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

Hanl, M.; Feller, F.; Honin, I.; Tan, K.; Schaeker-Huebner, L.; Bueckreiss, N.; Schiedel, M.; Guetschow, M.; Bendas, G.; Hansen, F. K.

doi:10.1101/2025.03.31.646177

bioRxiv · 10.1101/2025.03.31.646177

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

Hanl, M.·Feller, F.·Honin, I.·Tan, K.·Schaeker-Huebner, L.·Bueckreiss, N.·Schiedel, M.·Guetschow, M.·Bendas, G.·Hansen, F. K.

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Abstract

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

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Hanl, M., Feller, F., Honin, I., Tan, K., Schaeker-Huebner, L., Bueckreiss, N., Schiedel, M., Guetschow, M., Bendas, G., Hansen, F. K.. 2025-04-04. Target engagement studies and kinetic live-cell degradation assays enable the systematic characterization of HDAC6 PROTACs at endogenous protein expression levels. https://doi.org/10.1101/2025.03.31.646177

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