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Sci. STKE, 27 June 2006
Vol. 2006, Issue 341, p. pl3
[DOI: 10.1126/stke.3412006pl3]

PROTOCOLS

A High-Throughput Screening Method to Identify Small Molecule Inhibitors of Thyroid Hormone Receptor Coactivator Binding

Leggy A. Arnold1*, Eva Estébanez-Perpiñá2, Marie Togashi3, Anang Shelat1, Cory A. Ocasio4, Andrea C. McReynolds5, Phuong Nguyen3, John D. Baxter3, Robert J. Fletterick2, Paul Webb3, and R. Kiplin Guy1

1 St. Jude Children’s Research Hospital, Department of Chemical Biology and Therapeutics, 333 North Lauderdale Street, Memphis, TN 38105, USA.
2 University of California San Francisco, Department Biochemistry and Biophysics, 600 Sixteenth Street, San Francisco, CA 94143, USA.
3 University of California San Francisco, Diabetes Center and Department of Medicine, HSW 1210, 513 Parnassus Avenue, San Francisco, CA 94122, USA.
4 University of California, San Francisco, Department of Pharmaceutical Chemistry, 600 Sixteenth Street, San Francisco, CA 94143, USA.
5 University of California, San Francisco, Department of Pharmaceutical Chemistry, 1700 Fourth Street, Room 509, San Francisco, CA 94143, USA.

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.

*Address correspondence to: Alexander Arnold, St. Jude Children’s Research Hospital, Department of Chemical Biology and Therapeutics, 332 N. Lauderdale St., Memphis, TN 38105-2794, USA. Telephone, (901) 495 5803; fax, (901) 495-5715; e-mail, alexander.arnold{at}stjude.org

Citation: L. A. Arnold, E. Estébanez-Perpiñá, M. Togashi, A. Shelat, C. A. Ocasio, A. C. McReynolds, P. Nguyen, J. D. Baxter, R. J. Fletterick, P. Webb, R. K. Guy, A High-Throughput Screening Method to Identify Small Molecule Inhibitors of Thyroid Hormone Receptor Coactivator Binding. Sci. STKE 2006, pl3 (2006).

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