Preclinical Development of HIV-1 Vif Antagonists - Project 2

项目总结(见说明)：_ 该计划项目申请由一组研究人员组成，他们具有互补的研究实力和资源，将被用于开发对抗HIV-1 vif蛋白作用的新型小分子。项目2(对SAR的病毒学支持和抑制剂耐药性的机制)将主要支持项目1中的药物发现/SAR努力，以及在体外和体内识别与抑制剂耐药性有关的突变。项目2的具体职责包括： 在许可和非许可细胞中评估先导化合物及其类似物的抗病毒活性。 从项目1的合成孔径雷达演习中出现的类似物的抗病毒活性将在 允许细胞(病毒无关复制)和非允许细胞(病毒依赖复制)，并将被用来推动具有更强抗病毒活性的类似物的设计。 评估与病毒复制的中枢神经系统储存库相关的原代细胞中的抗病毒活性。巨噬细胞是中枢神经系统的主要细胞靶点，感染的巨噬细胞驱动病毒复制的神经致病表现。因此，VIF拮抗剂将在体外对原代巨噬细胞的抗病毒活性进行评估。 ？在体外和体内鉴定抑制物耐药性的机制。HIV-1将在非许可细胞中以增加优先Vif类似物的浓度进行传代，以产生抗药性病毒。此外，来自经抑制剂处理的猕猴的血浆病毒RNA(项目3)将被克隆和测序，以识别体内与耐药性有关的突变。VIF抑制剂的抗性对其他ARV类别敏感性的影响将与核心B一起进行。 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顺序，以分析猕猴的体内疗效。

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.