Biochemistry of HIV reverse transcriptase fidelity and inhibitor interactions

HIV逆转录酶保真度和抑制剂相互作用的生物化学

• 批准号: 9538330
• 负责人: JEFFREY J DESTEFANO
• 金额: $ 5万
• 依托单位: UNIV OF MARYLAND, COLLEGE PARK
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-09-15 至 2020-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9538330
• 关键词: 2' -deoxyadenosine; Acquired Immunodeficiency Syndrome; Address; Affinity; Animal Model; Antibodies; Antiviral Agents; Area; Base Sequence; Binding; Biochemical; Biochemistry; Biological Assay; Biosensor; Cell Culture Techniques; Cell model; Cells; Chemicals; Collaborations; Complex; Crystallization; DNA; DNA biosynthesis; Data; Development; Diagnostic; Drug Delivery Systems; Drug resistance; Drug toxicity; Drug usage; Effectiveness; Environment; Enzyme-Linked Immunosorbent Assay; Enzymes; Evolution; Frequencies; Funding; Future; Generations; Genetic Enhancement; Genetic Materials; Genetic Variation; Goals; Grant; HIV; HIV Infections; Health; Highly Active Antiretroviral Therapy; Human; Immune response; In Vitro; Innate Immune System; Lead; Letters; Light; Link; Literature; Measurement; Methodology; Methods; Modeling; Modification; Molecular Biology; Morphologic artifacts; Mutagenesis; Mutate; Mutation; Nevirapine; Nucleic Acid Binding; Nucleic Acids; Nucleosides; Nucleotides; Patients; Pharmaceutical Preparations; Pharmacotherapy; Physiological; Population; Predisposition; Process; Production; Property; Proteins; RNA; RNA-Directed DNA Polymerase; Resistance; Resolution; Retroviridae; Reverse Transcriptase Inhibitors; Reverse Transcription; Role; Science; Structure; System; Technology; Testing; Therapeutic; Time; Tube; Universities; Vaccines; Viral; Virus; Virus Replication; Vulnerable Populations; Work; Zidovudine; aptamer; base; combat; cost; crosslink; design; drug efficacy; efavirenz; enzyme activity; experimental study; falls; genetic variant; in vitro Assay; inhibitor/antagonist; innovation; novel; novel strategies; novel therapeutics; nuclease; public health relevance; resistance mutation; response; success; tool; zidovudine triphosphate

 DESCRIPTION (provided by applicant): Attempts to combat HIV have been hampered due to the virus's ability to rapidly mutate and produce genetic variants that can circumvent the immune response and resist drug therapy. The tremendous genetic diversity of HIV has greatly complicated the already daunting task of producing an effective vaccine for a virus capable of spreading through cell to cell contact and concealing its genetic material in the proviral state within populations of dormant reservoir cells. Highly Active Anti-Retroviral Therapy (HAART) has been a powerful tool for treating HIV patients but it has not effectively reached the most vulnerable populations, while resistance to many of the drugs used in HAART has inevitably emerged. Alternative approaches will be necessary in the future to help control and overcome HIV infections and AIDS. This application aims to contribute to these goals by more clearly defining the mechanism of HIV reverse transcriptase (RT) fidelity and examining the effect of physiological conditions on fidelity and the utilization of commonly used RT inhibitors. Experiments that show how RT biochemistry occurs under cellular conditions indicate that current literature on RT fidelity and interactions with drugs like AZT and ddC is misleading. Results from the proposed experiments will more clearly define how RT works in the cell making it easier to develop and evaluate potential new drugs. A novel approach for crystallization of HIV RT using primer-template mimics that bind very tightly to RT is also demonstrated in collaboration with Dr. Eddy Arnold's group at Rutgers. This method allows for the first time, rapid crystallization of RT in the absence of cross-linking and leads to the formation of catalytically active crystals that can be treated with RT inhibitors to investigate the structure o the RT-inhibitor complexes. Information can be used for structure-based design of novel RT inhibitors. Aptamers (nucleic acids that bind extremely tightly to target proteins) can potentially be used for diagnostic and therapeutic applications, such as replacements of antibodies in biochemical assays (e.g. ELISA), utilization as biosensors, as tools for studying virus molecular biology, and as models for antiviral drugs. Aptamers specific to HIV and other retroviruses have been shown to inhibit virus replication in cell and animal models. In this proposal, a new class of aptamers to HIV RT that are composed of "Xeno" nucleic acids (XNA) will be tested for viral inhibition along with the novel primer-template mimicking aptamers used for crystallization above. XNAs are composed of nucleotide mimics that resemble normal nucleotides but contain unique chemical and structural modification that differentiate them from normal nucleotides. XNA aptamers are unique because the unnatural structure of the nucleoside is less susceptible to degradative enzymes and other harsh conditions, while they are also less likely to be recognized by the innate immune system than current RNA and DNA aptamers.

 描述（由申请人提供）：由于病毒能够快速突变并产生可以绕过免疫反应和抵抗药物治疗的遗传变异，因此对抗艾滋病毒的努力受到阻碍。艾滋病毒的巨大遗传多样性使本已令人生畏的任务变得更加复杂，即为一种能够通过细胞与细胞接触传播并将其遗传物质以前病毒状态隐藏在休眠储存细胞群体中的病毒生产有效疫苗。高活性抗逆转录病毒疗法（HAART）一直是治疗艾滋病毒患者的有力工具，但它没有有效地达到最脆弱的人群，而对HAART中使用的许多药物的耐药性已不可避免地出现。今后需要采取其他办法，帮助控制和克服艾滋病毒感染和艾滋病。本申请旨在通过更清楚地定义HIV逆转录酶（RT）保真度的机制，并检查生理条件对保真度的影响以及常用RT抑制剂的利用率来实现这些目标。显示RT生物化学如何在细胞条件下发生的实验表明，目前关于RT保真度和与AZT和ddC等药物相互作用的文献是误导性的。实验结果将更清楚地定义RT在细胞中的工作方式，从而更容易开发和评估潜在的新药。与罗格斯大学的Eddy Arnold博士的研究小组合作，还证明了一种使用与RT紧密结合的引物模板模拟物结晶HIV RT的新方法。这种方法允许第一次，快速结晶RT在交联的情况下，并导致催化活性晶体的形成，可以用RT抑制剂处理，以研究结构的RT-抑制剂复合物。这些信息可用于基于结构的新型RT抑制剂的设计。适体（与靶蛋白极紧密结合的核酸）可能 可用于诊断和治疗应用，例如在生物化学测定（例如ELISA）中替代抗体，用作生物传感器，用作研究病毒分子生物学的工具，以及用作抗病毒药物的模型。对HIV和其他逆转录病毒特异性的适体已经显示出在细胞和动物模型中抑制病毒复制。在这一建议中，一类新的 由“Xeno”核酸（XNA）组成的HIV RT适体将与用于上述结晶的新型引物-模板模拟适体一起沿着测试病毒抑制。XNA由类似于正常核苷酸的核苷酸模拟物组成，但含有独特的化学和结构修饰，使其与正常核苷酸区分开来。XNA适体是独特的，因为核苷的非天然结构对降解酶和其他苛刻条件不太敏感，同时它们也不太可能被先天免疫系统识别，而不是目前的RNA和DNA适体。