Structure and Function of Integrase

Integrase的结构和功能

• 批准号: 8463943
• 负责人: ANNA MARIE SKALKA
• 金额: $ 41.95万
• 依托单位: RESEARCH INST OF FOX CHASE CAN CTR
• 依托单位国家: 美国
• 财政年份: 1997
• 资助国家: 美国
• 起止时间: 1997-08-01 至 2017-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8463943
• 关键词: AIDS therapy; AIDS/HIV problem; Active Sites; Address; Affect; Amino Acid Substitution; Architecture; Avian Sarcoma Viruses; Binding; Biological Assay; Catalysis; Chemicals; Clinical; Clinical Trials; Complement; Coupled; DNA; DNA Binding; Data; Data Analyses; Deuterium; Development; Drug Targeting; Enzymes; FDA approved; Fluorescence; Funding; HIV; HIV Integrase; HIV drug resistance; HIV-1; Hydrogen; Integrase; Integrase Inhibitors; Knowledge; Lead; Length; Maps; Mass Spectrum Analysis; Measures; Methods; Modeling; Molecular Conformation; Monitor; Pharmaceutical Preparations; Positioning Attribute; Property; Proteins; Publishing; Reaction; Research; Roentgen Rays; Role; Shapes; Site; Solutions; Spumavirus; Structural Models; Structure; Testing; Therapeutic; Viral; Viral Physiology; Virus; base; crosslink; design; dimer; drug development; in vitro activity; inhibitor/antagonist; insight; monomer; mutant; novel strategies; novel therapeutics; physical model; prototype; research study; screening; single-molecule FRET; small molecule libraries; success; viral DNA; viral resistance

DESCRIPTION (provided by applicant): Integrase (IN) is one of three virus-encoded enzymes that are essential for retroviral replication and a validated target for the development of drugs t treat HIV/AIDS. Although there has been success in developing clinically useful drugs that block the final step in the integration reaction, the so-called strand transfer inhibitors, there is a continuing need to augment or replace existing IN therapeutics as viral resistance is encountered. A detailed knowledge of all aspects of the structure, assembly, and catalysis by HIV-1 IN, will reveal unexploited vulnerabilities and novel strategies for inhibiting this critical enzyme. In the current funding period, we applied small angle X-ray scattering (SAXS) and protein-protein cross-linking methods to obtain the first experimentally-derived models of full-length unliganded apo-IN monomers and dimers in solution, using avian sarcoma virus (ASV) IN. The results revealed a dimer architecture (called a reaching dimer) that was previously unsuspected. The configuration of the reaching dimer resembles that of the viral DNA-binding, "inner" dimer in the crystal structure of the prototype foamy virus (PFV) IN. From these and other data, we have constructed a structural model for an HIV IN reaching dimer, which we hypothesize is pre-positioned to interact with viral DNA ends. In Aim 1 of this competitive renewal we propose to test this model by determining the solution structures of monomers, dimers, and tetramers of HIV IN, using methods successfully employed with ASV IN. We will identify the interactions that stabilize HIV dimers and determine the effects of substrate binding on their conformation. In Aim 2 we will identify compounds that alter the stability of HIV apo-IN dimers and inhibit the conformational changes that are required for IN function. The results of our studies will provide critical new information concerning HIV IN structure and function, and contribute to the design of new, allosterically-acting drugs that can complement the active site inhibitors now in clinical use.

描述(申请人提供)：整合酶(IN)是逆转录病毒复制所必需的三种病毒编码酶之一，也是治疗艾滋病毒/艾滋病药物开发的有效靶点。尽管已经成功地开发了临床上有用的药物来阻断整合反应的最后一步，即所谓的链转移抑制剂，但由于遇到病毒耐药性，仍有必要继续加强或取代现有的IN疗法。对HIV-1 IN的结构、组装和催化的所有方面的详细知识将揭示未利用的脆弱性和抑制这一关键的新策略 酵素。在目前的资助期间，我们应用小角X射线散射(SAXS)和蛋白质-蛋白质交联法获得了第一个以禽肉瘤病毒(ASV)IN为模板的全长去连接apo-IN单体和二聚体的实验模型。结果揭示了一种此前未曾被怀疑的二聚体结构(称为到达二聚体)。到达的二聚体的构型类似于病毒DNA结合的二聚体，在泡沫病毒(PFV)IN原型的晶体结构中是“内部”二聚体。根据这些和其他数据，我们已经构建了HIV IN到达二聚体的结构模型，我们假设它是预先定位的，可以与病毒DNA末端相互作用。在这次竞争性更新的目标1中，我们建议通过确定HIV IN的单体、二聚体和四聚体的溶液结构来测试这一模型，使用ASV IN成功采用的方法。我们将确定稳定HIV二聚体的相互作用，并确定底物结合对其构象的影响。在目标2中，我们将确定改变HIV载脂蛋白二聚体稳定性的化合物，并抑制IN功能所需的构象变化。我们的研究结果将提供有关HIV IN结构和功能的关键新信息，并有助于设计新的变构作用药物，以补充目前临床使用的活性部位抑制剂。