ABSTRACT
This report documents the first example of a specific inhibitor of protein kinases with preferential binding to the activated kinase conformation: 5H-benzo[4,5]cyclohepta[1,2-b]pyridin-5-one 11r (MK-8033), a dual c-Met/Ron inhibitor under investigation as a treatment for cancer. The design of 11r was based on the desire to reduce time-dependent inhibition of CYP3A4 (TDI) by members of this structural class. A novel two-step protocol for the synthesis of benzylic sulfonamides was developed to access 11r and analogues. We provide a rationale for the observed selectivity based on X-ray crystallographic evidence and discuss selectivity trends with additional examples. Importantly, 11r provides full inhibition of tumor growth in a c-Met amplified (GTL-16) subcutaneous tumor xenograft model and may have an advantage over inactive form kinase inhibitors due to equal potency against a panel of oncogenic activating mutations of c-Met in contrast to c-Met inhibitors without preferential binding to the active kinase conformation.
Subject(s)
Benzocycloheptenes/metabolism , Benzocycloheptenes/pharmacology , Drug Discovery , Proto-Oncogene Proteins c-met/antagonists & inhibitors , Proto-Oncogene Proteins c-met/metabolism , Receptor Protein-Tyrosine Kinases/antagonists & inhibitors , Sulfonamides/metabolism , Sulfonamides/pharmacology , Animals , Benzocycloheptenes/chemistry , Cell Line, Tumor , Dogs , Enzyme Activation/drug effects , Female , Humans , Mice , Models, Molecular , Protein Conformation , Protein Kinase Inhibitors/chemistry , Protein Kinase Inhibitors/metabolism , Protein Kinase Inhibitors/pharmacology , Proto-Oncogene Proteins c-met/chemistry , Rats , Substrate Specificity , Sulfonamides/chemistry , Xenograft Model Antitumor AssaysABSTRACT
c-Met is a transmembrane tyrosine kinase that mediates activation of several signaling pathways implicated in aggressive cancer phenotypes. In recent years, research into this area has highlighted c-Met as an attractive cancer drug target, triggering a number of approaches to disrupt aberrant c-Met signaling. Screening efforts identified a unique class of 5H-benzo[4,5]cyclohepta[1,2-b]pyridin-5-one kinase inhibitors, exemplified by 1. Subsequent SAR studies led to the development of 81 (MK-2461), a potent inhibitor of c-Met that was efficacious in preclinical animal models of tumor suppression. In addition, biochemical studies and X-ray analysis have revealed that this unique class of kinase inhibitors binds preferentially to the activated (phosphorylated) form of the kinase. This report details the development of 81 and provides a description of its unique biochemical properties.
Subject(s)
Antineoplastic Agents/chemical synthesis , Benzocycloheptenes/chemical synthesis , Pyridines/chemical synthesis , Receptor Protein-Tyrosine Kinases/antagonists & inhibitors , Animals , Antineoplastic Agents/pharmacokinetics , Antineoplastic Agents/pharmacology , Benzocycloheptenes/pharmacokinetics , Benzocycloheptenes/pharmacology , Cell Line, Tumor , Crystallography, X-Ray , Dogs , Drug Screening Assays, Antitumor , Female , Haplorhini , Humans , Mice , Mice, Nude , Models, Molecular , Mutation , Neoplasm Transplantation , Phosphorylation , Protein Binding , Pyrazoles/chemical synthesis , Pyrazoles/pharmacokinetics , Pyrazoles/pharmacology , Pyridines/pharmacokinetics , Pyridines/pharmacology , Rats , Receptor Protein-Tyrosine Kinases/genetics , Structure-Activity Relationship , Sulfonamides/chemical synthesis , Sulfonamides/pharmacokinetics , Sulfonamides/pharmacology , Transplantation, HeterologousABSTRACT
The receptor tyrosine kinase c-Met is implicated in oncogenesis and is the target for several small molecule and biologic agents in clinical trials for the treatment of cancer. Binding of the hepatocyte growth factor to the cell surface receptor of c-Met induces activation via autophosphorylation of the kinase domain. Here we describe the structural basis of c-Met activation upon autophosphorylation and the selective small molecule inhibiton of autophosphorylated c-Met. MK-2461 is a potent c-Met inhibitor that is selective for the phosphorylated state of the enzyme. Compound 1 is an MK-2461 analog with a 20-fold enthalpy-driven preference for the autophosphorylated over unphosphorylated c-Met kinase domain. The crystal structure of the unbound kinase domain phosphorylated at Tyr-1234 and Tyr-1235 shows that activation loop phosphorylation leads to the ejection and disorder of the activation loop and rearrangement of helix αC and the G loop to generate a viable active site. Helix αC adopts a orientation different from that seen in activation loop mutants. The crystal structure of the complex formed by the autophosphorylated c-Met kinase domain and compound 1 reveals a significant induced fit conformational change of the G loop and ordering of the activation loop, explaining the selectivity of compound 1 for the autophosphorylated state. The results highlight the role of structural plasticity within the kinase domain in imparting the specificity of ligand binding and provide the framework for structure-guided design of activated c-Met inhibitors.
Subject(s)
Protein Kinase Inhibitors/chemistry , Proto-Oncogene Proteins/antagonists & inhibitors , Proto-Oncogene Proteins/chemistry , Receptor Protein-Tyrosine Kinases/antagonists & inhibitors , Receptor Protein-Tyrosine Kinases/chemistry , Animals , Cell Line , Crystallography, X-Ray , Drug Design , Humans , Phosphorylation , Protein Binding , Protein Structure, Secondary , Protein Structure, Tertiary , Proto-Oncogene Proteins/genetics , Proto-Oncogene Proteins/metabolism , Receptor Protein-Tyrosine Kinases/genetics , Receptor Protein-Tyrosine Kinases/metabolism , Spodoptera , Structure-Activity Relationship , c-Mer Tyrosine KinaseABSTRACT
A family of beta-substituted histidine-containing peptides has been synthesized to probe the effect of noncovalent conformational rigidification on catalyst enantioselectivity. Unambiguous enhancement of enantioselectivity in the conjugate addition of azide to alpha,beta-unsaturated carboxylate derivatives has been achieved, enabling application to a sequential asymmetric azidation/cycloaddition for the synthesis of optically enriched triazoles and triazolines.