ABSTRACT
By targeting an extended region of the conventional 'DFG-out' pocket of p38alpha, while minimizing interactions with the specificity pocket and eliminating interactions with the adenine binding site, we are able to design and synthesize a number of pyrazole-urea based DFG-out p38alpha inhibitors with good potencies, and excellent selectivity.
Subject(s)
Mitogen-Activated Protein Kinase 14/metabolism , Phenylurea Compounds/chemistry , Protein Kinase Inhibitors/chemistry , Pyrazoles/chemistry , Adenine/metabolism , Amino Acid Motifs , Amino Acid Sequence , Binding Sites , Humans , Microsomes/metabolism , Mitogen-Activated Protein Kinase 14/antagonists & inhibitors , Phenylurea Compounds/chemical synthesis , Phenylurea Compounds/pharmacology , Protein Kinase Inhibitors/pharmacology , Pyrazoles/chemical synthesis , Pyrazoles/pharmacology , Structure-Activity RelationshipABSTRACT
The activation of receptor tyrosine kinases (RTKs) is tightly regulated through a variety of mechanisms. Kinetic studies show that activation of c-Kit RTK occurs through an inter-molecular autophosphorylation. Phosphopeptide mapping of c-Kit reveals that 14-22 phosphates are added to each mol of wild-type (WT) c-Kit during the activation. Phosphorylation sites are found on the JM, kinase insert (KID), c-terminal domains and the activation loop (A-loop), but only the sites on the JM domain contribute to the kinase activation. The A-loop tyrosine (Y(823)) is not phosphorylated until very late in the activation (>90% completion), indicating that the A-loop phosphorylation is not required for c-Kit activation. A sunitinib-resistant mutant D816H that accelerates auto-activation by 184-fold shows no phosphorylation on the A-loop tyrosine after full activation. A loss-of-phosphorylation mutation Y823F remains fully competent in auto-activation. Similar to WT and D816H, the unactivated Y823F mutant binds sunitinib and imatinib with high affinity (K(D) = 5.9 nM). But unlike the WT and D816H where the activated enzymes lose the ability to bind the two drugs, activated Y823F binds the two inhibitors effectively. These observations suggest that the A-loop of activated Y823F remains flexible and can readily adopt unactivated conformations to accommodate DFG-out binders.
Subject(s)
Antineoplastic Agents/metabolism , Drug Resistance, Neoplasm , Enzyme Inhibitors/metabolism , Indoles/metabolism , Phosphotyrosine/physiology , Protein Interaction Domains and Motifs/physiology , Proto-Oncogene Proteins c-kit/metabolism , Pyrroles/metabolism , Amino Acid Substitution , Benzamides , Catalytic Domain , Enzyme Activation , Humans , Imatinib Mesylate , Kinetics , Microchemistry/methods , Models, Biological , Mutant Proteins/chemistry , Mutant Proteins/isolation & purification , Mutant Proteins/metabolism , Peptide Mapping , Phosphorylation , Piperazines/metabolism , Protein Binding , Proto-Oncogene Proteins c-kit/antagonists & inhibitors , Proto-Oncogene Proteins c-kit/chemistry , Proto-Oncogene Proteins c-kit/genetics , Pyrimidines/metabolism , Recombinant Fusion Proteins/antagonists & inhibitors , Recombinant Fusion Proteins/chemistry , Recombinant Fusion Proteins/isolation & purification , Recombinant Fusion Proteins/metabolism , SunitinibABSTRACT
The design, synthesis and utility of fluorescence probes that bind to the DFG-out conformation of p38alpha kinase are described. Probes that demonstrate good affinity for p38alpha, have been identified and one of the probes, PF-04438255, has been successfully used in an high throughput screening (HTS) assay to identify two novel non-classical p38alpha inhibitors. In addition, a cascade activity assay was utilized to validate the selective binding of these non-classical kinase inhibitors to the unactive form of the enzyme.
Subject(s)
Fluorescent Dyes/chemical synthesis , Mitogen-Activated Protein Kinase 14/metabolism , Protein Kinase Inhibitors/chemical synthesis , Binding Sites , Computer Simulation , Crystallography, X-Ray , Fluorescent Dyes/chemistry , Fluorescent Dyes/pharmacology , High-Throughput Screening Assays , Kinetics , Mitogen-Activated Protein Kinase 14/chemistry , Naphthalenes/chemistry , Naphthalenes/pharmacology , Protein Kinase Inhibitors/chemistry , Protein Kinase Inhibitors/pharmacology , Pyrazoles/chemistry , Pyrazoles/pharmacology , Structure-Activity RelationshipABSTRACT
Most gastrointestinal stromal tumors (GISTs) exhibit aberrant activation of the receptor tyrosine kinase (RTK) KIT. The efficacy of the inhibitors imatinib mesylate and sunitinib malate in GIST patients has been linked to their inhibition of these mutant KIT proteins. However, patients on imatinib can acquire secondary KIT mutations that render the protein insensitive to the inhibitor. Sunitinib has shown efficacy against certain imatinib-resistant mutants, although a subset that resides in the activation loop, including D816H/V, remains resistant. Biochemical and structural studies were undertaken to determine the molecular basis of sunitinib resistance. Our results show that sunitinib targets the autoinhibited conformation of WT KIT and that the D816H mutant undergoes a shift in conformational equilibrium toward the active state. These findings provide a structural and enzymologic explanation for the resistance profile observed with the KIT inhibitors. Prospectively, they have implications for understanding oncogenic kinase mutants and for circumventing drug resistance.
