Novel α,β-unsaturated hydroxamic acid derivatives overcome cisplatin resistance

A series of α,β-unsaturated hydroxamic acid derivatives as novel HDAC inhibitors (HDACi) with structural modifications of the connecting unit and the CAP group was synthesized. The in vitro evaluation against the human cancer cell lines A2780 and Cal27 identified 6e and 7j as the most potent compounds regarding HDAC inhibitory activity and inhibition of proliferation. Isoform profiling against HDAC2, 4, 6 and 8 revealed a preference for HDAC2 and 6 for both compounds in contrast to the pan HDACi panobinostat. 6e and 7j enhanced significantly cisplatin-induced cytotoxicity in a combination treatment mediated by increased apoptosis induction and caspase-3/7 activation. The interaction between 6e or 7j and cisplatin was highly synergistic and more pronounced for the cisplatin resistant subline Cal27CisR. IC₅₀ values of cisplatin were even lower in Cal27CisR pretreated with 6e or 7j than for the parental cell line Cal27. Based on our findings, the novel dual class I/HDAC6 inhibitors could serve as an option to overcome cisplatin resistance with fewer side effects in comparison to panobinostat.     

组蛋白去乙酰基转移酶2在疾病中的研究进展

HDAC2是Ⅰ类组蛋白去乙酰基转移酶,能够促进激素和依赖细胞因子的信号转导.HDAC2是口腔癌、前列腺癌、卵巢癌、子宫内膜癌以及胃癌特异的肿瘤标记物;对肌肉和心脏相关疾病,胚胎发育、神经功能都有重要影响.药物研发和药物介入技术选择HDAC2为精确的靶标已经证明是一个有效的治疗方法.本文对HDAC2在正常和相关疾病中的作用进行了总结.     

His/GST-HDAC1在大肠杆菌中表达的研究

目的:对His/GST-HDAC1在大肠杆菌BL-21中的表达进行研究。方法:HDAC1的完整基因片断被克隆到pColdⅠ载体和pGEX载体上,并在其N末端分别联有His和GST;采用大肠杆菌BL21对HDAC1进行表达;采用亲和色谱对HDAC1进行纯化;用SDS-PAGE和蛋白质印迹来验证表达和纯化效果;对HDAC1活性进行测定。结果:多数HDAC1存在于大肠杆菌BL-21细胞裂解液的沉淀组分和纯化过程中的未结合组分中,小部分HDAC1可从细胞裂解液的上清液中得以纯化,但未显现出酶活性。用FPLC对HDAC1进行进一步分离,结果表明,HDAC1发生了分子聚集,使得它们的分子量大于正常分子量。结论:活性His/GST-HDAC1不能用大肠杆菌BL21成功表达。     

Acetanilide and bromoacetyl-lysine derivatives as activators for human histone deacetylase 8

In the current study, seven compounds (i.e. 1–7) were found to be novel activators for the N^ε-acetyl-lysine deacetylation reaction catalyzed by human histone deacetylase 8 (HDAC8). When assessed with the commercially available HDAC8 peptide substrate Fluor-de-Lys®-HDAC8 that harbors the unnatural 7-amino-4-methylcoumarin (AMC) residue immediately C-terminal to the N^ε-acetyl-lysine residue to be deacetylated, our compounds exhibited comparable activation potency to that of TM-2-51, the strongest HDAC8 activator reported in the current literature. However, when assessed with an AMC-less peptide substrate derived from the native HDAC8 non-histone substrate protein Zinc finger protein ZNF318, while our compounds were all found to be able to activate HDAC8 deacetylation reaction, TM-2-51 was found not to be able to. Our compounds also seemed to be largely selective for HDAC8 over other classical HDACs. Moreover, treatment with the strongest activator among our compounds (i.e. 7) was found to decrease the K_M of the above AMC-less HDAC8 substrate, while nearly maintaining the k_cat of the HDAC8-catalyzed deacetylation on this substrate.     

Discovery of histone deacetylase 8 selective inhibitors

We have developed an efficient method for synthesizing candidate histone deacetylase (HDAC) inhibitors in 96-well plates, which are used directly in high-throughput screening. We selected building blocks having hydrazide, aldehyde and hydroxamic acid functionalities. The hydrazides were coupled with different aldehydes in DMSO. The resulting products have the previously identified ‘cap/linker/biasing element’ structure known to favor inhibition of HDACs. These compounds were assayed without further purification. HDAC8-selective inhibitors were discovered from this novel collection of compounds.     

Inhibitors of histone deacetylase 6 based on a novel 3-hydroxy-isoxazole zinc binding group

Histone deacetylase 6 (HDAC6) is an established drug target for cancer treatment. Inhibitors of HDAC6 based on a hydroxamic acid zinc binding group (ZBG) are often associated with undesirable side effects. Herein, we describe the identification of HDAC6 inhibitors based on a completely new 3-hydroxy-isoxazole ZBG. A series of derivatives decorated with different aromatic or heteroaromatic linkers, and various cap groups were synthesised and biologically tested. In vitro tests demonstrated that some compounds are able to inhibit HDAC6 with good potency, the best candidate reaching an IC₅₀ of 700 nM. Such good potency obtained with a completely new ZBG make these compounds particularly attractive. The effect of the most active inhibitors on the acetylation levels of histone H3 and α- tubulin and their anti-proliferative activity of DU145 cells were also investigated. Docking studies were performed to evaluate the binding mode of these new derivatives and discuss structure-activity relationships.     

A homogeneous cellular histone deacetylase assay suitable for compound profiling and robotic screening

Most cellular assays that quantify the efficacy of histone deacetylase (HDAC) inhibitors measure hyperacetylation of core histone proteins H3 and H4. Here we describe a new approach, directly measuring cellular HDAC enzymatic activity using the substrate Boc-K(Ac)-7-amino-4-methylcoumarin (AMC). After penetration into HeLa cervical carcinoma or K562 chronic myeloid leukemia cells, the deacetylated product Boc-K-AMC is formed which, after cell lysis, is cleaved by trypsin, finally releasing the fluorophor AMC. The cellular potency of suberoylanilide hydroxamic acid, LBH589, trichostatin A, and MS275 as well-known HDAC inhibitors was determined using this assay. IC(50) values derived from concentration-effect curves correlated well with EC(50) values derived from a cellomics array scan histone H3 hyperacetylation assay. The cellular HDAC activity assay was adapted to a homogeneous format, fully compatible with robotic screening. Concentration-effect curves generated on a Tecan Genesis Freedom workstation were highly reproducible with a signal-to-noise ratio of 5.7 and a Z' factor of 0.88, indicating a very robust assay. Finally, a HDAC-inhibitor focused library was profiled in a medium-throughput screening campaign. Inhibition of cellular HDAC activity correlated well with cytotoxicity and histone H3 hyperacetylation in HeLa cells and with inhibition of human recombinant HDAC1 in a biochemical assay. Thus, by using Boc-K(Ac)-AMC as a cell-permeable HDAC substrate, the activity of various protein lysine-specific deacetylases including HDAC1-containing complexes is measurable in intact cells in a simple and homogeneous manner.     

Histone deacetylase modulators provided by Mother Nature

Protein acetylation status results from a balance between histone acetyltransferase and histone deacetylase (HDAC) activities. Alteration of this balance leads to a disruption of cellular integrity and participates in the development of numerous diseases, including cancer. Therefore, modulation of these activities appears to be a promising approach for anticancer therapy. Histone deacetylase inhibitors (HDACi) are epigenetically active drugs that induce the hyperacetylation of lysine residues within histone and non-histone proteins, thus affecting gene expression and cellular processes such as protein–protein interactions, protein stability, DNA binding and protein sub-cellular localization. Therefore, HDACi are promising anti-tumor agents as they may affect the cell cycle, inhibit proliferation, stimulate differentiation and induce apoptotic cell death. Over the last 30 years, numerous synthetic and natural products, including a broad range of dietary compounds, have been identified as HDACi. This review focuses on molecules from natural origins modulating HDAC activities and presenting promising anticancer activities.     

Combination of isoform-selective histone/protein deacetylase inhibitors improves Foxp3+ T-regulatory cell function

Comment on: Beier UH, et al. Sci Signal 2012; 5:ra45.     

Up‐regulation of HDACs,a harbinger of uraemic endothelial dysfunction,is prevented by defibrotide

Endothelial dysfunction is an earlier contributor to the development of atherosclerosis in chronic kidney disease (CKD), in which the role of epigenetic triggers cannot be ruled out. Endothelial protective strategies, such as defibrotide (DF), may be useful in this scenario. We evaluated changes induced by CKD on endothelial cell proteome and explored the effect of DF and the mechanisms involved. Human umbilical cord vein endothelial cells were exposed to sera from healthy donors (n = 20) and patients with end‐stage renal disease on haemodialysis (n = 20). Differential protein expression was investigated by using a proteomic approach, Western blot and immunofluorescence. HDAC1 and HDAC2 overexpression was detected. Increased HDAC1 expression occurred at both cytoplasm and nucleus. These effects were dose‐dependently inhibited by DF. Both the HDACs inhibitor trichostatin A and DF prevented the up‐regulation of the endothelial dysfunction markers induced by the uraemic milieu: intercellular adhesion molecule‐1, surface Toll‐like receptor‐4, von Willebrand Factor and reactive oxygen species. Moreover, DF down‐regulated HDACs expression through the PI3/AKT signalling pathway. HDACs appear as key modulators of the CKD‐induced endothelial dysfunction as specific blockade by trichostatin A or by DF prevents endothelial dysfunction responses to the CKD insult. Moreover, DF exerts its endothelial protective effect by inhibiting HDAC up‐regulation likely   through PI3K/AKT.     

可逆的组蛋白乙酰化调控p16的表达

p16^INK4a通过抑制CDK4/6的活性而在细胞周期进行中发挥重要的作用,研究发现,组蛋白乙酰转移酶p300能促进p16^INK4a启动子活性,而组蛋白去乙酰化酶HDAC3/4能够逆转由p300介导的p16^INK4a启动子活性的增加,HDAC3/4能够降低p16^INK4a mRNA和蛋白质的水平．染色质免疫沉淀(ChIP)实验结果表明转染p300表达质粒能够逆转由HDAC3/4介导的p16^INK4a启动子组蛋白的低乙酰化状态．此外,免疫荧光实验结果表明HDAC4的核质穿梭起着重要的作用．免疫印迹和染色质免疫沉淀实验证明HDAC的抑制剂丁酸钠盐(NaBu)能通过诱导组蛋白的高乙酰化而促进p16^INK4a的表达．基于这些实验结果,推测出可逆的组蛋白乙酰化参与p16^INK4a基因转录调控的模型．     

Synthesis and anticancer activities of thieno[3,2-d]pyrimidines as novel HDAC inhibitors

A series of thieno[3,2-d]pyrimidines bearing a hydroxamic acid moiety as novel HDAC inhibitors were designed and synthesized. The structures of the new synthesized compounds were confirmed using IR, ¹H, ¹³C NMR spectrum. Compounds 11–13 showed potent inhibitory activities against HDACs with IC₅₀ values at 0.38, 0.49 and 0.61 μM. Most of target compounds displayed strong anti-proliferative activity by a MTT assay on three human cancer cell lines including HCT-116, MCF-7 and HeLa. Compound 11, having potent inhibitory activities against HDACs, induced apoptosis and G2/M cell cycle arrest in HCT-116 cell line.     

Novel N-hydroxyfurylacrylamide-based histone deacetylase (HDAC) inhibitors with branched CAP group (Part 2)

Histone deacetylases (HDACs) are significant enzymes involved in tumor genesis and development. Herein, we report a series of novel N-hydroxyfurylacryl-amide-based HDAC inhibitors, which are marked by introducing branched hydrophobic groups as the capping group. The inhibitory activity of the synthesized compounds against HDACs and several tumor cell lines are firstly determined. Fifteen compounds with promising activities are selected for further evaluation of target selectivity profile against recombinant human HDAC1, HDAC4 and HDAC6. Compounds 10a, 10b, 10d and 16a exhibit outstanding selectivity against HDAC6. Analysis of HDAC4 X-ray structure and HDAC1, HDAC6 homology model indicates that these enzyme differ significantly in the rim near the surface of the active site. Although TSA has been known as a pan-HDAC inhibitor, it exhibits outstanding selectivity for HDAC6 over HDAC4. For further physicochemical properties study, six compounds are chosen for determination of their physicochemical properties including log D_7.4 and aqueous solubility. The results suggest that compounds with a smaller framework and with hydrophilicgroups are likely to have better aqueous solubility.     

Development and characterization of a CNS-penetrant benzhydryl hydroxamic acid class IIa histone deacetylase inhibitor

We have identified a potent, cell permeable and CNS penetrant class IIa histone deacetylase (HDAC) inhibitor 22, with >500-fold selectivity over class I HDACs (1,2,3) and ～150-fold selectivity over HDAC8 and the class IIb HDAC6 isoform. Dose escalation pharmacokinetic analysis demonstrated that upon oral administration, compound 22 can reach exposure levels in mouse plasma, muscle and brain in excess of cellular class IIa HDAC IC₅₀ levels for ～8?h. Given the interest in aberrant class IIa HDAC function for a number of neurodegenerative, neuromuscular, cardiac and oncology indications, compound 22 (also known as CHDI-390576) provides a selective and potent compound to query the role of class IIa HDAC biology, and the impact of class IIa catalytic site occupancy in vitro and in vivo.