High-Throughput Screening for Multifunctional Nef Inhibitors: Targeting HIV through Revitalizing Immune Defense Mechanisms

项目总结： 目前的抗逆转录病毒药物可以控制艾滋病毒感染，但不能治愈这种疾病。抗击艾滋病的欠发达地区 药物发现是为了重振和利用免疫系统的力量来对抗甚至消除艾滋病毒。 HIV-1Nef蛋白调节宿主免疫，促进疾病进展为艾滋病。两个最令人惊讶的- Nef的重要功能是表面下调CD4和下调I类主要组织。 配伍复合体(MHC-I)。NEF对CD4的作用确保正确处理 病毒，病毒的包膜蛋白。这一行动还有助于病毒隐藏表位并通过抗病毒抗体逃避检测。 体内，从而避免抗体依赖的细胞毒性(ADCC)，这是由宿主的自然 用来清除感染的杀伤细胞。此外，Nef还扰乱了另一种基本的免疫识别机制 通过下调MHC-I。NEF在这里的作用是阻止抗原提呈，这是由MHC-I在 感染细胞的表面。因此，由于细胞表面缺乏病毒抗原，感染 隐藏在免疫监视中，从而成功逃脱细胞毒性T细胞的杀伤。考虑到Nef的 阻断这些关键免疫机制的能力，可想而知，Nef的抑制剂可能逆转这种重要的免疫机制。 吞噬，这应该能够检测并可能清除宿主免疫系统的感染。 相信这一假设，我们的目标是通过抑制Nef来开发抗逆转录病毒药物，这是我们的长期目标 进球。在这个项目中，我们受到了我们最近的结构发现的启发，Nef使用一个共同的结合位点来 执行上述两个功能。这很令人兴奋，因为我们可能会使用一种药物来 阻断这两种基本功能，这可能会恢复抗击甚至消除艾滋病毒的巨大免疫力。 我们建议使用一种平行的方法来鉴定能够与这个多功能位点结合的抑制剂。 NEF。独一无二的是，我们设计了我们的药物筛选实验，使Nef能够在生物上采用Ac. 由于它的共选细胞伙伴的存在，即网状蛋白AP复合体的存在而导致的活性构象。我们的AP- Proach是创新的，因为我们的设计确保了Nef上基材结合口袋的正确形成 涉及目标多功能站点。我们的具体目标是：1)找出能阻断CD4下调的抑制剂。 由Nef提供的。在这里，我们将使用基于体外荧光偏振的高通量筛选(HTS) 试验和基于细胞的CD4下调试验，以寻找阻断CD4结合和 下调；2)确定可阻断Nef下调MHC-I的抑制剂。在这里，我们将使用类似的flo- 荧光偏振HTS法筛选可破坏MHC-ICD结合的Nef抑制剂 至Nef和clathrin AP1。这项工作的成功完成将产生Nef的双功能抑制剂， 可作为候选药物进一步开发成新颖的、现实世界中具有无与伦比的抗逆转录病毒药物 治疗潜力。

PROJECT SUMMARY: Current antiretrovirals control HIV infections but do not cure the disease. An underdeveloped area in anti-HIV drug discovery is to revitalize and harness the power of the immune system to combat and even eliminate HIV. The HIV-1 Nef protein modulates host immunity to promote disease progression to AIDS. Two of the most sig- nificant functions of Nef are surface downregulation of CD4 and surface downregulation of class I major histo- compatibility complex (MHC-I). Nef’s action on CD4 ensures proper processing of an important component of the virus, the viral Env protein. This action also helps the virus conceal epitopes and escape detection by anti- bodies, thereby avoiding antibody-dependent cellular cytotoxicity (ADCC) that is mediated by the host’s natural killer cells for clearing the infection. In addition, Nef disrupts another essential immune recognition mechanism by downregulating MHC-I. Nef’s action here blocks antigen presentation, which is mediated by MHC-I at the surface of the infected cells. Consequently, due to the lack of viral antigen on the cell surface, infections are hidden from immune surveillance and thus successfully evade the killing by the cytotoxic T cells. Given Nef’s abilities to block these key immune mechanisms, it is conceivable that inhibitors of Nef may reverse such ma- nipulation, which should enable detection and possibly clearance of the infection by the host immune system. Convinced by this hypothesis, we aim to develop antiretrovirals through Nef inhibition, which is our long-term goal. In this project, we are inspired by our recent structural findings that Nef uses a common binding site to execute both functions described above. This is exciting because we could potentially use a single drug to block both essential functions, which may restore tremendous immune power to fight and even eliminate HIV. We propose to use a paralleled approach to identify inhibitors capable of binding to this multifunctional site on Nef. Uniquely, we have designed our drug screening experiments in a way that Nef can adopt biologically ac- tive conformations due to the presence of its coopted cellular partners, namely clathrin AP complexes. Our ap- proach is innovative because our design ensures proper formation of the substrate-binding pockets on Nef that involve the targeted multifunctional site. Our specific aims are: 1) identify inhibitors that block CD4 downregula- tion by Nef. Here we will use both an in vitro fluorescence polarization-based, high throughput screening (HTS) assay and a cell-based CD4 downregulation assay to search for Nef inhibitors that block CD4 binding and downregulation; 2) identify inhibitors that block MHC-I downregulation by Nef. Here we will use a similar fluo- rescence polarization-based HTS assay for screening inhibitors of Nef that can disrupt the binding of MHC-ICD to Nef and clathrin AP1. Successful completion of this work will yield dual-functional inhibitors of Nef, which may serve as drug candidates to be further developed into novel, real world antiretrovirals with unparalleled therapeutic potentials.