Probing functional HIV-1 envelope glycoprotein conformations with novel potent CD4-mimetic compounds

项目总结/摘要 人免疫缺陷病毒（HIV-1）包膜糖蛋白（Env）三聚体（（gp 120-gp 41）3）与宿主结合 受体，CD 4和CCR 5/CXCR 4，并介导病毒进入靶细胞。CD 4结合诱导 亚稳态Env三聚体的大规模构象变化，导致从预触发的， 从“闭合”构象到更“开放”构象。CD 4诱导的Env三聚体的开放允许 gp 120亚基结合CCR 5/CXCR 4共受体，使病毒和靶细胞膜融合 由gp 41跨膜亚基。 CD 4-模拟化合物（CD 4 mcs）是与高度保守的口袋（CD 4 mcs）结合的小有机分子。 Phe-43腔），靠近CD 4的结合位点。CD 4 mcs竞争性阻断CD 4结合， 过早地触发Env中类似于由CD 4诱导的构象变化。在没有 在表达辅受体的靶细胞中，这些过早激活的Env迅速且不可逆地变成非- 不降低在亚抑制浓度下，CD 4 mcs打开Env以结合抗体， 获得中和或细胞毒性潜力。CD 4 mcs可以与抗体协同作用，减少HIV-1感染者的死亡。 在人源化小鼠中的细胞储库和保护猴免受异源SHIV粘膜攻击。 占据gp 120“前庭”的CD 4 mc支架通向Phe-43腔，是一个关键的决定因素 这些化合物的抗病毒潜力。替换原型的四甲基哌啶支架 具有茚满支架的CD 4 mcs为合理设计的抗病毒效力的改善打开了大门， 宽度尽管如此，一些具有明显可接近的Phe-43空腔的主要HIV-1毒株仍然相对 抗目前铅茚满CD 4 mcs。我们最近发现了新的CD 4 mc类似物与吲哚啉 这些支架在抗病毒效力和广度上表现出令人印象深刻的增加。在这个提案中，我们利用 在这一发现的基础上，设计了对二氢吲哚CD 4 mcs的进一步改进，以研究其机制 其抗病毒效力的基础，并确定自然HIV-1 Env变异如何影响病毒耐药性 与CD 4 mcs和病毒复制适应性的关系。在这些研究过程中，我们将检验以下假设： 假设1：增加前庭中吲哚啉CD 4 mcs与保守gp 120元件的接触 将进一步增强其抗病毒的效力和广度;假设2：虽然CD 4 mc耐药在小 HIV-1毒株的一个亚群是由Phe-43空腔内的变异引起的， 菌株对CD 4 mcs的耐受性受Env可耐受性的差异（Env经历CD 4 mcs的倾向）控制。 构象变化）;和假设3：CD 4 mcs通过增强与Env三聚体的结合而获得效力， 导致连续的激活和失活，最终导致GP 120脱落。 通过测试这些假设所产生的知识将有助于提高抗病毒功效的努力。 CD 4 mcs，并将增强其作为HIV-1 Env构象的小分子探针的实用性。

PROJECT SUMMARY/ABSTRACT The human immunodeficiency virus (HIV-1) envelope glycoprotein (Env) trimer ((gp120-gp41)3) binds to host receptors, CD4 and CCR5/CXCR4, and mediates the entry of the virus into the target cell. CD4 binding induces large-scale conformational changes in the metastable Env trimer, resulting in transitions from a pretriggered, “closed” conformation to more “open” conformations. The CD4-induced opening of the Env trimer allows the gp120 subunit to bind the CCR5/CXCR4 coreceptor, which enables the fusion of viral and target cell membranes by the gp41 transmembrane subunit. CD4-mimetic compounds (CD4mcs) are small organic molecules that bind to a highly conserved pocket (the Phe-43 cavity) on gp120, near the binding site for CD4. CD4mcs competitively block CD4 binding and prematurely trigger conformational changes in Env similar to those induced by CD4. In the absence of a coreceptor-expressing target cell, these prematurely activated Envs rapidly and irreversibly become non- functional. At sub-inhibitory concentrations, CD4mcs open Env to the binding of antibodies that consequently acquire neutralizing or cytotoxic potential. CD4mcs can synergize with antibodies to decrease the HIV-1-infected cell reservoir in humanized mice and to protect monkeys from a heterologous SHIV mucosal challenge. The CD4mc scaffold, which occupies the gp120 “vestibule” leading into the Phe-43 cavity, is a critical determinant of the antiviral potential of these compounds. Replacing the tetramethyl-piperidine scaffold of the prototypic CD4mcs with an indane scaffold opened the door to rationally designed improvements in antiviral potency and breadth. Nonetheless, some primary HIV-1 strains with apparently accessible Phe-43 cavities remain relatively resistant to current lead indane CD4mcs. We have recently identified novel CD4mc analogues with indoline scaffolds that demonstrate impressive increases in antiviral potency and breadth. In this proposal, we capitalize on this discovery to design further improvements in the indoline CD4mcs, to investigate the mechanisms underlying their antiviral potency, and to determine how natural HIV-1 Env variation influences virus resistance to CD4mcs and viral replication fitness. In the course of these studies, we will test the following hypotheses: Hypothesis 1: Increasing the contacts of the indoline CD4mcs with conserved gp120 elements in the vestibule will further enhance their antiviral potency and breadth; Hypothesis 2: Although CD4mc resistance in a small subset of HIV-1 strains results from variation within the Phe-43 cavity, the sensitivity of most primary HIV-1 strains to CD4mcs is governed by differences in Env triggerability (the propensity of the Env to undergo conformational change); and Hypothesis 3: CD4mcs gain potency by enhanced binding to the Env trimer, leading to sequential activation and inactivation that ultimately result in gp120 shedding. The knowledge generated by testing these hypotheses will assist efforts to improve the antiviral efficacy of CD4mcs and will enhance their utility as small-molecule probes of HIV-1 Env conformation.