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A High-Capacity Scintillation Proximity Assay for the Discovery and Evaluation of ZAP-70 Tandem SH2 Domain Antagonists

Abbott Laboratories, 100 Abbott Park Road, Abbott Park, IL 60064

Usha P. Warrior

Abbott Laboratories, 100 Abbott Park Road, Abbott Park, IL 60064

Hosup Yoon

Abbott Laboratories, 100 Abbott Park Road, Abbott Park, IL 60064

Karl W. Mollison

Abbott Laboratories, 100 Abbott Park Road, Abbott Park, IL 60064

Stevan W. Djuric

Abbott Laboratories, 100 Abbott Park Road, Abbott Park, IL 60064

James M. Trevillyan

Abbott Laboratories, 100 Abbott Park Road, Abbott Park, IL 60064

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 ¹²⁵I-labeled, diphosphorylated peptide corresponding to the human T-cell receptor -1 immunoglobulin receptor family tyrosine-based activation motif (ITAM). ZAP-70 tandem SH2 domain peptide was biotinylated and bound to streptavidin-coated SPA beads. ¹²⁵I-labeled -1 ITAM ([¹²⁵I]--1 ITAM) bound to immobilized ZAP-70 tandem SH2 domain peptide in a saturable, time- and peptide concentration-dependent fashion. Unlabeled diphosphorylated -1 ITAM competed binding with an ICso value equal to approximately 10-15 nM. Binding of -1 ITAM 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 [¹²⁵I]--1 ITAM 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 [¹²⁵I]--1 ITAM 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.

Journal of Biomolecular Screening, Vol. 3, No. 2, 139-144 (1998)
DOI: 10.1177/108705719800300208


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