title: High-throughput fluorescence polarization assay for chemical library screening against anti-apoptotic Bcl-2 family member Bfl-1.
abstract: Overexpression of the anti-apoptotic Bcl-2 family proteins occurs commonly in human cancers. Bfl-1 is highly expressed in some types of malignant cells, contributing significantly to tumor cell survival and chemoresistance. Therefore, it would be desirable to have chemical antagonists of Bfl-1. To this end, we devised a fluorescence polarization assay (FPA) using Bfl-1 protein and fluorescein-conjugated Bid BH3 peptide, which was employed for high-throughput screening of chemical libraries. Approximately 66 000 compounds were screened for the ability to inhibit BH3 peptide binding to Bfl-1, yielding 14 reproducible hits with ≥50% displacement. After dose-response analysis and confirmation using a secondary assay based on time-resolved fluorescence resonance energy transfer (TR-FRET), two groups of Bfl-1-specific inhibitors were identified, including chloromaleimide and sulfonylpyrimidine series compounds. FPAs generated for each of the six anti-apoptotic Bcl-2 proteins demonstrated selective binding of both classes of compounds to Bfl-1. Analogs of the sulfonylpyrimidine series were synthesized and compared with the original hit for Bfl-1 binding by both FPAs and TR-FRET assays. The resulting structure-activity relation analysis led to the chemical probe compound CID-2980973 (ML042). Collectively, these findings demonstrate the feasibility of using the HTS assay for discovery of selective chemical inhibitors of Bfl-1.
authors.affiliation: Sanford-Burnham Medical Research Institute, Program on Apoptosis and Cell Death Research, La Jolla, CA 92037, USA.
authors.forename: Dayong
authors.initials: D
authors.lastname: Zhai
authors.forename: Paulo
authors.initials: P
authors.lastname: Godoi
authors.forename: Eduard
authors.initials: E
authors.lastname: Sergienko
authors.forename: Russell
authors.initials: R
authors.lastname: Dahl
authors.forename: Xochella
authors.initials: X
authors.lastname: Chan
authors.forename: Brock
authors.initials: B
authors.lastname: Brown
authors.forename: Justin
authors.initials: J
authors.lastname: Rascon
authors.forename: Andrew
authors.initials: A
authors.lastname: Hurder
authors.forename: Ying
authors.initials: Y
authors.lastname: Su
authors.forename: Thomas D Y
authors.initials: TD
authors.lastname: Chung
authors.forename: Chaofang
authors.initials: C
authors.lastname: Jin
authors.forename: Paul
authors.initials: P
authors.lastname: Diaz
authors.forename: John C
authors.initials: JC
authors.lastname: Reed
mesh_terms: D004353:Drug Evaluation, Preclinical / Q000379:methods*; D005453:Fluorescence; D005454:Fluorescence Polarization / Q000379:methods; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods; D006367:HeLa Cells; D057166:High-Throughput Screening Assays / Q000379:methods*; D006801:Humans; D008301:Maleimides / Q000378:metabolism / Q000494:pharmacology; D015778:Minor Histocompatibility Antigens; D009369:Neoplasms / Q000378:metabolism; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000037:antagonists & inhibitors* / Q000187:drug effects / Q000378:metabolism*; D011743:Pyrimidines / Q000378:metabolism / Q000494:pharmacology; D054852:Small Molecule Libraries / Q000032:analysis*
chemical_list: C096816:BCL2-related protein A1; D008301:Maleimides; D015778:Minor Histocompatibility Antigens; D019253:Proto-Oncogene Proteins c-bcl-2; D011743:Pyrimidines; D054852:Small Molecule Libraries; C043592:maleimide; C030986:pyrimidine