title: A TR3/Nur77 peptide-based high-throughput fluorescence polarization screen for small molecule Bcl-B inhibitors.
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.
authors.affiliation: Burnham Institute for Medical Research, La Jolla, California 92037, USA.
authors.forename: Kenneth W
authors.initials: KW
authors.lastname: Yip
authors.forename: Paulo H C
authors.initials: PH
authors.lastname: Godoi
authors.forename: Dayong
authors.initials: D
authors.lastname: Zhai
authors.forename: Xochella
authors.initials: X
authors.lastname: Garcia
authors.forename: Jason F
authors.initials: JF
authors.lastname: Cellitti
authors.forename: Michael
authors.initials: M
authors.lastname: Cuddy
authors.forename: Motti
authors.initials: M
authors.lastname: Gerlic
authors.forename: Ya
authors.initials: Y
authors.lastname: Chen
authors.forename: Arnold
authors.initials: A
authors.lastname: Satterthwait
authors.forename: Stefan
authors.initials: S
authors.lastname: Vasile
authors.forename: Eduard
authors.initials: E
authors.lastname: Sergienko
authors.forename: John C
authors.initials: JC
authors.lastname: Reed
mesh_terms: D000595:Amino Acid Sequence; D002151:Calorimetry; D004268:DNA-Binding Proteins / Q000737:chemistry*; D004353:Drug Evaluation, Preclinical / Q000295:instrumentation / Q000379:methods*; D016650:Fluorescein-5-isothiocyanate / Q000494:pharmacology; D005454:Fluorescence Polarization / Q000379:methods*; D005982:Glutathione Transferase / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy; D008969:Molecular Sequence Data; D057105:Nuclear Receptor Subfamily 4, Group A, Member 1; D010455:Peptides / Q000737:chemistry*; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000037:antagonists & inhibitors*; D011987:Receptors, Steroid / Q000737:chemistry*
chemical_list: D004268:DNA-Binding Proteins; C539821:NR4A1 protein, human; D057105:Nuclear Receptor Subfamily 4, Group A, Member 1; D010455:Peptides; D019253:Proto-Oncogene Proteins c-bcl-2; D011987:Receptors, Steroid; D005982:Glutathione Transferase; D016650:Fluorescein-5-isothiocyanate