Yajing Yang

c-Met inhibitors with novel binding mode show activity against several hereditary papillary renal cell carcinoma-related mutations.

c-Met is a receptor tyrosine kinase often deregulated in human cancers, thus making it an attractive drug target. One mechanism by which c-Met deregulation leads to cancer is through gain-of-function mutations. Therefore, small molecules capable of targeting these mutations could offer therapeutic benefits for affected patients. SU11274 was recently described and reported to inhibit the activity of the wild-type and some mutant forms of c-Met, whereas other mutants are resistant to inhibition. We identified a novel series of c-Met small molecule inhibitors that are active against multiple mutants previously identified in hereditary papillary renal cell carcinoma patients. AM7 is active against wild-type c-Met as well as several mutants, inhibits c-Met-mediated signaling in MKN-45 and U-87 MG cells, and inhibits tumor growth in these two models grown as xenografts. The crystal structures of AM7 and SU11274 bound to unphosphorylated c-Met have been determined. The AM7 structure reveals a novel binding mode compared with other published c-Met inhibitors and SU11274. The molecule binds the kinase linker and then extends into a new hydrophobic binding site. This binding site is created by a significant movement of the C-helix and so represents an inactive conformation of the c-Met kinase. Thus, our results demonstrate that it is possible to identify and design inhibitors that will likely be active against mutants found in different cancers.

Discovery and Optimization of Triazolopyridazines as Potent and Selective Inhibitors of the c-Met Kinase.

Tumorigenesis is a multistep process in which oncogenes play a key role in tumor formation, growth, and maintenance. MET was discovered as an oncogene that is activated by its ligand, hepatocyte growth factor. Deregulated signaling in the c-Met pathway has been observed in multiple tumor types. Herein we report the discovery of potent and selective triazolopyridazine small molecules that inhibit c-Met activity.

Design, synthesis, and biological evaluation of potent c-Met inhibitors.

c-Met is a receptor tyrosine kinase that plays a key role in several cellular processes but has also been found to be overexpressed and mutated in different human cancers. Consequently, targeting this enzyme has become an area of intense research in drug discovery. Our studies began with the design and synthesis of novel pyrimidone 7, which was found to be a potent c-Met inhibitor. Subsequent SAR studies identified 22 as a more potent analog, whereas an X-ray crystal structure of 7 bound to c-Met revealed an unexpected binding conformation. This latter finding led to the development of a new series that featured compounds that were more potent both in vitro and in vivo than 22 and also exhibited different binding conformations to c-Met. Novel c-Met inhibitors have been designed, developed, and found to be potent in vitro and in vivo.

Discovery of a Potent, Selective, and Orally Bioavailable c-Met Inhibitor: 1-(2-Hydroxy-2-methylpropyl)-N-(5-(7-methoxyquinolin-4-yloxy)pyridin-2-yl)-5-methyl-3-oxo-2-phenyl-2,3-dihydro-1H-pyrazole-4-carboxamide (AMG 458)

Deregulation of the receptor tyrosine kinase c-Met has been implicated in human cancers. Pyrazolones with N-1 bearing a pendent hydroxyalkyl side chain showed selective inhibition of c-Met over VEGFR2. However, studies revealed the generation of active, nonselective metabolites. Blocking this metabolic hot spot led to the discovery of 17 (AMG 458). When dosed orally, 17 significantly inhibited tumor growth in the NIH3T3/TPR-Met and U-87 MG xenograft models with no adverse effect on body weight.

Structure-Based Design of Novel Class II c-Met Inhibitors: 2. SAR and Kinase Selectivity Profiles of the Pyrazolone Series

As part of our effort toward developing an effective therapeutic agent for c-Met-dependent tumors, a pyrazolone-based class II c-Met inhibitor, N-(4-((6,7-dimethoxyquinolin-4-yl)oxy)-3-fluorophenyl)-1,5-dimethyl-3-oxo-2-phenyl-2,3-dihydro-1H-pyrazole-4-carboxamide (1), was identified. Knowledge of the binding mode of this molecule in both c-Met and VEGFR-2 proteins led to a novel strategy for designing more selective analogues of 1. Along with detailed SAR information, we demonstrate that the low kinase selectivity associated with class II c-Met inhibitors can be improved significantly. This work resulted in the discovery of potent c-Met inhibitors with improved selectivity profiles over VEGFR-2 and IGF-1R that could serve as useful tools to probe the relationship between kinase selectivity and in vivo efficacy in tumor xenograft models. Compound 59e (AMG 458) was ultimately advanced into preclinical safety studies.

Discovery and optimization of potent and selective triazolopyridazine series of c-Met inhibitors

Deregulation of the receptor tyrosine kinase c-Met has been implicated in several human cancers and is an attractive target for small molecule drug discovery. We previously showed that O-linked triazolopyridazines can be potent inhibitors of c-Met. Herein, we report the discovery of a related series of N-linked triazolopyridazines which demonstrate nanomolar inhibition of c-Met kinase activity and display improved pharmacodynamic profiles. Specifically, the potent time-dependent inhibition of cytochrome P450 associated with the O-linked triazolopyridazines has been eliminated within this novel series of inhibitors. N-linked triazolopyridazine 24 exhibited favorable pharmacokinetics and displayed potent inhibition of HGF-mediated c-Met phosphorylation in a mouse liver PD model. Once-daily oral administration of 24 for 22days showed significant tumor growth inhibition in an NIH-3T3/TPR-Met xenograft mouse efficacy model.

In Vitro and In Vivo Activity of AMG 337, a Potent and Selective MET Kinase Inhibitor, in MET-Dependent Cancer Models

The MET receptor tyrosine kinase is involved in cell growth, survival, and invasion. Clinical studies with small molecule MET inhibitors have shown the role of biomarkers in identifying patients most likely to benefit from MET-targeted therapy. AMG 337 is an oral, small molecule, ATP-competitive, highly selective inhibitor of the MET receptor. Herein, we describe AMG 337 preclinical activity and mechanism of action in MET-dependent tumor models. These studies suggest MET is the only therapeutic target for AMG 337. In an unbiased tumor cell line proliferation screen (260 cell lines), a closely related analogue of AMG 337, Compound 5, exhibited activity in 2 of 260 cell lines; both were MET-amplified. Additional studies examining the effects of AMG 337 on the proliferation of a limited panel of cell lines with varying MET copy numbers revealed that high-level focal MET amplification (>12 copies) was required to confer MET oncogene addiction and AMG 337 sensitivity. One MET-amplified cell line, H1573 (>12 copies), was AMG 337 insensitive, possibly because of a downstream G12A KRAS mutation. Mechanism-of-action studies in sensitive MET-amplified cell lines demonstrated that AMG 337 inhibited MET and adaptor protein Gab-1 phosphorylation, subsequently blocking the downstream PI3K and MAPK pathways. AMG 337 exhibited potency in pharmacodynamic assays evaluating MET signaling in tumor xenograft models; >90% inhibition of Gab-1 phosphorylation was observed at 0.75 mg/kg. These findings describe the preclinical activity and mechanism of action of AMG 337 in MET-dependent tumor models and indicate its potential as a novel therapeutic for the treatment of MET-dependent tumors. Mol Cancer Ther; 15(7); 1568–79. ©2016 AACR.

Abstract 728: AMG 337, a novel, potent and selective MET kinase inhibitor, has robust growth inhibitory activity in MET-dependent cancer models

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Signaling through the MET receptor tyrosine kinase and its ligand, hepatocyte growth factor (HGF), promotes cell proliferation, survival, and invasion. Activation of MET signaling is a relatively common hallmark of a diverse range of human cancer types, and as a result, inhibition of MET signaling represents an attractive therapeutic opportunity for the treatment of cancer. In this study, we describe the characterization of AMG 337, a potent and highly selective small molecule ATP-competitive MET kinase inhibitor that demonstrates robust activity in MET-dependent cancer models. In enzymatic assays, AMG 337 inhibited MET kinase activity with an IC50 of 10 μM in all other tested cell lines. Further studies in an expanded panel of additional cancer cell lines derived from gastric, NSCLC, and esophageal cancer confirmed that the in-vitro anti-proliferative activity of AMG 337 correlated with amplification of MET. In those cell lines, treatment with AMG 337 inhibited downstream PI3K and MAPK signaling pathways, which translated into growth arrest as evidenced by an accumulation of cells in the G1 phase of the cell cycle, a concomitant reduction in DNA synthesis, and the induction of apoptosis. In vivo, oral administration of AMG 337 resulted in robust dose-dependent anti-tumor efficacy in MET amplified gastric cancer xenograft models, with inhibition of tumor growth consistent with the pharmacodynamic modulation of MET signaling. In conclusion, these findings illustrate the potential clinical utility of AMG 337 as a therapeutic agent for the treatment of tumors with evidence of dysregulated MET signaling, including MET amplification. A phase 1 clinical study is currently evaluating the safety, tolerability and pharmacokinectics of AMG 337 in patients with solid tumors. Citation Format: Paul E. Hughes, Yajing Yang, Karen Rex, Yihong Zhang, Paula J. Kaplan-Lefko, Sean Caenepeel, Jodi Moriguchi, Martin Broome, Deborah Choquette, Robert Radinsky, Richard Kendall, Angela Coxon, Isabelle Dussault. AMG 337, a novel, potent and selective MET kinase inhibitor, has robust growth inhibitory activity in MET-dependent cancer models. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 728. doi:10.1158/1538-7445.AM2014-728

Discovery of imidazopyridazines as potent Pim-1/2 kinase inhibitors

High levels of Pim expression have been implicated in several hematopoietic and solid tumor cancers, suggesting that inhibition of Pim signaling could provide patients with therapeutic benefit. Herein, we describe our progress towards this goal using a screening hit (rac-1) as a starting point. Modification of the indazole ring resulted in the discovery of a series of imidazopyridazine-based Pim inhibitors exemplified by compound 22m, which was found to be a subnanomolar inhibitor of the Pim-1 and Pim-2 isoforms (IC50 values of 0.024nM and 0.095nM, respectively) and to potently inhibit the phosphorylation of BAD in a cell line that expresses high levels of all Pim isoforms, KMS-12-BM (IC50=28nM). Profiling of Pim-1 and Pim-2 expression levels in a panel of multiple myeloma cell lines and correlation of these data with the potency of compound 22m in a proliferation assay suggests that Pim-2 inhibition would be advantageous for this indication.

Abstract 5396: Characterization of small molecule inhibitors of the PIM kinases in in vitro models of hematological malignancies

Proceedings: AACR 106th Annual Meeting 2015; April 18-22, 2015; Philadelphia, PA The three members of the Pim kinase family, Pim-1, -2, and -3, are established oncogenes and are attractive targets in hematological malignancies. We have developed multiple potent and selective scaffolds of pan-Pim inhibitors with picomolar enzymatic potency and nanomolar cellular potency. Using these agents, we have observed that the abolishment of Pim activity impairs tumor cell viability in multiple settings, both in vitro and in vivo. We have developed numerous assays to measure Pim protein levels and activity, including Pim downstream markers p-PDCD4 and p-BAD, that might be broadly applicable to human tissues. These assays have allowed us to establish a correlation between Pim protein levels and sensitivity to Pim inhibition across multiple tumor settings, in vitro. We have observed that all tested multiple myeloma (MM) cell lines express high levels of Pim-2 protein and that pan-Pim inhibition impairs viability in 90% of these lines and induces apoptosis in a subset. In acute myelogenous leukemia (AML) cell lines, sensitivity to Pim inhibition significantly correlates with Pim-1 protein expression. Numerous diffuse, large B-cell lymphoma (DLBCL) cell lines have high Pim levels and many are sensitive to Pim inhibition. We have also assessed Pim expression and activity in human tumor and normal tissues. Studies performed with myeloma cells isolated from patient bone marrow aspirates have revealed elevated Pim-2 protein levels as well as sensitivity to ex vivo dosing with Pim inhibitors, as evidenced by inhibition of PDCD4 phosphorylation. Primary patient samples from numerous other hematological tumors have also been found to have high Pim-1 or Pim-2 protein levels. To expand the possible utility of Pim inhibitors in the clinic, we have combined our molecules with numerous clinical agents, including dexamethasone, carfilzomib, and PI3K inhibitors, across multiple settings, in vitro. In all indications surveyed, we have observed that the combination of Pim molecules and these agents can lead to synergistic effects on cell viability, apoptosis and pathway signaling. In some cases, cell lines that show mild or no response to either single agent alone are sensitive to combination treatment. Collectively, our data provide a rationale for the development of Pim kinase inhibitors for use either as monotherapy or in combination with other agents in diverse tumor settings. Citation Format: Christine E. Sastri, Nadia Guerrero, Dongyin Yu, Bethany Mattson, Ken Dellamaggiore, Yajing Yang, Paul Hughes, Hui-Ling Wang, Victor Cee, Brian A. Lanman, Liping Pettus, Anthony B. Reed, Bin Wu, Ryan Wurz, Andrew Tasker, Li-Ya Huang, Daniel Branstetter, Karen Rex, Jeffrey Winston, Teresa L. Burgess, Richard Kendall, J Russell Lipford. Characterization of small molecule inhibitors of the PIM kinases in in vitro models of hematological malignancies. [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 5396. doi:10.1158/1538-7445.AM2015-5396