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 ≥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 K_d 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)

Keywords

Bcl-B TR3 Nur77 fluorescence polarization high-throughput screen

References

Green DR , Evan GI : A matter of life and death. Cancer Cell 2002 ;1:19-30.

Danial NN , Korsmeyer SJ : Cell death: critical control points. Cell 2004; 116:205-219.

Levine B. : Eating oneself and uninvited guests: autophagy-related pathways in cellular defense. Cell 2005;120:159-162.

Reed JC : Drug insight: cancer therapy strategies based on restoration of endogenous cell death mechanisms. Nat Clin Pract Oncol 2006;3:388-398.

O'Reilly LA , Strasser A. : Apoptosis and autoimmune disease. Inflamm Res 1999;48:5-21.

Cory S. , Huang DC , Adams JM : The Bcl-2 family: roles in cell survival and oncogenesis. Oncogene 2003;22:8590-8607.

Reed JC : Proapoptotic multidomain Bcl-2/Bax-family proteins: mechanisms, physiological roles, and therapeutic opportunities. Cell Death Differ 2006;13:1378-1386.

Reed JC , Pellecchia M. : Apoptosis-based therapies for hematologic malignancies. Blood 2005;106:408-418.

Fesik SW : Insights into programmed cell death through structural biology. Cell 2000;103:273-282.

10.

Leone M. , Zhai D. , Sareth S. , Kitada S. , Reed JC , Pellecchia M. : Cancer prevention by tea polyphenols is linked to their direct inhibition of antiapoptotic Bcl-2-family proteins. Cancer Res 2003;63:8118-8121.

11.

Oltersdorf T. , Elmore SW , Shoemaker AR , Armstrong RC , Augeri DJ , Belli BA , Bruncko M. , et al: An inhibitor of Bcl-2 family proteins induces regression of solid tumours. Nature 2005;435:677-681.

12.

Wang JL , Liu D. , Zhang ZJ , Shan S. , Han X. , Srinivasula SM , et al: Structure-based discovery of an organic compound that binds Bcl-2 protein and induces apoptosis of tumor cells. Proc Natl Acad Sci U S A 2000;97:7124-7129.

13.

Kitada S. , Leone M. , Sareth S. , Zhai D. , Reed JC , Pellecchia M. : Discovery, characterization, and structure-activity relationships studies of proapoptotic polyphenols targeting B-cell lymphocyte/leukemia-2 proteins . J Med Chem 2003;46:4259-4264.

14.

Moll UM , Marchenko N. , Zhang XK : p53 and Nur77/TR3-transcription factors that directly target mitochondria for cell death induction. Oncogene 2006;25:4725-4743.

15.

Li H. , Kolluri SK , Gu J. , Dawson MI , Cao X. , Hobbs PD , et al: Cytochrome c release and apoptosis induced by mitochondrial targeting of nuclear orphan receptor TR3. Science 2000;289:1159-1164.

16.

Lin B. , Kolluri SK , Lin F. , Liu W. , Han YH , Cao X. , et al: Conversion of Bcl-2 from protector to killer by interaction with nuclear orphan receptor Nur77/TR3. Cell 2004;116:527-540.

17.

Luciano F. , Krajewska M. , Ortiz-Rubio P. , Krajewski S. , Zhai D. , Faustin B. , et al: Nur77 converts phenotype of Bcl-B, an antiapoptotic protein expressed in plasma cells and myeloma. Blood 2007;109:3849-3855.

18.

Krajewska M. , Kitada S. , Winter JN , Variakojis D. , Lichtenstein A. , Zhai D. , et al: Bcl-B expression in human epithelial and non-epithelial malignancies. Clin Cancer Res 2008;14:3011-3021.

19.

Zhai D. , Jin C. , Satterthwait AC , Reed JC : Comparison of chemical inhibitors of antiapoptotic Bcl-2-family proteins . Cell Death Differ 2006;13:1419-1421.

20.

Vogel M. , Bukau B. , Mayer MP : Allosteric regulation of Hsp70 chaperones by a proline switch. Mol Cell 2006;21:359-367.

21.

Coval ML : Analysis of Hill interaction coefficients and the invalidity of the Kwon and Brown equation. J Biol Chem 1970;245:6335-6336.

22.

Pope AJ , Haupts UM , Moore KJ : Homogeneous fluorescence readouts for miniaturized high-throughput screening: theory and practice. Drug Discov Today 1999 ;4:350-362.

23.

Riddle SM , Vedvik KL , Hanson GT , Vogel KW : Time-resolved fluorescence resonance energy transfer kinase assays using physiological protein substrates: applications of terbium-fluorescein and terbium-green fluorescent protein fluorescence resonance energy transfer pairs. Anal Biochem 2006;356:108-116.

24.

Rega MF , Reed JC , Pellecchia M. : Robust lanthanide-based assays for the detection of anti-apoptotic Bcl-2-family protein antagonists. Bioorg Chem 2007;35:113-120.

25.

Kadkhodayan S. , Elliott LO , Mausisa G. , Wallweber HA , Deshayes K. , Feng B. , et al: Evaluation of assay technologies for the identification of protein-Peptide interaction antagonists. Assay Drug Dev Technol 2007;5:501-513.

26.

Banks P. , Gosselin M. , Prystay L. : Impact of a red-shifted dye label for high throughput fluorescence polarization assays of G protein-coupled receptors. J Biomol Screen 2000;5:329-334.