A high-throughput screening approach to discover potential colorectal cancer chemotherapeutics: repurposing drugs to identify novel disruptors of 14-3-3 proteins

Selectively inducing apoptosis of cancer cells is an effective therapeutic strategy, but the success of existing chemotherapeutics is compromised by emergent tumor cell resistance and systemic off-target effects. Therefore, the discovery of new pro-apoptotic compounds with minimal systemic side effects remains an urgent need. 14-3-3 proteins are molecular scaffolds that serve as important regulators of cell survival. We previously demonstrated that 14-3-3ζ can sequester BAD, a pro-apoptotic member of the BCL-2 protein family, in the cytoplasm to inhibit the induction of apoptosis. Despite 14-3-3ζ being a critical regulator of cell survival, the identification of molecules that potently disrupt 14-3-3ζ actions has yet to materialize as a chemotherapeutic approach. Herein, we established a BRET-based, high-throughput drug screening approach (Z'-score = 0.52) to identify molecules that disrupt the binding of 14-3-3ζ to a BAD-derived fragment containing serine residues critical for their interactions. A drug library containing 1971 compounds was used for screening, and the capacity of identified hits to induce cell death was examined in NIH-3T3 fibroblasts and colorectal cancer cell lines, HT-29 and Caco-2. These results were mechanistically supported by both in silico structural analysis that suggest the possible mode of binding and direct biophysical measurements that demonstrate concentration-dependent target engagement. Terfenadine, penfluridol, and lomitapide have potential to either be repurposed as chemotherapeutics, or more likely, used as starting points for novel lead development. The described assay cascade demonstrates the feasibility of both expanding on these compounds and identifying novel disruptors of 14-3-3ζ to develop pro-apoptotic agents to treat pathogenic aberrant cell growth.