title: A high-throughput screening method to identify small molecule inhibitors of thyroid hormone receptor coactivator binding.
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.
authors.affiliation: St. Jude Children's Research Hospital, Department of Chemical Biology and Therapeutics, 333 North Lauderdale Street, Memphis, TN 38105-2794, USA. alexander.arnold@stjude.org
authors.forename: Leggy A
authors.initials: LA
authors.lastname: Arnold
authors.forename: Eva
authors.initials: E
authors.lastname: Estébanez-Perpiñá
authors.forename: Marie
authors.initials: M
authors.lastname: Togashi
authors.forename: Anang
authors.initials: A
authors.lastname: Shelat
authors.forename: Cory A
authors.initials: CA
authors.lastname: Ocasio
authors.forename: Andrea C
authors.initials: AC
authors.lastname: McReynolds
authors.forename: Phuong
authors.initials: P
authors.lastname: Nguyen
authors.forename: John D
authors.initials: JD
authors.lastname: Baxter
authors.forename: Robert J
authors.initials: RJ
authors.lastname: Fletterick
authors.forename: Paul
authors.initials: P
authors.lastname: Webb
authors.forename: R Kiplin
authors.initials: RK
authors.lastname: Guy
mesh_terms: D000123:Acetyltransferases / Q000037:antagonists & inhibitors* / Q000378:metabolism; D001667:Binding, Competitive; D004305:Dose-Response Relationship, Drug; D015195:Drug Design; D004353:Drug Evaluation, Preclinical / Q000379:methods*; D005454:Fluorescence Polarization / Q000379:methods*; D005786:Gene Expression Regulation / Q000187:drug effects; D005982:Glutathione Transferase / Q000032:analysis; D051548:Histone Acetyltransferases; D006801:Humans; D007202:Indicators and Reagents; D008970:Molecular Weight; D056921:Nuclear Receptor Coactivator 3; D009994:Osmolar Concentration; D010777:Photochemistry; D011485:Protein Binding; D011988:Receptors, Thyroid Hormone / Q000187:drug effects* / Q000378:metabolism; D012399:Rotation; D012680:Sensitivity and Specificity; D015534:Trans-Activators / Q000037:antagonists & inhibitors* / Q000378:metabolism
chemical_list: D007202:Indicators and Reagents; D011988:Receptors, Thyroid Hormone; D015534:Trans-Activators; D000123:Acetyltransferases; D051548:Histone Acetyltransferases; C538866:NCOA3 protein, human; D056921:Nuclear Receptor Coactivator 3; D005982:Glutathione Transferase