Unusual zinc-binding mode of HDAC6-selective hydroxamate inhibitors.

Histone deacetylases (HDACs) regulate myriad cellular processes by catalyzing the hydrolysis of acetyl-l-lysine residues in histone and nonhistone proteins. The Zn2+-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 Danio rerio HDAC6 (henceforth simply "HDAC6") complexed with compounds that selectively inhibit HDAC6 while maintaining nanomolar inhibitory potency: N-hydroxy-4-[(N(2-hydroxyethyl)-2-phenylacetamido)methyl)-benzamide)] (HPB), ACY-1215 (Ricolinostat), and ACY-1083. These structures reveal that an unusual monodentate Zn2+ 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 Zn2+-binding conformers for the inhibitor: a major conformer (70%) with canonical bidentate hydroxamate-Zn2+ coordination geometry and a minor conformer (30%) with monodentate hydroxamate-Zn2+ 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.

Synthesis and antiproliferative properties of a new ceramide analog of varacin.

A benzopentasulfane was synthesized in 8 steps with a ceramide attached through an amide bond to the 7-position of the heterocycle structure. The anticancer activity of this synthetic ceramide-benzopolysulfane drug conjugate was analyzed against five human cancer cell lines MDA-MB-231 (breast), DU145 (prostate), MIA PaCa-2 (pancreas), HeLa (cervix), and U251 (glioblastoma). The ceramide-benzopolysulfane conjugate had IC50 values ranging from 10 to >20 µM with complete cell killing at 12.5 µM for MDA-MB-231 and 20 µM for DU145 and HeLa cells. The ceramide-benzopolysulfane conjugate had IC50 values 1.8 and 4.0 times lower than a PEG benzopolysulfane, N-(2-(2-(2-methoxyethoxy)ethoxy)ethyl)benzo[f][1,2,3,4,5]-pentathiepine-7-carboxamide, for MDA-MB-231 and DU145 cells, respectively. The parent "unsubstituted" benzopolysulfane, o-C6H4S5, had IC50 values 4.2 times lower and 2.7 times higher than the ceramide benzopolysulfane for MDA-MB-231 and DU145 cells, respectively. The results indicate that the polysulfur linkage is needed for activity since benzenedithiol, o-C6H4(SH)2, had IC50 values greater than 30 µM with little effect on MDA-MB-231 and DU145 cells. Thus, to account for the bioactivity, a bimolecular reaction of cellular thiol with the ceramide benzopolysulfane is a proposed followed by thiozone (S3) extrusion.

Creation of a histone deacetylase 6 inhibitor and its biological effects [corrected].

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.

Deoxypolypeptides bind and cleave RNA.

We have prepared L- and D-deoxypolypeptides (DOPPs) by selective reduction of appropriately protected polyhistidines with borane, reducing the carbonyl groups to methylenes. The result is a chiral polyamine, not amide, with a mainly protonated backbone and chirally mounted imidazolylmethylene side chains that are mostly unprotonated at neutrality because of the nearby polycationic backbone. We found that, in contrast with the D-octahistidine DOPP, the L-octahistidine DOPP is able to cooperatively bind to a D-polyuridylic acid RNA; this is consistent with results of previous studies showing that, relative to D-histidine, L-histidine is able to more strongly bind to RNA. The L-DOPP was also a better catalyst for cleaving the RNA than the D-DOPP, consistent with evidence that the L-DOPP uses its imidazole groups for catalysis, in addition to the backbone cations, but the D-DOPP does not use the imidazoles. The L-DOPP bifunctional process probably forms a phosphorane intermediate. This is a mechanism we have proposed for models of ribonuclease cleavage and for the ribonuclease A enzyme itself, based on our studies of the cleavage and isomerization of UpU catalyzed by imidazole buffers as well as other relevant studies. This mechanism contrasts with earlier, generally accepted ribonuclease cleavage mechanisms where the proton donor coordinates with the oxygen of the leaving group as the 2-hydroxyl of ribose attacks the unprotonated phosphate.

Development of a histone deacetylase 6 inhibitor and its biological effects.

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.

A hand-held fiber-optic implement for the site-specific delivery of photosensitizer and singlet oxygen.

We have constructed a fiber optic device that internally flows triplet oxygen and externally produces singlet oxygen, causing a reaction at the (Z)-1,2-dialkoxyethene spacer group, freeing a pheophorbide sensitizer upon the fragmentation of a reactive dioxetane intermediate. The device can be operated and sensitizer photorelease observed using absorption and fluorescence spectroscopy. We demonstrate the preference of sensitizer photorelease when the probe tip is in contact with octanol or lipophilic media. A first-order photocleavage rate constant of 1.13 h(-1) was measured in octanol where dye desorption was not accompanied by readsorption. When the probe tip contacts aqueous solution, the photorelease was inefficient because most of the dye adsorbed on the probe tip, even after the covalent ethene spacer bonds have been broken. The observed stability of the free sensitizer in lipophilic media is reasonable even though it is a pyropheophorbide-a derivative that carries a p-formylbenzylic alcohol substituent at the carboxylic acid group. In octanol or lipid systems, we found that the dye was not susceptible to hydrolysis to pyropheophorbide-a, otherwise a pH effect was observed in a binary methanol-water system (9:1) at pH below 2 or above 8.

Photosensitizer drug delivery via an optical fiber.

An optical fiber has been developed with a maneuverable mini-probe tip that sparges O(2) gas and photodetaches pheophorbide (sensitizer) molecules. Singlet oxygen is produced at the probe tip surface which reacts with an alkene spacer group releasing sensitizer upon fragmentation of a dioxetane intermediate. Optimal sensitizer photorelease occurred when the probe tip was loaded with 60 nmol sensitizer, where crowding of the pheophorbide molecules and self-quenching were kept to a minimum. The fiber optic tip delivered pheophorbide molecules and singlet oxygen to discrete locations. The 60 nmol sensitizer was delivered into petrolatum; however, sensitizer release was less efficient in toluene-d(8) (3.6 nmol) where most had remained adsorbed on the probe tip, even after the covalent alkene spacer bond had been broken. The results open the door to a new area of fiber optic-guided sensitizer delivery for the potential photodynamic therapy of hypoxic structures requiring cytotoxic control.

Synthesis, characterization, mechanism of decomposition, and antiproliferative activity of a class of PEGylated benzopolysulfanes structurally similar to the natural product varacin.

Benzopolysulfanes, 4-CH(3)(OCH(2)CH(2))(3)NHC(O)-C(6)H(4)-1,2-S(x) (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, C(6)H(4)S(x) (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 S(3) transfer reaction was observed in the presence of norbornene; no S(2) 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 IC(50) values 1.2-5.8 times lower than the parent "unsubstituted" benzopolysulfanes. Complete cell killing was observed for the PEG polysulfanes at 4 microM for PC3 and DU145 cells and at 12 muM 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.

Fiber-optic singlet oxygen [1O2 (1Delta(g))] generator device serving as a point selective sterilizer.

Traditionally, Type II heterogeneous photo-oxidations produce singlet oxygen via external irradiation of a sensitizer and external supply of ground-state oxygen. A potential improvement is reported here. A hollow-core fiber-optic device was developed with an "internal" supply of light and flowing oxygen, and a porous photosensitizer-end capped configuration. Singlet oxygen was delivered through the fiber tip. The singlet oxygen steady-state concentration in the immediate vicinity of the probe tip was ca 20 fm by N-benzoyl-DL-methionine trapping. The device is portable and the singlet oxygen-generating tip is maneuverable, which opened the door to simple disinfectant studies. Complete Escherichia coli inactivation was observed in 2 h when the singlet oxygen sensitizing probe tip was immersed in 0.1 mL aqueous samples of 0.1-4.4 x 10(7) cells. Photobleaching of the probe tip occurred after ca 12 h of use, requiring baking and sensitizer reloading steps for reuse.

Regioselective (biomimetic) synthesis of a pentasulfane from ortho-benzoquinone.

A mechanism is proposed for the formation of cyclic 5,6,7,8,9-pentathiabenzocycloheptene-1,2-diol, 4, from the reaction of o-benzoquinone with reduced elemental sulfur, H2Sx. 1,6-conjugate addition to the quinone is favored over 1,4-conjugate addition. Hydrogen bonding to the quinone oxygen enhances the nucleophilicity of H2Sx by facilitating the removal of the S-H proton. We propose that initially formed 3-polysulfidobenzene-diol intermediates are oxidized to their corresponding quinones and closure of the polysulfur ring subsequently takes place at the C3-C4 bond leading to 4. A possible mechanism for the formation of the pentasulfur linkage in 4 is discussed, which is the key moiety found in a number of natural products.