NIH Molecular Libraries Probe Production Network [MLPCN]
NIH Chemical Genomics Center [NCGC]

MLPCN Grant: DK058080
Assay Provider: R. Kip Guy, St. Jude Children's Research Hospital

NCGC Assay Overview:

Thyroid receptor (TR) regulates many homeostatic processes including basal metabolism, cardiovascular function, body weight, and lipid trafficking. TR modulators are potential therapeutics for obesity and hyperlipidemias but current thyroid analogs have undesirable side effects, particularly cardiac stimulation. To identify inhibitors that specifically prevent the interaction of TR with the steroid receptor coregulator 2 (SRC2), a fluorescence polarization assay was screened. This assay detects interaction of the ligand-binding domain of human TRb with a Texas Red labeled SRC2 peptide, corresponding to a 20 amino acid region of the nuclear receptor interaction domain (Arnold et al., 2006). Small molecule inhibitors that block the interaction of TR and SRC2 are detected by a decrease in fluorescence polarization. Total fluorescence (555 nm excitation and 632 nm emission) was also measured to identify potential fluorescent and light absorbing compounds. Such compounds may be artifacts that interfere with the fluorescence polarization assay.

The assay was screened against the MLSMR in a quantitative high-throughput screen (qHTS) of six library concentrations where FP (AID 1469) and FP total fluorescence (AID 1479) readouts were collected. The total fluorescence measurement for the FP assay served as a counterscreen to identify fluorescent compounds. The titration-response data were curve-fit and classified to identify actives and nascent SAR analysis was performed. Compounds with quality curve-fit in the FP measurement and little or no activity in the total fluorescence readout were selected for follow up. The follow up compounds were tested in the original screening assay (AID 2490) as well as an orthogonal assay where FP was measured by a fluorescein fluoroprobe (AID 2487).

A nitrosulfonyl benzoate series that showed good performance in the follow-up assays and suitable chemical tractability was chosen for probe optimization. Analogs from this series were tested in several secondary assays to further confirm their activity and determine the selectivity of TR-beta inhibition, including an Alpha Screen (AID 2444), a cell based gene reporter assay for transcription inhibition (AID 2479), a cell viability assay that measures cytotoxicity (AID 2447), and other nuclear receptor assays: PPAR-gamma (AID 2449), VDR (AID 2455) and AR (AID 2448) for selectivity. These assays indicated the nitrosulfonyl benzoate series were selective inhibitors of TR-beta and SRC-2 interaction in vitro. The most active compound (ML151) from this series was nominated as the probe for TR inhibition.

Arnold, L. A.; Estebanez-Perpina, E.; Togashi, M.; Shelat, A.; Ocasio, C. A.; McReynolds, A. C.; Nguyen, P.; Baxter, J. D.; Fletterick, R. J.; Webb, P.; Guy, R. K. A high-throughput screening method to identify small molecule inhibitors of thyroid hormone receptor coactivator binding. Sci STKE 2006, p 13.

Please refer to other AIDs (1469, 1479, 2490, 2487, 2479, 2444, 2447, 2449, 2455, 2448) for detailed assay protocols.

This summary is written for the purposes of summarizing the probe activities from the project. MLSCN probes are given a score of 100. Molecules in the prior art are given a score of 80. Other, less active molecules in the same chemical series as the probe molecules are given a score of 50. Molecules pending validation are given a score of 10. Inactive analogues from these series are given a score of 0.

Grant Number: DK058080