Novel targets for discovering peptide inhibitors of HIV-1 replication

发现 HIV-1 复制肽抑制剂的新靶点

• 批准号: 7929386
• 负责人: Xinhong Dong
• 金额: $ 20.75万
• 依托单位: MEHARRY MEDICAL COLLEGE
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-09-01 至 2012-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7929386
• 关键词: AIDS therapy; AIDS/HIV problem; Anti-HIV Agents; Anti-Retroviral Agents; Antiviral Agents; Binding; Binding Sites; Biochemical; Biological Assay; C-terminal; CD4 Positive T Lymphocytes; Cell Line; Cells; Chemicals; Competitive Binding; Data; Development; Epidemic; Flow Cytometry; Fluorescence Microscopy; Gagging; HIV; HIV Infections; HIV-1; Human; In Vitro; Infection Control; Kinetics; Label; Libraries; Mammalian Cell; Mediating; Mutagenesis; Peptide Library; Peptides; Pharmaceutical Preparations; Proteins; Research Priority; Resistance; Retroviridae; Role; Series; Staging; Surface Plasmon Resonance; T-Lymphocyte; Testing; Therapeutic; Validation; Virus Assembly; Vision; base; design; effective therapy; filamin; in vivo; inhibitor/antagonist; macrophage; novel; particle; protein aminoacid sequence; protein protein interaction; prototype; public health relevance; research study; resistant strain; synthetic peptide; uptake

DESCRIPTION (provided by applicant): The continual emergence of HIV strains that are resistant to currently approved anti-HIV drugs is an increasing threat to the effective treatment of HIV infection and control of the HIV/AIDS epidemic. Therefore, the discovery and development of new anti-HIV drugs with novel antiviral mechanisms and targets are urgently needed. The long-term objective of this application is to develop a novel class of anti-HIV drugs representing novel chemical entities targeting late stages of the HIV-1 replication cycle. Our recent studies demonstrate a novel protein- protein interaction between HIV-1 Gag and host filamin A, which is involved in late stages of the HIV-1 replication cycle in a productive manner. Disruption of the interaction redistributes Gag subcellular localization and inhibits particle release. These data suggest that the Gag-filamin A interaction could be developed as targets for HIV therapeutics. In this application, we hypothesize that small synthetic peptides, containing the binding site required for the interaction, might block specifically the interaction resulting in the impaired virus assembly and release. Experiments designed in this application will be performed to test this central hypothesis. In specific aim 1, the binding site for Gag and filamin A will be defined by mutagenesis, as well as in vitro and in vivo binding studies. Surface plasmon resonance (SPR) will be used to evaluate the binding kinetics of the interaction. Experiments in specific aim 2 will be designed to identify peptide candidates to specifically block the interaction. Gag- and filamin A-based libraries with overlapping peptide sequences covering the binding site and its surrounding region will be generated. Screen for peptide candidates against libraries will be performed using direct binding and competitive binding inhibition assays. The binding dynamics of peptide candidates with target proteins will be characterized. In specific aim 3, the role of peptide candidates on virus assembly and release will be examined. Cellular uptake and targeting validation of peptide candidates, which are mediated by Tat peptide, will be evaluated by fluorescence microscopy and flow cytometry. The role of peptide candidates on HIV-1 assembly and release will be determined in human T cell lines, and primary human CD4+ T cells and macrophages. Taken together, these studies will not only provide new sight into retrovirus-host interaction, but also impact the HIV/AIDS therapy by developing novel peptide inhibitors targeting the Gag-filamin A interaction. PUBLIC HEALTH RELEVANCE: The discovery of novel anti-retroviral drugs with new mechanisms is a high research priority due to the rapid emergence of isolates resistant to currently approved therapeutics. The information gained from our studies to identify and characterize peptide candidates targeting late stages of the HIV-1 replication cycle will impact HIV therapeutics. Our studies will take important steps toward developing the prototype of a novel class of anti-HIV inhibitors.

描述（由申请人提供）：对目前批准的抗艾滋病毒药物具有耐药性的艾滋病毒毒株的不断出现，对有效治疗艾滋病毒感染和控制艾滋病毒/艾滋病流行病构成越来越大的威胁。因此，迫切需要发现和开发具有新的抗病毒机制和靶点的新型抗hiv药物。这项申请的长期目标是开发一类新的抗hiv药物，代表针对HIV-1复制周期晚期的新型化学实体。我们最近的研究表明，HIV-1 Gag和宿主丝蛋白a之间存在一种新的蛋白-蛋白相互作用，这种相互作用以一种多产的方式参与HIV-1复制周期的后期阶段。相互作用的破坏重新分配Gag亚细胞定位并抑制颗粒释放。这些数据表明gag -丝蛋白A相互作用可以作为HIV治疗的靶点。在这一应用中，我们假设含有相互作用所需结合位点的小合成肽可能特异性地阻断相互作用，导致病毒组装和释放受损。本应用程序中设计的实验将用于检验这一中心假设。在具体目标1中，Gag和丝蛋白A的结合位点将通过诱变以及体外和体内结合研究来确定。表面等离子体共振（SPR）将用于评价相互作用的结合动力学。特定目标2中的实验将设计用于识别候选肽以特异性阻断相互作用。基于Gag和丝蛋白的文库将产生覆盖结合位点及其周围区域的重叠肽序列。筛选针对文库的候选肽将使用直接结合和竞争结合抑制试验进行。候选肽与靶蛋白的结合动力学将被表征。在特定目标3中，将研究候选肽在病毒组装和释放中的作用。由Tat肽介导的候选肽的细胞摄取和靶向验证将通过荧光显微镜和流式细胞术进行评估。候选肽对HIV-1组装和释放的作用将在人T细胞系、原代人CD4+ T细胞和巨噬细胞中确定。总之，这些研究不仅将为逆转录病毒-宿主相互作用提供新的视角，而且还将通过开发针对gag -丝蛋白A相互作用的新型肽抑制剂来影响HIV/AIDS的治疗。