A high-throughput screening approach to discover potential colorectal cancer chemotherapeutics: Repurposing drugs to disrupt 14-3-3 protein-BAD interactions

Selectively inducing apoptosis in cancer cells is an effective therapeutic strategy, but the reality of success of existing chemotherapeutics is compromised by emergent tumor cell resistance and systemic off-target effects. Therefore, the discovery of new classes of 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. Our previous study 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 being a critical mechanism of cell survival, it is unclear whether disrupting 14-3-3 protein:BAD interactions could be harnessed as a chemotherapeutic approach. Herein, we established a BRET-based, high-throughput drug screening approach (Z’-score = 0.52) capable of identifying molecules that can disrupt 14-3-3ζ:BAD interactions. An FDA-approved 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. Our in vitro results suggest that terfenadine, penfluridol, and lomitapide could be potentially repurposed for treating colorectal cancer. An in silico structural analysis, validated by grounding in the experimental data, provides insight into specific molecular interactions and highlights proposed binding modes that can be further modified to refine the affinity and selectivity of identified hits. This multi-modal screening method demonstrates the feasibility of identifying pro-apoptotic agents that can be applied towards conditions where aberrant cell growth or function are key determinants of disease pathogenesis.