MeFluHyA: a novel fluorescent screening tool for high-throughput identification of HDAC6-selective inhibitors

The cytosolic histone deacetylase 6 (HDAC6) plays a key role not only in cancer but also in neurodegenerative diseases, including amyotrophic lateral sclerosis (ALS) peripheral neuropathies. Pharmacological inhibition as well as genetic silencing of HDAC6 is able to rescue several defects. Therefore, developing pharmacological compounds targeting this enzyme is of crucial interest. Here, we report the design, synthesis, and characterization of Methyl Fluorescent Hydroxamic Acid (MeFluHyA), a novel fluorescent HDAC6-selective probe designed for cell imaging. By integrating a Cy5 fluorophore into a phenyl hydroxamic acid scaffold, MeFluHyA shows binding to the catalytically active CD2 domain of HDAC6 without significantly inhibiting its deacetylating function in the sub-micromolar range. Biochemical enzymatic activity assays confirmed its selectivity over other HDAC isoforms. Fluorescent imaging studies in HeLa cells demonstrated strong colocalization with HDAC6-eGFP and a commercial HDAC6 antibody. Competitive binding assays revealed that MeFluHyA effectively identifies known HDAC6 inhibitors, with reduced probe-binding serving as a readout for successful target engagement. MeFluHyA is a cell-permeable, fast and easy to use probe that can be added organism-independent, and its red-shifted fluorophore makes it compatible with multiplex, live and fixed imaging. Overall, we provide a novel tool for screening platforms aimed at identifying novel HDAC6-targeting inhibitors.