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

作者：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

来源：Journal of Biomolecular Screening, 2014, 19(8): 1201-1211.

DOI：10.1177/1087057114536227

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

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%26apos;-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.

• 出版日期2014-9