Structure-based optimization of click-based histone deacetylase inhibitors

Previously, we reported a click-chemistry based approach to the synthesis of a novel class of histone deacetylase (HDAC) inhibitors [1]. The lead compound NSC746457 was found to be as potent as SAHA (Vorinostat). Further optimization of NSC746457 by using the HDAC2-TSA crystal structure is described herein. Docking of NSC746457 into HDAC2 binding domain suggested that the hydrophobic residue Phe210 flanking the cap-group binding-motif could be exploited for structural optimization. Substitution on the methylene group of cinnamic cap region led to identification of more potent HDAC inhibitors: isopropyl derivative 5 and tert-butyl derivative 6, with an IC₅₀ value of 22 nM and 18 nM, respectively.     

Design, synthesis and primary activity assay of tripeptidomimetics as histone deacetylase inhibitors with linear linker and branched cap group

A novel series of tripeptidomimetics with spiro ring containing sulfur atoms as cap group and linear carbochain as linker was designed and synthesized as HDACs inhibitors. Several compounds possessed more potent HDAC8 inhibitory activity than clinically used drug SAHA, although their HDAC1 inhibitory activities and anti-proliferative activities against human breast cancer cell lines (MCF-7, MDA-MB-231) and prostate cancer cell line (PC-3) were not satisfactory. Among them, compounds 11l and 11k showed excellent potency against HDAC8 (IC₅₀ values were 0.021 ± 0.004 μM and 0.035 ± 0.007 μM, respectively, whereas SAHA was 0.70 ± 0.12 μM), and good selectivity over HDAC1. Up to now, few hydroxamic acid derivatives with linear linker were reported to possess HDAC8 selectivity over HDAC1.     

Novel amide derivatives as inhibitors of histone deacetylase: design, synthesis and SAR

Enzymatic inhibition of histone deacetylase (HDAC) activity is emerging as an innovative and effective approach for the treatment of cancer. A series of novel amide derivatives have been synthesized and evaluated for their ability to inhibit human HDACs. Multiple compounds were identified as potent HDAC inhibitors (HDACi), with IC(50) values in the low nanomolar (nM) range against enzyme activity in HeLa cell extracts and sub-microM for their in vitro anti-proliferative effect on cell lines. The introduction of an unsaturated linking group between the terminal aryl ring and the amide moiety was the key to obtain good potency. This approach yielded compounds such as (E)-N-[6-(hydroxyamino)-6-oxohexyl]-3-(7-quinolinyl)-2-propenamide (27) (HDAC IC(50) 8 nM) which showed potent in vivo activity in the P388 mouse leukemia syngeneic model (an increased lifespan (ILS) of 111% was obtained).     

Histone-deacetylase inhibition and butyrate formation: Fecal slurry incubations with apple pectin and apple juice extracts

OBJECTIVE: Butyrate plays a major role among the short-chained fatty acids formed by the microbial flora of the colon. It is considered to be an important nutrient of the colon mucosa and has been shown to trigger differentiation and apoptosis of colon-derived cells in culture. Inhibition of histone deacetylase (HDAC) seems to play a central role in these effects. Butyrate was thus suggested to act as a chemopreventive metabolite that can prevent the occurrence of colorectal cancer, one of the most abundant types of cancer in Western industrialized countries. Some polymeric carbohydrates such as pectin, resistant to digestion in the small intestine, have been shown to serve as substrates for butyrate formation by the microflora of the colon. METHODS: In this study we investigated fermentation supernatants (FSs) from incubations of human fecal slurry with apple pectin and with polyphenol-rich apple juice extracts (AJEs). RESULTS: We found that FSs from fermentations with pectin were rich in butyrate and very active in HDAC inhibition in nuclear extracts prepared from the colon tumor cell lines HT-29 and Caco-2 and in intact HeLa Mad 38 cells bearing a reporter gene driven by HDAC inhibition. The butyrate levels explained most of the HDAC-inhibitory potency in FSs from pectin-rich fermentations. FSs from fermentations with AJEs showed lower butyrate yields but comparable HDAC inhibition. Combined incubations of pectin with AJEs led to effects similar to those with FSs from incubations with pectin as the only substrate added. These effects could not be explained by a direct HDAC-inhibitory potency of AJEs. Furthermore, the FSs were not cytotoxic at the HDAC-inhibitory concentrations. CONCLUSION: These findings suggest that butyrate is the most relevant HDAC inhibitor formed in fermentations of human fecal slurry with apple pectin, whereas addition of AJEs leads to the formation of butyrate and other, yet unknown, HDAC inhibitors.     

Discovery of polyoxometalate-based HDAC inhibitors with profound anticancer activity in vitro and in vivo

We obtained 5 positive novel histone deacetylase inhibitors (HDACIs) from a polyoxometalate (POM) library by using a cell-based screening system targeting the p21 gene promoter. Among them, PAC-320, a new tri-organic-tin-substitute germanotungstate, displayed remarkable extracellular inhibitory activity. Meanwhile, the crystal structure of PAC-320 was characterized by X-ray crystallography. PAC-320 could stably exist under physiological conditions as revealed by UV spectrum, CV and TG. PAC-320 possessed a strong inhibitory effect to intracellular HDAC activity. More significantly, PAC-320 inhibited the growth of a variety of cancer cells, and exhibited remarkable anticancer effect in a hepatocarcinoma H22 cell mice model. This study revealed, for the first time, that the HDAC inhibitory activity is a mechanism by which POMs exert their anticancer effect.     

Cloning,purification, and homology modeling of Histone deacetylase in Leishmania donovani

Neglected diseases, such as leishmaniasis, are still a major health problem in poor countries. To date, there is a severe lack of effective, safe, and affordable treatment for leishmaniasis. Currently, there are very limited chemotherapeutic options, and the development of vaccines is still underway. Hence, novel therapeutic strategies need to be developed against leishmanial parasites. Histone deacetylases (HDACs), silent regulators of many critical pathways, have been validated as potential therapeutic targets in cancer and several parasitic diseases. In the present work, we have isolated and characterized biologically active Zn²⁺-dependent HDAC protein from leishmania that can be studied further as a potential anti-leishmanial drug target to develop new therapies against neglected diseases. The nucleotide sequence of the HDAC gene with no intervening sequence was amplified, cloned in a pET-28a vector, and later transformed into the BL21(DE3) competent E. coli bacterial cells. After transformation, the cells were cultured and induced with 0.6 mM of IPTG to express histidine-tagged HDAC protein (LD_HDAC), which was later purified using nickel affinity chromatography. The approximate protein size confirmed with the help of 10% SDS–PAGE was ~48.0 kDa. The enzymatic assay using the purified protein confirmed it as biologically active. A three dimensional structure of LD_HDAC was modeled using the crystal structure of HDAC2 protein of Homo sapiens (PDB ID: 6G3O). This protein can be utilized for the screening of Leishmania-specific HDAC inhibitors.     

Synthesis and evaluation of aliphatic-chain hydroxamates capped with osthole derivatives as histone deacetylase inhibitors

Our previous studies have demonstrated that osthole, a Chinese herbal compound, could be incorporated into the hydroxycinnamide scaffold of LBH-589, a potent HDAC inhibitor, as an effective hydrophobic cap; the resulting compounds showed significant potency against several HDAC isoforms. Here, we presented a series of osthole derivatives fused with the aliphatic-hydroxamate core of suberoylanilide hydroxamic acid (SAHA), a clinically-approved HDAC inhibitor. Several compounds showed potent activity against nuclear HDACs. Further assays against individual HDAC isoforms revealed that some compounds showed not only SAHA-like activity towards HDAC1, -4 and -6, they inhibited HDAC8 by log difference than SAHA and thus exhibited a broader HDAC inhibition spectrum. Among them, compound 6g showed potent antiproliferative effect on several human cancer cell lines.     

β-羟基丁酸通过抑制HDAC1/3上调β样淀粉肽处理的SH-SY5Y细胞TrkA的表达

目的 探讨β-羟基丁酸(β-hydroxybutyrate,BHB)对β样淀粉肽(β-amyloidpeptide,Aβ)处理的神经细胞保护作用及其可能的机制,为防治阿尔茨海默病(Alzheimer’s disease,AD)提供依据。 方法 将体外培养的SH-SY5Y细胞进行分组,单纯BHB组、BHB干预组细胞以终浓度为5 mM BHB预处理3 h,再向Aβ处理组、BHB干预组细胞加入终浓度为20 μM的Aβ,同时设立对照组,24 h后收集细胞;采用qRT-PCR、Western Blot法检测各组细胞TrkA、HDAC1和HDAC3 mRNA及其蛋白相对表达水平。以siRNA沉默HDAC1/3,分析细胞TrkA mRNA及其蛋白相对表达水平。 结果 与对照组相比,单纯BHB组细胞的TrkA mRNA及其蛋白相对表达水平明显升高(P<0.05)、HDAC1和HDAC3 mRNA及其蛋白相对表达水平显著降低(P<0.05),Aβ组细胞TrkA mRNA及其蛋白相对表达水平显著降低(P<0.01)、HDAC1和HDAC3 mRNA及其蛋白相对表达水平显著升高(P<0.01);与Aβ组相比,BHB干预组TrkAmRNA及其蛋白相对表达水平显著升高(P<0.01)、 HDAC1和HDAC3 mRNA及其蛋白相对表达水平显著降低(P<0.01)。沉默HDAC1/3后细胞TrkA mRNA及其蛋白相对表达水平显著升高(P<0.05)。 结论 BHB可通过抑制HDAC1/3,上调Aβ处理的SH-SY5Y细胞TrkA的表达。     

宫颈病变中HDAC1的表达以及与HPV16感染的关系

目的:探讨HDAC1的异常表达以及HPV16感染与宫颈癌发病的相关性。方法:用PCR技术及免疫组织化学(SP)方法检测110例慢性宫颈炎组织及宫颈上皮内瘤样变组织和宫颈鳞癌组织中HPV16感染以及HDAC1的表达。结果:在慢性宫颈炎组、CINⅠ组、CINⅡ组、CINⅢ组和宫颈癌组中HPV16的感染率分别为10.0%、36.4%、50.0%、66.7%、75.0%,组间差异有统计学意义(P<0.05);HDAC1阳性表达率分别为0、27.3%、41.7%、54.2%和70.0%,各组间差异有统计学意义(P<0.05)。HPV16感染组的HDAC1阳性表达率高于HPV16未感染组,差异有统计学意义(P<0.05)。结论:HPV16感染可能是通过提高组蛋白去乙酰化酶的水平抑制转录,在宫颈癌的发生、发展中起作用。     

HDAC1在燃煤污染型砷中毒患者血液及皮肤组织中的转录及表达

[目的]了解组蛋白去乙酰化酶1（HDAC1）在燃煤污染型砷中毒患者血液及皮肤组织中的转录及表达,探讨其在砷中毒发生、发展乃至癌变中的作用。[方法]在贵州省燃煤污染型地方性砷中毒病区,选择68例地方性砷中毒（以下简称“砷中毒”）确诊患者（轻度24例、中度28例、重度16例）为研究对象,其中40例经过皮肤病理学诊断,分为一般病变组（20例）、癌前病变组（14例）及癌变组（6例）。在距病区约12km的非砷暴露村,选择23例居民作为对照组。在知情同意的原则下,采集上述被调查者的外周血,采用实时荧光定量PCR（FQ．PCR）检测血中HDAC1 mRNA的表达。另收集自愿接受手术治疗的61例砷中毒患者皮肤组织标本（一般病变34例、癌前病变21例,癌变6例）和15例正常皮肤组织标本,采用免疫组织化学（IHC）法检测砷中毒患者皮肤及对照皮肤组织中HDAC1蛋白的表达。[结果]HDAC1 mRNA在对照组及轻、中、重度砷中毒患者外周血中表达的平均值分别为0．44457（0．37093—0．63904）、0．49286（0．08018～1．12747）、0．45185（0．24413～0．87641）和0．56676（0．30678～0．84471）,差异无统计学意义（χ^2=1．099,P〉0．05）;HDAC1 mRNA在一般病变组、癌前病变组和癌变组患者外周血中表达的平均值分别为0．32081（0．16723～0．83972）、0．50552（0．28280～1．43397）和0．80479（0．92123～1．89946）,差异无统计学意义（,=1．982,P〉0．05）。HDAC1蛋白在一般病变组、癌前病变组以及癌变组阳性表达率分别为100％、100％和83．33％,与对照组（46．67％）比较表达增强（P〈0．01）。[结论]HDAC1参与了砷中毒的发生发展,其蛋白表达增强可能是砷中毒发生的早期事件。     

基于生物信息学途径探讨阿托伐他汀钙抗肿瘤作用的分子机制

目的基于生物信息学途径探讨与阿托伐他汀钙抗肿瘤作用相关的分子机制及生物信号通路。方法人脐静脉内皮细胞株EA.hy926分为两组,对照组加0.01%二甲基亚砜(DMSO),实验组加阿托伐他汀钙(10-5 mol/L,以0.01%DMSO溶解),共同孵育24 h,提取总RNA,用Affymetrix U133 plus 2.0全基因组表达芯片检测两组对EA.hy926细胞基因表达谱的影响。用SAM软件筛选两组之间的差异基因,应用基因富集度分析(gene set enrichment analysis,GSEA)、DAVID基因功能聚类分析软件进行通路富集分析,应用the Connectivity Map(c Map)数据库对芯片数据进行分析,并对肿瘤相关信号通路的靶基因进行RT-PCR及Westernblot验证。结果基因表达谱芯片分析显示,实验组与对照组相比,获得差异表达基因649个,其中上调基因295个,下调基因354个;GSEA富集分析提示,上调了Kruppel样转录因子等血管保护基因,下调了CCNA2、CCNE2、CCNB1和CCNB2等细胞周期相关基因,并经过RT-PCR验证。通过Cmap分析,筛选到与MS-275、trichostatin A等组蛋白去乙酰化酶抑制剂及白藜芦醇等药物有较高的相似性。结论基于生物信息学的研究发现,阿托伐他汀钙可能发挥着类似组蛋白去乙酰化酶抑制剂(HDAC inhibitors)和G1/S(开始)期及G2/M(有丝分裂)期细胞周期抑制剂的作用,为阿托伐他汀钙作为抗肿瘤药物的研究提供了可行性。     

组蛋白去乙酰化酶2基因在苯并（a）芘致神经细胞凋亡中的量效和时效表达

[目的]通过体外培养细胞途径研究组蛋白去乙酰化酶2（HDAC2）基因在苯并（a）芘[B（a）P]所致神经细胞凋亡中的量效和时效表达。[方法]大鼠原代培养的神经元细胞分为两批：第一批分4个组,以B（a）P同时加入s9对细胞染毒,分别为二甲基亚砜（DMSO）组、低剂量组、中剂量组、高剂量组,使其终浓度为0、10、20、40gmol／L,继续培养48h;第二批分5个组,以20gmol／LB（a）P染毒,分别于染毒0、6、12、24、48h处理细胞。用流式细胞术检测神经细胞的凋亡率,实时荧光定量PCR法检测caspase-3,HDAC2基因的量效和时效表达情况。[结果]①流式细胞术结果显示,与DMSO组相比,中、高剂量组神经细胞的凋亡率明显增加（P〈O．05）;与低剂量组相比,高剂量组凋亡率增加（P〈0．05）。与0h组相比,染毒24、48h组神经细胞的凋亡率明显增加（P〈0．05）。②与DMSO组相比,高剂量组caspase-3、HDAC2mRNA的表达量明显增加（P〈0．01）;与低剂量组相比,高剂量组caspase-3mRNA表达量明显增加（P〈0．05）。与0h组相比,染毒48h组caspase-3、HDAC2mRNA表达量明显升高（P〈0．01,P〈0．05）。[结论]B（a）P可引起神经细胞凋亡,HDAC2基因表达上升,该基因可能在B（a）P所导致的神经细胞凋亡中起重要作用。     

玻璃化冻融助孕胎盘DNMT1、GCN5、HDAC1表达研究

目的研究玻璃化冻融囊胚助孕妇女胎盘组织中DNMT1、GCN5和HDAC1表达水平,评估该项助孕技术的安全性。方法收集2014年5月-2016年5月间在福建省妇幼保健院以玻璃化冻融囊胚助孕分娩的女性以及同时期自然妊娠女性共100例的胎盘标本,并统计其病例资料;采用免疫组化法检测、比较其胎盘中DNMT1、GCN5和HDAC1蛋白的表达情况。结果玻璃化冻融囊胚助孕组胎盘显著大于自然妊娠组(P<0.05);但其DNMT1、GCN5和HDAC1蛋白在表达率、表达强度、免疫组织化学评分方面比较差异无统计学意义(P>0.05)。玻璃化冻融囊胚助孕组较自然妊娠组有较高的先兆流产率和剖宫产率,差异具有统计学意义(P<0.05);而两组病例在分娩孕周、早产率、妊娠期并发症、妊娠合并症发生率方面比较差异无统计学意义(P>0.05)。结论玻璃化冻融技术不改变胎盘中DNMT1、GCN5,和HDAC1的表达,是一种安全有效的助孕技术。     

大豆皂甙Bb对食管癌细胞凋亡的影响及其机制研究

目的探讨大豆皂甙Bb(SSBb)抑制人食管癌细胞Eca-9706生长、诱导凋亡的效应及其机制。方法用噻唑蓝比色法测不同浓度SSBb对Eca-9706细胞生长的抑制率;缺口末端标记法检测Eca-9706细胞的凋亡率;免疫组织化学染色法和免疫印迹法检测人食管癌Eca-9706细胞在SSBb作用下c-met、VEGF、cyclinD1、NF-κB、PTEN、HDAC1、caspase3蛋白表达的变化。结果与对照组相比,SSBb处理的Eca-9706细胞的生长抑制率、凋亡率增加,caspase3、PTEN的免疫反应性和印迹信号增强,cyclinD1、c-met、VEGF、NF-κB、HDAC1的免疫反应性和印迹信号减弱(P0.01)。结论 SSBb对人食管癌Eca-9706细胞增殖有抑制作用,可降低Eca-9706细胞中的c-met和VEGF恶性标志的过高表达,并通过抑制HDAC1、NF-κB,激活PTEN和caspase3信号传导途径诱导食管癌细胞凋亡。     

香烟对大鼠肺白介素-8、谷胱甘肽及组蛋白去乙酰化酶2的影响

目的：探讨香烟暴露及戒烟对大鼠肺白介素-8、谷胱甘肽（GSH）及组蛋白去乙酰化酶2（HDAC2）的影响。方法：健康雄性SD大鼠40只随机分对照组、吸烟组（1月、2月组）、戒烟组（1月、2月组）。每组8只,吸烟组及戒烟组大鼠每天给予烟熏两次,吸烟组烟熏1月、2月取材;戒烟组烟熏2月后再继续饲养1月和2月后取材,分别检测支气管肺泡灌洗液（BALF）中白介素-8和GSH、肺组织HDAC2的表达。结果：比较吸烟组、戒烟组和对照组BALF中白介素-8含量升高、GSH浓度下降,戒烟后GSH渐升高,但仍不能达到正常;吸烟组肺组织HDAC2含量逐渐降低,戒烟后HDAC2又逐渐升高,但与对照组比较,仍有下降。结论：香烟暴露可诱导气道氧化应激反应,导致GSH减少,HDAC2减少,戒烟可有所改善。