Histone deacetylase (HDAC) inhibitors used in the clinic typically contain a hydroxamate zinc-binding group (ZBG). However, more recent work has shown that the use of alternative ZBGs, and, in particular, the heterocyclic oxadiazoles, can confer higher isoenzyme selectivity and more favorable ADMET profiles. Herein, we report on the synthesis and biochemical, crystallographic, and computational characterization of a series of oxadiazole-based inhibitors selectively targeting the HDAC6 isoform. Surprisingly, but in line with a very recent finding reported in the literature, a crystal structure of the HDAC6/inhibitor complex revealed that hydrolysis of the oxadiazole ring transforms the parent oxadiazole into an acylhydrazide through a sequence of two hydrolytic steps. An identical cleavage pattern was also observed both in vitro using the purified HDAC6 enzyme as well as in cellular systems. By employing advanced quantum and molecular mechanics (QM/MM) and QM calculations, we elucidated the mechanistic details of the two hydrolytic steps to obtain a comprehensive mechanistic view of the double hydrolysis of the oxadiazole ring. This was achieved by fully characterizing the reaction coordinate, including identification of the structures of all intermediates and transition states, together with calculations of their respective activation (free) energies. In addition, we ruled out several (intuitively) competing pathways. The computed data (ΔG‡ ≈ 21 kcal·mol-1 for the rate-determining step of the overall dual hydrolysis) are in very good agreement with the experimentally determined rate constants, which a posteriori supports the proposed reaction mechanism. We also clearly (and quantitatively) explain the role of the -CF3 or -CHF2 substituent on the oxadiazole ring, which is a prerequisite for hydrolysis to occur. Overall, our data provide compelling evidence that the oxadiazole warheads can be efficiently transformed within the active sites of target metallohydrolases to afford reaction products possessing distinct selectivity and inhibition profiles.

• MeSH
• Histone Deacetylase 6 chemistry   MeSH
• Hydrolysis MeSH
• Histone Deacetylase Inhibitors * pharmacology   MeSH
• Hydroxamic Acids chemistry   MeSH
• Oxadiazoles * MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Histone Deacetylase 6 MeSH
• Histone Deacetylase Inhibitors * MeSH
• Hydroxamic Acids MeSH
• Oxadiazoles * MeSH

Histone deacetylase 6 (HDAC6) is a unique member of the HDAC family of enzymes due to its complex domain organization and cytosolic localization. Experimental data point toward the therapeutic use of HDAC6-selective inhibitors (HDAC6is) for use in both neurological and psychiatric disorders. In this article, we provide side-by-side comparisons of hydroxamate-based HDAC6is frequently used in the field and a novel HDAC6 inhibitor containing the difluoromethyl-1,3,4-oxadiazole function as an alternative zinc-binding group (compound 7). In vitro isotype selectivity screening uncovered HDAC10 as a primary off-target for the hydroxamate-based HDAC6is, while compound 7 features exquisite 10,000-fold selectivity over all other HDAC isoforms. Complementary cell-based assays using tubulin acetylation as a surrogate readout revealed approximately 100-fold lower apparent potency for all compounds. Finally, the limited selectivity of a number of these HDAC6is is shown to be linked to cytotoxicity in RPMI-8226 cells. Our results clearly show that off-target effects of HDAC6is must be considered before attributing observed physiological readouts solely to HDAC6 inhibition. Moreover, given their unparalleled specificity, the oxadiazole-based inhibitors would best be employed either as research tools in further probing HDAC6 biology or as leads in the development of truly HDAC6-specific compounds in the treatment of human disease states.

• Keywords
• histone deacetylase, inhibitor profiling, metallohydrolase, nanoBRET, tubulin/histone acetylation,
• MeSH
• Acetylation MeSH
• Histone Deacetylase 6 * antagonists & inhibitors   MeSH
• Histone Deacetylases * metabolism   MeSH
• Histone Deacetylase Inhibitors * chemistry   pharmacology   MeSH
• Hydroxamic Acids * chemistry   pharmacology   MeSH
• Humans MeSH
• Cell Line, Tumor MeSH
• Oxadiazoles * chemistry   pharmacology   MeSH
• Protein Processing, Post-Translational MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• HDAC10 protein, human MeSH Browser
• Histone Deacetylase 6 * MeSH
• Histone Deacetylases * MeSH
• Histone Deacetylase Inhibitors * MeSH
• Hydroxamic Acids * MeSH
• Oxadiazoles * MeSH