Potent c-Met Inhibitors Research Articles

Discovery of LAH-1 as potent c-Met inhibitor for the treatment of non-small cell lung cancer

ABSTRCTDysregulated HGF/c-Met pathway has been implicated in multiple human cancers and has become an attractive target for cancer intervention. Herein, we report the discovery of N-(3-fluoro-4-((2-(3-hydroxyazetidine-1-carboxamido)pyridin-4-yl)oxy)phenyl)-1-(4-fluorophenyl)-4-methyl-6-oxo-1,6-dihydropyridazine-3-carboxamide (LAH-1), which demonstrated nanomolar MET kinase activity as well as desirable antiproliferative activity, especially against EBC-1 cells. Mechanism studies confirmed the effects of LAH-1 on modulation of HGF/c-Met pathway, induction of cell apoptosis, inhibition on colony formation as well as cell migration and invasion. In addition, LAH-1 also showed desirable in vitro ADME properties as well as acceptable in vivo PK parameters. The design, synthesis, and characterisation of LAH-1 are described herein.

EXTH-56. CSF PENETRATION COMPARISON OF TWO ORAL FORMULATIONS OF APL-101 IN A PRE-CLINICAL NON-HUMAN PRIMATE MODEL

Abstract Vebreltinib (APL-101) is a potent c-Met inhibitor which has shown efficacy in murine pre-clinical models and is under clinical trial evaluation for several cancer types, including adult high-grade glioma. The capsule formulation (APL-101-Cap) was reformulated as a pill (APL-101-Pill) with the expectation of increasing bioavailability. We determined and compared the plasma pharmacokinetics (PK) and cerebrospinal fluid (CSF) penetration of APL-101-Cap and APL-101-Pill in a non-human primate (NHP) model. METHODS: Seven animals, previously developed as serial CSF access models, were utilized across all studies. One NHP was studied with both formulations. Both formulations were administered orally as a single drug/single dose in serial studies, followed by serial plasma and CSF sample collections from 0-96 hours. Dosages: APL-101-Cap 3.25 mg/kg (n = 3) or 6.5 mg/kg (n = 4) and APL-101-Pill 5 mg/kg (n = 4). The resulting plasma concentrations from each formulation were quantified by LC-MS/MS. PK parameters were calculated via noncompartmental methods, and results were analyzed to assess CNS penetration (AUCCSF:AUCPlasma) and compare formulations. RESULTS: Mean (range) for plasma and CSF PK exposure [AUCLAST (hr*ng/mL)] reported. APL-101-Cap plasma exposure, regardless of dose, was more variable than APL-101-Pill. APL-101-Pill had lower exposure in both plasma (AUC 1893.0 (841-2865)] and CSF [AUC 27.7 (5.0-65.0)] than APL-101-Cap regardless of dose [(3.25 mg/kg): plasma AUC 9621.0 (1331-23132) and CSF AUC 688.0 (24-1352); 6.5 mg/kg: plasma AUC 6208.0 (1424-19800) and CSF AUC 606.0 (34-1179)]. CNS penetrance was higher with APL-101-Cap (3.2% [0.5-5.8%] at 3.25mg/kg; 4.2% (2.4-6%) at 6.5 mg/kg), but also more variable when compared to APL-101-Pill (1.2% [0.6-2.3%]). For APL-101-Cap, one animal at 3.25 mg/kg and two animals at 6.25 mg/kg had undetectable CSF concentrations. CONCLUSION: In this NHP model, single-dose oral administration of APL-101-Pill resulted in lower, but less variable plasma and CSF exposure compared to APL-101-Cap. APL-101-Cap CSF exposure was unaffected by dose escalation.

Design, synthesis and biological evaluation of sulfonylamidines as potent c-Met inhibitors by enhancing hydrophobic interaction.

The dysregulation of c-Met kinase has emerged as a significant contributing factor for the occurrence, progression, poor clinical outcomes and drug resistance of various human cancers. In our ongoing pursuit to identify promising c-Met inhibitors as potential antitumor agents, a docking study of the previously reported c-Met inhibitor 7 revealed a large unoccupied hydrophobic pocket, which could present an opportunity for further exploration of structure-activity relationships to improve the binding affinity with the allosteric hydrophobic back pocket of c-Met. Herein we performed structure-activity relationship and molecular modeling studies based on lead compound 7. The collective endeavors culminated in the discovery of compound 21j with superior efficacy to 7 and positive control foretinib by increasing the hydrophobic interaction with the hydrophobic back pocket of c-Met active site.

Type II c-Met inhibitors: molecular insight into crucial interactions for effective inhibition.

The c-Met tyrosine kinase plays an important role in human cancers. Preclinical studies demonstrated that c-Met is over-expressed, mutated and amplified in a variety of human tumor types and design of more potent c-Met inhibitors is a priority. In this study, 14 molecular dynamics simulations of potent type II c-Met inhibitors were run to resolve the critical interactions responsible for high affinity of ligands towards c-Met considering the essential flexibility of protein-ligand interactions. Residues Phe1223 and Tyr1159, involved in pi-pi interactions were recognized as the most effective residues in the ligand binding in terms of binding free energies. Hydrogen bond interaction with Met1160 was also found necessary for effective type II ligand binding to c-Met.

Identification of Iminium Intermediates Generation in the Metabolism of Tepotinib Using LC-MS/MS: In Silico and Practical Approaches to Bioactivation Pathway Elucidation.

Tepotinib (Tepmetko™, Merck) is a potent inhibitor of c-Met (mesenchymal−epithelial transition factor). In March 2020, tepotinib (TEP) was approved for use in Japan for the treatment of patients who suffered from non-small cell lung cancers (NSCLC) harboring an MET exon 14 skipping alteration and have progressed after platinum-based therapy. Practical and in silico experiments were used to screen for the metabolic profile and reactive intermediates of TEP. Knowing the bioactive center and structural alerts in the TEP structure helped in making targeted modifications to improve its safety. First, the prediction of metabolism vulnerable sites and reactivity metabolic pathways was performed using the StarDrop WhichP450™ module and the online Xenosite reactivity predictor tool, respectively. Subsequently, in silico data were used as a guide for the in vitro practical work. Second, in vitro phase I metabolites of TEP were generated from human liver microsome (HLM) incubations. Testing for the generation of unstable reactive intermediates was performed using potassium cyanide as a capturing agent forming stable cyano adduct that can be characterized and identified using liquid chromatography tandem mass spectrometry (LC-MS/MS). Third, in silico toxicity assessment of TEP metabolites was performed, and structural modification was proposed to decrease their side effects and to validate the proposed bioactivation pathway using the DEREK software. Four TEP phase I metabolites and four cyano adducts were characterized. The reactive intermediate generation mechanism of TEP may provide an explanation of its adverse reactions. The piperidine ring is considered a structural alert for toxicity as proposed by the DEREK software and a Xenosite reactivity model, which was confirmed by practical experiments. Steric hindrance or isosteric replacement at α-carbon of the piperidine ring stop the bioactivation sequence that was confirmed using the DEREK software. More drug discovery studies can be performed using this perception permitting the design of new drugs with an increased safety profile. To our knowledge, this is the first study for the identification of in vitro phase I metabolites and reactive intermediates in addition to toxicological properties of the metabolites for TEP that will be helpful for the evaluation of TEP side effects and drug–drug interactions in TEP-treated patients.

Dual potent c-Met and ALK inhibitors: from common feature pharmacophore modeling to structure based virtual screening

Everyday plenty of people succumb to various forms of cancer across the world and it stands as one of the main reasons of death in our today’s life. Receptor tyrosine kinases (RTKs) are a class of receptors involved in cancer progression. Since aberrant signaling has critical roles in cancer, both c-Met and ALK enzymes are regarded as attractive oncology targets for therapeutic objects. A number of potent dual inhibitors of c-Met and ALK are reported in literature that in the present work we based them to construct multiple common feature pharmacophore models and then applied them for ligand-based virtual screening. The score values of the models ranged from 22.489 to 28.169. The retrieved compounds from virtual screening were subjected to the docking study and the interaction pattern of common hits between two enzymes with high predicted affinity has been investigated. To this end, common hit compound ZINC000223394281 (z1) was directed to the molecular dynamics study and the results indicated that the hydrogen bond interaction between this compound and Asp1222 was mostly stable during the equilibrium time range. The life time of hydrogen bond made between the complex of ALK and Met1199 was also stable in 63%.

Clustering and Sampling of the c-Met Conformational Space: A Computational Drug Discovery Study.

c-Met kinase plays a critical role in a myriad of human cancers, and a massive scientific work was devoted to design more potent inhibitors. In this study, 16 molecular dynamics simulations of different complexes of potent c-Met inhibitors with U-shaped binding mode were carried out regarding the dynamic ensembles to design novel potent inhibitors. A cluster analysis was performed, and the most representative frame of each complex was subjected to the structure-based pharmacophore screening. The GOLD docking program investigated the interaction energy and pattern of output hits from the virtual screening. The most promising hits with the highest scoring values that showed critical interactions with c-Met were presented for ADME/Tox analysis. The screening yielded 45,324 hits that all of them were subjected to the docking studies and 10 of them with the highest-scoring values having diverse structures were presented for ADME/Tox analyses. The results indicated that all the hits shared critical Pi-Pi stacked and hydrogen bond interactions with Tyr1230 and Met1160 respectively.

Critical c-Met-inhibitor interactions resolved from molecular dynamics simulations of different c-Met complexes

Overexpression or deregulation of c-Met enzyme has been reported to be associated with the formation and development of various types of human cancers, so an intense scientific work was devoted to design more potent inhibitors. We ran 17 molecular dynamics simulations of potent type I c-Met inhibitors to investigate the critical and stable interactions regarding the inherent flexibility of protein and ligand via dynamics ensembles. Protein-ligand interactions of hydrogen bond, pi-pi stacked and hydrophobic have been investigated throughout the equilibrium time to suggest critical pharmacophores. Also, the fluctuations of amino acid residues were monitored by root mean square fluctuation (RMSF) measure. MM-PBSA approach was also used to estimate the binding free energy of different complexes. The pi-pi stacked interaction of c-Met inhibitors with Tyr1230 or both Tyr1230 and Tyr1159 was shown critical for a good inhibitory activity and was defined as the most important interaction type in terms of binding energy. Almost in all codes, ligands showed van der Waals interactions with hydrophobic residues of ATP site. In addition, hydrogen bond interaction with Met1160 was found necessary for a ligand being a potent inhibitor of c-Met. RMSF analysis showed that Met1160 and Pro1158 which make the key hydrogen bond interactions and Tyr1230 that contributes in pi-pi stacked interactions with the ligands were quite fixed during ligand binding.

Synthesis of novel 6,7-dimethoxy-4-anilinoquinolines as potent c-Met inhibitors

HGF/c-Met signalling pathway plays an important role in the development of cancers. A series of 6,7-dimethoxy-4-anilinoquinolines possessing benzimidazole moiety were synthesised and identified as potent inhibitors of the tyrosine kinase c-Met. Their in vitro biological activities against three cancer cell lines (A549, MCF-7, and MKN-45) were also evaluated. Most of these compounds exhibited moderate to remarkable potency. Among them, compound 12n showed the most potent inhibitory activity against c-Met with IC50 value of 0.030 ± 0.008 µM and it also showed excellent anticancer activity against the tested cancer cell lines at low micromolar concentration. Molecular docking verified the results and revealed the possible binding mode of the most promising compound 12n into the ATP-binding site of c-Met kinase.

Molecular simulation studies on the binding activity and selectivity of 3-amino-phenyl-5-chloro-pyrimidine-2, 4-diamine derivatives in complexes with kinases c-Met and ALK

ABSTRACTReceptor tyrosine kinases c-Met and ALK have been demonstrated to be important therapeutic targets for cancer therapy. However, selectivity and drug resistance could hinder the development of their corresponding inhibitors. In this study, three compounds with similar scaffold were examined to study activity and selectivity mechanism towards c-Met or ALK by utilising a combined approach of computational techniques, including flexible dock, molecular electrostatic potential (MESP) calculations, molecular dynamic (MD) simulation, and binding free-energy calculation. Molecular simulation provides us new chemical insights into steric and electronic complementarities of these inhibitors to target binding sites. The computed binding free energies were consistent with the changing trend of experimental affinities on c-Met and ALK. H-bond with Asp169 and hydrophobic interaction with Phe36 of c-Met, respectively, could be crucial for the binding affinity of an inhibitor binding to c-Met. Meanwhile, for inhibitor–ALK complex, both H-bond interactions with Arg28 and Met101 and hydrophobic interactions with Leu30, Val38, and Leu158 could enhance the bioactivity and selectivity. The present work may provide a structural understanding of molecular mechanism expected to be valuable for the guidelines of the development of new potent c-Met or ALK selective inhibitors.

Abstract LB-194: Therapeutic efficacy of ABN401, a highly potent and selective c-MET inhibitor, on the basis of diagnostic test for biomarkers in MET-amplified cancers

Abstract The receptor tyrosine kinase c-MET regulates essential cellular processes in tissue remodeling and homeostasis, and mammalian development. Although c-MET is an important in normal physiology, the dysregulation of c-MET signaling has been strongly implicated in tumorigenic transformation, tumor growth, angiogenesis, invasiveness, and metastasis. The aberrant activation of c-MET, particularly in tumors with gene amplification and mutations, reported in a wide variety of cancers. In approximately 10% of gastric cancer, MET is amplified and is activated in the ligand-independent pathway. MET-amplified cancer cell lines are dependent on its signaling for survival and proliferation. That being so, c-MET is attractive target and a number of clinical trials are ongoing. However, some clinical trials were failed due to inappropriate patient selection criteria such as low cut-off. To select patients who respond to c-MET inhibitor, the selection criteria by using both IHC and FISH is necessary. In this study, we determined the efficacy of ABN401 based on histopathologic and genetic analysis using cell lines and patient-derived xenograft (PDX) models for gastric cancer and other c-MET related cancers as well. The cytotoxic effect of ABN401 was evaluated in six gastric cancer cell lines and one normal fibroblast cell as control by using cell viability assay. c-MET and its downstream signaling were assessed by western blot assay. In vivo anti-tumor therapeutic efficacy of ABN401 was evaluated in two gastric cancer cell xenograft models and eight PDX models. MET copy number, c-MET protein expression and genetic aberration were estimated by diagnostic test such as IHC and FISH, NGS, and array CGH in cell lines and PDX models. Our results revealed that ABN401 showed over 90% of cytotoxicity in five gastric cancer cell lines (SNU5, SNU620, SNU638, Hs746T, MKN45) and inhibited auto-phosphorylation of the c-MET as well as its downstream signal. Except for SNU638, the other four cell lines were MET copy number > 10, MET/CEP7 > 5, and c-MET expression (IHC 3+). Moreover, ABN401 significantly suppressed tumor growth in three of eight PDX models with MET copy number >5. MET/CEP7 > 5 and c-MET expression level (IHC 3+). However, ABN401 had no effect in gastric PDX model with MET copy number 5, MET/CEP7 ratio > 5, c-MET protein level IHC 2+ and esophagus PDX model with MET copy number 6, MET/CEP7 ratio = 2.2, IHC 1+. Both in vivo and in vitro experiment suggest that MET copy number and c-MET expression level affects effectiveness of c-MET inhibitor ABN401, which alert us the importance of cut-off. Based on this study, we conclude that it would be possible to make a conjecture in future clinical trials by establishing the characteristics of each sample and find the correlation with the efficacy of c-MET inhibitor. Citation Format: Jooseok Kim, Kyungsoo Jung, Kyungeui Park, Minji Seo, Yoonla Choi, Junyoung Choi, Sungyoul Hong, Youngkee Shin. Therapeutic efficacy of ABN401, a highly potent and selective c-MET inhibitor, on the basis of diagnostic test for biomarkers in MET-amplified cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr LB-194.

Abstract 4792: Preclinical evaluation of GST-HG161, a potent and highly selective c-MET inhibitor

Abstract Objective: Hepatocellular carcinoma (HCC) is the third leading cause of cancer-related deaths worldwide. Studies indicate that aberrant activation of mesenchymal-epithelial transition factor (c-MET) is associated with HCC development. Blocking c-MET signaling shows antitumor efficacy in patients with HCC. Thus, c-MET has been an attractive therapeutic target for HCC. We disclose here for the first time a potent and selective c-MET inhibitor GST-HG161 and show the antitumor efficacy data in preclinical models of HCC. Method: MHCC97H HCC cell line with c-MET overexpression was used to evaluate the anti-proliferative activity of GST-HG161 in a 120-hour viability assay. Selectivity was evaluated with a panel of cell lines without overexpression of c-MET. Daily oral administration of GST-HG161 doses of 1, 3, 10 and 30mg/kg was used to evaluate its in vivo antitumor efficacy in two animal models of HCC with c-MET overexpression, MHCC97H xenograft (CDX) model and LI-03-0317 xenograft (PDX) model. The c-MET protein levels and gene copy number in LI-03-0317 PDX were assessed by immunohistochemistry (IHC) and fluorescence in situ hybridization (FISH), respectively. High c-MET expression in LI-03-0317 PDX was determined by the presence of tumor cells with membrane staining of 3+ intensity. Effect of GST-HG161 on c-MET signaling inhibition was assessed by western blot analysis of downstream effector proteins including p-MET, p-AKT and p-ERK. Result: GST-HG161 displayed potent anti-proliferative activity in MHCC97H HCC cell line (IC50 = 5 nM) while showed little activity in the cell lines without c-MET overexpression. It significantly inhibited tumor growth at tested doses (3, 10 and 30mg/kg) (P<0.05) in both MHCC97H CDX model and LI-03-0317 PDX model. In MHCC97H CDX model, western blot analysis showed that GST-HG161 potently inhibited p-MET and downstream effector proteins p-AKT and p-ERK in the tumor tissue, indicating that the tumor growth inhibition in this model was mediated through inhibition of c-MET. Conclusion: GST-HG161 is a potent and highly selective c-MET inhibitor. Its excellent antitumor efficacy as shown in the HCC mouse models provide us with confidence that GST-HG161 has high potential in the clinic for treating patients of HCC with high c-MET activity. Citation Format: Xiongbin Xu, Gang Li, Lihong Hu, Junguo Hao, Charles Z. Ding, Shuhui Chen. Preclinical evaluation of GST-HG161, a potent and highly selective c-MET inhibitor [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4792.

Abstract A097: Phase II trial of the c-Met inhibitor tepotinib in patients with advanced non-small cell lung cancer harboring MET exon 14-skipping mutations

Abstract Background: Approximately 3-4% of non-small cell lung cancers (NSCLCs) harbor activating mutations of the MET proto-oncogene that cause exon 14 skipping (METex14) and accumulation of c-Met lacking a juxtamembrane domain. Such tumors are sensitive to nonselective kinase inhibitors with activity against c-Met, so selective c-Met inhibitors may have greater efficacy and/or tolerability. This single-arm phase II trial (NCT02864992) is investigating the efficacy and safety of the potent and selective c-Met inhibitor tepotinib in patients (pts) with NSCLC harboring METex14. Methods: Eligible patients (pts) are adults with stage IIIB/IV NSCLC and METex14 mutations identified in tumor and/or circulating tumor DNA (ctDNA) in plasma (liquid biopsy) by a central laboratory. Pts with EGFR-activating mutations, ALK rearrangements, or >2 lines of prior therapy are excluded. Pts receive tepotinib 500 mg QD on a 21-day cycle until disease progression, intolerable toxicity, or withdrawal from treatment for other reasons. Primary endpoint: objective response rate (ORR). Secondary endpoints: progression-free and overall survival, safety, pharmacokinetics, and quality of life. Recruitment of up to 120 (minimum 60 tumor, 60 ctDNA METex14-positive) pts in Europe, USA, and Japan is planned. Results: Five pts (age 55-77 years; 4 stage IV, 1 stage IIIB; all male; 4 Caucasian, 1 Asian) have been enrolled. Pts have currently completed 0-8 cycles of tepotinib therapy. Most adverse events observed to date have been grade 1/2 in severity, mostly unrelated to tepotinib treatment. One grade ≥3 tepotinib-related AE has occurred: grade 3 increased serum amylase. One pt has died due to his disease. Of the four pts with post-baseline tumor evaluations, two have confirmed partial response, one unconfirmed partial response, and one (with only one post-baseline assessment) stable disease. Conclusions: Initial data suggest that tepotinib 500 mg QD has promising activity in METex14 NSCLC. The safety profile of tepotinib appears favorable. Recruitment to the trial is ongoing. Citation Format: Enriqueta Felip, Remi Veillon, Santiago Viteri, Jürgen Scheele, Rolf Bruns, Paul K. Paik. Phase II trial of the c-Met inhibitor tepotinib in patients with advanced non-small cell lung cancer harboring MET exon 14-skipping mutations [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr A097.

Identification of 3-substituted-6-(1-(1H-[1,2,3]triazolo[4,5-b]pyrazin-1-yl)ethyl)quinoline derivatives as highly potent and selective mesenchymal-epithelial transition factor (c-Met) inhibitors via metabolite profiling-based structural optimization

c-Met/HGF signaling pathway plays an important role in cancer progression, and it was considered to be related to poor prognosis and drug resistance. Based on metabolite profiling of (S)-7-fluoro-6-(1-(6-(1-methyl-1H-pyrazol-4-yl)-1H-imidazo[4,5-b]pyrazin-1-yl)ethyl)quinoline (1), a series of 2-substituted or 3-substituted-6-(1-(1H-[1,2,3]triazolo[4,5-b]pyrazin-1-yl)ethyl)quinoline derivatives was rationally designed and evaluated. Most of the 3-substituted derivatives not only exhibited potent activities in both enzymatic and cellular assays, but also were stable in liver microsomes among different species (human, rat and monkey). SAR investigation revealed that introducing of N-methyl-1H-pyrazol-4-yl group at the 3-position of quinoline moiety is beneficial to improve the inhibitory potency, especially in the cellular assays. The influence of fluorine atom at 7-position or 5, 7-position of quinoline moiety and substituents at the 6-position of triazolo[4,5-b]pyrazine core on overall activity is not very significant. Racemate 14, an extremely potent and exquisitely selective c-Met inhibitor, demonstrated favorable pharmacokinetic properties in rats, no significant AO metabolism, and effective tumor growth inhibition in c-Met overexpressed NSCLC (H1993 cell line) and gastric cancer (SNU-5 cell line) xenograft models. Docking analysis indicated that besides the typical interactions of most selective c-Met inhibitors, the intramolecular halogen bond and additional hydrogen bond interactions with kinase are beneficial to the binding. These results may provide deep insight into potential structural modifications for developing potent c-Met inhibitors.

P3.02b-117 Phase Ib Results from a Study of Capmatinib (INC280) + EGF816 in Patients with EGFR-Mutant Non-Small Cell Lung Cancer (NSCLC): Topic: EGFR RES

Among patients with EGFR-mutant NSCLC who progress on EGFR tyrosine kinase inhibitors (EGFR-TKIs), the most common (50%) resistance mechanism is secondary T790M mutation. cMET dysregulation is the second most common mechanism, with amplification occurring in 5‒22% of resistant patients. This study evaluates targeting these two mechanisms to overcome acquired resistance to EGFR-TKIs. Capmatinib (INC280) is a highly selective, potent cMET inhibitor with clinical activity in patients with cMET dysregulation. EGF816 is an irreversible EGFR-TKI that selectively inhibits T790M and EGFR-activating mutations, with antitumor activity in EGFRT790M-mutated NSCLC. In this open-label Phase Ib/II study, capmatinib was combined with EGF816 in patients with EGFR-mutated, EGFR-TKI resistant NSCLC. The Phase Ib primary objective is estimation of the maximum tolerated dose (MTD)/recommended Phase II dose (RP2D) of the combination using an adaptive Bayesian logistic regression model. Eligible patients (≥18 years; ECOG PS ≤2) must have documented EGFR-mutated (exon19del and/or L858R) NSCLC and documented progression (RECIST v1.1) while on EGFR-TKI treatment. Patients received capmatinib (starting dose 200 mg BID) plus EGF816 (starting dose 50 mg QD). At the data cut-off (Aug 1, 2016), 33 patients were enrolled at five capmatinib BID/EGF816 QD mg dose levels (200/50 [n=4]; 200/100 [n=5]; 400/75 [n=3]; 400/100 [n=16]; 400/150 mg [n=5]); 18/33 (55%) patients discontinued treatment, mainly (13 [39%] patients) due to disease progression. Dose-limiting toxicities (DLTs) occurred in 4 patients: in 1 patient at the 200/50 dose level (increased alanine aminotransferase), 1 patient at the 400/100 dose level (anaphylactic reaction), and 2 patients at the 400/150 dose level (pyrexia, maculopapular rash, and allergic dermatitis). The most frequent (≥30%) any-grade adverse events (AEs), regardless of causality, were nausea (55%), peripheral edema (45%), increased amylase (42%), increased blood creatinine (36%), decreased appetite and diarrhea (both 30%). The most frequent (>10%) Grade ≥3 AEs were maculopapular rash (18% [mainly in the 400/150 cohort]) and increased amylase (12%). Capmatinib and EGF816 exposure increased with dose; preliminary data indicate a ∼35% increase in EGF816 exposure (AUC) at steady state when co-administered with the capmatinib RP2D, compared with single-agent exposure. The investigator-assessed overall response rate was 42% (2/33 complete responses; 12/33 partial responses) across all dose levels and 50% (8/16 patients) at the 400/100 dose level, regardless of molecular status of resistance. The RP2D of the combination was declared as capmatinib 400 mg BID + EGF816 100 mg QD. Preliminary antitumor activity was observed across dose levels, independent of T790M status.

Design, synthesis and biological evaluation of c-Met kinase inhibitors bearing 2-oxo-1,2-dihydroquinoline scaffold

A series of 2-oxo-1,2-dihydroquinoline-containing c-Met inhibitors were designed, synthesized and evaluated for their in vitro activities targeting c-Met. Most compounds showed high potency against c-Met with IC50 values in the single-digit nM range. Among these compounds, two target compounds, namely 1h and 1n, stood out as the most potent c-Met inhibitors with IC50s of 0.6 and 0.7nM, respectively. And 1a exhibited higher potency than BMS-777607 did with respect to the inhibition of cell proliferation. The introduction of electron-donating substituent was favorable for the activities of the compounds to some extent. Furthermore, molecular docking studies also gave encouraging results that supported this work.

Design, synthesis and biological evaluation of novel 4-phenoxy-6,7-disubstituted quinolines possessing (thio)semicarbazones as c-Met kinase inhibitors

In continuing our efforts to identify small molecules able to inhibit c-Met kinase, three series of novel 6,7-disubstituted-4-phenoxyquinoline derivatives (23a–w, 26a–d and 30a–d) bearing (thio)semicarbazone scaffold were designed, synthesized and evaluated for their cytotoxicity. The biological data revealed that most compounds exhibited moderate-to-excellent activity against HT-29, MKN-45, A549 cancer cell lines and relative poor potency toward MDA-MB-231 cell as well as hardly any cytotoxicity in normal PBL cell. Eleven compounds were further examined for their inhibitory activity against c-Met kinase and three compounds (23h, 23n and 26a) demonstrated good inhibitory activity. This work resulted in the discovery of a potent c-Met inhibitor 23n, bearing 2-hydroxy-3-allylphenyl group at R2 moiety, as a valuable lead molecule, which possessed remarkable cytotoxicity and high selectivity against A549 and HT-29 cell lines with IC50 values of 11nM and 27nM. Besides, it displayed excellent c-Met kinase inhibition on a single-digital nanomolar level (IC50=1.54nM). Meanwhile, the results from preliminarily in vivo study reflected that compound 23n showed promising overall PK profiles, consistent with the efficacy in both MKN-45 and HT-29 tumor xenograft mice model. These results clearly indicated that compound 23n is a potent and highly selective c-Met inhibitor and its favorable in vitro and in vivo profiles warrant further investigation.

Abstract LB-C22: Acquired resistance to the cMET inhibitor savolitinib in lung cancer models through EGFR/mTOR/MYC deregulation and adoption of PIM signaling

Abstract Lung cancer is the most common cause of cancer death globally with a significant, unmet need for more efficacious treatments. Aberrant receptor tyrosine kinase (RTK) signaling is a well-documented driver of disease onset and progression in multiple cancer types, including non-small cell lung cancer (NSCLC), where the cMET RTK contributes to tumor progression, maintenance and resistance to targeted therapies. Here, we explore the therapeutic potential of the potent and selective cMET inhibitor savolitinib (volitinib, AZD6094, HMPL-504) in NSCLC and begin to elucidate mechanisms of acquired savolitinib resistance in preclinical models. Using in vitro proliferation assays and immunoblot analysis, we determine that savolitinib rapidly inhibits cMET auto-phosphorylation/activation and reduces the viability of NSCLC cell lines NCI-H1993 and EBC-1 with a GI50 of 4.20 nM and 2.14 nM, respectively. In vivo, once daily treatment of NCI-H1993 xenografts with 3.0 mg/kg savolitinib significantly slows tumor growth, whereas treatment of EBC-1 xenografts with 30.0 mg/kg results in tumor stasis. Importantly, we observe tumor regressions in a patient-derived xenograft model of a NSCLC lymph node metastasis, HLXF-036LN, dosed with savolitinib 50.0 mg/kg once daily. Pharmacodynamic analysis of in vitro and in vivo models shows that savolitinib sensitivity correlates with blockade of PI3K/AKT and MAPK signaling, and interestingly, with cMYC (MYC) protein down-regulation. To elucidate mechanisms of acquired resistance in NSCLC, we generated savolitinib resistance in vitro using the NCI-H1993 and EBC-1 cell lines and further sub-cloned resistant NCI-H1993 cells to study the heterogeneity of resistance mechanisms. Using small-molecule screening, phospho-protein arrays and interrogation of signaling pathway activity by immunoblot, we identify 1) deregulated mTORC1/2 signaling and 2) the uncoupling of MYC expression from cMET activation as commonly contributing to resistance in all clones tested. RNA interference (siRNA) and MYC over-expression experiments confirm the novel finding that sustained MYC expression can partially drive resistance to a tyrosine kinase inhibitor such as savolitinib. Additionally, we identify clone-specific resistance mechanisms arising via a previously-described switch to EGFR dependence or by our novel finding of a de novo requirement for PIM signaling. Taken together, this work demonstrates the preclinical efficacy of savolitinib in NSCLC and provides an initial characterization of potential resistance mechanisms, identifying core resistance targets and clone-specific vulnerabilities that could be exploited to counter acquired savolitinib resistance that may emerge in the clinic. Citation Format: Ryan E. Henry, Evan R. Barry, Brendon Ladd, Aleksandra Markovets, Garry J. Beran, Yongxin Ren, Feng Zhou, Lillian Castriotta, Ammar Adam, Weiguo Qing, Weiguo Su, Edwin Clark, Celina M. D'Cruz, Alwin Schuller. Acquired resistance to the cMET inhibitor savolitinib in lung cancer models through EGFR/mTOR/MYC deregulation and adoption of PIM signaling. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr LB-C22.

Abstract 782: Modulation of receptor tyrosine kinase signaling by cMET inhibitor, INC280, in cMETmut/amp gastric carcinoma cell line Hs746.T

Abstract Aberrant Receptor Tyrosine Kinase (RTK) activity is one of the hallmarks of cancer. The genetic alternations of ABL and EGFR are classical examples of RTK-dependent oncogenesis. Recent data suggest that non mutated RTK signaling can form feedback pathways to serve as resistant mechanism against targeted therapies. Here, we examined the regulation of RTK signaling by a potent cMET inhibitor, INC280 (enzymatic EC50 against c-MET = 0.13 nM), in a cMET-dependent human gastric tumor model Hs746.T. Hs746.T is a heavily cMET dependent cell line with both cMET amplication (12.9 copies) and mutation (G-T mutation at slice donor site to cause juxtamembrane domain deletion). This model is exquisitely sensitive to cMET inhibitor, INC280 in vitro (GI50 = 9 nM) and in vivo. The treatment of INC280 lead to significant inhibition of pcMET. Furthermore, the treatment of INC280 resulted in the suppression of pEGFR, though INC280 does not actively inhibit EGFR (enzymatic EC50 against EGFR > 10 microM). This result supports prior reports that cMET and EGFR can form physical complex, with cMET modulating EGFR activation. Consequently, INC280 inhibited downstream signaling nodes of pAKT, pS6RP, pMEK and pERK in Hs746.T. To examine the RTK signaling underlying possible treatment relapse, we treated Hs746.T cells chronically in vitro under increasing concentrations of INC280. After 8 weeks of treatment, Hs746.T-R cells were generated that grew at 600 nM of INC280. RTK signaling was examined in Hs746.T-R cells under treatment of INC280. As expected, cMET signaling was significantly curtailed in Hs746.T-R cells, as was pEGFR. Interestingly, pPDGFRb was elevated in Hs746.T-R, compared to the parental cell line. We hypothesize that the increased signaling through PDGFRb may serve as resistance mechanism to INC280 in Hs746.T-R cells. In summary, we have examined RTK signaling in the cMET-dependent Hs746.T cell line. Our finding supported the notion of RTK cross-talk between cMET and EGFR in this setting. Additionally, our data reveal that additional RTK signaling (PDGFRb) can be induced under chronic treatment of INC280 to possibly mediate resistance to cMET inhibition. Citation Format: Maria C. Pinzon-Ortiz, Xianhui Rong, Jinsheng Liang, Hui Qin Wang, Alan Huang, Robert Schlegel, Z. Alexander Cao. Modulation of receptor tyrosine kinase signaling by cMET inhibitor, INC280, in cMETmut/amp gastric carcinoma cell line Hs746.T. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 782. doi:10.1158/1538-7445.AM2015-782

Design, Synthesis, and Biological Evaluation of Novel Imidazo[1,2-a]pyridine Derivatives as Potent c-Met Inhibitors.

A series of imidazo[1,2-a]pyridine derivatives against c-Met was designed by means of bioisosteric replacement. In this study, a selective, potent c-Met inhibitor, 22e was identified, with IC50 values of 3.9 nM against c-Met kinase and 45.0 nM against c-Met-addicted EBC-1 cell proliferation, respectively. Compound 22e inhibited c-Met phosphorylation and downstream signaling across different oncogenic forms in c-Met overactivated cancer cells and model cells. Compound 22e significantly inhibited tumor growth (TGI = 75%) with good oral bioavailability (F = 29%) and no significant hERG inhibition. On the basis of systematic metabolic study, the pathway of all possible metabolites of 22e in liver microsomes of different species has been proposed, and a major NADPH-dependent metabolite 33 was generated by liver microsomes. To block the metabolic site, 42 was designed and synthesized for further evaluation. Taken together, the imidazo[1,2-a]pyridine scaffold showed promising pharmacological inhibition of c-Met and warrants further investigation.