RESUMO
The synthesis and biochemical characterization of AX4697, a fluorescent, bisindolylmaleimide-derived probe for PKCalpha and beta, is described. AX4697 was able to quantify changes in PKC expression in drug-treated Jurkat cells and was shown to covalently label PKCalpha on C619, a residue that sits just outside the active site.
Assuntos
Corantes Fluorescentes/síntese química , Corantes Fluorescentes/farmacologia , Indóis/síntese química , Indóis/farmacologia , Maleimidas/síntese química , Maleimidas/farmacologia , Proteína Quinase C-alfa/metabolismo , Proteína Quinase C/metabolismo , Domínio Catalítico , Desenho de Fármacos , Corantes Fluorescentes/química , Humanos , Indóis/química , Células Jurkat , Maleimidas/química , Estrutura Molecular , Proteína Quinase C beta , Relação Estrutura-AtividadeRESUMO
The design and synthesis of AX7574, a microcystin-derived probe for serine/threonine phosphatases, is described. A key step in the synthesis was the conjugation under basic conditions of a tetramethylrhodamine 1,3-diketone derivative to the arginine side chain present in microcystin-LR. The resulting conjugate specifically labeled the active site of protein phosphatases 1 (PP-1) with a 1:1 stoichiometry and IC50 of 4.0 nM. AX7574 was used to isolate and identify PP-1, PP-2A, PP-4, and PP-6 in Jurkat cells. Finally, AX7574 was able to record changes in the phosphatase activity levels of calyculin A treated Jurkat cells versus untreated control cells.
