Inhibiting Immune Evasion by HIV-1 Nef to Facilitate Eradication

描述(由申请人提供)：HIV-1附属蛋白的免疫逃避可能导致宿主免疫未能控制感染，并可能使根除方法复杂化。我们将检验这一假设，即抑制宿主免疫的病毒逃避可以有助于治疗策略。最近的数据表明，病毒基因表达的重新激活不足以导致潜伏感染HIV-1细胞的细胞死亡-死亡需要细胞毒性T淋巴细胞(CTL)的杀伤活性。因此，我们的战略重点是HIV-1 Nef，它提供病毒逃避CTL活性。NEF阻止I类MHC到达细胞膜，降低细胞表面病毒抗原的浓度，并抑制CTL对感染细胞的杀伤。干扰Nef的这种活性应该会增强CTL，并促进从宿主中根除感染细胞。原则上，这一策略适用于表达低水平病毒抗原的“储藏”细胞，以及CD阳性的记忆T细胞，在这些细胞中，潜伏病毒通过药物重新激活。 在这项提议的R21阶段，我们将验证Nef在病毒抵抗根除中的重要性，方法是证明从潜伏期重新激活病毒的原始T细胞显示MHC-I表面水平降低，并且这种减少是Nef依赖的，并与对CTL介导的杀伤的抵抗有关。同时，我们将利用我们最近关于Nef如何在分子和结构水平上调节MHC-I的知识来识别能够抑制这种活性的小分子。我们将利用我们的X射线结晶学模型来研究由 NEF，MHC-I链的细胞质结构域，以及？目的是为了设计原型多肽抑制剂，并设计一种能够识别小分子先导化合物的高通量筛选方法。 在R33阶段，我们将扩大我们的药物发现方法，包括一种新的计算方法，将显示氨基酸R基团的半刚性支架分子与络合物的结构相匹配。我们将通过这种方法以及我们的高通量筛选，使用基于细胞的二级筛选来评估线索，并从结构上对其进行优化。最后，我们将在体外、体外和体内环境中使用这些小分子来建立抑制Nef介导的免疫逃避可以促进病毒根除。

DESCRIPTION (provided by applicant): Immune evasion by HIV-1 accessory proteins likely contributes to the failure of host immunity to control the infection and may complicate approaches to eradication. We will test the hypothesis that inhibiting viral evasion of host immunity can contribute to a curative treatment strategy. Recent data suggest that reactivation of viral gene expression is not sufficient to cause the death of cells latently infected with HIV-1 cell-death requires the killing-activity of cytotoxic T lymphocytes (CTL). Consequently, our strategy focuses on HIV-1 Nef, which provides viral evasion of CTL-activity. Nef prevents class I MHC from reaching the plasma membrane, decreasing the concentration of viral antigens at the cell surface and inhibiting the killing of infected cells by CTL. Interference with this activity o Nef should empower CTL and facilitate the eradication of infected cells from the host. In principle, this strategy applies to "reservoir" cells expressing low levels of viral antigens as well as to CD-positive memory T cells in which latent virus is reactivated pharmacologically. During the R21 phase of this proposal, we will validate the importance of Nef in viral resistance to eradication by showing that primary T cells in which virus is reactivated from latency display reduced surface levels of MHC-I and that this reduction is Nef-dependent and associated with resistance to CTL-mediated killing. Concurrently, we will use our recent knowledge of how Nef modulates MHC-I at the molecular and structural levels to identify small molecules capable of inhibiting this activity. We will exploit our X-ray crystallographic model of the complex formed by Nef, the cytoplasmic domain of the MHC-I ?-chain, and the ? subunit of the endosomal clathrin adaptor AP1 to design prototypic peptide inhibitors and to devise a high throughput screen capable of identifying small molecule lead compounds. During the R33 phase, we will broaden our approach to drug discovery by including a novel computational method that matches semi-rigid scaffold molecules displaying amino acid R groups to the structure of the complex. We will evaluate leads from this approach as well as from our high throughput screen using cell- based secondary screens and optimize them structurally. Lastly, we will use these small molecules in in vitro, ex vivo, and in vivo settings o establish that inhibiting Nef-mediated immune evasion can facilitate viral eradication.