Preclinical Development of HIV-1 Vif Antagonists - Project 2

项目概要（见说明）：_ 该计划项目申请包括一个具有互补研究优势和资源的研究人员团队，将用于开发拮抗HIV-1 vif蛋白作用的新型小分子。项目2（SAR和抑制剂耐药机制的病毒学支持）将主要支持项目1中的药物发现/SAR工作，以及在体外和体内鉴定赋予抑制剂耐药的突变。项目2的具体职责包括： 评价先导化合物及其类似物在允许和非允许细胞中的抗病毒活性。 将比较项目1 SAR试验中出现的类似物的抗病毒活性， 允许细胞（VIF非依赖性复制）和非允许细胞（VIF依赖性复制），并将用于驱动具有更有效的抗病毒活性的类似物的设计。 在与病毒复制的CNS储库相关的原代细胞中评价抗病毒活性。巨噬细胞是CNS中的主要细胞靶点，并且感染的巨噬细胞驱动病毒复制的神经致病性表现。因此，将在体外评价vif拮抗剂在原代巨噬细胞中的抗病毒活性。 ？体外和体内抑制剂耐药机制的鉴定。HIV-1将在非允许细胞中以递增浓度的优先Vif类似物传代，以衍生抑制剂抗性病毒。此外，将克隆和测序来自经塞洛托治疗的猕猴的血浆病毒RNA（项目3），以鉴定体内赋予耐药性的突变。将使用核心B研究vif抑制剂耐药性对其他ARV类别敏感性的影响。 项目2中概述的病毒学研究将指导SAR研究，并推动类似物的优先级排序，以进一步分析猕猴体内疗效。

PROJECT SUMMARY (See Instructions): _ This program project application comprises a team of investigators with complimentary research strengths and resources that will be harnessed for the development of novel small molecules that antagonize the action of the HIV-1 vif protein. Project 2 (Virologic support for SAR and mechanisms of inhibitor resistance) will predominantly support drug discovery/SAR efforts in Project 1 as well as identifying mutations conferring inhibitor resistance in vitro and in vivo. Specific responsibilities of Project 2 include: ¿ Evaluation of antiviral activity of lead compounds and their analogs in permissive and non-permissive cells. The antiviral activities of analogs emerging from the SAR exercise of Project 1 will be compared in permissive cells (vif-independent replication) and non-permissive cells (vif-dependent replication) and will be used to drive the design of analogs with more potent antiviral activity. ¿ Evaluation of antiviral activity in primary cells relevant to CNS reservoirs of viral replication. Macrophage are the principle cellular targets in the CNS and infected macrophage drive the neuropathogenic manifestations of viral replication. Therefore, vif antagonists will be evaluated for antiviral activity in primary macrophage in vitro. ¿ Identification of mechanisms governing inhibitor resistance in vitro and in vivo. HIV-1 will be passaged in non-permissive cells in increasing concentrations of prioritized Vif analogs in order to derive inhibitor resistant virus. In addition, plasma viral RNA from inhibitor-treated macaques (Project 3) will be cloned and sequenced to identify mutations conferring resistance in vivo. Impact of vif-inhibitor resistance on sensitivity to other ARV classes will be conducted with Core B. The virologic studies outlined in Project 2 will guide the SAR studies and drive the prioritization of analogs that go forward for analysis of in vivo efficacy in macaques.