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

Histone deacetylase inhibition modulates histone acetylation at gene promoter regions and affects genome-wide gene transcription in <i>Schistosoma mansoni</i>

<div><p>Background</p><p>Schistosomiasis is a parasitic disease infecting hundreds of millions of people worldwide. Treatment depends on a single drug, praziquantel, which kills the <i>Schistosoma</i> spp. parasite only at the adult stage. HDAC inhibitors (HDACi) such as Trichostatin A (TSA) induce parasite mortality <i>in vitro</i> (schistosomula and adult worms), however the downstream effects of histone hyperacetylation on the parasite are not known.</p><p>Methodology/Principal findings</p><p>TSA treatment of adult worms <i>in vitro</i> increased histone acetylation at H3K9ac and H3K14ac, which are transcription activation marks, not affecting the unrelated transcription repression mark H3K27me3. We investigated the effect of TSA HDACi on schistosomula gene expression at three different time points, finding a marked genome-wide change in the transcriptome profile. Gene transcription activity was correlated with changes on the chromatin acetylation mark at gene promoter regions. Moreover, combining expression data with ChIP-Seq public data for schistosomula, we found that differentially expressed genes having the H3K4me3 mark at their promoter region in general showed transcription activation upon HDACi treatment, compared with those without the mark, which showed transcription down-regulation. Affected genes are enriched for DNA replication processes, most of them being up-regulated. Twenty out of 22 genes encoding proteins involved in reducing reactive oxygen species accumulation were down-regulated. Dozens of genes encoding proteins with histone reader motifs were changed, including SmEED from the PRC2 complex. We targeted SmEZH2 methyltransferase PRC2 component with a new EZH2 inhibitor (GSK343) and showed a synergistic effect with TSA, significantly increasing schistosomula mortality.</p><p>Conclusions/Significance</p><p>Genome-wide gene expression analyses have identified important pathways and cellular functions that were affected and may explain the schistosomicidal effect of TSA HDACi. The change in expression of dozens of histone reader genes involved in regulation of the epigenetic program in <i>S</i>. <i>mansoni</i> can be used as a starting point to look for possible novel schistosomicidal targets.</p></div

ChIP-qPCR targeting H3K9ac and total H3 at promoter region of differentially expressed genes.

<p>Chromatin immunoprecipitation (ChIP) was performed with antibodies anti-H3K9ac (A) or anti-H3 (B) and DNA from schistosomula treated for 12 h with 1 μM TSA (solid bars) or with vehicle (open bars). DNA from the promoter regions of the indicated selected genes that was present in the immunoprecipitated fraction was quantified by qPCR with specific primers. Results are presented as % input DNA at the indicated target promoter region normalized by % input DNA at the promoter of the non-expressed reference gene <i>Sm</i>Val19 (Smp_123090), as described in the Methods. Three independent biological replicates were analyzed. Statistical significance of enrichment was evaluated using t-test; asterisk indicates <i>p</i>-value ≤ 0.05.</p

Cellular functions misregulated by HDACi after 48 h schistosomula treatment.

<p>IPA analysis pointed to the “cell movement of smooth muscle cells” cellular function (A) that could be functionally limited due to the down-regulated transcription of all involved genes. (B) The cellular function “control of the quantity of reactive oxygen species (ROS)” may be decreased in TSA-treated parasites due to the fact that 20 out of 22 genes that encode proteins responsible for reducing free radical generation are down-regulated in schistosomula upon TSA treatment, and 2 out of 4 genes that increase ROS are up-regulated. Genes down-regulated by the HDACi treatment are colored in green and genes up-regulated are colored in red. Arrows indicate the predicted effect downstream of the gene according to the gene expression pattern; blue arrow shows a predicted inhibition of the function indicated at the center; orange arrow shows a predicted increase of the cellular function indicated at the center, and yellow arrows show inconsistent prediction of the function according to the gene expression pattern. <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0005539#pntd.0005539.s005" target="_blank">S5 Table</a> gives the corresponding Smp gene information for each gene symbol present in this figure.</p

Histone H3 post-translational modifications profile in <i>S</i>. <i>mansoni</i> under HDAC inhibitor effect.

<p>Western blotting of the enriched histone protein fractions extracted from <i>S</i>. <i>mansoni</i> adult worms is shown for three biological replicates. Each biological replicate consisted of worms treated for 24 h with 1 μM TSA (lanes 2, 4 and 6) or with vehicle (lanes 1, 3 and 5). Blots were developed with (A) anti-H3K9ac, (B) anti-H3K14ac, and (C) anti-H3K27me3 antibodies. Additionally, antibody anti-H3 was used as a sample loading normalizer. Molecular weight marker ladder (L) is indicated. The graph at the lower part of each panel shows the mean intensity of the bands for the three biological replicates, obtained by extracting the intensity values of scanned images; for each sample, the intensity of the modified histone band was normalized by the intensity of histone H3. Mean ± SD is shown; t-test was applied and statistically significant <i>p</i>-values ≤ 0.05 are indicated with an asterisk.</p

Synergistic effect of HDACi and EZH2i on schistosomula viability.

<p>(A) Schistosomula were treated with drugs in five different conditions: control with vehicle (DMSO + Ethanol); 20 μM GSK343; 50 μM GSK343; 1 μM TSA and 1 μM TSA + 20 μM GSK343. Parasites were observed in the microscope for four days, and photographed with light and fluorescence microscopy. The number of dead parasites (stained in red with propidium iodide) was counted and compared with the total number of parasites (counted in the light microscope). The percentages of live viable schistosomula were calculated along the four days, with a different pool of parasites being stained and analyzed independently at each day. The experiment was performed with six biological replicates, and the mean +/- SD is shown; for each biological replicate, two technical replicates were obtained, and at least 100 schistosomula were counted for each technical replicate. Significant decrease in viability when comparing GSK343 alone and GSK343 plus TSA was assessed with a two-way ANOVA statistical test; asterisks indicate <i>p</i>-value <i>≤</i> 0.001. (B) Typical images at day 4 are shown. The calibration bar shows 100 μm.</p

Gene ontology categories enrichment for sustained differentially expressed genes detected as up-regulated or down-regulated in schistosomula, which were consistently affected across all the three time points of treatment with HDAC inhibitor (TSA).

<p>Gene ontology categories enrichment for sustained differentially expressed genes detected as up-regulated or down-regulated in schistosomula, which were consistently affected across all the three time points of treatment with HDAC inhibitor (TSA).</p

Enriched gene network involving DNA replication, recombination and repair affected by HDACi treatment of schistosomula.

<p>Ingenuity Pathway Analysis (IPA) identified of a significantly enriched gene network of differentially expressed genes, following a 24 h treatment of schistosomula with TSA. The network is associated with DNA replication, recombination and repair, and includes relevant genes such as those encoding the minichromosome maintenance (MCM) complex, Polymerases and Prereplication complex organization (ORC proteins). Up-regulated genes affected by TSA are colored in red and down-regulated genes are colored in green; shading intensity is according to the Log₂ ratio (treated/control) value. Solid lines indicate direct interactions and dashed lines indicate indirect interactions between molecules. <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0005539#pntd.0005539.s004" target="_blank">S4 Table</a> gives the corresponding Smp gene information for each gene symbol present in this figure.</p

Docking pose of GSK343 in SmEZH2 and specific structural interactions of ligands.

<p>(A) Homology model of the SET domain of SmEZH2 colored red superimposed with hEHZ2 template colored blue, with docking pose of GSK343 (yellow) at the secondary pocket of SmEZH2. The orange spheres represent SO₄ groups and grey spheres represent zinc ions. (B) Blown-up image of the secondary pocket of SmEZH2. Two-dimensional schematic overview of structural interactions observed in the SmEZH2 secondary pocket with (C) GSK343 or (D) cofactor SAM are shown at distances lower than 3.5 Å, with hydrophobic contacts represented by a red arc with spokes radiating towards the ligand atoms they contact and hydrogen bonds indicated by green dashed lines between the atoms involved.</p