of some of these key enzymes, including Src, as proof-of-concept lead compounds or potential clinical candidates for the prevention of diseases associated with increased bone resorption, such as osteoporosis and metastatic bone disease. Although bisphosphonates have been prescribed with proven and improving efficacy for the prevention of bone loss for >30 years, adverse effects, such as upper gastrointestinal tract symptoms, and the requirement to take them at least 2 hours before food have limited patient compliance. Thus, with growing knowledge of the pathways regulating osteoclast function and the appreciation that some of these are active also in tumor cells, drug companies have made efforts to identify small-molecular lead compounds for development into new therapeutic agents for the prevention of bone loss with efficacy that matches or supersedes that of bisphosphonates. In this article, we review our current understanding of the signaling pathways that regulate osteoclast formation, activation, and survival with specific reference to the role of Src tyrosine kinase and downstream signaling and highlight in a variety of models of increased bone resorption the effects of Src kinase inhibitors that have been targeted to bone to limit potential adverse effects on other cells.

Future anti-catabolic therapeutic targets in bone disease.

Understanding of the regulation of bone catabolism has advanced significantly over the past two decades with the identification of key enzymes that regulate osteoclast formation, activation, and survival following their knockout in mice or recognition of mutations in humans. This led to the discovery of specific inhibitors of some of these key enzymes as proof-of-concept lead compounds or potential clinical candidates for the prevention of osteoporosis and other diseases associated with increased bone resorption. Bisphosphonates have been the major therapeutic agents prescribed for the prevention of bone loss in a variety of pathologic conditions for over 30 years. More potent amino bisphosphonates have increased efficacy than earlier drugs, but side effects such as upper gastrointestinal symptoms and the requirement to take them at least 2 h before food have limited patient compliance. This, coupled with the growing knowledge of the pathways regulating osteoclast function, has driven efforts to identify small molecular lead compounds that could be developed into new therapeutic agents with efficacy that matches or supersedes that of bisphosphonates for the prevention of bone loss. In this article, we review briefly the effects of specific inhibitors of bone resorption that have been developed to date and highlight in a variety of models of increased bone resorption the effects of Src kinase inhibitors that have been targeted to bone to limit potential unwanted side effects on other cells.

AP23846, a novel and highly potent Src family kinase inhibitor, reduces vascular endothelial growth factor and interleukin-8 expression in human solid tumor cell lines and abrogates downstream angiogenic processes.

c-Src is frequently activated in human malignancies, including colon, breast, and pancreatic carcinomas. Several recent studies have shown that activation of Src family kinases leads to tumor progression and metastasis by increasing cellular migration and invasion, promoting cell growth and survival, and deregulating expression of proangiogenic molecules. Therefore, selective inhibitors of Src are being developed for cancer therapy. In this study, we characterize the biological effects of the novel ATP-based Src family kinase inhibitor, AP23846, in tumor cells with high Src activity. As a lead compound, AP23846 is a potent c-Src kinase inhibitor (IC50 approximately 0.5 nmol/L in vitro, approximately 10-fold more potent than PP2, the most widely used commercially available Src family kinase inhibitor). At concentrations of 1 micromol/L, AP23846 led to complete Src inhibition for 48 hours in cells. No cytotoxicity was observed under these conditions, although proliferation rates were slower. Therefore, this was an excellent inhibitor to examine Src-regulated signaling pathways in tumor cells. AP23846 reduced cellular migration, vascular endothelial growth factor, and interleukin-8 in a dose-dependent fashion in pancreatic adenocarcinoma cells grown in vitro. Correspondingly, cell culture supernatants from L3.6pl pancreatic adenocarcinoma cells pretreated with AP23846 failed to promote migration of hepatic endothelial cells in vitro and failed to support angiogenesis into gel foams implanted s.c. in mice in vivo. These results suggest that Src inhibitors affect biological properties of tumor progression and may be useful as cancer therapeutic agents in more advanced disease.

Novel Small Molecule Inhibitors of Choline Kinase Identified by Fragment-Based Drug Discovery.

Choline kinase α (ChoKα) is an enzyme involved in the synthesis of phospholipids and thereby plays key roles in regulation of cell proliferation, oncogenic transformation, and human carcinogenesis. Since several inhibitors of ChoKα display antiproliferative activity in both cellular and animal models, this novel oncogene has recently gained interest as a promising small molecule target for cancer therapy. Here we summarize our efforts to further validate ChoKα as an oncogenic target and explore the activity of novel small molecule inhibitors of ChoKα. Starting from weakly binding fragments, we describe a structure based lead discovery approach, which resulted in novel highly potent inhibitors of ChoKα. In cancer cell lines, our lead compounds exhibit a dose-dependent decrease of phosphocholine, inhibition of cell growth, and induction of apoptosis at low micromolar concentrations. The druglike lead series presented here is optimizable for improvements in cellular potency, drug target residence time, and pharmacokinetic parameters. These inhibitors may be utilized not only to further validate ChoKα as antioncogenic target but also as novel chemical matter that may lead to antitumor agents that specifically interfere with cancer cell metabolism.

The Potent ALK Inhibitor Brigatinib (AP26113) Overcomes Mechanisms of Resistance to First- and Second-Generation ALK Inhibitors in Preclinical Models.

PURPOSE: Non-small cell lung cancers (NSCLCs) harboring ALK gene rearrangements (ALK+) typically become resistant to the first-generation anaplastic lymphoma kinase (ALK) tyrosine kinase inhibitor (TKI) crizotinib through development of secondary resistance mutations in ALK or disease progression in the brain. Mutations that confer resistance to second-generation ALK TKIs ceritinib and alectinib have also been identified. Here, we report the structure and first comprehensive preclinical evaluation of the next-generation ALK TKI brigatinib. EXPERIMENTAL DESIGN: A kinase screen was performed to evaluate the selectivity profile of brigatinib. The cellular and in vivo activities of ALK TKIs were compared using engineered and cancer-derived cell lines. The brigatinib-ALK co-structure was determined. RESULTS: Brigatinib potently inhibits ALK and ROS1, with a high degree of selectivity over more than 250 kinases. Across a panel of ALK+ cell lines, brigatinib inhibited native ALK (IC50, 10 nmol/L) with 12-fold greater potency than crizotinib. Superior efficacy of brigatinib was also observed in mice with ALK+ tumors implanted subcutaneously or intracranially. Brigatinib maintained substantial activity against all 17 secondary ALK mutants tested in cellular assays and exhibited a superior inhibitory profile compared with crizotinib, ceritinib, and alectinib at clinically achievable concentrations. Brigatinib was the only TKI to maintain substantial activity against the most recalcitrant ALK resistance mutation, G1202R. The unique, potent, and pan-ALK mutant activity of brigatinib could be rationalized by structural analyses. CONCLUSIONS: Brigatinib is a highly potent and selective ALK inhibitor. These findings provide the molecular basis for the promising activity being observed in ALK+, crizotinib-resistant patients with NSCLC being treated with brigatinib in clinical trials. Clin Cancer Res; 22(22); 5527-38. ©2016 AACR.

Discovery of Brigatinib (AP26113), a Phosphine Oxide-Containing, Potent, Orally Active Inhibitor of Anaplastic Lymphoma Kinase.

In the treatment of echinoderm microtubule-associated protein-like 4 (EML4)-anaplastic lymphoma kinase positive (ALK+) non-small-cell lung cancer (NSCLC), secondary mutations within the ALK kinase domain have emerged as a major resistance mechanism to both first- and second-generation ALK inhibitors. This report describes the design and synthesis of a series of 2,4-diarylaminopyrimidine-based potent and selective ALK inhibitors culminating in identification of the investigational clinical candidate brigatinib. A unique structural feature of brigatinib is a phosphine oxide, an overlooked but novel hydrogen-bond acceptor that drives potency and selectivity in addition to favorable ADME properties. Brigatinib displayed low nanomolar IC50s against native ALK and all tested clinically relevant ALK mutants in both enzyme-based biochemical and cell-based viability assays and demonstrated efficacy in multiple ALK+ xenografts in mice, including Karpas-299 (anaplastic large-cell lymphomas [ALCL]) and H3122 (NSCLC). Brigatinib represents the most clinically advanced phosphine oxide-containing drug candidate to date and is currently being evaluated in a global phase 2 registration trial.

Novel bone-targeted Src tyrosine kinase inhibitor drug discovery.

Bone-targeted Src tyrosine kinase (STK) inhibitors have recently been developed for the treatment of osteoporosis and cancer-related bone diseases. The concept of bone targeting derives from bisphosphonates, and from the evolution of such molecules in terms of therapeutic efficacy for the treatment of bone disorders. Interestingly, some of the earliest bisphosphonates were recognized for their ability to inhibit calcium carbonate precipitation (scaling) by virtue of their affinity to chelate calcium. This chelating property was subsequently exploited in the development of bisphosphonate analogs as inhibitors of the bone-resorbing cells known as osteoclasts, giving rise to breakthrough medicines, such as Fosamax (for the treatment of osteoporosis) and Zometa (for the treatment of osteoporosis and bone metastases). Relative to these milestone achievements, there is a tremendous opportunity to explore beyond the limited chemical space (functional group diversity) of such bisphosphonates to design novel bone-targeting moieties, which may be used to develop other classes of promising small-molecule drugs affecting different biological pathways. Here, we review studies focused on bone-targeted inhibitors of STK, a key enzyme in osteoclast-dependent bone resorption. Two strategies are described relative to bone-targeted STK inhibitor drug discovery: (i) the development of novel Src homology (SH)-2 inhibitors incorporating non-hydrolyzable phosphotyrosine mimics and exhibiting molecular recognition and bone-targeting properties, leading to the in vivo-effective lead compound AP-22408; and (ii) the development of novel ATP-based Src kinase inhibitors incorporating bone-targeting moieties, leading to the in vivo-effective lead compound AP-23236. In summary, AP-22408 and AP-23236, which differ mechanistically by virtue of blocking Src-dependent non-catalytic or catalytic activities in osteoclasts, exemplify ARIAD Pharmaceuticals' structure-based design of novel bone-targeted lead compounds, successfully achieving in vivo proof-of-concept and providing the framework for the next-generation molecules that have further advanced, in terms of preclinical studies, for the treatment of osteoporosis and related bone diseases, including osteolytic bone metastases.

SRC homology-2 inhibitors: peptidomimetic and nonpeptide.

The structural and functional characterization of Src homology-2 (SH2) domains and their relationship to catalytic proteins (e.g., kinases, phosphatases, and lipases) or non-catalytic proteins (e.g., upstream adapters, and downstream transcription factors) has significantly impacted our understanding of signal transduction pathways and the identification of promising therapeutic targets for drug discovery. Such SH2-containing proteins are known to be intimately involved in the regulation of a number of cellular processes, including growth, mitogenesis, motility, metabolism, and gene transcription. Molecular recognition and biochemical selectivity exists for various SH2 domains based on their binding to phosphotyrosine (pTyr) and contiguous C-terminal amino acids of cognate protein 'partners' in a sequence-dependent manner (i.e., -pTyr-AA(1)-AA(2)-AA(3)-) which result in the formation of signal transduction protein complexes in cells. In recent years, drug discovery efforts have advanced peptidomimetic and nonpeptide inhibitors of such protein-protein interactions based on mimicking pTyr-containing peptide ligands as well as SH2 structure-based de novo design of nonpeptide templates that can capture key binding sites on the target protein. Noteworthy are peptidomimetic and nonpeptide inhibitors of Src, Lck, Grb2, PI-3K, and Zap70 from pioneering efforts that led to the first examples of cellularly and in vivo active SH2 inhibitors. This mini-review highlights key achievements in SH2 inhibitor drug discovery with an emphasis on peptidomimetic and nonpeptide lead compounds in terms of structure-based design, key chemical and biological properties, and proof-of-concept studies relative to further defining the role(s) of SH2 domains in signal transduction processes, cellular functions, and in vivo disease models.

Regulation of bone remodeling and emerging breakthrough drugs for osteoporosis and osteolytic bone metastases.

Major advances have been made in the past 10 years in our understanding of the molecular basis of bone cell formation and bone remodeling. For example, the discovery of osteoprotegerin, the decoy receptor and inhibitor of receptor activator of NF-kappaB ligand (RANKL), and the RANKL/receptor activator of NF-kappaB (RANK) signaling pathway that is essential for osteoclastogenesis, has helped clarify the mechanisms regulating osteoclast formation, activation, and survival. PTH, like most other osteoclast stimulating factors, promotes RANKL production by osteoblast/stromal cells when they are exposed to it continuously, but when given intermittently it stimulates bone formation and reduces fracture risk in postmenopausal women. This anabolic effect is associated with increased expression of insulin-like and fibroblast growth factors and decreased osteoblast apoptosis. Src tyrosine kinase is essential for osteoclast activation and also negatively regulates osteoblast activity. Thus, it is a well-validated therapeutic target for the prevention of postmenopausal and other forms of bone loss. Preliminary in vitro and in vivo studies of specifically designed, bone targeted, non-peptide Src inhibitors have shown that these compounds inhibit bone resorption and stimulate new bone formation. The design of drugs using structure/function approaches such as this should lead to the development of novel therapeutics that could be used to counteract the negative effects of chronic renal failure on the skeleton.

Fragment growing and linking lead to novel nanomolar lactate dehydrogenase inhibitors.

Lactate dehydrogenase A (LDH-A) catalyzes the interconversion of lactate and pyruvate in the glycolysis pathway. Cancer cells rely heavily on glycolysis instead of oxidative phosphorylation to generate ATP, a phenomenon known as the Warburg effect. The inhibition of LDH-A by small molecules is therefore of interest for potential cancer treatments. We describe the identification and optimization of LDH-A inhibitors by fragment-based drug discovery. We applied ligand based NMR screening to identify low affinity fragments binding to LDH-A. The dissociation constants (K(d)) and enzyme inhibition (IC(50)) of fragment hits were measured by surface plasmon resonance (SPR) and enzyme assays, respectively. The binding modes of selected fragments were investigated by X-ray crystallography. Fragment growing and linking, followed by chemical optimization, resulted in nanomolar LDH-A inhibitors that demonstrated stoichiometric binding to LDH-A. Selected molecules inhibited lactate production in cells, suggesting target-specific inhibition in cancer cell lines.

Ponatinib (AP24534), a multitargeted pan-FGFR inhibitor with activity in multiple FGFR-amplified or mutated cancer models.

Members of the fibroblast growth factor receptor family of kinases (FGFR1-4) are dysregulated in multiple cancers. Ponatinib (AP24534) is an oral multitargeted tyrosine kinase inhibitor being explored in a pivotal phase II trial in patients with chronic myelogenous leukemia due to its potent activity against BCR-ABL. Ponatinib has also been shown to inhibit the in vitro kinase activity of all four FGFRs, prompting us to examine its potential as an FGFR inhibitor. In Ba/F3 cells engineered to express activated FGFR1-4, ponatinib potently inhibited FGFR-mediated signaling and viability with IC(50) values <40 nmol/L, with substantial selectivity over parental Ba/F3 cells. In a panel of 14 cell lines representing multiple tumor types (endometrial, bladder, gastric, breast, lung, and colon) and containing FGFRs dysregulated by a variety of mechanisms, ponatinib inhibited FGFR-mediated signaling with IC(50) values <40 nmol/L and inhibited cell growth with GI(50) (concentration needed to reduce the growth of treated cells to half that of untreated cells) values of 7 to 181 nmol/L. Daily oral dosing of ponatinib (10-30 mg/kg) to mice reduced tumor growth and inhibited signaling in all three tumor models examined. Importantly, the potency of ponatinib in these models is similar to that previously observed in BCR-ABL-driven models and plasma levels of ponatinib that exceed the IC(50) values for FGFR1-4 inhibition can be sustained in patients. These results show that ponatinib is a potent pan-FGFR inhibitor and provide strong rationale for its evaluation in patients with FGFR-driven cancers.

Structural mechanism of the Pan-BCR-ABL inhibitor ponatinib (AP24534): lessons for overcoming kinase inhibitor resistance.

The BCR-ABL inhibitor imatinib has revolutionized the treatment of chronic myeloid leukemia. However, drug resistance caused by kinase domain mutations has necessitated the development of new mutation-resistant inhibitors, most recently against the T315I gatekeeper residue mutation. Ponatinib (AP24534) inhibits both native and mutant BCR-ABL, including T315I, acting as a pan-BCR-ABL inhibitor. Here, we undertook a combined crystallographic and structure-activity relationship analysis on ponatinib to understand this unique profile. While the ethynyl linker is a key inhibitor functionality that interacts with the gatekeeper, virtually all other components of ponatinib play an essential role in its T315I inhibitory activity. The extensive network of optimized molecular contacts found in the DFG-out binding mode leads to high potency and renders binding less susceptible to disruption by single point mutations. The inhibitory mechanism exemplified by ponatinib may have broad relevance to designing inhibitors against other kinases with mutated gatekeeper residues.

Crizotinib-resistant mutants of EML4-ALK identified through an accelerated mutagenesis screen.

Activating gene rearrangements of anaplastic lymphoma kinase (ALK) have been identified as driver mutations in non-small-cell lung cancer, inflammatory myofibroblastic tumors, and other cancers. Crizotinib, a dual MET/ALK inhibitor, has demonstrated promising clinical activity in patients with non-small-cell lung cancer and inflammatory myofibroblastic tumors harboring ALK translocations. Inhibitors of driver kinases often elicit kinase domain mutations that confer resistance, and such mutations have been successfully predicted using in vitro mutagenesis screens. Here, this approach was used to discover an extensive set of ALK mutations that can confer resistance to crizotinib. Mutations at 16 residues were identified, structurally clustered into five regions around the kinase active site, which conferred varying degrees of resistance. The screen successfully predicted the L1196M, C1156Y, and F1174L mutations, recently identified in crizotinib-resistant patients. In separate studies, we demonstrated that crizotinib has relatively modest potency in ALK-positive non-small-cell lung cancer cell lines. A more potent ALK inhibitor, TAE684, maintained substantial activity against mutations that conferred resistance to crizotinib. Our study identifies multiple novel mutations in ALK that may confer clinical resistance to crizotinib, suggests that crizotinib's narrow selectivity window may underlie its susceptibility to such resistance and demonstrates that a more potent ALK inhibitor may be effective at overcoming resistance.