A TR3/Nur77 Peptide-Based High-Throughput Fluorescence Polarization Screen for Small Molecule Bcl-B InhibitorsBurnham Institute for Medical Research, La Jolla, California
Burnham Institute for Medical Research, La Jolla, California
Burnham Institute for Medical Research, La Jolla, California
San Diego Center for Chemical Genomics, Burnham Institute for Medical Research, La Jolla, California
Burnham Institute for Medical Research, La Jolla, California
Burnham Institute for Medical Research, La Jolla, California
Burnham Institute for Medical Research, La Jolla, California
Burnham Institute for Medical Research, La Jolla, California
Burnham Institute for Medical Research, La Jolla, California
San Diego Center for Chemical Genomics, Burnham Institute for Medical Research, La Jolla, California
San Diego Center for Chemical Genomics, Burnham Institute for Medical Research, La Jolla, California
Burnham Institute for Medical Research, La Jolla, California, jreed{at}burnham.org
Nuclear receptor TR3/Nur77/NR4A1 binds several antiapoptotic Bcl-2-family proteins (Bcl-B, Bcl-2, Bfl-1) in a non-BH3-dependent manner. A 9-amino-acid peptide derived from full-length TR3 with polyarginine tail (TR3-r8) recapitulates TR3's binding specificity, displaying high affinity for Bcl-B. TR3-r8 peptide was used to screen for small molecule Bcl-B inhibitors. A fluorescence polarization assay (FPA) employing fluorescein isothiocyanate (FITC)-labeled TR3-r8 peptide (FITC-TR3-r8) and Bcl-B protein was optimized, with nonfluorescent TR3-r8 serving to demonstrate reversible, competitive binding. Approximately 50,000 compounds were screened at 3.75 mg/L, yielding 145 reproducible hits with
Key Words: Bcl-B • TR3 • Nur77 • fluorescence polarization • high-throughput screen
This version was published on August
1, 2008 Journal of Biomolecular Screening, Vol. 13, No. 7,
665-673 (2008) |
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50% FITC-TR3-r8 displacement (a confirmed hit rate of 0.29%). After dose-response analyses and counterscreening with an unrelated FITC-based FPA, 6 candidate compounds remained. Nuclear magnetic resonance (NMR) showed that 2 of these compounds bound Bcl-B, but not glutathione S-transferase (GST) control protein. One Bcl-B-binding compound was unable to displace FITClabeled BH3 peptides from Bcl-B, confirming a unique binding mechanism compared with traditional antagonists of antiapoptotic Bcl-2-family proteins. This compound bound Bcl-B with Kd 1.94 ± 0.38 µM, as determined by isothermal titration calorimetry. Experiments using Bcl-B overexpressing HeLa cells demonstrated that this compound induced Bcl-B-dependent cell death. The current FPA represents a screen that can identify noncanonical inhibitors of Bcl-2-family proteins. (Journal of Biomolecular Screening 2008:665-673)