Subunit Assembly and Substrate Interactions in HIV-1 RT

HIV-1 RT 中的亚基组装和底物相互作用

基本信息

批准号：
7367969
负责人：
MARY D BARKLEY
金额：
$ 23.84万
依托单位：
CASE WESTERN RESERVE UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2006
资助国家：
美国
起止时间：
2006-03-15 至 2010-02-28
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7367969
关键词：
Acquired Immunodeficiency Syndrome Active Sites Adverse effects Antiviral Therapy Arts Binding Biochemical Biological Assay C-terminal Chromosomes Class Clinical Complex Cysteine DBL Oncoprotein DNA DNA Binding DNA-Directed DNA Polymerase Development Dimerization Dissociation Drug effect disorder Drug resistance Energy Transfer Engineering Enzymes Equilibrium Fluorescence Fluorescence Anisotropy Fluorescent Probes Future Gel Generations Genomics Goals HIV HIV Infections HIV Integrase HIV Protease HIV therapy HIV-1 Individual Investigation Kinetics Knowledge Laboratories Life Ligands Link Measurement Measures Methods Molecular Molecular Conformation Molecular Target Monitor Mutation Nevirapine Nucleotides Numbers Pharmaceutical Preparations Pliability Polymerase Process Protein Conformation Proteins RNA RNA primers RNA-Directed DNA Polymerase Rate Reaction Research Personnel Reverse Transcriptase Inhibitors Ribonuclease H Site Solutions Specificity Structure Substrate Interaction Techniques Testing Therapeutic Time United States base conformational conversion design dimer efavirenz enzyme activity high throughput screening improved inhibitor/antagonist insight monomer mutant non-nucleoside reverse transcriptase inhibitors polymerization programs protein protein interaction research study resistance mechanism sedimentation equilibrium single molecule stop flow technique stopped-flow fluorescence

项目摘要

The goal of this proposal is to investigate protein-ligand and protein-protein interactions in HIV-1 reverse transcriptase (RT). Being a key player in HIV infection, RT is the molecular target of the majority of AIDS drugs. Ligands studied will include five nonnucleoside reverse transcriptase inhbitiors (NNRTI) as well as normal substrates. RTs studied will include up to five drug resistance mutants in addition to wild type. The enzyme copies the single-stranded genomic RNA into a double-stranded DMAprovirus, which is subsequently integrated into the host chromosome by HIV integrase. RT has RNA- and DMA-dependent DMA polymerase and RNase H activities. The biologically active enzyme is a heterodimer of p66 and p51 subunits. The p51 subunit is derived from p66 subunit by HIV protease. The proposal applies biochemical and biophysical techniques, including powerful site-specific fluorescence techniques, to fundamental investigations of proposed inhibition mechanisms and protein conformations that may reveal new targets for antiviral therapy. This laboratory is presently one of few using solution biophysical techniques to study RT. Specifically, we propose to: 1. Characterize inhibitor binding to RT. 2. Determine inhibitor effects on individual steps in DMA polymerization. 3. Investigate inhibitor effects on conformation and dynamics of RT subunits and assembly.

该提案的目标是研究 HIV-1 逆转中的蛋白质-配体和蛋白质-蛋白质相互作用转录酶（RT）。作为 HIV 感染的关键因素，RT 是大多数艾滋病的分子靶标药物。研究的配体将包括五种非核苷逆转录酶抑制剂 (NNRTI) 以及正常基材。除野生型外，所研究的 RT 还将包括多达 5 种耐药突变体。这酶将单链基因组 RNA 复制成双链 DMAprovirus，即随后通过 HIV 整合酶整合到宿主染色体中。 RT 具有 RNA 和 DMA 依赖性 DMA 聚合酶和 RNase H 活性。生物活性酶是p66和p51的异二聚体亚单位。 p51 亚基是由 HIV 蛋白酶从 p66 亚基衍生而来。本提案适用生化和生物物理技术，包括强大的位点特异性荧光技术，以基础对所提出的抑制机制和蛋白质构象的研究可能揭示新的靶标抗病毒治疗。该实验室是目前为数不多的使用溶液生物物理技术来研究 RT 的实验室之一。具体来说，我们建议： 1. 表征抑制剂与 RT 的结合。 2. 确定抑制剂对 DMA 聚合中各个步骤的影响。 3. 研究抑制剂对 RT 亚基和组装的构象和动力学的影响。