ABSTRACT
Variously substituted indolin-2-ones were synthesized and evaluated for activity against KDR, Flt-1, FGFR-1 and PDGFR. Extension at the 5-position of the oxindole ring with ethyl piperidine (compound 7i) proved to be the most beneficial for attaining both biochemical and cellular potencies. Further optimization of 7i to balance biochemical and cellular potencies with favorable ADME/ PK properties led to the identification of 8h, a compound with a clean CYP profile, acceptable pharmacokinetic and toxicity profiles, and robust efficacy in multiple xenograft tumor models.
Subject(s)
Drug Design , Indoles/chemical synthesis , Piperidines/chemical synthesis , Protein Kinase Inhibitors/chemical synthesis , Receptor Protein-Tyrosine Kinases/antagonists & inhibitors , Animals , Binding Sites , Cell Line, Tumor , Crystallography, X-Ray , Cytochrome P-450 CYP3A/metabolism , Female , Half-Life , Humans , Indoles/pharmacokinetics , Indoles/therapeutic use , Lung/drug effects , Lung/metabolism , Mice , Neoplasms/drug therapy , Piperidines/pharmacokinetics , Piperidines/therapeutic use , Protein Kinase Inhibitors/pharmacokinetics , Protein Kinase Inhibitors/therapeutic use , Protein Structure, Tertiary , Rats , Receptor Protein-Tyrosine Kinases/metabolism , Structure-Activity Relationship , Transplantation, HeterologousABSTRACT
Tyrosine 984 in the juxtamembrane region of the insulin receptor, between the transmembrane helix and the cytoplasmic tyrosine kinase domain, is conserved among all insulin receptor-like proteins from hydra to humans. Crystallographic studies of the tyrosine kinase domain and proximal juxtamembrane region reveal that Tyr-984 interacts with several other conserved residues in the N-terminal lobe of the kinase domain, stabilizing a catalytically nonproductive position of alpha-helix C. Steady-state kinetics measurements on the soluble kinase domain demonstrate that replacement of Tyr-984 with phenylalanine results in a 4-fold increase in kcat in the unphosphorylated (basal state) enzyme. Moreover, mutation of Tyr-984 in the full-length insulin receptor results in significantly elevated receptor phosphorylation levels in cells, both in the absence of insulin and following insulin stimulation. These data demonstrate that Tyr-984 plays an important structural role in maintaining the quiescent, basal state of the insulin receptor. In addition, the structural studies suggest a possible target site for small molecule activators of the insulin receptor, with potential use in the treatment of noninsulin-dependent diabetes mellitus.
Subject(s)
Cell Membrane/metabolism , Receptor, Insulin/chemistry , Receptor, Insulin/physiology , Tyrosine/chemistry , Amino Acid Sequence , Animals , Binding Sites , Crystallography, X-Ray , Diabetes Mellitus, Type 2/metabolism , Humans , Kinetics , Models, Molecular , Molecular Sequence Data , Phosphorylation , Protein Binding , Protein Structure, Tertiary , Receptor, Insulin/metabolism , TransfectionABSTRACT
Muscle-specific kinase (MuSK) is a receptor tyrosine kinase expressed selectively in skeletal muscle. During neuromuscular synapse formation, agrin released from motor neurons stimulates MuSK autophosphorylation in the kinase activation loop and in the juxtamembrane region, leading to clustering of acetylcholine receptors. We have determined the crystal structure of the cytoplasmic domain of unphosphorylated MuSK at 2.05 A resolution. The structure reveals an autoinhibited kinase domain in which the activation loop obstructs ATP and substrate binding. Steady-state kinetic analysis demonstrates that autophosphorylation results in a 200-fold increase in k(cat) and a 10-fold decrease in the K(m) for ATP. These studies provide a molecular basis for understanding the regulation of MuSK catalytic activity and suggest that an additional in vivo component may contribute to regulation via the juxtamembrane region.
