Target engagement studies and kinetic live-cell degradation assays enable the systematic characterization of HDAC6 PROTACs at endogenous protein expression levels

Histone deacetylase 6 (HDAC6) is an important drug target for the treatment of cancer, inflammation, and neurodegenerative disorders. In recent years, the development of proteolysis-targeting chimeras (PROTACs) has emerged to achieve the chemical knockdown of HDAC6. Consequently, there is an urgent need to develop efficient methods for target engagement studies and to enable a thorough characterization of the degradation efficiency and kinetics of HDAC6 PROTACs. In this work, we present a simple NanoBRET assay to assess HDAC6 cellular target engagement using a HeLaHDAC6-HiBiT cell line that stably expresses the LgBiT protein. For this purpose, we successfully designed, synthesized, characterized, and utilized the cell permeable TAMRA-based fluorescent ligand 5. The key advantage of this NanoBRET assay using HeLaHDAC6-HiBiT cells is the endogenously tagged HDAC6, allowing us to study binding of inhibitors in a near-native environment. Furthermore, we succeeded in establishing a system for kinetic live cell monitoring of HDAC6 degradation. The analysis of the degradation kinetics of a set of HDAC6 PROTACs provided detailed insights into their degradation efficiency and will be helpful for the development of improved HDAC6 degraders in the future. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=98 SRC="FIGDIR/small/646177v2_ufig1.gif" ALT="Figure 1"> View larger version (27K): org.highwire.dtl.DTLVardef@147069forg.highwire.dtl.DTLVardef@3d64eorg.highwire.dtl.DTLVardef@1eac700org.highwire.dtl.DTLVardef@aedc3d_HPS_FORMAT_FIGEXP M_FIG C_FIG