A Highly Selective, Cell-Permeable Fluorescent Probe for Imaging Histone Deacetylase 6 in Live Cells

Abstract

Histone Deacetylase 6 (HDAC6) plays a crucial role in diverse cellular processes, including cytoskeletal regulation, protein quality control, and stress responses; and its dysregulation is linked to multiple cancers and neurodegenerative disorders, making it a key therapeutic target. However, a detailed understanding of its dynamic functions has been limited by the lack of chemical tools for its visualization in living systems. Utilizing the highly selective inhibitor Nexturastat A as a targeting scaffold, we developed 6SiR-C3-NextA probe incorporating a bright, photostable, far-red silicon-rhodamine fluorophore. Biochemical and cellular assays show that 6SiR-C3-NextA binds to HDAC6 with high affinity (Kdapp = 21 {+/-} 4 nM) and selectivity over other HDAC enzymes, as validated using a developed panel of engineered cell lines expressing individual human HDACs. The probe is cell-permeable, exhibits low cytotoxicity, compatible with super-resolution technique and enables the visualization of endogenous HDAC6 across multiple cell lines. We demonstrate its utility by performing imaging of HDAC6s association with the microtubule network and its dynamic recruitment to stress granules in living cells.