Discovery of MYC/NSD3S Protein–Protein Interaction Negative Modulators with a Live Cell Nanoluciferase Protein-Fragment Complementation uHTS Assay

Abstract Protein–protein interactions (PPIs) are central to physiological and pathophysiological processes. Identifying PPI modulators is the first step toward the development of new treatments. c-MYC (MYC), a key oncogene, interacts with NSD3S, stabilizing its structure and enhancing the oncogenic effect. In this work, we developed the NanoPCA assay in live cells in a 1536-well uHTS format to monitor the MYC/NSD3S PPI. We validated the platform by screening 12,000 compounds. The actions of several dose-dependent modulators were confirmed. Using orthogonal assays (immunoprecipitation, MYC reporter, and viability), we identified ethoxysanguinarine and withaferin A as potential negative modulators. Additionally, PPI in silico modeling and identification of key interaction points (hotspots) were performed to understand how the compounds may bind, revealing their potential ability to block the MYC/NSD3S PPI. These represent initial chemical tools that disrupt the MYC/NSD3S PPI signal in live cell assays and biochemical lysates, laying the foundation for targeted therapies against MYC/NSD3S-dependent cancers.