RESUMEN
Dysferlinopathies, which are muscular diseases caused by mutations in the dysferlin gene, remain serious medical problems due to the lack of therapeutic agents. Herein, we report the design, synthesis, and structure-activity relationships of a 2,6-disubstituted 3H-imidazo[4,5-b]pyridine series, which was identified from the phenotypic screening of chemicals that increase the level of dysferlin in myocytes differentiated from patient-derived induced pluripotent stem cells (iPSCs). Optimization studies with cell-based phenotypic assay led to the identification of a highly potent compound, 19, with dysferlin elevation effects at double-digit nanomolar concentrations. In addition, the molecular target of our chemical series was identified as tubulin, through a tubulin polymerization assay and a competitive binding assay using a photoaffinity labeling probe.
Asunto(s)
Imidazoles/química , Distrofia Muscular de Cinturas/tratamiento farmacológico , Piridinas/química , Moduladores de Tubulina/uso terapéutico , Sitios de Unión , Diferenciación Celular , Proliferación Celular/efectos de los fármacos , Diseño de Fármacos , Disferlina/metabolismo , Células Hep G2 , Humanos , Imidazoles/farmacología , Imidazoles/uso terapéutico , Células Madre Pluripotentes Inducidas/citología , Células Madre Pluripotentes Inducidas/metabolismo , Simulación del Acoplamiento Molecular , Distrofia Muscular de Cinturas/patología , Proteína MioD/metabolismo , Miocitos Cardíacos/citología , Miocitos Cardíacos/metabolismo , Estructura Terciaria de Proteína , Piridinas/farmacología , Piridinas/uso terapéutico , Relación Estructura-Actividad , Tubulina (Proteína)/química , Tubulina (Proteína)/metabolismo , Moduladores de Tubulina/química , Moduladores de Tubulina/farmacologíaRESUMEN
The human DEAD (Asp-Glu-Ala-Asp) box protein DDX41, a member of the DEXDc helicase family, has nucleic acid-dependent ATPase and RNA and DNA translocase and unwinding activities. DDX41 is affected by somatic mutations in sporadic cases of myeloid neoplasms as well as in a biallelic fashion in 50% of patients with germline DDX41 mutations. The R525H mutation in DDX41 is thought to play important roles in the development of hereditary myelodysplastic syndrome and acute myelocytic leukemia. In this study, human DDX41 and its R525H mutant (R525H) were expressed in Escherichia coli and purified. The ATPase activities of the recombinant DDX41 and R525H proteins were dependent on both ATP and double-stranded DNA (dsDNA), such as poly(dG-dC) and poly(dA-dT). High-throughput screening was performed with a dsDNA-dependent ATPase assay using the human R525H proteins. After hit confirmation and counterscreening, several small-molecule inhibitors were successfully identified. These compounds show DDX41-selective inhibitory activities.