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Journal of Biomolecular Screening
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Article

A TR3/Nur77 Peptide-Based High-Throughput Fluorescence Polarization Screen for Small Molecule Bcl-B Inhibitors

Kenneth W. Yip, Paulo H. C. Godoi, Dayong Zhai, Xochella Garcia, Jason F. Cellitti, Michael Cuddy, Motti Gerlic, Ya Chen, Arnold Satterthwait, Stefan Vasile, Eduard Sergienko, and John C. Reed*

Burnham Institute for Medical Research

* To whom correspondence should be addressed. E-mail: reedoffice{at}burnham.org.


  Abstract
Nuclear receptor TR3/Nur77/NR4A1 binds several antiapoptotic Bcl-2-family proteins (Bcl-B, Bcl-2, Bfl-1) in a non-BH3dependent 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-TR3r8) 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 ≥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 FITC-labeled 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-Bdependent cell death. The current FPA represents a screen that can identify noncanonical inhibitors of Bcl-2-family proteins. (Journal of Biomolecular Screening XXXX:xx-xx)

First published on July 14, 2008, doi:10.1177/1087057108320918

Journal of Biomolecular Screening 2008;13:665.

A more recent version of this article appeared on August 1, 2008

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