A Novel\nConfocal Scanning Protein–Protein Interaction\nAssay (PPI-CONA) Reveals Exceptional Selectivity and Specificity of\nCC0651, a Small Molecule Binding Enhancer of the Weak Interaction\nbetween the E2 Ubiquitin-Conjugating Enzyme CDC34A and Ubi

Protein–protein interactions (PPIs) are some of\nthe most\nchallenging target classes in drug discovery. Highly sensitive detection\ntechniques are required for the identification of chemical modulators\nof PPIs. Here, we introduce PPI confocal nanoscanning (PPI-CONA),\na miniaturized, microbead based high-resolution fluorescence imaging\nassay. We demonstrate the capabilities of PPI-CONA by detecting low\naffinity ternary complex formation between the human CDC34A ubiquitin-conjugating\n(E2) enzyme, ubiquitin, and CC0651, a small molecule enhancer of the\nCDC34A–ubiquitin interaction. We further exemplify PPI-CONA\nwith an E2 enzyme binding study on CC0651 and a CDC34A binding specificity\nstudy of a series of CC0651 analogues. Our results indicate that CC0651\nis highly selective toward CDC34A. We further demonstrate how PPI-CONA\ncan be applied to screening very low affinity interactions. PPI-CONA\nholds potential for high-throughput screening for modulators of PPI\ntargets and characterization of their affinity, specificity, and selectivity.