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：
9064995
负责人：
JEFFREY J DESTEFANO
金额：
$ 28.5万
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/9064995
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 Materials Genetic Variation Goals Grant HIV HIV Infections Health Highly Active Antiretroviral Therapy Human Immune response Immune system In Vitro 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 crosslink design drug efficacy efavirenz enzyme activity falls genetic variant in vitro Assay inhibitor/antagonist innovation novel novel strategies novel therapeutics nuclease public health relevance research study 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（由适用提供）：由于病毒能够快速突变和产生可以避免免疫响应和抵抗药物治疗的遗传变异的能力，试图对抗HIV的尝试受到阻碍。艾滋病毒的巨大遗传多样性极大地使已经艰巨的任务复杂化，即为能够通过细胞传播到细胞接触的病毒而产生有效的疫苗，并在休眠储层细胞种群中隐藏其遗传物质。高度活跃的抗逆转录病毒疗法（HAART）是治疗艾滋病毒患者的强大工具，但尚未有效地达到最脆弱的人群，而对HAART使用的许多药物的抵抗力不可避免地出现了。将来需要采取替代方法来帮助控制和克服艾滋病毒感染和艾滋病。该应用程序旨在通过更清楚地定义艾滋病毒逆转录酶（RT）保真度的机制来促进这些目标，并检查物理条件对忠诚度的影响以及对常用的RT抑制剂的利用。实验表明RT生物化学是如何在细胞条件下发生的，表明当前有关RT保真度以及与AZT和DDC等药物相互作用的文献具有误导性。提出的实验的结果将更清楚地定义RT在细胞中的工作原理，从而使开发和评估潜在的新药更容易。还与Rutgers的Eddy Arnold博士的小组合作证明了一种与RT非常紧密结合的引物模仿模拟物结晶的新型方法。该方法首次允许在没有交联的情况下快速结晶RT，并导致形成催化活性晶体，可以用RT抑制剂处理，以研究RT抑制剂复合物的结构。信息可用于基于结构的新型RT抑制剂设计。 apatamers（与靶蛋白非常紧密结合的核酸）可能有可能用于诊断和治疗应用，例如在生化测定中替代抗体（例如ELISA），用作BIOSENORS，作为研究病毒分子生物学的工具，以及作为抗病毒药的模型。特异于HIV和其他逆转录病毒的ATAMER已显示可抑制细胞和动物模型中的病毒复制。在此提案中，一个新的类由“ Xeno”核酸（XNA）组成的HIV RT的适体将测试病毒抑制作用，以及模仿上述结晶的新型底漆 - 模板。 XNA由类似于正常核骨的核骨化模拟物组成，但包含独特的化学和结构修饰，可将它们与正常核骨化物区分开。 XNA适体是独一无二的，因为核骨化的不自然结构不易受到降解酶和其他危害条件的影响，而与当前的RNA和DNA ApaTamer相比，先天免疫系统识别它们的可能性也较小。