Publication Date
1-1-2023
Journal
Methods in Enzymology
DOI
10.1016/bs.mie.2022.11.001
PMID
36764757
PMCID
PMC11102804
PubMedCentral® Posted Date
5-19-2024
PubMedCentral® Full Text Version
Author MSS
Published Open-Access
yes
Keywords
Proteolysis, Proteins, Ubiquitin-Protein Ligases, PROTAC, Target Engagement, Nano-BRET, Binding Constant, Relative Intracellular Accumulation Coefficient
Abstract
In recent years, Proteolysis Targeting Chimera (PROTAC) technology has emerged as one of the most promising approaches to remove disease-associated proteins by utilizing cells' own destruction machinery. To achieve successful degradation of a protein of interest (POI), the heterobifunctional PROTAC molecules must penetrate into the cells first, followed by target engagement and formation of the POI-PROTAC-E3 ligase complex. Based on this understanding, the assessment of cell permeability and in cell target engagement are of great importance to evaluate the efficacy of PROTAC candidates. PROTAC molecules can be classified as non-covalent and covalent, and covalent PROTACs can be further divided into irreversible and reversible covalent. Here, we present a high-throughput assay to prioritize different types of BTK PROTACs by measuring their intracellular accumulation quantitatively, using kinase binding assays and the NanoBRET target engagement platform.
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