Inhibiting Immune Evasion by HIV-1 Nef to Facilitate Eradication

描述（由申请方提供）：HIV-1辅助蛋白的免疫逃避可能导致宿主免疫无法控制感染，并可能使根除方法复杂化。我们将检验抑制病毒逃避宿主免疫力可能有助于治愈性治疗策略的假设。最近的数据表明，病毒基因表达的再激活不足以导致潜伏感染HIV-1的细胞死亡，细胞死亡需要细胞毒性T淋巴细胞（CTL）的免疫活性。因此，我们的策略集中在HIV-1 Nef，它提供病毒逃避CTL活性。Nef阻止I类MHC到达质膜，降低细胞表面的病毒抗原浓度并抑制CTL对感染细胞的杀伤。干扰Nef的这种活性应该增强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“储库”细胞以及CD阳性记忆T细胞，其中潜伏病毒被重新激活。 在本提案的R21阶段，我们将通过显示病毒从潜伏期重新激活的原代T细胞显示MHC-I表面水平降低，并且这种降低是Nef依赖性的，并且与CTL介导的杀伤抗性相关，来验证Nef在病毒耐药性中对根除的重要性。同时，我们将利用我们最近的知识，Nef如何调节MHC-I在分子和结构水平，以确定能够抑制这种活动的小分子。我们将利用我们的X射线晶体学模型的复合物形成的， Nef，MHC-I的胞质结构域？链，而？本发明的目的在于设计原型肽抑制剂并设计能够鉴定小分子先导化合物的高通量筛选。 在R33阶段，我们将通过包括一种新的计算方法来拓宽我们的药物发现方法，该方法将显示氨基酸R基团的半刚性支架分子与复合物的结构相匹配。我们将评估来自这种方法的线索以及来自我们使用基于细胞的二次筛选的高通量筛选的线索，并在结构上优化它们。最后，我们将在体外、离体和体内环境中使用这些小分子，以确定抑制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.