PEPTIDE-BASED APPROACHES TO ANTAGONISM AND MECHANISM OF HIV-1 ENVELOPE

这一多学科POI计划项目中的项目2的总体目标是开发HIV-1进入抑制剂，通过使用多肽和蛋白质科学策略设计病毒进入拮抗剂，在病毒遇到宿主细胞之前针对环境尖峰进行靶向。此外，衍生的多肽拮抗剂以及从母体多肽产生的多肽模拟物将被用作机制探针，以确定HLV-1 Env复合体失活的脆弱性。最近，我们发现了一系列多肽三氮唑，它们的靶标是GP 120上的保守足迹，它与CD4结合位点有显著重叠。这些抑制剂以纳摩尔亲和力结合gp120，并在负责与病毒进入所需的两种细胞受体结合的部位阻断相互作用。序列最小化、非天然氨基酸取代、核磁共振和计算机模拟的结合揭示了三唑类多肽中的一个核心药效团，它由三个氨基酸残基组成。生物物理和功能研究表明，这些抑制剂稳定了GP 120的部分结构状态，并通过变构机制破坏了介导病毒进入所需的Env复合体的构象转变。此外，我们最近发现，多肽三氮唑可能是通过构象作用，在没有宿主细胞的情况下导致GP 120脱落，从而导致病毒不可逆转的失活。此外，值得注意的是，含有游离硫基的三唑变异体破坏了HIV-1被膜的结构完整性，导致病毒内衣壳蛋白p24的泄漏。展望未来，这些结果将被用于设计和验证模拟多肽和小分子Env拮抗剂来灭活病毒。为了实现本计划项目的总体目标，项目2将追求以下三个具体目标：[1)设计、合成和开发结构简化的HIV-1被膜和宿主细胞受体相互作用的多肽和模拟多肽拮抗剂；[2)绘制多肽和模拟多肽拮抗剂的gpl20结合位点图，以定义由多肽三氮唑引发的GP 120的构象转变；以及[3]确定多肽三唑和多肽模拟抑制剂对Env蛋白和病毒颗粒结构和功能的机制影响，并据此描绘病毒Env对拮抗和灭活的脆弱性。由项目2开发的基于多肽的拮抗剂将对该计划项目了解环境蛋白进入机器的结构机制的总体努力做出重大贡献，这反过来又有望为艾滋病预防和干预提供新的策略。

The overarching goal of Project 2 within this multidisciplinary POI Program Project is the development of HIV- 1 entry inhibitors that target the Env spike before the virus encounters the host cell by employing peptide and protein science strategies to design viral entry antagonists. In addition, the derived peptide antagonists, as well as the peptidomimetics emerging from the parent peptides, will be utilized as mechanistic probes to define the vulnerabilities ofthe HlV-1 Env complex to deactivation. Recently, we have discovered a family of peptide triazoles that target a conserved footprint, on gp 120, which overlaps significantly with the CD4 binding site. These inhibitors bind gp120 with nanomolar affinity and block interactions at the sites responsible for binding with both of the cellular receptors required for viral entry. A combination of sequence minimization, non-natural amino acid substitutions, nuclear magnetic resonance and computational simulations have revealed a core pharmacophore in the peptide triazoles that consists of three amino acid residues. Biophysical and functional studies suggest that the inhibitors stabilize a partially structured state of gp 120 and, through an allosteric mechanism, disrupt the conformational transitions of Env complex required to mediate viral entry. In addition, we recently discovered that peptide triazoles, likely by conformational effects, cause gp 120 shedding in the absence of host cells and consequent irreversible inactivation of virus. Further, and of considerable interest, peptide triazole variants containing a free sulfiiydryl group disrupt the structural integrity of HIV-1 envelope membrane, leading to leakage of the intra-viral capsid protein p24. Going forward, these results will be employed to design and validate peptidomimetic and small molecule Env antagonists that inactivate virus. To achieve the overall goals of this Program Project, three Specific Aims will be pursued by Project 2: [1] design, synthesize and develop structure-simplified peptide and peptidomimetic antagonists of HIV-1 envelope and host cell receptor interactions; [2] map the gpl20 binding site for the peptide and peptidomimetic antagonists that define the conformational transitions of gp 120 triggered by peptide triazoles; and [3] determine the mechanistic effects of peptide triazole and peptidomimetic inhibitors on Env protein and virus particle structure and function, and from this understanding, delineate vulnerabilities of the virus Env to antagonism and inactivation. The peptide-based antagonists developed by Project 2 will contribute substantially to the overall effort of this Program Project to understand structural mechanisms of the Env protein entry machine, which in turn holds the promise of providing new tactics for both AIDS prevention and intervention.