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：
7197365
负责人：
MARY D BARKLEY
金额：
$ 23.78万
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/7197365
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 Proviruses 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

DESCRIPTION (provided by applicant): 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 DMA provirus, 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.

描述（由申请人提供）：该提案的目的是研究蛋白质 - 配体和 HIV-1逆转录酶（RT）中的蛋白质蛋白相互作用。作为艾滋病毒感染的关键参与者，RT是大多数艾滋病药物的分子靶标。所研究的配体将包括五个非核苷逆转录酶无限型（NNRTI）以及正常底物。研究的RTS除了野生型外，还包括多达五个耐药性突变体。酶将单链基因组RNA复制到双链DMA病毒中，随后通过HIV积分将其集成到宿主染色体中。 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亚基的异二聚体。 p51亚基是由HIV蛋白酶从p66亚基中得出的。该建议将生化和生物物理技术（包括强大的位点特异性荧光技术）应用于对拟议的抑制机制和蛋白质构象的基本研究，这些研究可能揭示了抗病疗法的新靶标。目前，该实验室是使用溶液生物物理技术来研究RT的少数实验室之一。具体而言，我们提出：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。