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J Biomol Screen. 2014 Sep;19(8):1201-11. doi: 10.1177/1087057114536227. Epub 2014 May 27.

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

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Sanford Burnham Medical Research Institute, La Jolla, CA, USA
Sanford Burnham Medical Research Institute, La Jolla, CA, USA.
National Cancer Institute, Frederick, MD, USA.
Center for Drug Design, University of Minnesota, Minneapolis, MN, USA.
Department of Chemistry, University of Minnesota, Minneapolis, MN, USA.
Institute for Therapeutics Discovery and Development, Department of Medicinal Chemistry, University of Minnesota, Minneapolis, MN, USA.
Sanford-Burnham Medical Research Institute at Lake Nona, Orlando, FL, USA.
Harbor Branch Oceanographic Institute at Florida Atlantic University, Fort Pierce, FL, USA.
Sanford Burnham Medical Research Institute, La Jolla, CA, USA Roche Pharmaceuticals, Basel, Switzerland.


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.


Bcl-2 family; apoptosis; bioassay-guided fractionation; fluorescence polarization; natural product extracts; ultra-high-throughput screening

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