Friday, June 3, 2022

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

N. Connor Payne, Semer Maksoud, Bakhos A. Tannous, Ralph Mazitschek

Cell Chemical Biology, 2022

DOI: https://doi.org/10.1016/j.chembiol.2022.05.003

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.




Comprehensive Exploration of Isocitrate Dehydrogenase (IDH) Mutations: Tumorigenesis, Drug Discovery, and Covalent Inhibitor Advances

Conghao Gai, Hairong Zeng ,  Haoming Xu, Xiaoyun Chai, Yan Zou, Chunlin Zhuang, Guangbo Ge, Qingjie Zhao  European Journal of Medicinal Chem...