Novel Small Molecule Inhibitors of HIV

描述（由申请人提供）：HIV-1病毒粒子感染因子（Vif）是HIV-1在CD4+ t细胞和巨噬细胞等自然靶细胞中复制所必需的重要调节蛋白。我们和其他人最近的研究表明，Vif是一种关键的病毒对抗防御，可以特异性中和由人类APOBEC3G （A3G）和相关胞苷脱氨酶介导的先天抗病毒防御机制。这些抗病毒蛋白诱导新合成的负链病毒DNA中胞嘧啶对尿嘧啶的致命修饰，导致在缺乏逆转录病毒Vif蛋白的情况下产生无功能病毒。Vif劫持细胞Cullin5 （Cul5）、拉长素b和拉长素，形成病毒E3泛素连接酶，靶向A3G进行多泛素化和降解。我们已经鉴定出N，N，N‘，N’- tetrakis -（2-吡啶基甲基）-乙二胺（TPEN）作为Vif的小分子抑制剂。该项目的总体目标是进一步表征新的小分子Vif抑制剂及其衍生物，以恢复有效的先天抗病毒防御。我们特别建议1。研究TPEN对多种HIV-1亚型和HIV-2亚型Vif功能的影响。我们已经观察到TPEN是HIV-1 Vif （NL4-3）功能的有效抑制剂，它允许抗病毒胞苷脱氨酶A3G甚至抑制野生型HIV-1的感染性。目前尚不清楚TPEN是否能抑制多种HIV-1亚型的Vif分子。我们将构建HIV-1和HIV-2 Vif分子所有主要亚型的表达载体，并测试TPEN对这些Vif分子的影响。我们还将确定TPEN是否可以抑制表达内源性A3G的原代CD4+ T细胞中的HIV-1复制。在此目的下提出的研究旨在进一步证实TPEN抗hiv活性的作用。2. 观察TPEN衍生物对A3G病毒Vif功能及抗病毒活性的影响。我们已经观察到TPEN是HIV-1 Vif功能的有效抑制剂，它允许抗病毒哺乳动物胞苷脱氨酶APOBEC3G （A3G）甚至抑制野生型HIV-1的感染性。目前尚不清楚TPEN是否仅通过锌螯合作用抑制Vif，因为另一种膜透性锌螯合剂氯喹诺没有效果。为了解决这个问题，我们将评估不具有结合锌能力的TPEN衍生物的抗vif活性。改变疏水、亲水性或带电性质的TPEN衍生物也将评估其抗vif功能。在此目的下提出的研究旨在进一步表征TPEN抗hiv活性的机制，并确定具有广泛治疗窗口的更有效的抗vif抑制剂。公共卫生相关性：HIV-1病毒粒子感染因子（Vif）是HIV-1在CD4+ t细胞和巨噬细胞等自然靶细胞中复制所必需的重要调节蛋白。我们和其他人最近的研究表明，Vif是一种关键的病毒对抗防御，可以特异性中和由人类APOBEC3G （A3G）和相关胞苷脱氨酶介导的先天抗病毒防御机制。我们已经鉴定出一种小分子Vif抑制剂。该项目的总体目标是进一步表征新的小分子Vif抑制剂及其衍生物，以恢复有效的先天抗病毒防御。

DESCRIPTION (provided by applicant): The HIV-1 virion infectivity factor (Vif) is an essential regulatory protein required for HIV- 1 replication in natural target cells such as CD4+ T-cells and macrophages. Recent studies from our group and others have demonstrated that Vif is a critical viral counter defense that specifically neutralizes an innate antiviral defense mechanism, mediated by human APOBEC3G (A3G) and related cytidine deaminases. These antiviral proteins induce lethal modification of cytosines to uracils in newly synthesized minus-strand viral DNA, resulting in the production of non-functional viruses in the absence of the retroviral Vif protein. Vif hijacks cellular Cullin5 (Cul5), ElonginB, and ElonginC to form a viral E3 ubiquitin ligase that targets A3G for polyubiquitination and degradation. We have identified N,N,N',N'-Tetrakis-(2-pyridylmethyl)-Ethylenediamine (TPEN) as a small molecule inhibitor of Vif. The overall goal of this project is to further characterize the novel small molecule Vif inhibitor and its derivatives that restore the potent innate antiviral defense. In particular we propose 1. To examine the effect of TPEN on Vif functions from multiple HIV-1 subtypes as well as HIV-2. We have observed that TPEN is a potent inhibitor of HIV-1 Vif (NL4-3) function which allows the anti-viral cytidine deaminase A3G to suppress even the wild-type HIV-1 infectivity. It is not clear whether TPEN could inhibit Vif molecules from a wide range of HIV-1 subtypes. We will construct expression vectors for all the major subtypes of HIV-1 and HIV-2 Vif molecules and test the effect of TPEN on these Vif molecules. We will also determine whether TPEN could inhibit HIV-1 replication in primary CD4+ T cells which express endogenous A3G. Studies proposed in this aim are designed to further substantiate the effect of TPEN anti-HIV activity. 2. To examine the effect of derivatives of TPEN on Vif function and the anti-viral activity of A3G. We have observed that TPEN is a potent inhibitor of HIV-1 Vif function which allows the anti-viral mammalian cytidine deaminase APOBEC3G (A3G) to suppress even the wild-type HIV-1 infectivity. It is not clear whether TPEN inhibits Vif solely through zinc chelation, since another membrane permeable zinc chelator, chloroquinol, was not effective. To address this issue, we will evaluate TPEN derivatives that do not have the ability to bind zinc for anti-Vif activity. TPEN derivatives that have altered hydrophobic, hydrophilic, or charged properties will also be evaluated for their anti-Vif function. Studies proposed in this aim are designed to further characterize the mechanism of TPEN anti-HIV activity and to identify more potent anti-Vif inhibitors with a broad therapeutic window. PUBLIC HEALTH RELEVANCE: The HIV-1 virion infectivity factor (Vif) is an essential regulatory protein required for HIV- 1 replication in natural target cells such as CD4+ T-cells and macrophages. Recent studies from our group and others have demonstrated that Vif is a critical viral counter defense that specifically neutralizes an innate antiviral defense mechanism, mediated by human APOBEC3G (A3G) and related cytidine deaminases. We have identified a small molecule inhibitor of Vif. The overall goal of this project is to further characterize the novel small molecule Vif inhibitor and its derivatives that restore the potent innate antiviral defense.