Functional Classification of Zn2+-Dependent Histone Deacetylase Homologs and Variants Using Chemical Profiling

Zinc-dependent histone deacetylases (HDACs) are found in all species, ranging from bacteria to eukaryotes. They catalyze the removal of acetyl groups or various organic acid residues, including fatty acids, from proteins or polyamines. These enzymes are located in various cellular compartments, and their function is modulated by their ability to form different protein complexes. HDACs are prominent targets in the study of human diseases and infections, but they have also recently been considered as targets for fungicides. The active sites of HDACs are highly conserved, both structurally and sequentially. Therefore, the development of isoenzyme-selective inhibitors poses an exceptionally great challenge. In this study, a chemical profiling approach was used to classify human HDAC isoenzymes, bacterial homologs, and mutant variants by function and to identify specific ligand-binding patterns. While the recognition profiles generally reflected established HDAC classes, HDAC8 exhibited significant differences from HDAC1–3. In contrast, HDAC6 demonstrated substantial similarity with HDAC1–3 despite their low sequence identity, distinguishing itself from the sequentially closely related HDAC10. These findings demonstrate that sequence similarity alone is not a reliable predictor of ligand recognition and underscore the importance of local active-site architecture and physicochemical properties. Zebrafish HDAC10 closely resembles human HDAC10, supporting its use as a functional surrogate, while bacterial homologs display distinct profiles that suggest opportunities for species-selective inhibitor development. A single active-site substitution in class IIa HDAC4 (H976Y) shifted its recognition profile from HDAC class IIa toward class I. Engineered HDAC11 variants showed substantially improved recombinant expression and comparable stability while retaining wild-type-like ligand recognition. This supports their use in screening, biophysical, and structural studies. Together, these results establish ligand recognition profiling as a practical approach for functionally classifying HDACs and guiding the development of selective inhibitors.