Synthesis and Biological Investigation of Oxazole Hydroxamates as Highly Selective Histone Deacetylase 6 (HDAC6) Inhibitors

Histone deacetylase 6 (HDAC6) catalyzes the removal of an acetyl group from lysine residues of several non-histone proteins. Here we report the preparation of thiazole-, oxazole-, and oxadiazole-containing biarylhydroxamic acids by a short synthetic procedure. We identified them as selective HDAC6 inhibitors by investigating the inhibition of recombinant HDAC enzymes and the protein acetylation in cells by Western blotting (tubulin vs histone acetylation). The most active compounds exhibited nanomolar potency and high selectivity for HDAC6. For example, an oxazole hydroxamate inhibits HDAC6 with an IC₅₀ of 59 nM and has a selectivity index of >200 against HDAC1 and HDAC8. This is the first report showing that the nature of a heterocycle directly connected to a zinc binding group (ZBG) can be used to modulate subtype selectivity and potency for HDAC6 inhibitors to such an extent. We rationalize the high potency and selectivity of the oxazoles by molecular modeling and docking

Synthesis and Biological Investigation of Oxazole Hydroxamates as Highly Selective Histone Deacetylase 6 (HDAC6) Inhibitors

Histone deacetylase 6 (HDAC6) catalyzes the removal of an acetyl group from lysine residues of several non-histone proteins. Here we report the preparation of thiazole-, oxazole-, and oxadiazole-containing biarylhydroxamic acids by a short synthetic procedure. We identified them as selective HDAC6 inhibitors by investigating the inhibition of recombinant HDAC enzymes and the protein acetylation in cells by Western blotting (tubulin vs histone acetylation). The most active compounds exhibited nanomolar potency and high selectivity for HDAC6. For example, an oxazole hydroxamate inhibits HDAC6 with an IC₅₀ of 59 nM and has a selectivity index of >200 against HDAC1 and HDAC8. This is the first report showing that the nature of a heterocycle directly connected to a zinc binding group (ZBG) can be used to modulate subtype selectivity and potency for HDAC6 inhibitors to such an extent. We rationalize the high potency and selectivity of the oxazoles by molecular modeling and docking

Supporting Information from Isoform-selective HDAC1/6/8 inhibitors with an imidazo-ketopiperazine cap containing stereochemical diversity

Experimental procedures for compound synthesis, biological assays and molecular dockin

Alkoxyurea-Based Histone Deacetylase Inhibitors Increase Cisplatin Potency in Chemoresistant Cancer Cell Lines

The synthesis and biological evaluation of potent hydroxamate-based dual HDAC1/6 inhibitors with modest HDAC6 preference and a novel alkoxyurea connecting unit linker region are described. The biological studies included the evaluation of antiproliferative effects and HDAC inhibitory activity in the human ovarian cancer cell line A2780, the human squamous carcinoma cell line Cal27, and their cisplatin resistant sublines A2780CisR and Cal27CisR. The three most potent compounds <b>1g</b>–<b>i</b> showed IC₅₀ values in the low μM and sub-μM range. <b>1g</b>–<b>i</b> revealed low nM IC₅₀ values for HDAC6 with up to 15-fold preference over HDAC1, >3500-fold selectivity over HDAC4, and >100-fold selectivity over HDAC8. Furthermore, their ability to enhance cisplatin sensitivity was analyzed in Cal27 and Cal27CisR cells. Notably, a 48 h preincubation of <b>1g</b>–<b>i</b> significantly enhanced the antiproliferative effects of cisplatin in Cal27 and Cal27CisR. <b>1g</b>–<b>i</b> interacted synergistically with cisplatin. These effects were more pronounced for the cisplatin resistant subline Cal27CisR

Supporting Information from Isoform-selective HDAC1/6/8 inhibitors with an imidazo-ketopiperazine cap containing stereochemical diversity

Experimental procedures for compound synthesis, biological assays and molecular dockin

Discovery of Inhibitors of Schistosoma mansoni HDAC8 by Combining Homology Modeling, Virtual Screening, and in Vitro Validation

Schistosomiasis, caused by S. mansoni, is a tropical disease that affects over 200 million people worldwide. A novel approach for targeting eukaryotic parasites is to tackle their dynamic epigenetic machinery that is necessary for the extensive phenotypic changes during their life cycle. We recently identified S. mansoni histone deacetylase 8 (smHDAC8) as a potential target for antiparasitic therapy. Here we present results from a virtual screening campaign on smHDAC8. Besides hydroxamates, several sulfonamide-thiazole derivatives were identified by a target-based virtual screening using a homology model of smHDAC8. In vitro testing of 75 compounds identified 8 hydroxamates as potent and lead-like inhibitors of the parasitic HDAC8. Solving of the crystal structure of smHDAC8 with two of the virtual screening hits confirmed the predicted binding mode

Structure-Based Design and Synthesis of Novel Inhibitors Targeting HDAC8 from Schistosoma mansoni for the Treatment of Schistosomiasis

Schistosomiasis is a major neglected parasitic disease that affects more than 265 million people worldwide and for which the control strategy consists of mass treatment with the only available drug, praziquantel. In this study, a series of new benzohydroxamates were prepared as potent inhibitors of Schistosoma mansoni histone deacetylase 8 (smHDAC8). Crystallographic analysis provided insights into the inhibition mode of smHDAC8 activity by these 3-amidobenzohydroxamates. The newly designed inhibitors were evaluated in screens for enzyme inhibitory activity against schistosome and human HDACs. Twenty-seven compounds were found to be active in the nanomolar range, and some of them showed selectivity toward smHDAC8 over the major human HDACs (1 and 6). The active benzohydroxamates were additionally screened for lethality against the schistosome larval stage using a fluorescence-based assay. Four of these showed significant dose-dependent killing of the schistosome larvae and markedly impaired egg laying of adult worm pairs maintained in culture

Ligand-triggered gating of the smHDAC8 active site pocket.

<p>Close-up view of the active sites of the (A) smHDAC8/SAHA, (B) hHDAC8/SAHA (PDB 1T69), (C) smHDAC8/M344, and (D) hHDAC8/M344 (PDB 1T67) complexes shown as ribbon and sticks (upper panels), surface view (middle panels), and side cut-away surface view (lower panels). The conformational changes of smHDAC8 Y99 and F151 compared to hHDAC8 Y100 and F152 strongly influence the binding modes of the SAHA and M344 inhibitors to these enzymes.</p

smHDAC8 is a functional acetyl-L-lysine deacetylase that is essential for parasite infectivity.

<p>(A) Down-regulation of <i>smHDAC8</i> decreases the number of adult worms recovered from infected mice. (B) The number of recovered eggs from the livers of infected mice is decreased. ‘*’, p<0.05; ‘**’, p<0.01. (C) Comparison of smHDAC8 and hHDAC8 deacetylase activities. Data indicate the average of relative deacetylase activity (hHDAC8 = 100%). Error bars represent the standard deviations (SD). (D) Close-up view of hHDAC8 active site. D101 and Y306, which participate in the hHDAC8 catalytic mechanism, and M274, which is replaced by a histidine in smHDAC8, are displayed. (E) Deacetalyse activities of smHDAC8 wild-type (wt), D100A, Y341F, and H292M mutants. Data indicate the average of relative deacetylase activity (smHDAC8 wt = 100%).</p

smHDAC8 adopts a canonical HDAC fold with specific external loops.

<p>(A) Structure-based sequence alignment of schistosome, mouse and human HDAC8 proteins. Sequences similarities are shown by levels of blue. Secondary structure elements found in smHDAC8 and hHDAC8 are shown above and below the alignment, respectively. Residues that could not be built in densities are depicted with a black dotted line. Important residues that participate in the specificity of the smHDAC8 active site are labeled with triangles. The numbering indicated above the alignment corresponds to smHDAC8. For clarity, the first thirteen residues of mouse and human HDAC8 have been removed. (B) Superposition of native smHDAC8 (green) and SAHA-inhibited hHDAC8 (blue; PDB 1T69) structures. Both enzymes adopt the same fold. smHDAC8 sequence insertions form specific external loops and C-terminus (colored in pink). The orange sphere represents the catalytic zinc ion (Zn). (C,D) Ribbon representations of smHDAC8 (C) and hHDAC8 (D) structures. Both enzymes adopt the same fold and their catalytic zinc ion is found at the same position.</p