An assay method for evaluating chemical selectivity of agonists for insulin signaling pathways based on...

An optical method for evaluating the physiologically relevant agonist and antagonist selectivity of an insulin signaling pathway based on an insulin-dependent on/off switching of phosphorylation of a target peptide via insulin receptor is described. Insulin receptor serves as a binding for insulin and a given insulin receptor-binding peptide as a target for an insulin-receptor complex. Upon binding of insulin to its receptor, the insulin receptor undergoes autophosphorylation which enables the receptor to have a kinase activity and phosphorylate various substrates. The phosphorylated tyrosine in the substrate was measured with a monoclonal anti-phosphotyrosine antibody. As the target substrate for insulin receptor, a Y939 peptide consisting of 12 amino acid residues derived from insulin receptor substrate 1 (IRS-1) was used. The present assay method involves different sequential steps: (1) immobilization of a biotin-coupled Y939 peptide on an avidin coated 96-well plate via biotin-avidin complexation; (2) insulin-dependent phosphorylation of the Y939 peptide by the insulin receptor; (3) enzymatic reaction and absorptiometric assay of the phosphorylated Y939 peptide using the anti-phosphotyrosine antibody labeled with horseradish peroxidase. An insulin-dependent absorbance was observed for insulin concentrations from 1.0 x 10(-10) to 1.0 x 10(-7) M, and it leveled off. The observed absorbance was explained to be due to an increase in the phosphorylated Y939 peptide caused by insulin and its receptor complexation. No signal was, however, induced by both vanadyl and vanadate ions at concentrations up to 1.0 x 10(-4) M; these results and previous intact cell level data taken together led to the conclusion that these ions did not induce phosphorylation of the Y939 peptide. Upon addition of tyrphostin 25, a specific inhibitor for insulin receptor kinase activity, phosphorylation of the Y939 peptide in the presence of 1.0 microM insulin was competitively inhibited over 1.0 x 10(-4) M tyrphostin 25. The present system thus exhibited "physiologically more relevant" agonist and antagonist selectivity, the principle of which is based in part on the insulin signal transduction rather than simply relying on the binding assay. The potential use of the present method for evaluating the selectivity of a wide range of agonists and antagonists toward the insulin signaling pathways is discussed.