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

IF 3.7 Q1 CHEMISTRY, MEDICINAL

ACS Pharmacology and Translational Science Pub Date : 2025-08-05 DOI:10.1021/acsptsci.5c00247

Maria Hanl, Felix Feller, Irina Honin, Kathrin Tan, Martina Miranda, Linda Schäker-Hübner, Nico Bückreiß, Matthias Schiedel, Michael Gütschow, Gerd Bendas and Finn K. Hansen*,

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引用次数: 0

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 HeLa^{HDAC6–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 HeLa^{HDAC6–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.

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靶标接合研究和动态活细胞降解测定使组蛋白去乙酰化酶6降解物的系统表征成为可能

组蛋白去乙酰化酶6 （HDAC6）是治疗癌症、炎症和神经退行性疾病的重要药物靶点。近年来，为了实现HDAC6的化学敲低，出现了靶向蛋白水解嵌合体（proteolysis-targeting chimeras, PROTACs）。因此，迫切需要开发有效的靶接触研究方法，并能够彻底表征HDAC6 PROTACs的降解效率和动力学。在这项工作中，我们提出了一种简单的NanoBRET检测方法，使用稳定表达LgBiT蛋白的HeLaHDAC6-HiBiT细胞系来评估HDAC6细胞靶点的结合情况。为此，我们成功地设计、合成、表征和利用了基于tamra的细胞渗透性荧光配体5。使用HeLaHDAC6-HiBiT细胞的NanoBRET试验的主要优势是内源性标记的HDAC6，使我们能够在接近天然的环境中研究抑制剂的结合。此外，我们成功地建立了HDAC6降解的动态活细胞监测系统。通过对一组HDAC6 PROTACs降解动力学的分析，可以更详细地了解其降解效率，并为今后改进HDAC6降解剂的开发提供帮助。

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来源期刊

ACS Pharmacology and Translational Science Medicine-Pharmacology (medical)

CiteScore

10.00

自引率

3.30%

发文量

133

期刊介绍： ACS Pharmacology & Translational Science publishes high quality, innovative, and impactful research across the broad spectrum of biological sciences, covering basic and molecular sciences through to translational preclinical studies. Clinical studies that address novel mechanisms of action, and methodological papers that provide innovation, and advance translation, will also be considered. We give priority to studies that fully integrate basic pharmacological and/or biochemical findings into physiological processes that have translational potential in a broad range of biomedical disciplines. Therefore, studies that employ a complementary blend of in vitro and in vivo systems are of particular interest to the journal. Nonetheless, all innovative and impactful research that has an articulated translational relevance will be considered. ACS Pharmacology & Translational Science does not publish research on biological extracts that have unknown concentration or unknown chemical composition. Authors are encouraged to use the pre-submission inquiry mechanism to ensure relevance and appropriateness of research.