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Development of a fluorescent-tagged kinase assay system for the detection and characterization of allosteric kinase inhibitors.
Simard, Jeffrey R; Getlik, Matthäus; Grütter, Christian; Pawar, Vijaykumar; Wulfert, Sabine; Rabiller, Matthias; Rauh, Daniel.
Afiliação
  • Simard JR; Chemical Genomics Centre of the Max Planck Society, Otto-Hahn-Strasse 15, D-44227 Dortmund, Germany.
J Am Chem Soc ; 131(37): 13286-96, 2009 Sep 23.
Article em En | MEDLINE | ID: mdl-19572644
ABSTRACT
Kinase disregulation disrupts the intricate network of intracellular signaling pathways and contributes to the onset of diseases such as cancer. Although several kinase inhibitors are on the market, inhibitor selectivity and drug resistance mutations persist as fundamental challenges in the development of effective long-term treatments. Chemical entities binding to less conserved allosteric sites would be expected to offer new opportunities for scaffold development. Because no high-throughput method was previously available, we developed a fluorescence-based kinase binding assay for identifying and characterizing ligands which stabilize the inactive kinase conformation. Here, we present a description of the development and validation of this assay using the serine/threonine kinase p38alpha. By covalently attaching fluorophores to the activation loop of the kinase, we were able to detect conformational changes and measure the K(d), k(on), and k(off) associated with the binding and dissociation of ligands to the allosteric pocket. We report the SAR of a synthesized focused library of pyrazolourea derivatives, a scaffold known to bind with high affinity to the allosteric pocket of p38alpha. Additionally, we used protein X-ray crystallography together with our assay to examine the binding and dissociation kinetics to characterize potent quinazoline- and quinoline-based type II inhibitors, which also utilize this binding pocket in p38alpha. Last, we identified the b-Raf inhibitor sorafenib as a potent low nanomolar inhibitor of p38alpha and used protein X-ray crystallography to confirm a unique binding mode to the inactive kinase conformation.
Assuntos

Texto completo: 1 Coleções: 01-internacional Base de dados: MEDLINE Assunto principal: Proteínas Quinases p38 Ativadas por Mitógeno / Inibidores de Proteínas Quinases / Avaliação Pré-Clínica de Medicamentos / Corantes Fluorescentes Tipo de estudo: Diagnostic_studies / Prognostic_studies Limite: Humans Idioma: En Revista: J Am Chem Soc Ano de publicação: 2009 Tipo de documento: Article País de afiliação: Alemanha

Texto completo: 1 Coleções: 01-internacional Base de dados: MEDLINE Assunto principal: Proteínas Quinases p38 Ativadas por Mitógeno / Inibidores de Proteínas Quinases / Avaliação Pré-Clínica de Medicamentos / Corantes Fluorescentes Tipo de estudo: Diagnostic_studies / Prognostic_studies Limite: Humans Idioma: En Revista: J Am Chem Soc Ano de publicação: 2009 Tipo de documento: Article País de afiliação: Alemanha