Development of a High Throughput Screen for Antagonists of Vif Dimerization

描述（由申请人提供）：艾滋病毒-1是艾滋病的病原体，目前全世界约有3300万人感染，仅北美就有190万人感染（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，在发展中国家尚未得到控制。世界各地耐药的艾滋病毒毒株的迅速出现，使确定新的药物靶点的创新方法成为一个优先事项，这可能导致一种新的抗逆转录病毒疗法。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APOBEC3G的破坏；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。介导亚基相互作用的Vif片段是已知的。发现这种蛋白-蛋白相互作用界面在感染野生型HIV的活细胞中是可接近的，这是一个独特的机会，因此，该结构域的肽模拟物的转导明显损害了病毒的复制。本研究旨在开发一种基于活细胞淬灭FRET试验的具有Vif多聚拮抗剂活性的小分子HTS初级筛选方法。该同质实验是基于Vif荧光蛋白嵌合体在HEK 293T细胞中的表达，通过Vif多聚化介导的荧光共振能量转移（FRET）实现距离依赖猝灭。破坏Vif多聚的化合物将产生增强的荧光信号。在大肠杆菌中，来自主筛的命中将进行正交的二次筛检。在这个实验中，内酰胺酶通过能量独立的双精氨酸易位途径分泌到细菌的塑料周围空间，这对于耐氨苄西林的生长是必不可少的。我们已经证明了内酰胺酶的转运依赖于Vif自缔合。来自二级筛选的命中将被验证：(1)通过感染性测试的抗病毒活性，(2)抑制差异表位标记Vif的共免疫沉淀的能力，以及保护APOBEC3G免受Vif依赖性降解的能力。通过该提案确定的化合物将成为解决新疗法任务的重要先导化合物，并为研究Vif的结构和功能提供新的研究试剂。

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.