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

作者：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^*

来源：Journal of Biomolecular Screening, 2008, 13(7): 665-673.

DOI：10.1177/1087057108320918

• Bcl-B
• TR3
• Nur77
• fluorescene polarization
• high-throughput screen

Nuclear receptor TR3/NR4A1 binds several antiapoptptic 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 polyargenine tail (TR3-r8) recaptulates 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 (FTIC)-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 >= 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 compounds was unable to displace FITC-labeled BH3 peptides from Bcl-2-family proteins. This compound bound Bcl-B with K-d 1.94 +/- 0.38 mu M, as determined by isothermal titration calorimetry. Experiments using Bcl-B overexpressing HeLa cells demonstrate 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.

• 出版日期2008-8
• 单位河北医科大学