Structural biasing elements for in-cell histone deacetylase paralog selectivity

We use the structural dissection of two 1,3-dioxanes with in-cell histone deacetylase (HDAC) paralog selectivity to identify key elements for selective HDAC inhibitors. We demonstrate that o-aminoanilides are inactive toward HDAC6 while apparently inhibiting deacetylases that act upon histone substrates. This finding has important clinical implications for the development of HDAC inhibitor-based treatments that do not interfere with microtubule dynamics associated with HDAC6. We also show that suberoylanilide hydroxamic acid (SAHA) alone is a nonparalog-selective HDAC inhibitor and that the 1,3-dioxane diversity appended to SAHA is essential for HDAC6 paralog selectivity.     

Design,Synthesis and Biological Evaluation of Novel Selective Thiol-based Histone Deacetylase(HDAC) VI InhibitorsBearing Indeno[1,2-c]pyrazole or Benzoindazole Scaffold

A series of thiol-based indeno[1,2-c]pyrazoles and benzoindazole compounds was designed and synthesized according to the structural specificity of histone deacetylase VI(HDAC6) and the structural characteristics of HDAC inhibitors. The inhibitory activities of the target compounds against HDAC6 and HDAC1 were screened by fluorescence analysis. Most of the target compounds showed moderate inhibitory activity against HDAC6(IC₅₀=44—598 nmol/L). Among them, compound A-4 displayed the highest selectivity against HDAC6 and similar inhibitory activity(IC₅₀=44 nmol/L) to that of the positive drug SAHA(IC₅₀=41 nmol/L) against HDAC6.     

Ligand release mechanisms and channels in histone deacetylases

Exploring the molecular channels of class I histone deacetylases (HDACs) with buried active sites are important to understand their structures and functionalities. In this work, we perform hybrid classical molecular dynamics and random acceleration molecular dynamics simulations to explore the B3N [i.e., (4‐(dimethylamino)N‐[7(hydroxyamino)‐7‐oxoheptyle] benzamide)] exit channels in the x‐ray crystal structures of HDAC3 and HDAC8 enzymes. Our simulations identify B3N release through four different channels in HDAC3 (denoted as A1, A2, B1, and B2) and HDAC8 (referred as A1, B1, B2, and B3) enzymes, among which egression through channel A1 is more predominant in both the enzymes. This mechanism is similar to ligand release in HDAC1 and HDAC2 described in our previous study and can be the fingerprint ligand release mechanisms in class I HDACs. Ligand release events through B channels, on the other hand, are different among HDAC3 and HDAC8, highlighting the significances of substituted residues in controlling the access to these channels This study reveals a novel aromatic gating mechanism elicited by TYR154‐TRP141‐TYR111 that controls the B3N access to all the B channels in HDAC8. The TRP141 in HDAC8 is substituted by LEU133 in HDAC3, which do not hinder the access to B channels in HDAC3. However, two hydrogen bonded barricades formed as ARG28‐GLY297‐GLY295‐GLY131 and TRP129‐ARG28‐ALA130‐LEU29‐TRP129 obstruct the B3N from exploring the B channels in HDAC3. The structural and dynamical characterizations of molecular channels and ligand unbinding mechanisms reported in this study provide novel structural insights and atomic level perspectives on HDAC3 and HDAC8 enzymes, thereby potentially aiding in the design of more specific HDAC inhibitors.Copyright © 2013 Wiley Periodicals, Inc.     

同源模建和分子对接研究HDAC1/HDAC8的选择性

组蛋白去乙酰化酶(HDACs)是近年来治疗肿瘤的重要靶标之一.由于HDACs包含多种亚型,且各亚型的生理功能存在一定的差异,其选择性抑制剂的开发已成为当前的研发热点.我们通过同源模建的HDAC1结构,与已有的HDAC8晶体结构的活性位点进行比较分析,探讨了对两者选择性有重要影响的残基,为基于受体的选择性抑制剂研究提供重要信息.同时选择了52个HDAC抑制剂,分别建立了HDAC1、HDAC8的活性值与对接打分值的线性回归模型.所建的HDAC1和HDAC8的线性构效关系模型的非交叉验证系数R2分别为0.82和0.80,表明具有一定的统计学意义.利用所建模型对已设计合成的化合物进行了预测,预测结果对HDAC1、HDAC8选择性抑制剂的优化改造提供了一定的指导意义.     

Identification of novel HDAC8 selective inhibitors through ligand and structure based studies: Exploiting the acetate release channel differences among class I isoforms

Histone deacetylases (HDACs) are key regulators of gene expression and have emerged as crucial therapeutic targets for cancer. Among the HDACs, inhibition of HDAC8 enzyme has been reported to be a novel strategy in the treatment of female-specific cancers. Most of the HDAC inhibitors discovered so far inhibit multiple HDAC isoforms causing toxicities in the clinic thus limiting their potential. Therefore, the discovery of isoform-selective HDAC8 inhibitors is highly desirable. In the present study, a combination of ligand and structure based drug design tools were utilized to build a statistically significant pharmacophore based 3D QSAR model with statistical parameters R²: 0.9964, and Q²: 0.7154, from a series of 31 known HDAC8 inhibitors. Top 1000 hits obtained from Virtual screening of Phase database were subjected to docking studies against HDAC8. Top 100 hits obtained were redocked into HDAC Class I (HDAC 1,2,3) and Class II isoforms (HDAC 4, 6) and rescored with XP Glide Score. Based on fitness score, XP glide score and interacting amino acid residues, five HDAC8 inhibitors (1–5) were selected for in vitro studies. The HDAC8 activity assay followed by enzyme kinetics clearly indicated Compounds 1, 2 and 3 to be potent HDAC8 selective inhibitors with IC₅₀ of 126 pM, 112 nM, and 442 nM respectively. These compounds were cytotoxic to HeLa cells where HDAC8 is overexpressed but not to normal cells, HEK293. Also, they were able to induce apoptosis by modulating Bax/Bcl2, cleavage of PARP and release of Cytochrome C. Molecular Dynamics simulations observed most favorable interaction patterns and presented a rationale for the activities of the identified compounds. Selectivity against HDAC8 was due to exploitation of the architectural difference in the acetate release channel among class I HDAC isoforms.     

HDAC对接研究:苯甲酰胺类抑制剂结合方式推测

通过计算机模拟的对接过程研究，发现了MS-275— 一种苯甲酰胺类的组蛋白去乙酰酶（HDAC）抑制剂与酶的可能的全新结合方式.这种结合方式与已经阐明的组蛋白去乙酰酶类似蛋白（HDLP）与曲古柳菌素A(trichostatin A, TSA)和suberoylanilide hydroxamic acid(SAHA)形成的复合物晶体结构中配体与酶的作用方式完全不同.从对接结果看，MS-275的作用靶点在酶活性口袋的最狭窄部位，而不是直接作用于锌离子.这似乎能够解释MS-275的低毒性特点，并且为设计和筛选全新的HDAC抑制剂提供了新思路.     

Limited proteolysis of human histone deacetylase 1

Background  

Histone deacetylase (HDAC) proteins are associated with cell proliferation, differentiation, apoptosis, and cancer. Specifically, HDAC1 is linked with cell growth, a hallmark of cancer formation. HDAC1 is a phosphoprotein and phosphorylation at S421 and S423 promotes HDAC1 enzymatic activity and protein association. While single and double point mutants of HDAC1 at S421 and S423 appear functionally similar, the evidence suggests that HDAC1 is phosphorylated simultaneously at both S421 and S423 in vivo. Additional experiments are necessary to probe the role of double phosphorylation of HDAC1 at S421 and S423.     

Profiling of Substrates for Zinc-dependent Lysine Deacylase Enzymes: HDAC3 Exhibits Decrotonylase Activity In Vitro

Systematic screening of the activities of the eleven human zinc-dependent lysine deacylases against a series of fluorogenic substrates as well as kinetic evaluation revealed substrates for screenings of histone deacetylases HDAC10 and HDAC11 at reasonably low enzyme concentrations. Furthermore, HDAC3 in complex with nuclear receptor corepressor?1 (HDAC3-NCoR1) was shown to harbor decrotonylase activity in?vitro.     

Target Design of Novel Histone Deacetylase 6 Selective Inhibitors with 2-Mercaptoquinazolinone as the Cap Moiety

Epigenetic alterations found in all human cancers are promising targets for anticancer therapy. In this sense, histone deacetylase inhibitors (HDACIs) are interesting anticancer agents that play an important role in the epigenetic regulation of cancer cells. Here, we report 15 novel hydroxamic acid-based histone deacetylase inhibitors with quinazolinone core structures. Five compounds exhibited antiproliferative activity with IC₅₀ values of 3.4–37.8 µM. Compound 8 with a 2-mercaptoquinazolinone cap moiety displayed the highest antiproliferative efficacy against MCF-7 cells. For the HDAC6 target selectivity study, compound 8 displayed an IC₅₀ value of 2.3 µM, which is 29.3 times higher than those of HDAC3, HDAC4, HDAC8, and HDAC11. Western blot assay proved that compound 8 strongly inhibited tubulin acetylation, a substrate of HDAC6. Compound 8 also displayed stronger inhibition activity against HDAC11 than the control drug Belinostat. The inhibitory mechanism of action of compound 8 on HDAC enzymes was then explored using molecular docking study. The data revealed a high binding affinity (−7.92 kcal/mol) of compound 8 toward HDAC6. In addition, dock pose analysis also proved that compound 8 might serve as a potent inhibitor of HDAC11.     

Apicidin选择性抑制ClassⅠ HDACs分子动力学研究

采用模拟方法研究组蛋白脱乙酰酶抑制剂(Apicidin)选择性抑制组蛋白去乙酰化酶(Histone deacetylases, HDACs)中的HDAC1和HDAC8. 通过HDAC8晶体结构同源模建HDAC1三维结构模型, 将Apicidin分别与HDAC1和HDAC8对接并进行分子动力学模拟, 结果表明, HDAC1活性口袋入口处的Arg270是Apicidin-HDAC1形成稳定结构的重要因素; HDAC1中Tyr303及His178与Apicidin形成2个持续存在的氢键, 而在HDAC8中未发现, 这是Apicidin选择性抑制HDAC1高于HDAC8的另一重要原因.     

磺胺基羟肟酸类HDAC抑制剂三维定量构效关系

组蛋白去乙酰化酶(HDAC)对染色质分布和基因调节起着重要的作用,也是治疗癌症和其它疾病的新靶点.羟肟酸类抑制剂是目前研究最多的组蛋白去乙酰化酶抑制剂.应用比较分子力场(CoMFA)法对一系列磺胺基羟肟酸类HDAC抑制剂进行了结构活性关系研究,得到的模型具有较高的交叉验证系数(q²=0.704).并在此基础上,建立了非交叉验证的偏最小二乘分析(PLS)模型.用该模型对随机选择的6个化合物组成的测试集进行了预测,得到了令人满意的结果,所建模型具有良好的预测能力.本研究对于设计高活性的HDAC抑制剂及抗癌药物都有指导意义.     

氧肟酸类组蛋白去乙酰化酶抑制剂的药效团模型研究

基于24个目前已知的氧肟酸类组蛋白去乙酰化酶抑制剂,我们运用Catalyst软件建立了一个三维药效团模型。其中,最好的药效团模型1,包含了四个化学特征（一个氢键供体,一个芳环和两个疏水基）,相关系数达到0.946,并由另外20个化合物进行了测试验证。我们第一次特征性描述了组蛋白去乙酰化酶的帽子（CAP）部分。我们的研究结果对于设计全新组蛋白去乙酰化酶抑制剂具有很好的指导作用。     

The HDAC/HSP90 Inhibitor G570 Attenuated Blue Light-Induced Cell Migration in RPE Cells and Neovascularization in Mice through Decreased VEGF Production

Age-related macular degeneration (AMD) occurs due to an abnormality of retinal pigment epithelium (RPE) cells that leads to gradual degeneration of the macula. Currently, AMD drug pipelines are endowed with limited options, and anti-VEGF agents stand as the dominantly employed therapy. Despite the proven efficacy of such agents, the evidenced side effects associated with their use underscore the need to elucidate other mechanisms involved and identify additional molecular targets for the sake of therapy improvement. The previous literature provided us with a solid rationale to preliminarily explore the potential of selective HDAC6 and HSP90 inhibitors to treat wet AMD. Rather than furnishing single-target agents (either HDAC6 or HSP90 inhibitor), this study recruited scaffolds endowed with the ability to concomitantly modulate both targets (HDAC6 and HSP90) for exploration. This plan was anticipated to accomplish the important goal of extracting amplified benefits via dual inhibition (HDAC6/HSP90) in wet AMD. As a result, G570 (indoline-based hydroxamate), a dual selective HDAC6-HSP90 inhibitor exerting its effects at micromolar concentrations, was pinpointed in the present endeavor to attenuate blue light-induced cell migration and retinal neovascularization by inhibiting VEGF production. In addition to the identification of a potential chemical tool (G570), the outcome of this study validates the candidate HDAC6-HSP90 as a compelling target for the development of futuristic therapeutics for wet AMD.     

Design,synthesis and antitumor evaluations of nucleoside base hydroxamic acid derivatives as DNMT and HDAC dual inhibitors

DNA methyl transferase(DNMT) and histone deacetylase(HDAC) are well recognized epigenetic targets for discovery of antitumor agents.In this study,we designed and synthesized a series of nucleoside base hydroxamic acid derivatives as DNMT and HDAC dual inhibitors.MTT assays and enzymatic inhibitory activity tests indicated that compound 204 exhibited potent DNMT1 and HDAC1/6 inhibitory potency simultaneously in enzymatic levels and at cellular levels,inducing hypomethylation of p16 and hyperacetylation of histones H3 K9 and H4 K8.Besides,204 remarkably inhibited proliferation against cancer cells U937 by prompting G0/G1 cell cycle arrest.Molecular docking models explained the functional mechanism of 204 inhibiting DNMT1 and HDAC.Preliminary studies on metabolic profiles revealed that 204 showed desirable stability in liver microsomes.Our study suggested that 204 inhibiting DNMT and HDAC concurrently can be a potential lead compound for epigenetic cancer therapy.     

Exploring inhibitor release pathways in histone deacetylases using random acceleration molecular dynamics simulations

Molecular channel exploration perseveres to be the prominent solution for eliciting structure and accessibility of active site and other internal spaces of macromolecules. The volume and silhouette characterization of these channels provides answers for the issues of substrate access and ligand swapping between the obscured active site and the exterior of the protein. Histone deacetylases (HDACs) are metal-dependent enzymes that are involved in the cell growth, cell cycle regulation, and progression, and their deregulations have been linked with different types of cancers. Hence HDACs, especially the class I family, are widely recognized as the important cancer targets, and the characterizations of their structures and functions have been of special interest in cancer drug discovery. The class I HDACs are known to possess two different protein channels, an 11 ? and a 14 ? (named channels A and B1, respectively), of which the former is a ligand or substrate occupying tunnel that leads to the buried active site zinc ion and the latter is speculated to be involved in product release. In this work, we have carried out random acceleration molecular dynamics (RAMD) simulations coupled with the classical molecular dynamics to explore the release of the ligand, N-(2-aminophenyl) benzamide (LLX) from the active sites of the recently solved X-ray crystal structure of HDAC2 and the computationally modeled HDAC1 proteins. The RAMD simulations identified significant structural and dynamic features of the HDAC channels, especially the key 'gate-keeping' amino acid residues that control these channels and the ligand release events. Further, this study identified a novel and unique channel B2, a subchannel from channel B1, in the HDAC1 protein structure. The roles of water molecules in the LLX release from the HDAC1 and HDAC2 enzymes are also discussed. Such structural and dynamic properties of the HDAC protein channels that govern the ligand escape reactions will provide further mechanistic insights into the HDAC enzymes, which, in the long run, have a potential to bring new ideas for developing more promising HDAC inhibitors as well as extend our atomic level understandings on their mechanisms of action.