Development\nof a Fluorescence Probe for High-Throughput\nScreening of Allosteric Inhibitors Targeting TRAP1

Tumor necrosis factor receptor-associated protein 1 (TRAP1)\nis\na molecular chaperone implicated in pro-tumorigenic pathways by regulating\nthe folding of substrate proteins (clients) within cancer cells. Recent\nresearch has pinpointed a potentially druggable allosteric site within\nthe client binding site (CBS) of TRAP1, suggesting this site might\noffer a more effective strategy for developing potent and selective\nTRAP1 inhibitors. However, the absence of reliable assay systems has\nhindered quantitative evaluation of inhibitors. In this study, we\nhave developed a fluorescent probe, Rho6TPP, designed to target the\nCBS. Utilizing fluorescence polarization-based high-throughput screening\nassays, Rho6TPP exhibits excellent signal-to-noise ratio (>20), <i>Z</i> factor (>0.6), and <i>Z</i>′ factor\n(>0.6).\nAdditionally, it facilitates comparative analysis of existing small\nmolecules and discovery of novel binders. MitoTam, a mitochondria-targeted\ntamoxifen, emerges as a potent CBS-targeting TRAP1 inhibitor. Our\nfindings highlight the potential of Rho6TPP as a crucial tool for\nadvancing the development of CBS-targeting TRAP1 inhibitors.