A direct high-throughput protein quantification strategy facilitates discovery and characterization of a celastrol-derived BRD4 degrader. Issue 8 (18th August 2022)

• Record Type: Journal Article
• Authors: Payne, N. Connor; Maksoud, Semer; Tannous, Bakhos A.; Mazitschek, Ralph
• Is Part Of: Cell chemical biology. Volume 29:Issue 8(2022)
• Page Start: 1333
• Page End: 1340.e5
• Publication Date: 2022-08-18
• Subjects: TR-FRET -- small molecules -- targeted protein degradation -- PROTAC -- degrader -- high-throughput -- protein quantification -- reversible -- covalent -- celastrol; Biochemistry -- Periodicals; 572.05
• DOI: 10.1016/j.chembiol.2022.05.003
• Languages: English
• ISSNs: 2451-9456

Abstract

Summary: 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 in a high-throughput format with unmodified cell lines. We furthermore validate our platform in the characterization of celastrol, a p- quinone methide-containing pentacyclic triterpenoid, as a broad cysteine-targeting E3 ubiquitin ligase warhead for potent and efficient targeted protein degradation. Graphical abstract: Highlights: Dual-mode TR-FRET assay for target protein abundance and ligand affinity profiling High-throughput quantification of unmodified endogenous proteins in cell lysate Celastrol is a reversible covalent warhead for redirecting E3 ligase activity Abstract : Payne et al. describe a generalizable high-throughput assay strategy for the direct, rapid quantification of endogenous target proteins in lysates from unmodified cells in 1 hour. Using this simple mix-and-read platform, the authors characterize celastrol as a potent E3-ligase-recruiting element for the development of proteolysis-targeting chimeras (PROTACs).