Ultra-High-Throughput Screening of Natural Product Extracts to Identify Proapoptotic Inhibitors of Bcl-2 Family Proteins

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...

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Bibliographic Details
Published in:	Journal of biomolecular screening 2014-09, Vol.19 (8), p.1201-1211
Main Authors:	Hassig, Christian A., Zeng, Fu-Yue, Kung, Paul, Kiankarimi, Mehrak, Kim, Sylvia, Diaz, Paul W., Zhai, Dayong, Welsh, Kate, Morshedian, Shana, Su, Ying, O’Keefe, Barry, Newman, David J., Rusman, Yudi, Kaur, Harneet, Salomon, Christine E., Brown, Susan G., Baire, Beeraiah, Michel, Andrew R., Hoye, Thomas R., Francis, Subhashree, Georg, Gunda I., Walters, Michael A., Divlianska, Daniela B., Roth, Gregory P., Wright, Amy E., Reed, John C.
Format:	Article
Language:	eng
Subjects:	bcl-X Protein - antagonists & inhibitors Biological Products - chemistry Caco-2 Cells Caspase 3 - metabolism Caspase 7 - metabolism Drug Screening Assays, Antitumor - methods Fluorescence Polarization - methods High-Throughput Screening Assays - instrumentation High-Throughput Screening Assays - methods Humans Miniaturization Molecular Targeted Therapy - methods Mycotoxins - isolation & purification Mycotoxins - pharmacology Proto-Oncogene Proteins c-bcl-2 - antagonists & inhibitors Solid Phase Extraction
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Summary:	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.
ISSN:	1087-0571 2472-5552 1552-454X 2472-5560