Small molecules targeting HDAC6: indirect microtubule assembly modulators.

<title>Abstract</title> <p> Protein lysine acetylation is a fundamental post-translational modification regulating diverse cellular processes, and its dysregulation is implicated in numerous human diseases. Histone deacetylase 6 (HDAC6) is a unique cytoplasmic deacetylase that primarily targets non-histone proteins, including α-tubulin, and plays critical roles in cytoskeletal organization, intracellular transport, and protein quality control. Due to its involvement in neurodegenerative disorders, cancer, inflammatory diseases, fibrosis, and cardiovascular and metabolic dysfunction, HDAC6 has emerged as an attractive therapeutic target. However, the development of highly selective HDAC6 inhibitors remains challenging because the catalytic pocket and zinc-coordinating residues are highly conserved across HDAC isoforms. In this study, we employed a computational approach combining pharmacophore-based virtual screening, molecular docking, binding site interaction analysis, and ADMET prediction to identify novel selective inhibitors of human HDAC6. From the Enamine compound library screened using the Pharmit service, seven compounds were selected based on their predicted high affinity for HDAC6 and low affinity for other human HDAC isoforms. Docking analysis revealed that these compounds coordinate the catalytic Zn²⁺ ion via alternative monodentate neutral O-donor zinc-binding groups (carbonyl or sulfoxide oxygen) and establish key interactions with residues in the L1 and L2 pockets (Leu749, Ser568, Phe620) that determine HDAC6 isoform selectivity, as well as with catalytically essential residues His611 and Tyr782. Experimental validation in primary porcine neuron cultures confirmed the biological activity of all seven compounds, with no observed cytotoxicity toward neural stem cells. Importantly, comparative analysis with plant tubulin-associated histone deacetylases, particularly <italic>Arabidopsis thaliana</italic> HDA14, revealed that six compounds exhibited high predicted affinity for these plant enzymes, recapitulating the binding pattern observed for human HDAC6. These findings demonstrate the potential of human HDAC6-selective inhibitors for selective targeting of plant tubulin deacetylases and provide a structural framework for the rational design of isoform-selective inhibitors across eukaryotic lineages. </p>