Affordable Access

deepdyve-link
Publisher Website

A TR3/Nur77 peptide-based high-throughput fluorescence polarization screen for small molecule Bcl-B inhibitors.

Authors
  • Yip, Kenneth W
  • Godoi, Paulo H C
  • Zhai, Dayong
  • Garcia, Xochella
  • Cellitti, Jason F
  • Cuddy, Michael
  • Gerlic, Motti
  • Chen, Ya
  • Satterthwait, Arnold
  • Vasile, Stefan
  • Sergienko, Eduard
  • Reed, John C
Type
Published Article
Journal
Journal of Biomolecular Screening
Publisher
SAGE Publications
Publication Date
Aug 01, 2008
Volume
13
Issue
7
Pages
665–673
Identifiers
DOI: 10.1177/1087057108320918
PMID: 18626112
Source
Medline
License
Unknown

Abstract

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 > or =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 microM, 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.

Report this publication

Statistics

Seen <100 times