A high-capacity scintillation proximity assay for the discovery and evaluation of ZAP-70 tandem SH2 domain antagonists

A scintillation proximity assay (SPA) is described, which quantitates the ability of compounds to inhibit the binding interaction of a select phosphopeptide with the tandem SH2 domains of the ZAP-70 protein tyrosine kinase, The method is based on the ability of a truncated ZAP-70 tandem SH2 domain-derived peptide to bind an I-125-labeled, diphosphorylated peptide corresponding to the human T-cell receptor zeta-1 immunoglobulin receptor family tyrosine-based activation motif (ITAM), ZAP-70 tandem SH2 domain peptide was biotinylated and bound to streptavidin-coated SPA beads. I-125-labeled zeta-1ITAM ([I-125]-zeta-1ITAM) bound to immobilized ZAP-70 tandem SH2 domain peptide in a saturable, time-and peptide concentration-dependent fashion. Unlabeled diphosphorylated zeta-1ITAM competed binding with an IC50 value equal to approximately 10-15 nM, Binding of zeta-1ITAM to the ZAP-70 tandem SH2 domain was dependent on the cooperative interaction of the dual phosphotyrosine residues. Unlabeled monotyrosyl-phosphorylated peptides failed to compete with [I-125]-zeta-1ITAM binding to ZAP-70 SH2 domain. Also, labeled monotyrosyl-phosphorylated peptides failed to associate with the ZAP-70 SH2 domain in direct binding studies, Association and dissociation binding kinetics were determined to be extremely rapid at room temperature, reaching equilibrium within 5 min. The K-d for [I-125]-zeta-1ITAM binding to ZAP-70 tandem SH2 domain peptide was determined by Scatchard analysis to be 1.5-2 nM, The SPA assay was adapted for automated, high-capacity screening, which allowed evaluation of 23,040 small molecular weight compounds per day, The assay is useful for both drug discovery and as a research tool for the study of binding interactions between signal-transducing molecules critical for T-cell activation.