Acylhydrazone inhibitors of HIV-1 ribonuclease H

HIV-1 核糖核酸酶 H 的酰腙抑制剂

• 批准号: 7849976
• 负责人: RIEKO ISHIMA
• 金额: $ 37.43万
• 依托单位: UNIVERSITY OF PITTSBURGH AT PITTSBURGH
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-06-01 至 2011-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7849976
• 关键词: Anti-HIV Agents; Anti-Infective Agents; Anti-Retroviral Agents; Binding; Binding Sites; Biochemical; CCR5 gene; Cells; Complex; DNA; DNA-Directed DNA Polymerase; Data; Databases; Development; Drug Industry; Drug usage; Enzymes; Escherichia coli; Essential Drugs; Genomics; Goals; HIV; HIV Infections; HIV Integrase Inhibitors; HIV drug resistance; HIV-1; HIV-1 Reverse Transcriptase; Hydrazones; Individual; Libraries; Microscopic; Molecular Conformation; Multi-Drug Resistance; Mutation; Patients; Peptide Hydrolases; Pharmaceutical Preparations; Phenotype; Polymerase; Prevalence; Program Development; Property; RNA; RNA-Directed DNA Polymerase; Research; Resistance; Resistance development; Resistance profile; Ribonuclease H; Screening procedure; Site; Structure; Therapeutic; Validation; Variant; Viral; Virus; analog; base; design; drug discovery; drug resistant virus; enzyme activity; improved; inhibitor/antagonist; interest; member; novel; pathogen; prevent; public health relevance

DESCRIPTION (provided by applicant): The increasing prevalence of drug resistant HIV compromises the continued utility of current HIV drugs. Virtually all clinically used drugs are directed at only two HIV targets, protease and reverse transcriptase (RT), thus one approach to the problem of HIV drug resistance is to develop therapeutics directed at novel HIV targets, since therapeutics directed at novel HIV targets will almost certainly be active against current drug- resistant virus strains. One such target is RT-associated ribonuclease H (RNaseH), an essential enzyme activity for HIV replication and the only HIV enzyme not yet targeted by any clinically used or pipeline therapeutics. We have identified acylhydrazones as interesting compounds in that certain analogs are potent inhibitors of both HIV-1 RT DNA polymerase and RT-RNaseH activities. We hypothesize that this bifunctional inhibition is due to binding of the inhibitor to two distinct sites on RT, one of which is in or near the RNaseH domain. We recently obtained a crystal structure of acylhydrazones in the RT polymerase (pol) domain, so detailed understanding of the interaction of acylhydrazones with the RT RNaseH domain will be invaluable for design of both new RNaseH inhibitors (RNaseHI) and new bifunctional inhibitors. To this end, we propose four Specific Aims, (1) to determine the structure of HIV-RT RNaseH in complex with acylhydrazone inhibitors. Detailed NMR studies of the interaction of acylhydrazones with an active isolated HIV-1 RT RNaseH domain will provide precise structural information of the inhibitor binding pocket in the RNaseH domain, the microscopic conformation of RNaseH-inhibitor interaction sites, and influence of inhibitor binding on RT-RNaseH structure; (2) to validate the RNaseHI binding pocket determined in Aim 1. The RNaseHI binding pocket determined in Aim 1 will be validated in the context of intact RT and HIV-1 virus by evaluating the effect of mutation of selected residues interacting with RNaseHI, in the RNaseH fragment (biochemical and NMR structural analyses), in intact RT, and in HIV-1; (3) to optimize the inhibitory potency of acylhydrazone and analog inhibitors. Several approaches will be used to identify and develop potent monofunctional (RNaseH-specific) and bifunctional inhibitors, including screening of a library of 5000 hydrazone derivatives and synthesis of new analogs based on structures of acylhydrazones in the RNaseH and pol site binding pockets; (4) to conduct detailed biochemical and virologic characterizations of new RNaseHI. The inhibitory properties of potent RNaseHI and bifunctional acylhydrazones from Aim 3 will be characterized with purified RT and in cell-based HIV replication studies (including resistance development). We hypothesize that bifunctional inhibitors may be preferable in the context of resistance development. Selected RNaseHI will be further evaluated in NMR structural studies to better define the binding pocket for RNaseHI. This structural information along with the crystal data will provide a firm basis for rational design of new inhibitors. PUBLIC HEALTH RELEVANCE: There are more than 20 drugs for the treatment of HIV infection, yet the virus continues to spread in the US and worldwide. Furthermore, drug resistant HIV is increasing, limiting treatment options for individuals infected with these strains. New drugs are essential. This project will explore compounds directed at a novel HIV target, ribonuclease H, with the goal of developing new drugs that will be active against existing drug resistant HIV.

描述(由申请人提供)：抗药性艾滋病毒的流行日益增加，影响了目前艾滋病毒药物的持续使用。几乎所有临床使用的药物都只针对两个HIV靶点，即蛋白酶和逆转录酶(RT)，因此解决HIV耐药性问题的一种方法是开发针对新的HIV靶点的疗法，因为针对新的HIV靶点的疗法几乎肯定会对当前的耐药病毒株起作用。其中一个靶点是逆转录酶相关的核糖核酸酶H(RNAseH)，它是艾滋病毒复制的关键酶活性，也是唯一尚未被任何临床使用或流水线疗法靶向的HIV酶。我们已经确定酰肼类化合物是有趣的化合物，因为某些类似物对HIV-1 RT DNA聚合酶和RT-RNAseH活性都有有效的抑制作用。我们假设这种双功能抑制是由于抑制剂与RT上的两个不同的位点结合，其中一个在RNAseH结构域或附近。我们最近获得了逆转录酶(Poll)结构域中酰肼类化合物的晶体结构，因此详细了解酰肼类化合物与RT RNAseH结构域的相互作用对于设计新的RNAseH抑制剂(RNaseHI)和新的双功能抑制剂具有重要意义。为此，我们提出了四个具体目标：(1)确定与酰肼类抑制剂形成的复合体中HIV-RT RNAseH的结构。对酰肼类化合物与活性分离的HIV-1 RT RNAseH结构域相互作用的详细核磁共振研究将提供RNAseH结构域中抑制剂结合口袋的精确结构信息，RNAseH-抑制剂相互作用位点的微观构象，以及抑制剂结合对RT-RNAseH结构的影响；(2)验证Aim 1中确定的RNaseHI结合口袋；(3)优化酰肼及其类似物的抑制效力。将使用几种方法来鉴定和开发有效的单功能(RNAseH特异性)和双功能抑制剂，包括筛选5000个肼类衍生物的文库，并根据RNAseH和POL位点结合口袋中的酰肼的结构合成新的类似物；(4)对新的RNaseHI进行详细的生化和病毒学表征。来自AIM 3的有效的RNaseHI和双官能酰肼的抑制特性将通过纯化的RT和基于细胞的HIV复制研究(包括耐药性发展)来表征。我们假设，在耐药性发展的背景下，双功能抑制剂可能是更好的。选定的RNaseHI将在核磁共振结构研究中进一步评估，以更好地定义RNaseHI的结合口袋。这些结构信息和晶体数据将为合理设计新的缓蚀剂提供坚实的基础。与公共卫生相关：目前有20多种药物用于治疗艾滋病毒感染，但该病毒仍在美国和全球范围内传播。此外，抗药性艾滋病毒正在增加，限制了感染这些毒株的个人的治疗选择。新药是必不可少的。该项目将探索针对新的艾滋病毒靶标--核糖核酸酶H的化合物，目标是开发对现有抗药性艾滋病毒具有积极作用的新药。