Please use this identifier to cite or link to this item: https://hdl.handle.net/10356/101779
Title: A high-capacity scintillation proximity assay for the discovery and evaluation of ZAP-70 tandem SH2 domain antagonists
Authors: Sheets, Michael P.
Warrior, Usha P.
Mollison, Karl W.
Djuric, Stevan W.
Trevillyan, James M.
Yoon, Ho Sup
Keywords: DRNTU::Science::Biological sciences::Molecular biology
Issue Date: 1998
Source: Sheets, M. P., Warrior, U. P., Yoon, H., Mollison, K. W.,Djuric, S. W., & Trevillyan, J. M. (1998). A High-Capacity Scintillation Proximity Assay for the Discovery and Evaluation of ZAP-70 Tandem SH2 Domain Antagonists. Journal of Biomolecular Screening, 3(2), 139-144.
Series/Report no.: Journal of biomolecular screening
Abstract: 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 ζ-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 ζ-1 ITAM ([125I]-ζ-1 ITAM) bound to immobilized ZAP-70 tandem SH2 domain peptide in a saturable, time- and peptide concentration-dependent fashion. Unlabeled diphosphorylated ζ-1 ITAM competed binding with an ICso value equal to approximately 10-15 nM. Binding of ζ-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]-ζ-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 Kd for [125I]-ζ-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.
URI: https://hdl.handle.net/10356/101779
http://hdl.handle.net/10220/18792
ISSN: 1087-0571
DOI: 10.1177/108705719800300208
Rights: © 1998 The Society for Biomolecular Screening.
Fulltext Permission: none
Fulltext Availability: No Fulltext
Appears in Collections:SBS Journal Articles

SCOPUSTM   
Citations 20

15
Updated on Jan 28, 2023

Web of ScienceTM
Citations 20

14
Updated on Jan 30, 2023

Page view(s) 10

692
Updated on Jan 30, 2023

Google ScholarTM

Check

Altmetric


Plumx

Items in DR-NTU are protected by copyright, with all rights reserved, unless otherwise indicated.