Development of a High Throughput Screen for Antagonists of Vif Dimerization

Vif 二聚化拮抗剂高通量筛选的开发

• 批准号: 8191259
• 负责人: Harold C Smith
• 金额: $ 3.23万
• 依托单位: OYAGEN, INC.
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-09-25 至 2011-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8191259
• 关键词: AIDS/HIV problem; Acquired Immunodeficiency Syndrome; Address; Ampicillin; Anti-Retroviral Agents; Antiviral Agents; Arginine; Bacteria; Binding; Biological Assay; C-terminal; Cell Extracts; Cells; Chimeric Proteins; Co-Immunoprecipitations; Code; DNA; Developing Countries; Development; Dimerization; Drug Delivery Systems; Drug Industry; Drug resistance; Epidemic; Epitopes; Escherichia coli; Fluorescence; Fluorescence Resonance Energy Transfer; Growth; HIV; HIV Infections; HIV-1; Health; Host Defense; Infection; Lactamase; Lead; Life; Link; Luciferases; Marketing; Mediating; Membrane; Mutation; North America; Pathway interactions; Peptide Signal Sequences; Peptides; Persons; Phase; Polyubiquitination; Positioning Attribute; Property; Proteins; Reagent; Reporter; Research; Resistance; Signal Transduction; Structure; Therapeutic; Twin Multiple Birth; Validation; Variant; Viral; Viral Packaging; Viral Physiology; Virion; Western Blotting; base; high throughput screening; in vivo; innovation; novel; novel therapeutics; particle; periplasm; prevent; protein protein interaction; resistant strain; small molecule; viral DNA

DESCRIPTION (provided by applicant): HIV-1 is the causative agent of AIDS, that presently infects approximately 33 million persons worldwide with 1.9 million infected persons in North America alone (http://www.unaids.org). Recent studies have shown that HIV/AIDS has become a global epidemic that is not under control in developing nations. The rapid emergence of drug-resistant strains of HIV throughout the world has placed a priority on innovative approaches for the identification of novel drug targets that may lead to a new class of anti-retroviral therapies. In this regard, the HIV protein known as Vif is essential for HIV infection because it binds to and induces the destruction of APOBEC3G; a broad antiviral hostdefense factor. Vif subunits interact to form multimers and this property has been shown to be necessary for HIV infectivity. The segment of Vif that mediates subunit interaction is known. A unique opportunity has presented itself in the finding this protein-protein interaction interface is accessible in living cells infected with wild type HIV and consequently, viral replication was markedly impaired by the transduction of peptide mimics of this domain. This proposal seeks to develop an HTS primary screen for small molecules that have Vif multimerization antagonist activity based of a live cell quenched FRET assay. This homogeneous assay is based on the expression of fluorescent protein chimeras of Vif in HEK 293T cells to achieve distance-dependent quenching through fluorescence resonance energy transfer (FRET) mediated by Vif multimerization. Compounds that disrupt Vif multimerization will yield an enhanced fluorescence signal. Hits from the primary screen will be subjected to an orthogonal secondary screen in E. coli. In this assay, the secretion of ¿-lactamase into the periplastic space of bacteria occurs through an energy-independent, twin-arginine translocation pathway and is essential for ampicilin-resistant growth. We have rendered ¿-lactamase transport dependent on Vif self-association. Hits from the secondary screen will be validated for their: (1) antiviral activity through infectivity assays, (2) ability to inhibit co-immunoprecipitation of differentially epitope tagged Vif and for their ability to protect APOBEC3G from Vif-dependent degradation. Compounds identified through this proposal will be important as lead compounds to address a mandate for novel therapeutics and also provide new research reagents to study the structure and function of Vif. PUBLIC HEALTH RELEVANCE: The rapid emergence of drug-resistant strains of HIV throughout the world has placed a priority on innovative approaches for the identification of novel drug targets that may lead to a new class of anti-retroviral therapies. The HIV protein known as Vif and its ability to self-associate is required for HIV infectivity. A unique opportunity has presented itself in the finding that the interface for Vif-Vif interaction is accessible in living cells infected with wild type HIV and that viral replication can be impaired in these cells by peptide mimics of the self-association domain. This proposal seeks to develop an HTS primary screen for small molecules that have Vif multimerization antagonist activity based of a live cell FRET assay and an orthogonal secondary screen in E. coli. Compounds identified by these assays will be validated for their antiviral activity and ability to rescue host cell defense. These compounds will satisfy a mandate for novel therapeutics and also provide new research reagents for studying the structure and function of Vif.

描述（由申请人提供）：HIV-1是AIDS的病原体，目前全世界约有3300万人感染，仅北美就有190万感染者（http：www.unaids.org）。最近的研究表明，艾滋病毒/艾滋病已成为一种全球流行病，在发展中国家没有得到控制。艾滋病毒耐药性菌株在世界各地迅速出现，这使人们优先重视创新办法，以确定可能导致新一类抗逆转录病毒疗法的新药物靶点。在这方面，被称为Vif的HIV蛋白对HIV感染至关重要，因为它结合并诱导APOBEC 3G的破坏; APOBEC 3G是一种广泛的抗病毒宿主防御因子。Vif亚基相互作用形成多聚体，这种特性已被证明是HIV感染所必需的。已知Vif中介导亚基相互作用的片段。发现这种蛋白质-蛋白质相互作用界面在感染野生型HIV的活细胞中是可接近的，因此，病毒复制被该结构域的肽模拟物的转导显著削弱，这是一个独特的机会。该提案旨在开发基于活细胞淬灭FRET测定的具有Vif多聚化拮抗剂活性的小分子的HTS初级筛选。该均相测定基于Vif的荧光蛋白嵌合体在HEK 293 T细胞中的表达，以通过Vif多聚化介导的荧光共振能量转移（FRET）实现距离依赖性淬灭。破坏Vif多聚化的化合物将产生增强的荧光信号。来自主屏幕的点击将在E中进行正交的二次屏幕。杆菌在该测定中，通过能量非依赖性双精氨酸易位途径将β-内酰胺酶分泌到细菌的塑料周隙中，并且对于氨苄青霉素抗性生长是必需的。我们已经使β-内酰胺酶的转运依赖于Vif自身结合。将验证来自二次筛选的命中物的以下方面：（1）通过感染性测定的抗病毒活性，（2）抑制差异表位标记的Vif的免疫共沉淀的能力以及其保护APOBEC 3G免于Vif依赖性降解的能力。通过该提案鉴定的化合物将作为先导化合物，以解决新疗法的任务，并提供新的研究试剂来研究Vif的结构和功能。 公共卫生关系：艾滋病毒耐药性菌株在世界各地迅速出现，这使人们优先重视创新办法，以确定可能导致新一类抗逆转录病毒疗法的新药物靶点。HIV蛋白质Vif及其自我结合的能力是HIV感染所必需的。一个独特的机会出现在这样的发现中，即Vif-Vif相互作用的界面在感染野生型HIV的活细胞中是可接近的，并且病毒复制可以在这些细胞中通过自缔合结构域的肽模拟物受损。该建议旨在开发基于活细胞FRET测定的具有Vif多聚化拮抗剂活性的小分子的HTS初级筛选和E.杆菌将验证通过这些测定鉴定的化合物的抗病毒活性和拯救宿主细胞防御的能力。这些化合物将满足新疗法的要求，并为研究Vif的结构和功能提供新的研究试剂。