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-d...
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sg-ntu-dr.10356-1017792020-03-07T12:24:53Z A high-capacity scintillation proximity assay for the discovery and evaluation of ZAP-70 tandem SH2 domain antagonists Sheets, Michael P. Warrior, Usha P. Mollison, Karl W. Djuric, Stevan W. Trevillyan, James M. Yoon, Ho Sup School of Biological Sciences DRNTU::Science::Biological sciences::Molecular biology 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. 2014-02-14T02:55:27Z 2019-12-06T20:44:26Z 2014-02-14T02:55:27Z 2019-12-06T20:44:26Z 1998 1998 Journal Article 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. 1087-0571 https://hdl.handle.net/10356/101779 http://hdl.handle.net/10220/18792 10.1177/108705719800300208 en Journal of biomolecular screening © 1998 The Society for Biomolecular Screening. |
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DRNTU::Science::Biological sciences::Molecular biology Sheets, Michael P. Warrior, Usha P. Mollison, Karl W. Djuric, Stevan W. Trevillyan, James M. Yoon, Ho Sup A high-capacity scintillation proximity assay for the discovery and evaluation of ZAP-70 tandem SH2 domain antagonists |
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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. |
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School of Biological Sciences |
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School of Biological Sciences Sheets, Michael P. Warrior, Usha P. Mollison, Karl W. Djuric, Stevan W. Trevillyan, James M. Yoon, Ho Sup |
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Article |
author |
Sheets, Michael P. Warrior, Usha P. Mollison, Karl W. Djuric, Stevan W. Trevillyan, James M. Yoon, Ho Sup |
author_sort |
Sheets, Michael P. |
title |
A high-capacity scintillation proximity assay for the discovery and evaluation of ZAP-70 tandem SH2 domain antagonists |
title_short |
A high-capacity scintillation proximity assay for the discovery and evaluation of ZAP-70 tandem SH2 domain antagonists |
title_full |
A high-capacity scintillation proximity assay for the discovery and evaluation of ZAP-70 tandem SH2 domain antagonists |
title_fullStr |
A high-capacity scintillation proximity assay for the discovery and evaluation of ZAP-70 tandem SH2 domain antagonists |
title_full_unstemmed |
A high-capacity scintillation proximity assay for the discovery and evaluation of ZAP-70 tandem SH2 domain antagonists |
title_sort |
high-capacity scintillation proximity assay for the discovery and evaluation of zap-70 tandem sh2 domain antagonists |
publishDate |
2014 |
url |
https://hdl.handle.net/10356/101779 http://hdl.handle.net/10220/18792 |
_version_ |
1681041070561951744 |