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

  • Cell Chem Biol. 2022 Aug 18;29(8):1333-1340.e5. doi: 10.1016/j.chembiol.2022.05.003.
N Connor Payne  1 ,  Semer Maksoud  2 ,  Bakhos A Tannous  2 ,  Ralph Mazitschek  3
Affiliations
  • 1. Center for Systems Biology, Massachusetts General Hospital, Boston, MA 02114, USA; Department of Chemistry & Chemical Biology, Harvard University, Cambridge, MA 02138, USA.
  • 2. Experimental Therapeutics and Molecular Imaging Unit, Department of Neurology, Neuro-Oncology Division, Massachusetts General Hospital, Harvard Medical School, Boston, MA 02129, USA.
  • 3. Center for Systems Biology, Massachusetts General Hospital, Boston, MA 02114, USA; Harvard T.H. Chan School of Public Health, Boston, MA 02115, USA; Broad Institute of MIT and Harvard, Cambridge, MA 02142, USA. Electronic address: [email protected].
PMID: 35649410 DOI: 10.1016/j.chembiol.2022.05.003
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 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.

Keywords
PROTAC; TR-FRET; celastrol; covalent; degrader; high-throughput; protein quantification; reversible; small molecules; targeted protein degradation.
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