Quantitative measurement of PROTAC intracellular accumulation.

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.