Development of a High Throughput Screen for Antagonists of Vif Dimerization

描述(申请人提供)：艾滋病毒-1是艾滋病的病原体，目前全球约有3,300万人感染，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/艾滋病已经成为一种全球流行病，在发展中国家无法得到控制。艾滋病毒耐药株在世界各地的迅速出现使确定新的药物靶标的创新方法成为优先事项，这些药物靶标可能导致一类新的抗逆转录病毒疗法。在这方面，被称为Vif的HIV蛋白对HIV感染是必不可少的，因为它结合并诱导APOBEC3G的破坏；APOBEC3G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，这一特性已被证明是艾滋病毒传染性所必需的。调节亚基相互作用的Vif片段是已知的。一个独特的机会出现在发现这种蛋白质-蛋白质相互作用界面在感染野生型HIV的活细胞中是可访问的，因此，病毒复制明显受到该结构域的多肽模拟物转导的影响。这项建议寻求开发基于活细胞猝灭FRET试验的具有Vif多聚化拮抗剂活性的小分子的HTS初级筛选。这种均一检测方法是基于Vif的荧光蛋白嵌合体在HEK 293T细胞中的表达，通过Vif多聚体介导的荧光共振能量转移(FRET)实现距离依赖性猝灭。破坏Vif多聚体的化合物将产生增强的荧光信号。来自一次筛选的命中将在大肠杆菌中经历一个正交的二次筛选。在这项试验中，内酰胺酶的分泌进入细菌的周质空间是通过能量不依赖的双精氨酸转位途径进行的，对于氨苄西林耐药生长是必不可少的。我们已经使内酰胺酶的运输依赖于Vif的自结合。来自二次筛选的点击将验证它们：(1)通过传染性分析的抗病毒活性，(2)抑制差异表位标记的Vif的免疫共沉淀的能力，以及它们保护APOBEC3G免受Vif依赖的降解的能力。通过这项提议确定的化合物将是重要的先导化合物，以满足新疗法的要求，并提供新的研究试剂来研究VIF的结构和功能。 与公共卫生相关：世界各地艾滋病毒耐药株的迅速出现使确定新的药物靶标的创新方法成为优先事项，这些药物靶标可能导致一类新的抗逆转录病毒疗法。被称为Vif的HIV蛋白及其自我关联能力是HIV传染性所必需的。一个独特的机会出现在发现Vif-Vif相互作用的界面在感染野生型HIV的活细胞中是可访问的，并且病毒在这些细胞中的复制可以通过自我结合结构域的多肽模拟而受到损害。这项建议旨在开发一种基于活细胞FRET试验的具有Vif多聚化拮抗剂活性的小分子的HTS一次筛选和在大肠杆菌中的正交二次筛选。通过这些检测确定的化合物将被验证其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。

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.