RESUMO
A series of dinucleoside 5-polyphosphates UpnU (n = 2-7) was synthesized. Their relative potencies as agonists at the G-protein-coupled receptors P2Y1, P2Y2, P2Y4, and P2Y6 were determined by intracellular calcium measurements using fluorescent imaging techniques. The correlation of phosphate chain length to activities at these receptors is discussed.
Assuntos
Fosfatos de Dinucleosídeos/metabolismo , Agonistas do Receptor Purinérgico P2 , Nucleotídeos de Uracila/metabolismo , Cálcio/metabolismo , Fosfatos de Dinucleosídeos/síntese química , Humanos , Ligação Proteica , Receptores Purinérgicos P2/metabolismo , Espectrometria de Fluorescência , Relação Estrutura-Atividade , Transdução Genética , Células Tumorais Cultivadas , Nucleotídeos de Uracila/síntese químicaRESUMO
Modern automated drug-screening can generate hundreds of inhibitor leads from diverse chemical sources in a short period of time. Traditional methods of inhibitor analysis are resource intensive and limit the number of inhibitors that can be analyzed for their mechanism of inhibition. This paper presents methods we have developed for rapid estimation of both potency and mechanism of potential inhibitor leads for a biochemically complex screening target (protein kinase C) using commercially available computer programs for statistical experimental design. Our findings indicate that, with careful choice of factor levels, statistical experimental design clearly identifies the various interactions of the assay components with inhibitors. Suitably plotted, the data can be used to examine the competitive nature of the inhibitor and can provide estimates of IC50 and Michaelis constants useful for planning further kinetic work. The techniques used are amenable to automation and should be useful for identifying inhibitors that may have only marginal potency, but exhibit desirable mechanistic profiles suitable for structural analoging efforts.
