Histone deacetylases (HDACs) regulate myriad cellular processes by catalyzing the hydrolysis of acetyl-l-lysine residues in histone and nonhistone proteins. The Zn-dependent class IIb enzyme HDAC6 regulates microtubule function by deacetylating α-tubulin, which suppresses microtubule dynamics and leads to cell cycle arrest and apoptosis. Accordingly, HDAC6 is a target for the development of selective inhibitors that might be useful in new therapeutic approaches for the treatment of cancer, neurodegenerative diseases, and other disorders. Here, we present high-resolution structures of catalytic domain 2 from HDAC6 (henceforth simply "HDAC6") complexed with compounds that selectively inhibit HDAC6 while maintaining nanomolar inhibitory potency:-hydroxy-4-[((2-hydroxyethyl)-2-phenylacetamido)methyl)-benzamide)] (HPB), ACY-1215 (Ricolinostat), and ACY-1083. These structures reveal that an unusual monodentate Zn coordination mode is exploited by sterically bulky HDAC6-selective phenylhydroxamate inhibitors. We additionally report the ultrahigh-resolution structure of the HDAC6-trichostatin A complex, which reveals two Zn-binding conformers for the inhibitor: a major conformer (70%) with canonical bidentate hydroxamate-Zn coordination geometry and a minor conformer (30%) with monodentate hydroxamate-Zn coordination geometry, reflecting a free energy difference of only 0.5 kcal/mol. The minor conformer is not visible in lower resolution structure determinations. Structural comparisons of HDAC6-inhibitor complexes with class I HDACs suggest active site features that contribute to the isozyme selectivity observed in biochemical assays.

Development of isoform-selective histone deacetylase (HDAC) inhibitors is important in elucidating the function of individual HDAC enzymes and their potential as therapeutic agents. Among the eleven zinc-dependent HDACs in humans, HDAC6 is structurally and functionally unique. Here, we show that a hydroxamic acid-based small-molecule N-hydroxy-4-(2-[(2-hydroxyethyl)(phenyl)amino]-2-oxoethyl)benzamide (HPOB) selectively inhibits HDAC6 catalytic activity in vivo and in vitro. HPOB causes growth inhibition of normal and transformed cells but does not induce cell death. HPOB enhances the effectiveness of DNA-damaging anticancer drugs in transformed cells but not normal cells. HPOB does not block the ubiquitin-binding activity of HDAC6. The HDAC6-selective inhibitor HPOB has therapeutic potential in combination therapy to enhance the potency of anticancer drugs.anticancer agents | epigenetics-based chemotherapy | drug discovery H istone deacetylase 6 (HDAC6) is unique among the eleven zinc-dependent HDACs in humans. HDAC6 is located in the cytoplasm, and it has two catalytic domains and an ubiquitinbinding domain at the C-terminal region (1-3). This study focused on the development of a HDAC6-selective inhibitor and its biological effects. The substrates of HDAC6 include nonhistone proteins such as α-tubulin, peroxiredoxin (PRX), cortactin, and heat shock protein 90 (Hsp90) but not histones (4-7). HDAC6 plays a key role in the regulation of microtubule dynamics including cell migration and cell-cell interactions. The reversible acetylation of Hsp90, a substrate of HDAC6, modulates its chaperone activity and, accordingly, the stability of survival and antiapoptotic factors, including epidermal growth factor receptor (EGFR), protein kinase AKT, proto-oncogene C-RAF, survivin, and other factors. HDAC6, through its ubiquitin-binding activity and interaction with other partner proteins, plays a role in the degradation of misfolded proteins by binding polyubiquitinated proteins and delivering them to the dynein and motor proteins for transport into aggresomes which are degraded by lysosomes (8-10). Thus, HDAC6 has multiple biological functions through deacetylasedependent and -independent mechanisms modulating many cellular pathways relevant to normal and tumor cell growth, migration, and death. HDAC6 is an attractive target for potential cancer treatment.There are several previous reports on the development of HDAC6-selective inhibitors (11)(12)(13)(14)(15). The most extensively studied is tubacin (16,17). Tubacin has non-drug-like qualities, high lipophilicity, and difficult synthesis and has proved to be more useful as a research tool rather than as a potential drug (18). We and others (12)(13)(14)(15)19) have developed HDAC6-selective inhibitors whose pharmacokinetics, toxicity, and efficacy make them potentially more useful than tubacin as therapeutic agents. ACY-1215, 2-(Diphenylamino)-N-(7-(hydroxyamino)-7-oxoheptyl)pyrimidine-5-carboxamide, a HDAC6-selective inhibitor, is currently being evaluated in clinical trial...

Benzopolysulfanes, 4-CH3(OCH2CH2)3NHC(O)-C6H4-1,2-Sx (x = 3–7, and 9) were synthesized with a PEG group attached through an amide bond and examined for water solubility, antitumor activity, and propensity to equilibrate and desulfurate. LCMS and HPLC data show the PEG pentasulfane ring structure predominates, and the tri-, tetra-, hexa-, hepta-, and nonasulfanes were present at very low concentrations. The presence of the PEG group improved water solubility by 50-fold compared to the unsubstituted benzopolysulfanes, C6H4Sx (x = 3, 5, and 7), based on intrinsic solubility measurements. Polysulfur linkages in the PEG compounds decomposed in the presence of ethanethiol and hydroxide ion. The PEG pentathiepin desulfurated rapidly and an S3 transfer reaction was observed in the presence of norbornene, no S2 transfer reaction was observed with 2,3-dimethylbutadiene. The antitumor activities of the PEG-substituted benzopolysulfane mixtures were analyzed against four human tumor cell lines PC3 (prostate), DU145 (prostate), MDA-MB-231 (breast), and Jurkat (T-cell leukemia). The PEG conjugated polysulfanes had IC50 values 1.2–5.8 times lower than the parent “unsubstituted” benzopolysulfanes. Complete cell killing was observed for the PEG polysulfanes with 4 μM for PC3 and DU145 cells, and with 12 μM for MDA-MB-231 cells. The results suggest that solubilization of the polysulfur linkage is a key parameter to the success of these compounds as drug leads.

We report the development of a potent, selective histone deacetylase 6 (HDAC6) inhibitor. This HDAC6 inhibitor blocks growth of normal and transformed cells but does not induce death of normal cells. The HDAC6 inhibitor alone is as effective as paclitaxel in anticancer activity in tumor-bearing mice.