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

基本信息

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
项目类别：
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&apos -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

项目摘要

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）：由于病毒具有快速突变的能力，并且基因变异可以规避免疫反应并抵抗药物治疗，因此对抗艾滋病毒的尝试受到阻碍。艾滋病毒基因的巨大多样性使本已艰巨的任务变得更加复杂。生产一种有效的病毒疫苗，能够通过细胞间的接触传播，并将其遗传物质隐藏在休眠储存细胞群中的原病毒状态，这是一种强大的方法。治疗艾滋病毒患者的工具，但尚未有效覆盖大多数人群，而对HAART中使用的许多药物的耐药性不可避免地出现，未来将需要替代方法来帮助弱势群体控制和克服艾滋病毒感染和艾滋病。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 抑制剂的利用来为实现这些目标做出贡献。实验表明，RT 生物化学在细胞条件下是如何发生的。 RT 的最新文献所提出的实验结果将更清楚地定义 RT 在细胞中的作用，从而更容易开发和评估潜在的新药物，这是一种使用引物模板结晶 HIV RT 的新方法。与罗格斯大学 Eddy Arnold 博士的团队合作也证明了与 RT 紧密结合的模拟物。这种方法首次允许 RT 在没有交联的情况下快速结晶，并导致催化形成。可以用 RT 抑制剂处理的活性晶体，以研究 RT 抑制剂复合物的结构。信息可用于新型 RT 抑制剂的结构设计。可用于诊断和治疗应用，例如在生化测定（例如 ELISA）中替代抗体、用作生物传感器、作为研究病毒分子生物学的工具以及作为 HIV 和其他逆转录病毒特异性抗病毒药物的模型。在该提案中，抑制病毒在细胞和动物模型中的复制。由“异种”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进行病毒抑制测试，XNA 由类似于正常核苷酸但含有独特化学物质的核苷酸模拟物组成。使它们与正常核苷酸不同的结构修饰是独特的，因为核苷的非天然结构不易受到降解酶和其他恶劣条件的影响，而它们。与现有的 RNA 和 DNA 适配体相比，它们也不太可能被先天免疫系统识别。