Structural studies of HIV-1 integration

HIV-1整合的结构研究

• 批准号: 7535222
• 负责人: Mamuka Kvaratskhelia
• 金额: $ 32.47万
• 依托单位: OHIO STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-01-01 至 2010-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7535222
• 关键词: Acids; Acquired Immunodeficiency Syndrome; Address; Affinity; Amino Acids; Antiviral Agents; Antiviral Therapy; Binding; Binding Sites; Biochemical; Biological Assay; Characteristics; Charge; Clinical; Complex; Coupled; DNA; DNA Binding; DNA Structure; Data; Disulfides; Enzymes; Fluorescence Resonance Energy Transfer; Foundations; Future Generations; HIV; HIV-1; HIV-1 integrase; Highly Active Antiretroviral Therapy; Human Chromosomes; In Vitro; Individual; Infection; Integrase; Integrase Inhibitors; Knowledge; Life Expectancy; Modeling; Molecular; Molecular Conformation; Molecular Models; Monitor; Nucleic Acids; Nucleoproteins; Patients; Peptide Hydrolases; Pharmaceutical Preparations; Phase; Platinum; Positioning Attribute; Potassium Permanganate; Printing; Process; Property; Proteins; RNA-Directed DNA Polymerase; Recombinants; Research; Research Personnel; Site; Site-Directed Mutagenesis; Structure; Surface; Testing; Toxic effect; Vertebral column; Viral; Virus; arginyllysine; clinically relevant; crosslink; design; foot; improved; in vivo; inhibitor/antagonist; inorganic phosphate; molecular modeling; monomer; novel; novel strategies; numb protein; programs; protein protein interaction; small molecule; therapy resistant; viral DNA

The practical benefits of treatment of AIDS patients with highly active anti-retroviral therapy (HAART) are manifested by substantial increase of the life expectancy of HIV infected individuals. The current therapies target two retroviral enzymes reverse transcriptase and protease. More recently, viral entry inhibitor has been developed. However, problems associated with the toxicity of currently available drugs and emergence of new infections initiated with HAART-resistant viruses, highlight the need to continually improve available inhibitors and develop new drugs against as yet unexploited targets. Integrase (IN) is one of the most promising new targets, as its function is essential to HIV replication. The recent advances of diketo acid inhibitors of HIV integration into phase 1 clinical trails confirm this notion. Biochemical studies indicated that these compounds specifically bind IN:viral DMAstructure and compete with target DMA binding. However, little is known regarding structural foundations that warrant formation of the ternary IN:viral DNA:DKA complex or how viral and host DMAs bind the IN multimer. The proposed research will address these important questions. In particular, we will employ novel experimental strategies to accomplish the following three specific aims. Specific Aim 1 will determine HIV-1 lntegrase:viral DNA contacts using domain selective cross-linking coupled with mass spectrometric foot-printing. This novel approach together with molecular modeling and site-directed mutagenesis studies should enable us to obtain detailed structural information as to how viral DNA binds IN multimer. Specific Aim 2 will employ protein and nucleic acid foot-printing to identify IN:host DNA contacts and to determine DNA conformation in the integration complex. The experimental results will be used to create a plausible molecular model for the functional nucleoprotein complex. Specific Aim 3 will characterize diketo acid (DKA) inhibitor binding site. Photo-affinity cross-linking coupled with mass spectrometric analysis will be utilized to identify new amino acid contacts to DKA; as well as disulfide DNA cross-linking and scintillation proximity assays will be used to dissect contributions of viral DNA end distortion for specific inhibitor binding. Taken together, we will obtain detailed structural information on biologically relevant IN:viral DNA:DKA complex. Such new and important structural data will facilitate the rational design of future generation inhibitors of potential clinical relevance.

治疗高度活性抗逆转录病毒治疗（HAART）的艾滋病患者的实际好处是 表现为艾滋病毒感染者的预期寿命的大幅提高。当前的疗法 靶向两个逆转录病毒酶逆转录酶和蛋白酶。最近，病毒进入抑制剂具有 已开发。但是，与当前可用药物和出现的毒性有关的问题 通过抗Haart抗性病毒发起的新感染，强调了不断改善可用的必要性 抑制剂并开发针对尚未开发的靶标的新药。集成酶（in）是最多的 有希望的新目标，因为其功能对于艾滋病毒复制至关重要。 Diketo Acid的最新进展 HIV整合到第1阶段临床跟踪中的抑制剂证实了这一概念。生化研究表明 这些化合物特异性结合：病毒DMAS结构并与靶DMA结合竞争。然而， 关于保证形成三元的结构基础，鲜为人知：病毒DNA：DKA 复合物或病毒和宿主DMA如何结合多聚体。拟议的研究将解决这些问题 重要问题。特别是，我们将采用新颖的实验策略来完成以下操作 三个具体目标。特定的目标1将确定HIV-1 LNTEGRASE：使用域选择性的病毒DNA接触 交联加和质谱脚印。这种新颖的方法与分子一起 建模和站点定向的诱变研究应使我们能够获得详细的结构信息作为 病毒DNA如何在多聚体中结合。特定的目标2将采用蛋白质和核酸脚印 识别：宿主DNA接触并确定整合复合物中的DNA构象。这 实验结果将用于为功能性核蛋白创建合理的分子模型 复杂的。特定的目标3将表征二甲酸（DKA）抑制剂结合位点。照片亲和力交联 结合质谱分析将用于鉴定与DKA的新氨基酸接触。也是如此 由于二硫键DNA交联和闪烁接近测定将用于剖析病毒的贡献 特异性抑制剂结合的DNA末端失真。综上所述，我们将获得详细的结构信息 关于生物学相关的：病毒DNA：DKA复合物。如此新的重要结构数据将促进 潜在临床相关性的未来一代抑制剂的合理设计。