Target Engagement Studies and Kinetic Live-Cell Degradation Assays Enable the Systematic Characterization of Histone Deacetylase 6 Degraders

  • ACS Pharmacol Transl Sci. 2025 Aug 5;8(9):3074-3089. doi: 10.1021/acsptsci.5c00247.
Maria Hanl  1 ,  Felix Feller  1 ,  Irina Honin  1 ,  Kathrin Tan  1 ,  Martina Miranda  1 ,  Linda Schäker-Hübner  1 ,  Nico Bückreiß  1 ,  Matthias Schiedel  2 ,  Michael Gütschow  3 ,  Gerd Bendas  1 ,  Finn K Hansen  1
Affiliations
  • 1. Department of Pharmaceutical and Cell Biological Chemistry, Pharmaceutical Institute, University of Bonn, An der Immenburg 4, Bonn 53121, Germany.
  • 2. Institute of Medicinal and Pharmaceutical Chemistry, Technische Universität Braunschweig, Beethovenstraße 55, Braunschweig 38106, Germany.
  • 3. Department of Pharmaceutical and Medicinal Chemistry, Pharmaceutical Institute, University of Bonn, An der Immenburg 4, Bonn 53121, Germany.
PMID: 40969900 DOI: 10.1021/acsptsci.5c00247
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.

Keywords
HDAC6; NanoBRET; PROTAC; degradation kinetics; targeted protein degradation.
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