Novel small-molecule inhibitors of HIV-1 Vif

描述(由申请人提供)：目前针对HIV-1的治疗受到多重耐药病毒发展和显著累积药物毒性的限制。因此，HIV-1研究的一个主要目标是开发具有新作用模式的新型抗逆转录病毒药物。HIV-1病毒粒子传染性因子(Vif)是HIV-1在原代CD4+T细胞和巨噬细胞中复制所必需的调节蛋白。最近具有里程碑意义的研究表明，VIF是一种关键的病毒对策，它特异性地对抗由人APOBEC3G(HA3G)及其相关蛋白介导的天然抗病毒防御机制。该项目的总体目标是将这些最新发现转化为新的小分子Vif抑制剂，以恢复这种重要的天然抗病毒防御。为此，我们开发了一种新颖而简便的VIF功能抑制剂功能增益筛选方法。我们的基于机制的检测利用hA3G-荧光素酶(LUC)报告结构来直接监测存在Vif和潜在Vif抑制剂的情况下hA3G的细胞水平。在这个第一阶段的SBIR项目中，我们将优化和验证我们的高通量筛选试验；然后，我们将通过自动化高通量筛选(HTS)从Progenics的超过300,000种小分子化合物的多样化化学库中识别特定的Vif抑制剂。将对“高质量命中”进行针对HIV-1伪病毒和正品HIV-1的活性检测。最后，将在一系列二次检测中进一步确定有效和特定的抑制剂的特征，以确定它们的作用机制和分子靶点。第一阶段SBIR项目的成功将被定义为发现一个或多个候选先导系列，这些系列具有以下特性：1)在hA3G-Luc稳定化试验中具有强大的活性，2)针对至少10个HIV-1分离株的不同小组具有强大的抗病毒活性(IC50中值为2？M)，3)证实了取消Vif功能的机制，以及4)最低的细胞毒性(CC50是IC50的20倍)。表现出这一特征的主要候选药物将在第二阶段项目中针对效力、特异性和生物利用度进行优化，以支持这种新的艾滋病毒-1治疗模式的人体测试。由于多重耐药病毒的发展和累积的药物毒性，医学上仍然迫切需要发现和开发新的抗逆转录病毒药物。这项提议的总体目标是利用HIV-1研究中的新发现，发现以病毒粒子传染性因子(VIF)为靶点的小分子化合物。VIF特异性抑制剂将有可能恢复由APOBEC3G和相关蛋白介导的人体自身强大的抗病毒防御机制。

DESCRIPTION (provided by applicant): Present therapies for HIV-1 are limited by the development of multidrug-resistant virus and by significant cumulative drug toxicities. A major goal of HIV-1 research, therefore, is the development of new classes of antiretroviral agents with novel modes of action. The HIV-1 virion infectivity factor (Vif) is an essential regulatory protein required for HIV-1 replication in primary CD4+ T-cells and macrophages. Recent landmark studies have demonstrated that Vif is a critical viral countermeasure that specifically combats an innate antiviral defense mechanism mediated by human APOBEC3G (hA3G) and related proteins. The overall goal of this project is to translate these recent discoveries into novel small-molecule Vif inhibitors that restore this important innate antiviral defense. To this end, we have developed a novel and facile gain-of-function screen for inhibitors of Vif function. Our mechanism-based assay utilizes an hA3G-luciferase (luc) reporter construct to directly monitor cellular levels of hA3G in the presence of Vif and potential Vif inhibitors. In this Phase I SBIR project, we will optimize and validate our high throughput screening assay; specific Vif inhibitors will then be identified from Progenics' diverse chemical library of over 300,000 small-molecule compounds via automated high throughput screening (HTS). "Quality hits" will be assayed for activity against HIV-1 pseudoviruses and against authentic HIV-1. Finally, potent and specific inhibitors will be characterized further in a battery of secondary assays to establish their mechanism of action and molecular target. Success in the Phase I SBIR project will be defined as the discovery of one or more candidate lead series that have the following properties: 1) potent activity in the hA3G-luc stabilization assay, 2) potent antiviral activity against a diverse panel of at least 10 HIV-1 isolates (median IC50 < 2¿M), 3) confirmed mechanism of abrogating Vif function, and 4) minimal cytotoxicity (CC50 20-fold greater than IC50). Lead candidates that exhibit this profile will be optimized for potency, specificity and bioavailability in the Phase II project to support human testing of this novel mode of HIV-1 therapy. Due to the development of multi-drug resistant virus and cumulative drug toxicities, there continues to be an urgent medical need to discover and develop new antiretrovirals. The overall goal of this proposal is to exploit new discoveries in HIV-1 research to discover small-molecule compounds that target the virion infectivity factor (Vif). Vif-specific inhibitors would have the potential to restore the body's own potent antiviral defense mechanism mediated by APOBEC3G and related proteins.