Live cell, kinetic monitoring enables study of complex formation and target ubiquitination induced by proteolysis targeting chimeras

Abstract: PROTACs continue to be an exciting therapeutic modality due to their possible use to regulate previously un-druggable targets such as transcription factors and non-enzymatic proteins. PROTACs consist of two ligands joined by a chemical linker that, ideally, will bring a target protein into proximity of a ubiquitin ligase. Subsequent transfer of ubiquitin to the target protein will mark it for degradation by the cell. Despite their promise, their is still much that needs to be learned about the mechanism of action of specific PROTACs. By characterizing PROTACs based on their ability to form the ternary complex of ligase, PROTAC and target and subsequently transfer of ubiquitin to the target, we can work toward developing PROTACs which are even more efficient in these crucial steps. Here we described results showing that using the proximity sensing ability of a BRET approach provide useful tools to detect either complex formation or ubiquitination. Furthermore, when this approach is paired with a microplate reader that can maintain cell health by regulating temperature and environmental gas levels, real-time data can be collected in the living cells. Initial data show that BRD4 tagged with a luciferase can be paired with a fluorescently tagged ligase to study complex formation or a fluorescently tagged ubiquitin to study ubiquitination. Both results show the expected PROTAC dose-dependent increase in BRET signal.