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

在这个多学科POI计划项目中，项目2的总体目标是开发HIV- 1进入抑制剂，通过采用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靶向Env尖峰。此外，衍生的肽拮抗剂，以及从母体肽出现的肽模拟物，将被用作机制探针，以确定HIV-1 Env复合物对失活的脆弱性。最近，我们已经发现了一个家庭的肽三唑，目标保守的足迹，糖蛋白120，这与CD 4结合位点显着重叠。这些抑制剂以纳摩尔亲和力结合gp 120，并在负责与病毒进入所需的两种细胞受体结合的位点阻断相互作用。序列最小化、非天然氨基酸取代、核磁共振和计算模拟的组合揭示了由三个氨基酸残基组成的肽三唑中的核心药效团。生物物理和功能研究表明，抑制剂稳定的部分结构状态的糖蛋白120，并通过变构机制，破坏介导病毒进入所需的Env复合物的构象转变。此外，我们最近发现，肽三唑，可能通过构象效应，导致糖蛋白120脱落，在没有宿主细胞和随后的病毒不可逆灭活。此外，相当令人感兴趣的是，含有游离巯基的肽三唑变体破坏HIV-1包膜膜的结构完整性，导致病毒内衣壳蛋白p24的泄漏。展望未来，这些结果将用于设计和验证抗病毒的肽模拟物和小分子Env拮抗剂。为了实现本计划项目的总体目标，项目2将追求三个具体目标：[1]设计、合成和开发HIV-1包膜和宿主细胞受体相互作用的结构简化的肽和肽模拟物拮抗剂; [2]绘制肽和肽模拟物拮抗剂的gp 120结合位点，其定义肽三唑引发的gp 120构象转变;[3]设计、合成和开发HIV-1包膜和宿主细胞受体相互作用的结构简化的肽和肽模拟物拮抗剂。和[3]确定肽三唑和肽模拟物抑制剂对Env蛋白和病毒颗粒结构和功能的机制作用，并从这一理解出发，描述病毒Env对拮抗和失活的脆弱性。由项目2开发的基于肽的拮抗剂将大大有助于本计划项目的整体努力，以了解Env蛋白进入机器的结构机制，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.