Drug development for Vif-APOBEC3G in HIV-1/AIDS

Vif-APOBEC3G 治疗 HIV-1/AIDS 的药物开发

• 批准号: 7061978
• 负责人: David Kabat
• 金额: $ 36.91万
• 依托单位: OREGON HEALTH & SCIENCE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-12-01 至 2008-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7061978
• 关键词: DNA binding protein; aminohydrolases; antiAIDS agent; cytidine; drug discovery /isolation; enzyme inhibitors; enzyme linked immunosorbent assay; high throughput technology; human immunodeficiency virus 1; laboratory rabbit; method development; protein engineering; recombinant proteins; virus protein; yeasts

DESCRIPTION (provided by applicant): Recent studies by ourselves and others have substantially elucidated the role of the HIV-1 encoded viral infectivity factor (Vif) in neutralizing a potent antiviral system that occurs in lymphocytes and macrophages and some leukemic T cell lines. This antiviral system principally involves the cytidine deaminases APOBEC3G (A3G) and/or ASF, which are incorporated into HIV-1 cores where they lethally hypermutate newly synthesized viral reverse transcripts. Vif binds to A3G and ASF and induces their polyubiquitination and degradation, thereby eliminating them from infected cells and precluding their incorporation into HIV-1 progeny. Although researchers have used one Vif as a standard, HIV-1 Vifs are highly divergent and we have found that natural HIV-1 isolates encode Vifs that bind promiscuously to all APOBEC3 paralogs but have widely distinctive effects on their concentrations. In addition, the APOBECSs are coexpressed in different amounts and proportions in HIV-1 susceptible cells, and they broadly heterooligomerize to form a collaborative and inducible antiviral network. Our results suggest that Vif diversity is partly an adaptive response to the diversity of the APOBEC3 network in different cells and compartments, that it may play a critical role in HIV-1 pathogenesis, and that it should also be considered in anti-Vif drug development. Based on these results and on substantial preliminary evidence, we propose three substantial and synergistic aims: (1) Develop a robust series of screening platforms for identification of small molecules that inhibit diverse Vifs within mammalian cells. (2) We found that HIV-1 Vif is made in yeast as a soluble protein that associates with A3G. Produce substantial amounts of Vif, and its A3G-binding subdomain and A3G in the yeast Pichia pastoris as a resource to analyze inhibitor mechanisms, and to support the high throughput screening system of aim 3. (3) Optimize ELISA assays to screen and analyze compounds that inhibit Vif- A3G binding and to measure affinities of diverse Vifs for A3G and other cytidine deaminases. This program builds on recent insights to develop and to optimize novel inhibitor screening approaches and investigational resources for AIDS, as an essential prelude to high throughput screening for effective anti-Vif therapeutics.

描述（由申请人提供）：我们自己和其他人最近的研究已经基本上阐明了 HIV-1 编码的病毒感染因子 (Vif) 在中和淋巴细胞、巨噬细胞和一些白血病 T 细胞系中存在的有效抗病毒系统中的作用。该抗病毒系统主要涉及胞苷脱氨酶 APOBEC3G (A3G) 和/或 ASF，它们被整合到 HIV-1 核心中，在其中对新合成的病毒逆转录物进行致命性超突变。 Vif 与 A3G 和 ASF 结合，诱导它们的多泛素化和降解，从而将它们从受感染的细胞中消除，并阻止它们掺入 HIV-1 后代。尽管研究人员使用一种 Vif 作为标准，但 HIV-1 Vif 差异很大，我们发现天然 HIV-1 分离株编码的 Vif 与所有 APOBEC3 旁系同源物混杂结合，但对其浓度具有广泛独特的影响。此外，APOBECS 在 HIV-1 易感细胞中以不同的量和比例共表达，并且它们广泛异源寡聚化，形成协作和诱导型抗病毒网络。我们的结果表明，Vif 多样性在一定程度上是对不同细胞和区室中 APOBEC3 网络多样性的适应性反应，它可能在 HIV-1 发病机制中发挥关键作用，并且在抗 Vif 药物开发中也应考虑它。基于这些结果和大量初步证据，我们提出了三个实质性和协同目标：（1）开发一系列强大的筛选平台，用于鉴定抑制哺乳动物细胞内多种 Vif 的小分子。 (2) 我们发现 HIV-1 Vif 在酵母中作为与 A3G 结合的可溶性蛋白质产生。在酵母毕赤酵母中产生大量的 Vif 及其 A3G 结合子结构域和 A3G，作为分析抑制剂机制的资源，并支持目标 3 的高通量筛选系统。 (3) 优化 ELISA 测定，以筛选和分析抑制 Vif-A3G 结合的化合物，并测量不同 Vif 对 A3G 和其他胞苷脱氨酶的亲和力。该计划以最新见解为基础，开发和优化艾滋病的新型抑制剂筛选方法和研究资源，作为有效抗 Vif 疗法高通量筛选的重要前奏。