A direct high-throughput protein quantification strategy facilitates discovery and characterization of a celastrol-derived BRD4 degrader.

ABSTRACT: We describe a generalizable time-resolved Förster resonance energy transfer (TR-FRET)-based platform to profile the cellular action of heterobifunctional degraders (or proteolysis-targeting chimeras [PROTACs]) that is capable of both accurately quantifying protein levels in whole-cell lysates in less than 1 h and measuring small-molecule target engagement to endogenous proteins, here specifically for human bromodomain-containing protein 4 (BRD4). The detection mix consists of a single primary antibody targeting the protein of interest, a luminescent donor-labeled anti-species nanobody, and a fluorescent acceptor ligand. Importantly, our strategy can readily be applied to other targets of interest and will greatly facilitate the cell-based profiling of small-molecule inhibitors and PROTACs

SUBMITTER: Payne NC 

PROVIDER: S-EPMC9391279 | biostudies-literature | 2022 Aug

REPOSITORIES: biostudies-literature