Inhibition of histone deacetylase activity is a novel function of the antifolate drug methotrexate

Methotrexate (MTX) is a dihydrofolate reductase (DHFR) inhibitor widely used for treating human cancers, and overexpression of histone deacetylase (HDAC) is usually found in tumors. HDAC inhibitors (HDACi) can reactivate tumor suppressor genes and serve as potential anti-cancer drugs. In this study, we found that MTX shared structural similarity with some HDACi and molecular modeling showed that MTX indeed docks into the active site of HDLP, a bacterial homologue of HDAC. Subsequent in vitro assay demonstrated MTX’s inhibition on HDAC activity in human cancer cells. The global acetylation of histone H3 was also induced by MTX. Moreover, MTX inhibited immunoprecipitated HDAC1/2 activity but not their protein levels. This study provides evidence that MTX inhibits HDAC activity.     

Differentiation of eosinophilic leukemia EoL-1 cells into eosinophils induced by histone deacetylase inhibitors

EoL-1 cells differentiate into eosinophils in the presence of n-butyrate, but the mechanism has remained to be elucidated. Because n-butyrate can inhibit histone deacetylases, we hypothesized that the inhibition of histone deacetylases induces the differentiation of EoL-1 cells into eosinophils. In this study, using n-butyrate and two other histone deacetylase inhibitors, apicidin and trichostatin A, we have analyzed the relationship between the inhibition of histone deacetylases and the differentiation into eosinophils in EoL-1 cells. It was demonstrated that apicidin and n-butyrate induced a continuous acetylation of histones H4 and H3, inhibited the proliferation of EoL-1 cells without attenuating the level of FIP1L1-PDGFRA mRNA, and induced the expression of markers for mature eosinophils such as integrin beta7, CCR1, and CCR3 on EoL-1 cells, while trichostatin A evoked a transient acetylation of histones and induced no differentiation into eosinophils. These findings suggest that the continuous inhibition of histone deacetylases in EoL-1 cells induces the differentiation into mature eosinophils.     

Discovery of indole-3-butyric acid derivatives as potent histone deacetylase inhibitors

In discovery of HDAC inhibitors (HDACIs) with improved anticancer potency, structural modification was performed on the previous derived indole-3-butyric acid derivative. Among all the synthesised compounds, molecule I13 exhibited high HDAC inhibitory and antiproliferative potencies in the in vitro investigations. The IC₅₀ values of I13 against HDAC1, HDAC3, and HDAC6 were 13.9, 12.1, and 7.71 nM, respectively. In the cancer cell based screening, molecule I13 showed increased antiproliferative activities in the inhibition of U937, U266, HepG2, A2780, and PNAC-1 cells compared with SAHA. In the HepG2 xenograft model, 50 mg/kg/d of I13 could inhibit tumour growth in athymic mice compared with 100 mg/kg/d of SAHA. Induction of apoptosis was revealed to play an important role in the anticancer potency of molecule I13. Collectively, a HDACI (I13) with high anticancer activity was discovered which can be utilised as a lead compound for further HDACI design.     

Discovery of potent histone deacetylase inhibitors with modified phenanthridine caps

In discovery of novel HDAC inhibitory with anticancer potency, pharmacophores of phenanthridine were introduced to the structure of HDAC inhibitors. Fatty and aromatic linkers were evaluated for their solubility and activity. Both enzyme inhibitory and in vitro antiproliferative (against U937 cells) screening results revealed better activities of compounds with aromatic linker than molecules with fatty linker. Compared with SAHA (IC₅₀ values of 1.34, 0.14, 2.58, 0.67 and 18.17 µM), molecule Fb-4 exhibited 0.87, 0.09, 0.32, 0.34 and 17.37 µM of IC₅₀ values against K562, U266, MCF-7, U937 and HEPG2 cells, respectively. As revealed by cell cycle and apoptotic analysis, induction of G2/M phase arrest and apoptosis plays an important role in the inhibition of MCF-7 cells by Fb-4. Generally, a potent HDAC inhibitor was developed in the present study which could be utilised as a lead compound for further anticancer drug design.     

Development of novel ferulic acid derivatives as potent histone deacetylase inhibitors

Histone deacetylase inhibitors (HDACIs) offer a promising strategy for cancer therapy. The discovery of potent ferulic acid-based HDACIs with hydroxamic acid or 2-aminobenzamide group as zinc binding group was reported. The halogeno-acetanilide was introduced as novel surface recognition moiety (SRM). The majority of title compounds displayed potent HDAC inhibitory activity. In particular, FA6 and FA16 exhibited significant enzymatic inhibitory activities, with IC₅₀ values of 3.94 and 2.82 μM, respectively. Furthermore, these compounds showed moderate antiproliferative activity against a panel of human cancer cells. FA17 displayed promising profile as an antitumor candidate. The results indicated that these ferulic acid derivatives could serve as promising lead compounds for further optimization.     

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.     

Zinc binding groups for histone deacetylase inhibitors

Zinc binding groups (ZBGs) play a crucial role in targeting histone deacetylase inhibitors (HDACIs) to the active site of histone deacetylases (HDACs), thus determining the potency of HDACIs. Due to the high affinity to the zinc ion, hydroxamic acid is the most commonly used ZBG in the structure of HDACs. An alternative ZBG is benzamide group, which features excellent inhibitory selectivity for class I HDACs. Various ZBGs have been designed and tested to improve the activity and selectivity of HDACIs, and to overcome the pharmacokinetic limitations of current HDACIs. Herein, different kinds of ZBGs are reviewed and their features have been discussed for further design of HDACIs.     

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.     

Sensitization of mesothelioma to TRAIL apoptosis by inhibition of histone deacetylase: role of Bcl-xL down-regulation

The TNF-related apoptosis-inducing ligand (TRAIL) is an immunological inducer of apoptosis selectively killing many, but not all, cancer cells. Malignant mesothelioma (MM) is fatal neoplasia with no current treatment, most likely due to high resistance of MM cells towards inducers of apoptosis, including TRAIL. We studied whether inhibition of histone deacetylase (HDAC), recently shown to sensitize malignant cells to a variety of apoptogenic substances, renders MM cells susceptible to TRAIL. Indeed, sub-apoptotic doses of the HDAC inhibitor suberohydroxamic acid (SBHA) sensitized MM cells to TRAIL apoptosis. Of the apoptotic mediators tested, the anti-apoptotic protein Bcl-x(L) was strongly down-regulated by combined treatment of the cells with SBHA and TRAIL but not by the HDAC inhibitor alone, while little or no change in the expression of other Bcl-2 family members highly expressed in MM cells, including Mcl-1 and Bax, was observed. Our data suggest a cross-talk between HDAC inhibition and TRAIL that results in modulation of expression of specific apoptotic mediators, and point to the potential of their combinatorial use in treatment of TRAIL-resistant neoplastic disease.     

Downregulation of histone deacetylase 1 by microRNA-520h contributes to the chemotherapeutic effect of doxorubicin

Doxorubicin induces DNA damage to exert its anti-cancer function. Histone deacetylase 1 (HDAC1) can protect the genome from DNA damage. We found that doxorubicin specifically downregulates HDAC1 protein expression and identified HDAC1 as a target of miR-520h, which was upregulated by doxorubicin. Doxorubicin-induced cell death was impaired by exogenous HDAC1 or by miR-520h inhibitor. Moreover, HDAC1 reduced the level of γH2AX by preventing the interaction of doxorubicin with DNA. In summary, doxorubicin downregulates HDAC1 protein expression, by inducing the expression of HDAC1-targeting miR-520h, to exacerbate DNA–doxorubicin interaction. The upregulation of HDAC1 protein may contribute to drug resistance of human cancer cells and targeting HDAC1 is a promising strategy to increase the clinical efficacy of DNA damage-inducing chemotherapeutic drugs.     

Transcriptional regulation of cholesterol 24-hydroxylase by histone deacetylase inhibitors

The mechanistic basis for the tissue specific expression of cholesterol elimination pathways is poorly understood. To gain additional insight into this phenomenon we considered it of interest to investigate if epigenetic mechanisms are involved in the regulation of the brain-specific enzyme cholesterol 24-hydroxylase (CYP46A1), a key regulator of brain cholesterol elimination. We demonstrated a marked time-dependent derepression of the expression of CYP46A1, in response to treatment with the potent histone deacetylase (HDAC) inhibitor Trichostatin A. The pattern of expression of the genes in the genomic region surrounding CYP46A1 was found to be diametrically opposite in brain and liver. Intraperitoneal injection of HDAC inhibitors in mice led to a significant derepression of hepatic Cyp46a1 mRNA expression and tissue specific changes in Hmgcr and Cyp39a1 mRNA expression. These results are discussed in the context of the phenomenology of tissue specific cholesterol balance.     

Search for novel histone deacetylase inhibitors. Part II: Design and synthesis of novel isoferulic acid derivatives

Previously, we described the discovery of potent ferulic acid-based histone deacetylase inhibitors (HDACIs) with halogeno-acetanilide as novel surface recognition moiety (SRM). In order to improve the affinity and activity of these HDACIs, twenty seven isoferulic acid derivatives were described herein. The majority of title compounds displayed potent HDAC inhibitory activity. In particular, IF5 and IF6 exhibited significant enzymatic inhibitory activities, with IC₅₀ values of 0.73 ± 0.08 and 0.57 ± 0.16 μM, respectively. Furthermore, these compounds showed moderate antiproliferative activity against human cancer cells. Especially, IF6 displayed promising profile as an antitumor candidate with IC₅₀ value of 3.91 ± 0.97 μM against HeLa cells. The results indicated that these isoferulic acid derivatives could serve as promising lead compounds for further optimization.     

Copper induces histone hypoacetylation through directly inhibiting histone acetyltransferase activity

The abnormal accumulation of Cu2+ is closely correlated with the incidence of different diseases, such as Alzheimer's disease and Wilson disease. To study in vivo functions of Cu2+ will lead to a better understanding of the nature of these diseases. In the present study, effect of Cu2+ on histone acetylation was investigated in human hepatoma cells. Exposure of cells to Cu2+ resulted in a significant decrease of histone acetylation, as indicated by the decrease of the overall histone acetylation and the decrease of histone H3 and H4 acetylation. Since histone acetyltransferase (HAT) and histone deacetylase (HDAC) are the enzymes controlled the state of histone acetylation in vivo, we tested their contribution to the inhibition of Cu2+ on histone acetylation. One hundred nanomolar trichostatin A, the specific inhibitor of HDAC, did not attenuate the inhibitory effect of Cu2+ on histone acetylation. Combined with that Cu2+ showed no effect on the in vitro activity of HDAC, these results led to the conclusion that it is HAT, but not HDAC that is involved in Cu2+ -induced histone hypoacetylation. This conclusion was confirmed by the facts that (1) Cu2+ significantly inhibited the in vitro activity of HAT, (2) Cu2+ -treated cells possessed a lower HAT activity than control cells, and (3) 50 or 100 microM bathocuproine disulfonate, a chelator of Cu2+, significantly attenuated the inhibition of Cu2+ on HAT activity and histone acetylation in the similar pattern. Combined with that Cu2+ showed no or obvious cytotoxicity at 100 or 200 microM in human hepatoma cells, and the previous study that Cu2+ inhibits the histone H4 acetylation of yeast cells at nontoxic or toxic levels, the data presented here suggest that inhibiting histone acetylation is probably one general in vivo function of Cu2+, where HAT is its molecular target.     

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。     

An efficient synthesis of SK-658 and its analogs as potent histone deacetylase inhibitors

SK-658 is a potent histone deacetylase (HDAC) inhibitor that showed higher activity than SAHA due to the presence of extended hydrophobic group. We designed and synthesized thioester and SS-hybrid bearing SK-658 analogs as HDAC inhibitors. All the compounds were active in nano molar range and showed higher inhibitory activity than SAHA and SK-658. Among these, disulfide compounds showed the highest activity.