Novel inhibitors of human histone deacetylases: Design,synthesis and bioactivity of 3-alkenoylcoumarines

Histone deacetylases (HDACs) are well-established, promising targets for anticancer therapy due to their critical role in cancer development. Accordingly, an increasing number of HDAC inhibitors displaying cytotoxic effects against cancer cells have been reported. Among them, a large panel of chemical structures was described including coumarin-containing molecules. In this study, we described synthesis and biological activity of new coumarin-based derivatives as HDAC inhibitors. Among eight derivatives, three compounds showed HDAC inhibitory activities and antitumor activities against leukemia cell lines without affecting the viability of peripheral blood mononuclear cells from healthy donors.     

The interplay between microRNAs and histone deacetylases in neurological diseases

Neurological conditions, such as Alzheimer’s disease and stroke, represent a prevalent group of devastating illnesses with few treatments. Each of these diseases or conditions is in part characterized by the dysregulation of many genes, including those that code for microRNAs (miRNAs) and histone deacetylases (HDACs). Recently, a complex relationship has been uncovered linking miRNAs and HDACs and their ability to regulate one another. This provides a new avenue for potential therapeutics as the ability to reinstate a careful balance between miRNA and HDACs has lead to improved outcomes in a number of in vitro and in vivo models of neurological conditions. In this review, we will discuss recent findings on the interplay between miRNAs and HDACs and its implications for pathogenesis and treatment of neurological conditions, including amyotrophic lateral sclerosis, Alzheimer’s disease, Huntington’s disease and stroke.     

Role of class I and class II histone deacetylases in carcinoma cells using siRNA

The role of the individual histone deacetylases (HDACs) in the regulation of cancer cell proliferation was investigated using siRNA-mediated protein knockdown. The siRNA for HDAC3 and HDAC1 demonstrated significant morphological changes in HeLa S3 consistent with those observed with HDAC inhibitors. SiRNA for HDAC 4 or 7 produced no morphological changes in HeLa S3 cells. HDAC1 and 3 siRNA produced a concentration-dependent inhibition of HeLa cell proliferation; whereas, HDAC4 and 7 siRNA showed no effect. HDAC3 siRNA caused histone hyperacetylation and increased the percent of apoptotic cells. These results demonstrate that the Class I HDACs such as HDACs 1 and 3 are important in the regulation of proliferation and survival in cancer cells. These results and the positive preclinical results with non-specific inhibitors of the HDAC enzymes provide further support for the development of Class I selective HDAC inhibitors as cancer therapeutics.     

Sequence and expression analysis of histone deacetylases in rice

Histone acetylation levels are determined by the action of histone acetyltransferases and histone deacetylases (HDACs). Sequence similarity and profile searching tools were used to analyze the genome sequence of rice (Oryzae sativa) for genes encoding HDAC proteins. The rice RPD3/HDA1-family HDAC proteins can be divided into four classes based on sequence similarity and phylogenetic analysis of sequences obtained from the rice genome. The spatial expression pattern of rice HDACs genes indicated that some HDAC genes have different expression profiles. Furthermore, our analysis indicated that expression of HDA705, HDT701, and HDT702 could be affected by salicylic acid, jasmonic acid or abscisic acid. Expression of HDA714, SRT702, and SRT701 could be modulated by abiotic stresses, such as cold, mannitol and salt. These results indicate that different HDAC genes have distinct expression patterns and members of rice HDAC families may be involved in plant response to environmental stresses.     

Factors affecting the substrate specificity of histone deacetylases

Histone deacetylases (HDACs) catalyze the deacetylation of epsilon-acetyl-lysine residues within the N-terminal tail of core histones and thereby mediate changes in the chromatin structure and regulate gene expression in eukaryotic cells. So far, surprisingly little is known about the substrate specificities of different HDACs. Here, we prepared a library of fluorogenic tripeptidic substrates of the general format Ac-P(-2)-P(-1)-Lys(Ac)-MCA (P(-1), P(-2)=all amino acids except cysteine) and measured their HDAC-dependent conversion in a standard fluorogenic HDAC assay. Different HDAC subtypes can be ranked according to their substrate selectivity: HDAH > HDAC8 > HDAC1 > HDAC3 > HDAC6. HDAC1, HDAC3, and HDAC6 exhibit a similar specificity profile, whereas both HDAC8 and HDAH have rather distinct profiles. Furthermore, it was shown that second-site modification (e.g., phosphorylation) of substrate sequences as well as corepressor binding can modulate the selectivity of enzymatic substrate conversion.     

小鼠组蛋白去乙酰化酶2多肽抗血清的制备

目的利用人工合成小鼠组蛋白去乙酰化酶2（histone deacetylase 2,HDAC2）多肽制备特异性抗HDAC2抗血清,用于相关疾病的体内外诊断。方法根据HDAC2基因编码的氨基酸序列合成多肽,与载体偶联后免疫动物,所制备的抗血清用ELISA、Western blot及免疫组织化学方法鉴定。结果 ELISA检测表明所制备的抗血清可同多肽抗原发生阳性反应,效价1∶4 000;Western blot结果显示抗血清可与多肽抗原及APP/PS1转基因小鼠的脑组织发生反应;免疫血清1∶100,1∶200,1∶400,1∶800四个稀释度均能与小鼠脑组织中的HDAC2反应。结论所制备的多肽抗血清可识别组织及血清中的HDAC2,可应用于相关领域的体内外研究。     

Application of p21 and klf2 reporter gene assays to identify selective histone deacetylase inhibitors for cancer therapy

Novel 2-aminoanilide histone deacetylase (HDAC) inhibitors were designed to increase their contact with surface residues surrounding the HDAC active site compared to the contacts made by existing clinical 2-aminoanilides such as SNDX-275, MGCD0103, and Chidamide. Their HDAC selectivity was assessed using p21 and klf2 reporter gene assays in HeLa and A204 cells, respectively, which provide a cell-based readout for the inhibition of HDACs associated either with the p21 or klf2 promoter. A subset of the designed compounds selectively induced p21 over klf2 relative to the clinical reference compound SNDX-275. A representative lead compound from this subset had antiproliferative effects in cancer cells associated with induction of acetylated histone H4, endogenous p21, cell cycle arrest, and apoptosis. The p21- versus klf2-selective compounds described herein may provide a chemical starting point for developing clinically-differentiated HDAC inhibitors for cancer therapy.     

Design,synthesis and preliminary bioactivity studies of 1,2-dihydrobenzo[d]isothiazol-3-one-1,1-dioxide hydroxamic acid derivatives as novel histone deacetylase inhibitors

Histone deacetylase (HDAC) is a clinically validated target for antitumor therapy. In order to increase HDAC inhibition and efficiency, we developed a novel series of saccharin hydroxamic acids as potent HDAC inhibitors. Among them, compounds 11e, 11m, 11p exhibited similar or better HDACs inhibitory activity compared with the approved drug SAHA. Further biological evaluation indicated that compound 11m had potent antiproliferative activities against MDA-MB-231 and PC-3.     

The histone deacetylases Rpd3 and Hst1 antagonistically regulate de novo NAD+ metabolism in the budding yeast Saccharomyces cerevisiae

NAD⁺ is a cellular redox cofactor involved in many essential processes. The regulation of NAD⁺ metabolism and the signaling networks reciprocally interacting with NAD⁺-producing metabolic pathways are not yet fully understood. The NAD⁺-dependent histone deacetylase (HDAC) Hst1 has been shown to inhibit de novo NAD⁺ synthesis by repressing biosynthesis of nicotinic acid (BNA) gene expression. Here, we alternatively identify HDAC Rpd3 as a positive regulator of de novo NAD⁺ metabolism in the budding yeast Saccharomyces cerevisiae. We reveal that deletion of RPD3 causes marked decreases in the production of de novo pathway metabolites, in direct contrast to deletion of HST1. We determined the BNA expression profiles of rpd3Δ and hst1Δ cells to be similarly opposed, suggesting the two HDACs may regulate the BNA genes in an antagonistic fashion. Our chromatin immunoprecipitation analysis revealed that Rpd3 and Hst1 mutually influence each other’s binding distribution at the BNA2 promoter. We demonstrate Hst1 to be the main deacetylase active at the BNA2 promoter, with hst1Δ cells displaying increased acetylation of the N-terminal tail lysine residues of histone H4, H4K5, and H4K12. Conversely, we show that deletion of RPD3 reduces the acetylation of these residues in an Hst1-dependent manner. This suggests that Rpd3 may function to oppose spreading of Hst1-dependent heterochromatin and represents a unique form of antagonism between HDACs in regulating gene expression. Moreover, we found that Rpd3 and Hst1 also coregulate additional targets involved in other branches of NAD⁺ metabolism. These findings help elucidate the complex interconnections involved in effecting the regulation of NAD⁺ metabolism.     

Role of histone deacetylase 2 and its posttranslational modifications in cardiac hypertrophy

Cardiac hypertrophy is a form of global remodeling, although the initial step seems to be an adaptation to increased hemodynamic demands. The characteristics of cardiac hypertrophy include the functional reactivation of the arrested fetal gene program, where histone deacetylases (HDACs) are closely linked in the development of the process. To date, mammalian HDACs are divided into four classes: I, II, III, and IV. By structural similarities, class II HDACs are then subdivided into IIa and IIb. Among class I and II HDACs, HDAC2, 4, 5, and 9 have been reported to be involved in hypertrophic responses; HDAC4, 5, and 9 are negative regulators, whereas HDAC2 is a pro-hypertrophic mediator. The molecular function and regulation of class IIa HDACs depend largely on the phosphorylation-mediated cytosolic redistribution, whereas those of HDAC2 take place primarily in the nucleus. In response to stresses, posttranslational modification (PTM) processes, dynamic modifications after the translation of proteins, are involved in the regulation of the activities of those hypertrophy-related HDACs. In this article, we briefly review 1) the activation of HDAC2 in the development of cardiac hypertrophy and 2) the PTM of HDAC2 and its implications in the regulation of HDAC2 activity. [BMB Reports 2015; 48(3): 131-138]     

Cotargeting histone deacetylases and oncogenic BRAF synergistically kills human melanoma cells by necrosis independently of RIPK1 and RIPK3

Past studies have shown that histone deacetylase (HDAC) and mutant BRAF (v-Raf murine sarcoma viral oncogene homolog B1) inhibitors synergistically kill melanoma cells with activating mutations in BRAF. However, the mechanism(s) involved remains less understood. Here, we report that combinations of HDAC and BRAF inhibitors kill BRAF^V600E melanoma cells by induction of necrosis. Cotreatment with the HDAC inhibitor suberoylanilide hydroxamic acid (SAHA) or panobinostat (LBH589) and the BRAF inhibitor PLX4720 activated the caspase cascade, but caspases appeared dispensable for killing, in that inhibition of caspases did not invariably block induction of cell death. The majority of dying cells acquired propidium iodide positivity instantly when they became positive for Annexin V, suggesting induction of necrosis. This was supported by caspase-independent release of high-mobility group protein B1, and further consolidated by rupture of the plasma membrane and loss of nuclear and cytoplasmic contents, as manifested by transmission electron microscopic analysis. Of note, neither the necrosis inhibitor necrostatin-1 nor the small interference RNA (siRNA) knockdown of receptor-interacting protein kinase 3 (RIPK3) inhibited cell death, suggesting that RIPK1 and RIPK3 do not contribute to induction of necrosis by combinations of HDAC and BRAF inhibitors in BRAF^V600E melanoma cells. Significantly, SAHA and the clinically available BRAF inhibitor vemurafenib cooperatively inhibited BRAF^V600E melanoma xenograft growth in a mouse model even when caspase-3 was inhibited. Taken together, these results indicate that cotreatment with HDAC and BRAF inhibitors can bypass canonical cell death pathways to kill melanoma cells, which may be of therapeutic advantage in the treatment of melanoma.     

Cu2+ is required for pyrrolidine dithiocarbamate to inhibit histone acetylation and induce human leukemia cell apoptosis

Pyrrolidine dithiocarbamate (PDTC) has been considered as a potential anticancer drug due to its powerful apoptogenic effect towards cancer cells, where Cu(2+) plays a distinct yet undefined role. Here we report that Cu(2+) is critically needed for PDTC to inhibit histone acetylation in both human leukemia HL-60 cells and human hepatoma Hep3B cells. The inhibition of histone acetylation mainly resulted from the increase of intracellular Cu(2+), but was not due to the inhibition of NF-kappaB activity by PDTC-Cu(2+) since the combinations of Cu(2+) with SN50, MG132 (two known NF-kappaB inhibitors), or bathocuproine disulfonate (BCS, a specific Cu(2+) chelator that does not cross the plasma membrane), did not lead to obvious inhibition of histone acetylation. Histone acetyltransferase (HAT) and histone deacetylase (HDAC) are the enzymes controlling the state of histone acetylation in vivo. Cells exposed to PDTC-Cu(2+) showed a comparable decrease in histone acetylation levels in HL-60 cells in the absence or presence of the HDAC inhibitors, trichostatin A (TSA) or sodium butyrate (NaBu); the inhibition rates were about 45, 44 and 43%, respectively. PDTC-Cu(2+) had no effect on the activity of HDAC in vitro, but significantly inhibited the HAT activity both in HL-60 cells and in a cell-free in vitro system. PDTC-Cu(2+) also induced HL-60 cell apoptosis, and treating cells with TSA, NaBu or BCS significantly attenuated the apoptosis induced by PDTC-Cu(2+). Collectively, these results showed that inhibition of histone acetylation represents a distinct mechanism for the cytotoxicity of PDTC in the presence of Cu(2+), where HAT is its possible molecular target.