Novel small-molecule inhibitors of HIV-1 Vif

描述（由申请人提供）：目前针对HIV-1的治疗受到多药耐药病毒的发展和显著的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病毒粒子感染因子（Vif）是原代CD4+ t细胞和巨噬细胞中HIV-1复制所必需的重要调节蛋白。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，可以特异性对抗由人类APOBEC3G （hA3G）和相关蛋白介导的先天抗病毒防御机制。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，以恢复这种重要的先天抗病毒防御。为此，我们开发了一种新的、简便的Vif功能抑制剂的功能获得筛选方法。我们基于机制的分析利用hA3G-荧光素酶（luc）报告构建直接监测细胞中存在Vif和潜在Vif抑制剂时hA3G的水平。在第一阶段的SBIR项目中，我们将优化和验证我们的高通量筛选方法；然后，通过自动高通量筛选（HTS），从Progenics的30多万种小分子化合物的化学文库中鉴定出特定的Vif抑制剂。将检测“高质量命中”对HIV-1假病毒和真HIV-1病毒的活性。最后，强效和特异性抑制剂将在一系列二级分析中进一步表征，以确定其作用机制和分子靶点。I期SBIR项目的成功将被定义为发现一个或多个候选先导系列，这些先导系列具有以下特性：1)在hA3G-luc稳定试验中具有强大的活性，2)对至少10个HIV-1分离株具有强大的抗病毒活性（中位IC50 < 2¿M）， 3)已确认的废除Vif功能的机制，以及4)最小的细胞毒性（CC50比IC50高20倍）。表现出这种特征的候选药物将在II期项目中对效力、特异性和生物利用度进行优化，以支持这种新型HIV-1治疗模式的人体测试。由于多重耐药病毒的发展和药物毒性的累积，发现和开发新的抗逆转录病毒药物仍然是迫切的医疗需要。该提案的总体目标是利用HIV-1研究中的新发现，发现靶向病毒粒子感染因子（Vif）的小分子化合物。vif特异性抑制剂有可能恢复机体自身由APOBEC3G及相关蛋白介导的抗病毒防御机制。

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.