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


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.

Oncogenic KRAS G12C: Kinetic and Redox Characterization of Covalent Inhibition

Minh V. Huynh, Derek Parsonage, Tom E. Forshaw, Venkat R. Chirasani, G. Aaron Hobbs, Hanzhi Wu, Jingyun Lee, Cristina M. Furdui, Leslie B. P...