PF-06463922 is a potent and selective next-generation ROS1/ALK inhibitor capable of blocking crizotinib-resistant ROS1 mutations.

Oncogenic c-ros oncogene1 (ROS1) fusion kinases have been identified in a variety of human cancers and are attractive targets for cancer therapy. The MET/ALK/ROS1 inhibitor crizotinib (Xalkori, PF-02341066) has demonstrated promising clinical activity in ROS1 fusion-positive non-small cell lung cancer. However, emerging clinical evidence has shown that patients can develop resistance by acquiring secondary point mutations in ROS1 kinase. In this study we characterized the ROS1 activity of PF-06463922, a novel, orally available, CNS-penetrant, ATP-competitive small-molecule inhibitor of ALK/ROS1. In vitro, PF-06463922 exhibited subnanomolar cellular potency against oncogenic ROS1 fusions and inhibited the crizotinib-refractory ROS1(G2032R) mutation and the ROS1(G2026M) gatekeeper mutation. Compared with crizotinib and the second-generation ALK/ROS1 inhibitors ceritinib and alectinib, PF-06463922 showed significantly improved inhibitory activity against ROS1 kinase. A crystal structure of the PF-06463922-ROS1 kinase complex revealed favorable interactions contributing to the high-affinity binding. In vivo, PF-06463922 showed marked antitumor activity in tumor models expressing FIG-ROS1, CD74-ROS1, and the CD74-ROS1(G2032R) mutation. Furthermore, PF-06463922 demonstrated antitumor activity in a genetically engineered mouse model of FIG-ROS1 glioblastoma. Taken together, our results indicate that PF-06463922 has potential for treating ROS1 fusion-positive cancers, including those requiring agents with CNS-penetrating properties, as well as for overcoming crizotinib resistance driven by ROS1 mutation.

Mechanistic understanding of translational pharmacokinetic-pharmacodynamic relationships in nonclinical tumor models: a case study of orally available novel inhibitors of anaplastic lymphoma kinase.

The orally available novel small molecules PF06463922 [(10R)-7-amino-12-fluoro-2,10,16-trimethyl-15-oxo-10,15,16,17-tetrahydro-2H-8,4-(metheno)pyrazolo[4,3-h][2,5,11]benzoxadiazacyclotetradecine-3-carbonitrile] and PF06471402 [(10R)-7-amino-12-fluoro-2,10,16-trimethyl-15-oxo-10,15,16,17-tetrahydro-2H-8,4-(azeno)pyrazolo[4,3-h][2,5,11]benzoxadiazacyclo-tetradecine-3-carbonitrile] are second-generation anaplastic lymphoma kinase (ALK) inhibitors targeted to both naïve and resistant patients with non-small cell lung cancer (NSCLC) to the first-generation ALK inhibitor crizotinib. The objectives of the present study were to characterize and compare the pharmacokinetic-pharmacodynamic (PKPD) relationships of PF06463922 and PF06471402 for target modulation in tumor and antitumor efficacy in athymic mice implanted with H3122 NSCLC cells expressing a crizotinib-resistant echinoderm microtubule-associated protein-like 4 (EML4)-ALK mutation, EML4-ALK(L1196M). Furthermore, the PKPD relationships for these ALK inhibitors were evaluated and compared between oral administration and subcutaneous constant infusion (i.e., between different pharmacokinetic [PK] profiles). Oral and subcutaneous PK profiles of these ALK inhibitors were adequately described by a one-compartment PK model. An indirect response model extended with a modulator fit the time courses of PF06463922- and PF06471402-mediated target modulation (i.e., ALK phosphorylation) with an estimated unbound EC50,in vivo of 36 and 20 nM, respectively, for oral administration, and 100 and 69 nM, respectively, for subcutaneous infusion. A drug-disease model based on the turnover concept fit tumor growth curves inhibited by PF06463922 and PF06471402 with estimated unbound tumor stasis concentrations of 51 and 27 nM, respectively, for oral administration, and 116 and 70 nM, respectively, for subcutaneous infusion. Thus, the EC50,in vivo to EC60,in vivo estimates for ALK inhibition corresponded to the concentrations required tumor stasis in all cases, suggesting that the pharmacodynamic relationships of target modulation to antitumor efficacy were consistent among the ALK inhibitors, even when the PK profiles with different administration routes were considerably different.

Metabolism, excretion and pharmacokinetics of [14C]crizotinib following oral administration to healthy subjects.

1. Crizotinib (XALKORI®), an oral inhibitor of anaplastic lymphoma kinase (ALK) and mesenchymal-epithelial transition factor kinase (c-Met), is currently approved for the treatment of patients with non-small cell lung cancer that is ALK-positive. 2. The metabolism, excretion and pharmacokinetics of crizotinib were investigated following administration of a single oral dose of 250 mg/100 µCi [(14)C]crizotinib to six healthy male subjects. 3. Mean recovery of [(14)C]crizotinib-related radioactivity in excreta samples was 85% of the dose (63% in feces and 22% in urine). 4. Crizotinib and its metabolite, crizotinib lactam, were the major components circulating in plasma, accounting for 33% and 10%, respectively, of the 0-96 h plasma radioactivity. Unchanged crizotinib was the major excreted component in feces (∼ 53% of the dose). In urine, crizotinib and O-desalkyl crizotinib lactam accounted for ∼ 2% and 5% of the dose, respectively. Collectively, these data indicate that the primary clearance pathway for crizotinib in humans is oxidative metabolism/hepatic elimination. 5. Based on plasma exposure in healthy subjects following a single dose of crizotinib and in vitro potency against ALK and c-Met, the crizotinib lactam diastereomers are not anticipated to contribute significantly to in vivo activity; however, additional assessment in cancer patients is warranted.

Translational pharmacokinetic-pharmacodynamic modeling for an orally available novel inhibitor of anaplastic lymphoma kinase and c-Ros oncogene 1.

An orally available macrocyclic small molecule, PF06463922 [(10R)-7-amino-12-fluoro-2,10,16-trimethyl-15-oxo-10,15,16,17-tetrahydro-2H-8,4-(metheno)pyrazolo[4,3-h][2,5,11]benzoxadiazacyclotetradecine-3-carbonitrile], is a selective inhibitor of anaplastic lymphoma kinase (ALK) and c-Ros oncogene 1 (ROS1). The objectives of the present study were to characterize the pharmacokinetic-pharmacodynamic relationships of PF06463922 between its systemic exposures, pharmacodynamic biomarker (target modulation), and pharmacologic response (antitumor efficacy) in athymic mice implanted with H3122 non-small cell lung carcinomas expressing echinoderm microtubule-associated protein-like 4 (EML4)-ALK mutation (EML4-ALK(L1196M)) and with NIH3T3 cells expressing CD74-ROS1. In these nonclinical tumor models, PF06463922 was orally administered to animals with EML4-ALK(L1196M) and CD74-ROS1 at twice daily doses of 0.3-20 and 0.01-3 mg/kg per dose, respectively. Plasma concentration-time profiles of PF06463922 were adequately described by a one-compartment pharmacokinetic model. Using the model-simulated plasma concentrations, a pharmacodynamic indirect response model with a modulator sufficiently fit the time courses of target modulation (i.e., ALK phosphorylation) in tumors of EML4-ALK(L1196M)-driven models with EC50,in vivo of 36 nM free. A drug-disease model based on an indirect response model reasonably fit individual tumor growth curves in both EML4-ALK(L1196M)- and CD74-ROS1-driven models with the estimated tumor stasis concentrations of 51 and 6.2 nM free, respectively. Thus, the EC60,in vivo (52 nM free) for ALK inhibition roughly corresponded to the tumor stasis concentration in an EML4-ALK(L1196M)-driven model, suggesting that 60% ALK inhibition would be required for tumor stasis. Accordingly, we proposed that the EC60,in vivo for ALK inhibition corresponding to the tumor stasis could be considered a minimum target efficacious concentration of PF06463922 for cancer patients in a phase I trial.

Pharmacokinetic/pharmacodynamic modeling of crizotinib for anaplastic lymphoma kinase inhibition and antitumor efficacy in human tumor xenograft mouse models.

Crizotinib [Xalkori; PF02341066; (R)-3-[1-(2,6-dichloro-3-fluoro-phenyl)-ethoxy]-5-(1-piperidin-4-yl-1H-pyrazol-4-yl)-pyridin-2-ylamine] is an orally available dual inhibitor of anaplastic lymphoma kinase (ALK) and hepatocyte growth factor receptor. The objectives of the present studies were to characterize: 1) the pharmacokinetic/pharmacodynamic relationship of crizotinib plasma concentrations to the inhibition of ALK phosphorylation in tumors, and 2) the relationship of ALK inhibition to antitumor efficacy in human tumor xenograft models. Crizotinib was orally administered to athymic nu/nu mice implanted with H3122 non-small-cell lung carcinomas or severe combined immunodeficient/beige mice implanted with Karpas299 anaplastic large-cell lymphomas. Plasma concentration-time courses of crizotinib were adequately described by a one-compartment pharmacokinetic model. A pharmacodynamic link model reasonably fit the time courses of ALK inhibition in both H3122 and Karpas299 models with EC(50) values of 233 and 666 ng/ml, respectively. A tumor growth inhibition model also reasonably fit the time course of individual tumor growth curves with EC(50) values of 255 and 875 ng/ml, respectively. Thus, the EC(50) for ALK inhibition approximately corresponded to the EC(50) for tumor growth inhibition in both xenograft models, suggesting that >50% ALK inhibition would be required for significant antitumor efficacy (>50%). Furthermore, based on the observed clinical pharmacokinetic data coupled with the pharmacodynamic parameters obtained from the present nonclinical xenograft mouse model, >70% ALK inhibition was projected in patients with non-small-cell lung cancer who were administered the clinically recommended dosage of crizotinib, twice-daily doses of 250 mg (500 mg/day). The result suggests that crizotinib could sufficiently inhibit ALK phosphorylation for significant antitumor efficacy in patients.

Enzymatic characterization of c-Met receptor tyrosine kinase oncogenic mutants and kinetic studies with aminopyridine and triazolopyrazine inhibitors.

The c-Met receptor tyrosine kinase (RTK) is a key regulator in cancer, in part, through oncogenic mutations. Eight clinically relevant mutants were characterized by biochemical, biophysical, and cellular methods. The c-Met catalytic domain was highly active in the unphosphorylated state (k(cat) = 1.0 s(-1)) and achieved 160-fold enhanced catalytic efficiency (k(cat)/K(m)) upon activation to 425000 s(-1) M(-1). c-Met mutants had 2-10-fold higher basal enzymatic activity (k(cat)) but achieved maximal activities similar to those of wild-type c-Met, except for Y1235D, which underwent a reduction in maximal activity. Small enhancements of basal activity were shown to have profound effects on the acquisition of full enzymatic activity achieved through accelerating rates of autophosphorylation. Biophysical analysis of c-Met mutants revealed minimal melting temperature differences indicating that the mutations did not alter protein stability. A model of RTK activation is proposed to describe how a RTK response may be matched to a biological context through enzymatic properties. Two c-Met clinical candidates from aminopyridine and triazolopyrazine chemical series (PF-02341066 and PF-04217903) were studied. Biochemically, each series produced molecules that are highly selective against a large panel of kinases, with PF-04217903 (>1000-fold selective relative to 208 kinases) being more selective than PF-02341066. Although these prototype inhibitors have similar potencies against wild-type c-Met (K(i) = 6-7 nM), significant differences in potency were observed for clinically relevant mutations evaluated in both biochemical and cellular contexts. In particular, PF-02341066 was 180-fold more active against the Y1230C mutant c-Met than PF-04217903. These highly optimized inhibitors indicate that for kinases susceptible to active site mutations, inhibitor design may need to balance overall kinase selectivity with the ability to inhibit multiple mutant forms of the kinase (penetrance).

Nonclinical antiangiogenesis and antitumor activities of axitinib (AG-013736), an oral, potent, and selective inhibitor of vascular endothelial growth factor receptor tyrosine kinases 1, 2, 3.

PURPOSE: Axitinib (AG-013736) is a potent and selective inhibitor of vascular endothelial growth factor (VEGF) receptor tyrosine kinases 1 to 3 that is in clinical development for the treatment of solid tumors. We provide a comprehensive description of its in vitro characteristics and activities, in vivo antiangiogenesis, and antitumor efficacy and translational pharmacology data. EXPERIMENTAL DESIGN: The potency, kinase selectivity, pharmacologic activity, and antitumor efficacy of axitinib were assessed in various nonclinical models. RESULTS: Axitinib inhibits cellular autophosphorylation of VEGF receptors (VEGFR) with picomolar IC(50) values. Counterscreening across multiple kinase and protein panels shows it is selective for VEGFRs. Axitinib blocks VEGF-mediated endothelial cell survival, tube formation, and downstream signaling through endothelial nitric oxide synthase, Akt and extracellular signal-regulated kinase. Following twice daily oral administration, axitinib produces consistent and dose-dependent antitumor efficacy that is associated with blocking VEGFR-2 phosphorylation, vascular permeability, angiogenesis, and concomitant induction of tumor cell apoptosis. Axitinib in combination with chemotherapeutic or targeted agents enhances antitumor efficacy in many tumor models compared with single agent alone. Dose scheduling studies in a human pancreatic tumor xenograft model show that simultaneous administration of axitinib and gemcitabine without prolonged dose interruption or truncation of axitinib produces the greatest antitumor efficacy. The efficacious drug concentrations predicted in nonclinical studies are consistent with the range achieved in the clinic. Although axitinib inhibits platelet-derived growth factor receptors and KIT with nanomolar in vitro potencies, based on pharmacokinetic/pharmacodynamic analysis, axitinib acts primarily as a VEGFR tyrosine kinase inhibitor at the current clinical exposure. CONCLUSIONS: The selectivity, potency for VEGFRs, and robust nonclinical activity may afford broad opportunities for axitinib to improve cancer therapy.

An orally available small-molecule inhibitor of c-Met, PF-2341066, exhibits cytoreductive antitumor efficacy through antiproliferative and antiangiogenic mechanisms.

The c-Met receptor tyrosine kinase and its ligand, hepatocyte growth factor (HGF), have been implicated in the progression of several human cancers and are attractive therapeutic targets. PF-2341066 was identified as a potent, orally bioavailable, ATP-competitive small-molecule inhibitor of the catalytic activity of c-Met kinase. PF-2341066 was selective for c-Met (and anaplastic lymphoma kinase) compared with a panel of >120 diverse tyrosine and serine-threonine kinases. PF-2341066 potently inhibited c-Met phosphorylation and c-Met-dependent proliferation, migration, or invasion of human tumor cells in vitro (IC(50) values, 5-20 nmol/L). In addition, PF-2341066 potently inhibited HGF-stimulated endothelial cell survival or invasion and serum-stimulated tubulogenesis in vitro, suggesting that this agent also exhibits antiangiogenic properties. PF-2341066 showed efficacy at well-tolerated doses, including marked cytoreductive antitumor activity, in several tumor models that expressed activated c-Met. The antitumor efficacy of PF-2341066 was dose dependent and showed a strong correlation to inhibition of c-Met phosphorylation in vivo. Near-maximal inhibition of c-Met activity for the full dosing interval was necessary to maximize the efficacy of PF-2341066. Additional mechanism-of-action studies showed dose-dependent inhibition of c-Met-dependent signal transduction, tumor cell proliferation (Ki67), induction of apoptosis (caspase-3), and reduction of microvessel density (CD31). These results indicated that the antitumor activity of PF-2341066 may be mediated by direct effects on tumor cell growth or survival as well as antiangiogenic mechanisms. Collectively, these results show the therapeutic potential of targeting c-Met with selective small-molecule inhibitors for the treatment of human cancers.

Pharmacokinetic-pharmacodynamic modeling of biomarker response and tumor growth inhibition to an orally available cMet kinase inhibitor in human tumor xenograft mouse models.

(R)-3-[1-(2,6-Dichloro-3-fluoro-phenyl)-ethoxy]-5-(1-piperidin-4-yl-1H-pyrazol-4-yl)-pyridin-2-ylamine (PF02341066) was identified as an orally available, ATP-competitive small molecule inhibitor of cMet receptor tyrosine kinase. The objectives of the present studies were to characterize 1) the pharmacokinetic-pharmacodynamic relationship of the plasma concentrations of PF02341066 to cMet phosphorylation in tumor (biomarker) and 2) the relationship of cMet phosphorylation to antitumor efficacy (pharmacological response). Athymic mice implanted with GTL16 gastric carcinoma or U87MG glioblastoma xenografts were treated with PF02341066 once daily at doses selected to encompass ED(50) values. Plasma concentrations of PF02341066 were best described by a one-compartment pharmacokinetic model. A time-delay (hysteresis) was observed between the plasma concentrations of PF02341066 and the cMet phosphorylation response. A link model was therefore used to account for this hysteresis. The model fitted the time courses of cMet phosphorylation well, suggesting that the main reason for the hysteresis is a rate-limiting distribution from plasma into tumor. The EC(50) and EC(90) values were estimated to be 19 and 167 ng/ml, respectively. For tumor growth inhibition, the exponential tumor growth model fitted the time courses of individual tumor growth inhibition well. The EC(50) for the GTL16 tumor growth inhibition was estimated to be 213 ng/ml. Thus, the EC(90) for the inhibition of cMet phosphorylation corresponded to the EC(50) for the tumor growth inhibition, suggesting that near-complete inhibition of cMet phosphorylation (>90%) is required to significantly inhibit tumor growth (>50%). The present results will be helpful in determining the appropriate dosing regimen and in guiding dose escalation to rapidly achieve efficacious systemic exposure in the clinic.

Cytoreductive antitumor activity of PF-2341066, a novel inhibitor of anaplastic lymphoma kinase and c-Met, in experimental models of anaplastic large-cell lymphoma.

A t(2;5) chromosomal translocation resulting in expression of an oncogenic kinase fusion protein known as nucleophosmin-anaplastic lymphoma kinase (NPM-ALK) has been implicated in the pathogenesis of anaplastic large-cell lymphoma (ALCL). PF-2341066 was recently identified as a p.o. bioavailable, small-molecule inhibitor of the catalytic activity of c-Met kinase and the NPM-ALK fusion protein. PF-2341066 also potently inhibited NPM-ALK phosphorylation in Karpas299 or SU-DHL-1 ALCL cells (mean IC(50) value, 24 nmol/L). In biochemical and cellular screens, PF-2341066 was shown to be selective for c-Met and ALK at pharmacologically relevant concentrations across a panel of >120 diverse kinases. PF-2341066 potently inhibited cell proliferation, which was associated with G(1)-S-phase cell cycle arrest and induction of apoptosis in ALK-positive ALCL cells (IC(50) values, approximately 30 nmol/L) but not ALK-negative lymphoma cells. The induction of apoptosis was confirmed using terminal deoxyribonucleotide transferase-mediated nick-end labeling and Annexin V staining (IC(50) values, 25-50 nmol/L). P.o. administration of PF-2341066 to severe combined immunodeficient-Beige mice bearing Karpas299 ALCL tumor xenografts resulted in dose-dependent antitumor efficacy with complete regression of all tumors at the 100 mg/kg/d dose within 15 days of initial compound administration. A strong correlation was observed between antitumor response and inhibition of NPM-ALK phosphorylation and induction of apoptosis in tumor tissue. In addition, inhibition of key NPM-ALK signaling mediators, including phospholipase C-gamma, signal transducers and activators of transcription 3, extracellular signal-regulated kinases, and Akt by PF-2341066 were observed at concentrations or dose levels, which correlated with inhibition of NPM-ALK phosphorylation and function. Collectively, these data illustrate the potential clinical utility of inhibitors of NPM-ALK in treatment of patients with ALK-positive ALCL.

Noninvasive assessment of the role of cyclooxygenases in cardiovascular health: a detailed HPLC/MS/MS method.

A robust method for the routine quantitation of a selected group of urinary eicosanoid metabolites of interest to cardiovascular research in human and mouse is described and discussed. Included are the addition of stable isotope-labeled internal standards, solid phase extraction, and quantitation by liquid chromatography/tandem mass spectrometry using selected reaction monitoring (SRM) techniques.

The ALK/ROS1 Inhibitor PF-06463922 Overcomes Primary Resistance to Crizotinib in ALK-Driven Neuroblastoma.

UNLABELLED: Neuroblastomas harboring activating point mutations in anaplastic lymphoma kinase (ALK) are differentially sensitive to the ALK inhibitor crizotinib, with certain mutations conferring intrinsic crizotinib resistance. To overcome this clinical obstacle, our goal was to identify inhibitors with improved potency that can target intractable ALK variants such as F1174L. We find that PF-06463922 has high potency across ALK variants and inhibits ALK more effectively than crizotinib in vitro. Most importantly, PF-06463922 induces complete tumor regression in both crizotinib-resistant and crizotinib-sensitive xenograft mouse models of neuroblastoma, as well as in patient-derived xenografts harboring the crizotinib-resistant F1174L or F1245C mutations. These studies demonstrate that PF-06463922 has the potential to overcome crizotinib resistance and exerts unprecedented activity as a single targeted agent against F1174L and F1245C ALK-mutated xenograft tumors, while also inducing responses in an R1275Q xenograft model. Taken together, these results provide the rationale to move PF-06463922 into clinical trials for treatment of patients with ALK-mutated neuroblastoma. SIGNIFICANCE: The next-generation ALK/ROS1 inhibitor PF-06463922 exerts unparalleled activity in ALK-driven neuroblastoma models with primary crizotinib resistance. Our biochemical and in vivo data provide the preclinical rationale for fast-tracking the development of this agent in children with relapsed/refractory ALK-mutant neuroblastoma.

PF-06463922, an ALK/ROS1 Inhibitor, Overcomes Resistance to First and Second Generation ALK Inhibitors in Preclinical Models.

We report the preclinical evaluation of PF-06463922, a potent and brain-penetrant ALK/ROS1 inhibitor. Compared with other clinically available ALK inhibitors, PF-06463922 displayed superior potency against all known clinically acquired ALK mutations, including the highly resistant G1202R mutant. Furthermore, PF-06463922 treatment led to regression of EML4-ALK-driven brain metastases, leading to prolonged mouse survival, in a superior manner. Finally, PF-06463922 demonstrated high selectivity and safety margins in a variety of preclinical studies. These results suggest that PF-06463922 will be highly effective for the treatment of patients with ALK-driven lung cancers, including those who relapsed on clinically available ALK inhibitors because of secondary ALK kinase domain mutations and/or brain metastases.

Discovery of (10R)-7-amino-12-fluoro-2,10,16-trimethyl-15-oxo-10,15,16,17-tetrahydro-2H-8,4-(metheno)pyrazolo[4,3-h][2,5,11]-benzoxadiazacyclotetradecine-3-carbonitrile (PF-06463922), a macrocyclic inhibitor of anaplastic lymphoma kinase (ALK) and c-ros oncogene 1 (ROS1) with preclinical brain exposure and broad-spectrum potency against ALK-resistant mutations.

Although crizotinib demonstrates robust efficacy in anaplastic lymphoma kinase (ALK)-positive non-small-cell lung carcinoma patients, progression during treatment eventually develops. Resistant patient samples revealed a variety of point mutations in the kinase domain of ALK, including the L1196M gatekeeper mutation. In addition, some patients progress due to cancer metastasis in the brain. Using structure-based drug design, lipophilic efficiency, and physical-property-based optimization, highly potent macrocyclic ALK inhibitors were prepared with good absorption, distribution, metabolism, and excretion (ADME), low propensity for p-glycoprotein 1-mediated efflux, and good passive permeability. These structurally unusual macrocyclic inhibitors were potent against wild-type ALK and clinically reported ALK kinase domain mutations. Significant synthetic challenges were overcome, utilizing novel transformations to enable the use of these macrocycles in drug discovery paradigms. This work led to the discovery of 8k (PF-06463922), combining broad-spectrum potency, central nervous system ADME, and a high degree of kinase selectivity.

Design of potent and selective inhibitors to overcome clinical anaplastic lymphoma kinase mutations resistant to crizotinib.

Crizotinib (1), an anaplastic lymphoma kinase (ALK) receptor tyrosine kinase inhibitor approved by the U.S. Food and Drug Administration in 2011, is efficacious in ALK and ROS positive patients. Under pressure of crizotinib treatment, point mutations arise in the kinase domain of ALK, resulting in resistance and progressive disease. The successful application of both structure-based and lipophilic-efficiency-focused drug design resulted in aminopyridine 8e, which was potent across a broad panel of engineered ALK mutant cell lines and showed suitable preclinical pharmacokinetics and robust tumor growth inhibition in a crizotinib-resistant cell line (H3122-L1196M).

Substrate-specific conformational regulation of the receptor tyrosine kinase VEGFR2 catalytic domain.

The contributions of the phosphoacceptor and the catalytic domain context to protein kinase biology and inhibitor potency are routinely overlooked, which can lead to mischaracterization of inhibitor and receptor functions. The receptor tyrosine kinase vascular endothelial growth factor receptor-2 (VEGFR2) is studied as a model system using a series of phosphoacceptor substrates (k(cat)/K(m) 684-116,000 M(-1) s(-1)) to assess effects on catalysis and inhibitor binding. ATP-competitive inhibitor potency toward the VEGFR2 catalytic domain (VEGFR2-CD) varies with different phosphoacceptor substrates, which is unexpected because the phosphoacceptors do not affect K(m,ATP) values. Indazole-based inhibitors are up to 60-fold more potent with two substrates (gastrin, minigastrin) relative to the others. Thus there is a component of uncompetitive inhibition because a specific phosphoacceptor enhances potency but is not strictly required. This substrate-specific inhibitory potency enhancement correlates with phosphoacceptor active site saturation and is not observed with other related kinases. The effect is confined to a specific catalytic domain conformation because autophosphorylation eliminates the potency enhancement as does the addition of the juxtamembrane domain (20 amino acids). Indazole inhibitor structure-activity analysis reveals that the magnitude of potency enhancement correlates with the size of the substituent that binds in a regulatory region of the active site. VEGFR drugs profiled with VEGFR2-CD using minigastrin have potency well-correlated with inhibition of full-length, cellular VEGFR2 autophosphorylation, an indication that the minigastrin-induced conformation is biologically relevant. These findings raise the possibility that inhibitors directed toward a common target can have different biological effects based on the kinase-substrate complexes present in different cellular contexts.

Lessons from (S)-6-(1-(6-(1-methyl-1H-pyrazol-4-yl)-[1,2,4]triazolo[4,3-b]pyridazin-3-yl)ethyl)quinoline (PF-04254644), an inhibitor of receptor tyrosine kinase c-Met with high protein kinase selectivity but broad phosphodiesterase family inhibition leading to myocardial degeneration in rats.

The hepatocyte growth factor (HGF)/c-Met signaling axis is deregulated in many cancers and plays important roles in tumor invasive growth and metastasis. An exclusively selective c-Met inhibitor (S)-6-(1-(6-(1-methyl-1H-pyrazol-4-yl)-[1,2,4]triazolo[4,3-b]pyridazin-3-yl)ethyl)quinoline (8) was discovered from a highly selective high-throughput screening hit via structure-based drug design and medicinal chemistry lead optimization. Compound 8 had many attractive properties meriting preclinical evaluation. Broad off-target screens identified 8 as a pan-phosphodiesterase (PDE) family inhibitor, which was implicated in a sustained increase in heart rate, increased cardiac output, and decreased contractility indices, as well as myocardial degeneration in in vivo safety evaluations in rats. Compound 8 was terminated as a preclinical candidate because of a narrow therapeutic window in cardio-related safety. The learning from multiparameter lead optimization and strategies to avoid the toxicity attrition at the late stage of drug discovery are discussed.

Sensitivity of selected human tumor models to PF-04217903, a novel selective c-Met kinase inhibitor.

The c-Met pathway has been implicated in a variety of human cancers for its critical role in tumor growth, invasion, and metastasis. PF-04217903 is a novel ATP-competitive small-molecule inhibitor of c-Met kinase. PF-04217903 showed more than 1,000-fold selectivity for c-Met compared with more than 150 kinases, making it one of the most selective c-Met inhibitors described to date. PF-04217903 inhibited tumor cell proliferation, survival, migration/invasion in MET-amplified cell lines in vitro, and showed marked antitumor activity in tumor models harboring either MET gene amplification or a hepatocyte growth factor (HGF)/c-Met autocrine loop at well-tolerated dose levels in vivo. Antitumor efficacy of PF-04217903 was dose-dependent and showed a strong correlation with inhibition of c-Met phosphorylation, downstream signaling, and tumor cell proliferation/survival. In human xenograft models that express relatively high levels of c-Met, complete inhibition of c-Met activity by PF-04217903 only led to partial tumor growth inhibition (38%-46%) in vivo. The combination of PF-04217903 with Recepteur d'origine nantais (RON) short hairpin RNA (shRNA) knockdown in the HT29 model that also expresses activated RON kinase-induced tumor cell apoptosis and resulted in enhanced antitumor efficacy (77%) compared with either PF-04217903 (38%) or RON shRNA alone (56%). PF-04217903 also showed potent antiangiogenic properties in vitro and in vivo. Furthermore, PF-04217903 strongly induced phospho-PDGFRß (platelet-derived growth factor receptor) levels in U87MG xenograft tumors, indicating a possible oncogene switching mechanism in tumor cell signaling as a potential resistance mechanism that might compromise tumor responses to c-Met inhibitors. Collectively, these results show the use of highly selective inhibition of c-Met and provide insight toward targeting tumors exhibiting different mechanisms of c-Met dysregulation.

Discovery of a novel class of exquisitely selective mesenchymal-epithelial transition factor (c-MET) protein kinase inhibitors and identification of the clinical candidate 2-(4-(1-(quinolin-6-ylmethyl)-1H-[1,2,3]triazolo[4,5-b]pyrazin-6-yl)-1H-pyrazol-1-yl)ethanol (PF-04217903) for the treatment of cancer.

The c-MET receptor tyrosine kinase is an attractive oncology target because of its critical role in human oncogenesis and tumor progression. An oxindole hydrazide hit 6 was identified during a c-MET HTS campaign and subsequently demonstrated to have an unusual degree of selectivity against a broad array of other kinases. The cocrystal structure of the related oxindole hydrazide c-MET inhibitor 10 with a nonphosphorylated c-MET kinase domain revealed a unique binding mode associated with the exquisite selectivity profile. The chemically labile oxindole hydrazide scaffold was replaced with a chemically and metabolically stable triazolopyrazine scaffold using structure based drug design. Medicinal chemistry lead optimization produced 2-(4-(1-(quinolin-6-ylmethyl)-1H-[1,2,3]triazolo[4,5-b]pyrazin-6-yl)-1H-pyrazol-1-yl)ethanol (2, PF-04217903), an extremely potent and exquisitely selective c-MET inhibitor. 2 demonstrated effective tumor growth inhibition in c-MET dependent tumor models with good oral PK properties and an acceptable safety profile in preclinical studies. 2 progressed to clinical evaluation in a Phase I oncology setting.

Structure based drug design of crizotinib (PF-02341066), a potent and selective dual inhibitor of mesenchymal-epithelial transition factor (c-MET) kinase and anaplastic lymphoma kinase (ALK).

Because of the critical roles of aberrant signaling in cancer, both c-MET and ALK receptor tyrosine kinases are attractive oncology targets for therapeutic intervention. The cocrystal structure of 3 (PHA-665752), bound to c-MET kinase domain, revealed a novel ATP site environment, which served as the target to guide parallel, multiattribute drug design. A novel 2-amino-5-aryl-3-benzyloxypyridine series was created to more effectively make the key interactions achieved with 3. In the novel series, the 2-aminopyridine core allowed a 3-benzyloxy group to reach into the same pocket as the 2,6-dichlorophenyl group of 3 via a more direct vector and thus with a better ligand efficiency (LE). Further optimization of the lead series generated the clinical candidate crizotinib (PF-02341066), which demonstrated potent in vitro and in vivo c-MET kinase and ALK inhibition, effective tumor growth inhibition, and good pharmaceutical properties.