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.

Current Experimental Methods for Characterizing Protein–Protein Interactions

“…Such an FP assay also provides an opportunity for the detection of inhibitors targeting the PPI . For instance, an FP competition assay has been developed to identify inhibitors targeting the MDM2–p53 peptide interaction, to screen small molecules that inhibit the ZipA–FtsZ peptide interaction, to screen natural product extracts that disrupt the binding of antiapoptotic Bcl‐2 family members to Bim BH3 peptides . The advantages of an FP assay are its low cost, simple mix‐and‐read format without wash steps, and the high‐throughput screening (HTS) capacity of the assay when carried out in a multiwell plate (96/384/1,536).…”

Target protein-oriented isolation of Hes1 dimer inhibitors using protein based methods

“…It is essential to inhibit undesired PPIs in drug development, and bioactivity-guided isolation using the inhibitory activity of PPIs is an attractive method to obtain effective inhibitors. However, currently there are only a few examples of such approaches using PPI assay systems 22,23 . We previously developed a Hes1-Hes1 interaction fluorescent plate assay and reported the screening results of our natural products compound library ( Fig.…”

Natural Products as Pharmaceuticals and Sources for Lead Structures**Note: This chapter reflects the opinions of the authors, not necessarily those of the US Government

“…In a recent paper, a multidisciplinary group from academia and the NCI demonstrated that such a screening program was successful in identifying natural product extracts and subsequently purified natural products from the initial hits that interfered with some of the apoptotic Bcl-2 proteins [142]. In a recent paper, a multidisciplinary group from academia and the NCI demonstrated that such a screening program was successful in identifying natural product extracts and subsequently purified natural products from the initial hits that interfered with some of the apoptotic Bcl-2 proteins [142].…”

“…Thus, by use of sophisticated robotic systems using in vitro and in cellulo assays, Hassig et al were able to identify novel agents from an initial screen of close to 150,000 crude extracts from plants, microbes, and marine invertebrates [142]. In addition, secondary assays in a dose response format, including counter screens, both using in cellulo systems in 384 well plates and volumes of about 30 µl, were also necessary in order to confirm that results observed in the in vitro conditions could occur within the cell (transfer across membranes, etc.).…”