A high-throughput screening method to identify small molecule inhibitors of thyroid hormone receptor coactivator binding.

Leggy A. Arnold, Eva Estébanez-Perpiñá, Marie Togashi, Anang Shelat, Cory A. Ocasio, Andrea C. McReynolds, Phuong Nguyen, John D. Baxter, Robert J. Fletterick, Paul Webb, R. Kiplin Guy

Research output: Contribution to journalArticle

22 Scopus citations

Abstract

To provide alternative methods for regulation of gene transcription initiated by the binding of thyroid hormone (T3) to the thyroid receptor (TR), we have developed a high-throughput method for discovering inhibitors of the interaction of TR with its transcriptional coactivators. The screening method is based on fluorescence polarization (FP), one of the most sensitive and robust high-throughput methods for the study of protein-protein interactions. A fluorescently labeled coactivator is excited by polarized light. The emitted polarized light is a function of the molecular properties of the labeled coactivator, especially Brownian molecular rotation, which is very sensitive to changes in the molecular mass of the labeled complex. Dissociation of hormone receptor from fluorescently labeled coactivator peptide in the presence of small molecules can be detected by this competition method, and the assay can be performed in a high-throughput screening format. Hit compounds identified by this method are evaluated by several secondary assay methods, including a dose-response analysis, a semiquantitative glutathione-S-transferase assay, and a hormone displacement assay. Subsequent in vitro transcription assays can detect inhibition of thyroid signaling at low micromolar concentrations of small molecules in the presence of T3.

Original languageEnglish (US)
Pages (from-to)pl3
JournalScience's STKE : signal transduction knowledge environment
Volume2006
Issue number341
DOIs
StatePublished - Jun 27 2006

ASJC Scopus subject areas

  • Medicine(all)

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