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.

• Klíčová slova
• histone deacetylase, inhibitor profiling, metallohydrolase, nanoBRET, tubulin/histone acetylation,
• MeSH
• acetylace MeSH
• histondeacetylasa 6 * antagonisté a inhibitory   MeSH
• histondeacetylasy * metabolismus   MeSH
• inhibitory histondeacetylas * chemie   farmakologie   MeSH
• kyseliny hydroxamové * chemie   farmakologie   MeSH
• lidé MeSH
• nádorové buněčné linie MeSH
• oxadiazoly * chemie   farmakologie   MeSH
• posttranslační úpravy proteinů MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• HDAC10 protein, human MeSH Prohlížeč
• histondeacetylasa 6 * MeSH
• histondeacetylasy * MeSH
• inhibitory histondeacetylas * MeSH
• kyseliny hydroxamové * MeSH
• oxadiazoly * MeSH

Tubastatin A, a tetrahydro-γ-carboline-capped selective HDAC6 inhibitor (HDAC6i), was rationally designed 10 years ago, and has become the best investigated HDAC6i to date. It shows efficacy in various neurological disease animal models, as HDAC6 plays a crucial regulatory role in axonal transport deficits, protein aggregation, as well as oxidative stress. In this work, we provide new insights into this HDAC6i by investigating the molecular basis of its interactions with HDAC6 through X-ray crystallography, determining its functional capability to elevate the levels of acetylated α-tubulin in vitro and in vivo, correlating PK/PD profiles to determine effective doses in plasma and brain, and finally assessing its therapeutic potential toward psychiatric diseases through use of the SmartCube screening platform.

• Publikační typ
• časopisecké články MeSH

Histone deacetylase 6 (HDAC6) is a multidomain cytosolic enzyme having tubulin deacetylase activity that has been unequivocally assigned to the second of the tandem catalytic domains. However, virtually no information exists on the contribution of other HDAC6 domains on tubulin recognition. Here, using recombinant protein expression, site-directed mutagenesis, fluorimetric and biochemical assays, microscale thermophoresis, and total internal reflection fluorescence microscopy, we identified the N-terminal, disordered region of HDAC6 as a microtubule-binding domain and functionally characterized it to the single-molecule level. We show that the microtubule-binding motif spans two positively charged patches comprising residues Lys-32 to Lys-58. We found that HDAC6-microtubule interactions are entirely independent of the catalytic domains and are mediated by ionic interactions with the negatively charged microtubule surface. Importantly, a crosstalk between the microtubule-binding domain and the deacetylase domain was critical for recognition and efficient deacetylation of free tubulin dimers both in vitro and in vivo Overall, our results reveal that recognition of substrates by HDAC6 is more complex than previously appreciated and that domains outside the tandem catalytic core are essential for proficient substrate deacetylation.

• Klíčová slova
• cytoskeleton, histone deacetylase 6 (HDAC6), intrinsically disordered protein, microtubule-associated protein (MAP), post-translational modification, protein motif, protein-protein interaction, structure-function, substrate specificity, total internal reflection fluorescence (TIRF), tubulin,
• MeSH
• acetylace MeSH
• histondeacetylasa 6 metabolismus   MeSH
• katalytická doména MeSH
• lidé MeSH
• mikrotubuly metabolismus   MeSH
• proteinové domény fyziologie   MeSH
• sekvence aminokyselin MeSH
• substrátová specifita MeSH
• tubulin metabolismus   MeSH
• vazba proteinů MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• HDAC6 protein, human MeSH Prohlížeč
• histondeacetylasa 6 MeSH
• tubulin MeSH