Novel targets for discovering peptide inhibitors of HIV-1 replication

描述(申请人提供)：对目前批准的抗艾滋病毒药物产生抗药性的艾滋病毒毒株不断出现，对艾滋病毒感染的有效治疗和艾滋病毒/艾滋病流行的控制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抗HIV新药。这项应用的长期目标是开发一类新的抗艾滋病毒药物，代表针对HIV-1复制周期后期阶段的新化学实体。我们最近的研究表明，HIV-1 Gag和宿主细丝蛋白A之间存在一种新的蛋白质-蛋白质相互作用，这种相互作用以一种有效的方式参与了HIV-1复制周期的后期阶段。这种相互作用的中断会重新分布GAG的亚细胞定位，并抑制颗粒的释放。这些数据表明，Gag-细丝素A的相互作用可以作为HIV治疗的靶点。在这个应用中，我们假设包含相互作用所需结合位点的小合成肽可能特异性地阻止相互作用，导致受损的病毒组装和释放。在本应用程序中设计的实验将用于验证这一中心假设。在特定的目标1中，将通过突变以及体外和体内结合研究来确定Gag和细丝素A的结合位置。表面等离子体共振(SPR)将被用来评估相互作用的结合动力学。在特定目标2中的实验将被设计来识别特定阻断相互作用的候选多肽。将生成基于Gag和细丝蛋白A的文库，这些文库具有覆盖结合位点及其周围区域的重叠肽序列。将使用直接结合和竞争结合抑制分析来筛选针对文库的候选多肽。将对候选多肽与目标蛋白的结合动力学进行表征。在具体目标3中，将检查候选多肽在病毒组装和释放中的作用。由TAT多肽介导的候选多肽的细胞摄取和靶向性验证将通过荧光显微镜和流式细胞仪进行评估。候选多肽对HIV-1组装和释放的作用将在人类T细胞系、原代人类CD4+T细胞和巨噬细胞中确定。综上所述，这些研究不仅将为研究逆转录病毒与宿主的相互作用提供新的视角，而且将通过开发针对Gag-细丝素A相互作用的新型多肽抑制剂来影响HIV/AIDS的治疗。 公共卫生相关性：具有新机制的新型抗逆转录病毒药物的发现是一项高度优先的研究，因为对目前批准的治疗方法产生抗药性的分离株迅速出现。从我们的研究中获得的信息，以识别和表征针对HIV-1复制周期后期阶段的候选多肽，将影响HIV治疗。我们的研究将朝着开发一类新型抗HIV抑制剂的原型迈出重要的一步。

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.