title: Ultra-High-Throughput Screening of Natural Product Extracts to Identify Proapoptotic Inhibitors of Bcl-2 Family Proteins.
abstract: Antiapoptotic Bcl-2 family proteins are validated cancer targets composed of six related proteins. From a drug discovery perspective, these are challenging targets that exert their cellular functions through protein-protein interactions (PPIs). Although several isoform-selective inhibitors have been developed using structure-based design or high-throughput screening (HTS) of synthetic chemical libraries, no large-scale screen of natural product collections has been reported. A competitive displacement fluorescence polarization (FP) screen of nearly 150,000 natural product extracts was conducted against all six antiapoptotic Bcl-2 family proteins using fluorochrome-conjugated peptide ligands that mimic functionally relevant PPIs. The screens were conducted in 1536-well format and displayed satisfactory overall HTS statistics, with Z'-factor values ranging from 0.72 to 0.83 and a hit confirmation rate between 16% and 64%. Confirmed active extracts were orthogonally tested in a luminescent assay for caspase-3/7 activation in tumor cells. Active extracts were resupplied, and effort toward the isolation of pure active components was initiated through iterative bioassay-guided fractionation. Several previously described altertoxins were isolated from a microbial source, and the pure compounds demonstrate activity in both Bcl-2 FP and caspase cellular assays. The studies demonstrate the feasibility of ultra-high-throughput screening using natural product sources and highlight some of the challenges associated with this approach.
authors.affiliation: Sanford Burnham Medical Research Institute, La Jolla, CA, USA chassig@sanfordburnham.org.
authors.forename: Christian A
authors.initials: CA
authors.lastname: Hassig
authors.affiliation: Sanford Burnham Medical Research Institute, La Jolla, CA, USA.
authors.forename: Fu-Yue
authors.initials: FY
authors.lastname: Zeng
authors.affiliation: Sanford Burnham Medical Research Institute, La Jolla, CA, USA.
authors.forename: Paul
authors.initials: P
authors.lastname: Kung
authors.affiliation: Sanford Burnham Medical Research Institute, La Jolla, CA, USA.
authors.forename: Mehrak
authors.initials: M
authors.lastname: Kiankarimi
authors.affiliation: Sanford Burnham Medical Research Institute, La Jolla, CA, USA.
authors.forename: Sylvia
authors.initials: S
authors.lastname: Kim
authors.affiliation: Sanford Burnham Medical Research Institute, La Jolla, CA, USA.
authors.forename: Paul W
authors.initials: PW
authors.lastname: Diaz
authors.affiliation: Sanford Burnham Medical Research Institute, La Jolla, CA, USA.
authors.forename: Dayong
authors.initials: D
authors.lastname: Zhai
authors.affiliation: Sanford Burnham Medical Research Institute, La Jolla, CA, USA.
authors.forename: Kate
authors.initials: K
authors.lastname: Welsh
authors.affiliation: Sanford Burnham Medical Research Institute, La Jolla, CA, USA.
authors.forename: Shana
authors.initials: S
authors.lastname: Morshedian
authors.affiliation: Sanford Burnham Medical Research Institute, La Jolla, CA, USA.
authors.forename: Ying
authors.initials: Y
authors.lastname: Su
authors.affiliation: National Cancer Institute, Frederick, MD, USA.
authors.forename: Barry
authors.initials: B
authors.lastname: O'Keefe
authors.affiliation: National Cancer Institute, Frederick, MD, USA.
authors.forename: David J
authors.initials: DJ
authors.lastname: Newman
authors.affiliation: Center for Drug Design, University of Minnesota, Minneapolis, MN, USA.
authors.forename: Yudi
authors.initials: Y
authors.lastname: Rusman
authors.affiliation: Center for Drug Design, University of Minnesota, Minneapolis, MN, USA.
authors.forename: Harneet
authors.initials: H
authors.lastname: Kaur
authors.affiliation: Center for Drug Design, University of Minnesota, Minneapolis, MN, USA.
authors.forename: Christine E
authors.initials: CE
authors.lastname: Salomon
authors.affiliation: Department of Chemistry, University of Minnesota, Minneapolis, MN, USA.
authors.forename: Susan G
authors.initials: SG
authors.lastname: Brown
authors.affiliation: Department of Chemistry, University of Minnesota, Minneapolis, MN, USA.
authors.forename: Beeraiah
authors.initials: B
authors.lastname: Baire
authors.affiliation: Department of Chemistry, University of Minnesota, Minneapolis, MN, USA.
authors.forename: Andrew R
authors.initials: AR
authors.lastname: Michel
authors.affiliation: Department of Chemistry, University of Minnesota, Minneapolis, MN, USA.
authors.forename: Thomas R
authors.initials: TR
authors.lastname: Hoye
authors.affiliation: Institute for Therapeutics Discovery and Development, Department of Medicinal Chemistry, University of Minnesota, Minneapolis, MN, USA.
authors.forename: Subhashree
authors.initials: S
authors.lastname: Francis
authors.affiliation: Institute for Therapeutics Discovery and Development, Department of Medicinal Chemistry, University of Minnesota, Minneapolis, MN, USA.
authors.forename: Gunda I
authors.initials: GI
authors.lastname: Georg
authors.affiliation: Institute for Therapeutics Discovery and Development, Department of Medicinal Chemistry, University of Minnesota, Minneapolis, MN, USA.
authors.forename: Michael A
authors.initials: MA
authors.lastname: Walters
authors.affiliation: Sanford-Burnham Medical Research Institute at Lake Nona, Orlando, FL, USA.
authors.forename: Daniela B
authors.initials: DB
authors.lastname: Divlianska
authors.affiliation: Sanford-Burnham Medical Research Institute at Lake Nona, Orlando, FL, USA.
authors.forename: Gregory P
authors.initials: GP
authors.lastname: Roth
authors.affiliation: Harbor Branch Oceanographic Institute at Florida Atlantic University, Fort Pierce, FL, USA.
authors.forename: Amy E
authors.initials: AE
authors.lastname: Wright
authors.affiliation: Sanford Burnham Medical Research Institute, La Jolla, CA, USA Roche Pharmaceuticals, Basel, Switzerland.
authors.forename: John C
authors.initials: JC
authors.lastname: Reed
mesh_terms: D001688:Biological Products / Q000737:chemistry*; D018938:Caco-2 Cells; D053148:Caspase 3 / Q000378:metabolism; D053179:Caspase 7 / Q000378:metabolism; D004354:Drug Screening Assays, Antitumor / Q000379:methods; D005454:Fluorescence Polarization / Q000379:methods; D057166:High-Throughput Screening Assays / Q000295:instrumentation / Q000379:methods*; D006801:Humans; D008904:Miniaturization; D058990:Molecular Targeted Therapy / Q000379:methods; D009183:Mycotoxins / Q000302:isolation & purification / Q000494:pharmacology; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000037:antagonists & inhibitors*; D052616:Solid Phase Extraction; D051020:bcl-X Protein / Q000037:antagonists & inhibitors
chemical_list: D001688:Biological Products; D009183:Mycotoxins; D019253:Proto-Oncogene Proteins c-bcl-2; D051020:bcl-X Protein; D053148:Caspase 3; D053179:Caspase 7
keywords: Bcl-2 family; apoptosis; bioassay-guided fractionation; fluorescence polarization; natural product extracts; ultra-high-throughput screening