D-peptide inhibitors of HIV assembly and maturation

描述（由申请人提供）：HIV装配和成熟的D-肽抑制剂背景：需要对耐药性不敏感的新疗法来对抗全球HIV流行病。病毒组装和成熟仍然是一个重要的，但在很大程度上未开发的抗病毒靶点。HIV组装和成熟在蛋白质水平上通过HIV Gag多蛋白及其亚结构域（如基质蛋白（MA）和衣壳蛋白（CA））的自缔合来介导。已显示衍生自MA和CA或选自噬菌体文库的肽能够在体外阻断病毒组装和成熟。然而，这些基于肽的组装/成熟抑制剂的抗病毒效果不同，这在很大程度上是由于它们在活细胞的富含蛋白酶的环境中的稳定性差-肽治疗剂的固有缺点。完全由D-氨基酸组成的肽，即，D-肽对蛋白水解具有抗性，这转化为与常规L-肽相比大大改善的生物利用度和降低的免疫原性。因此，D-肽理想地适合作为用于治疗开发的先导药物化合物。目的/假设：我们试图通过化学蛋白合成和镜像噬菌体展示的方法，开发基于D肽的HIV MA和CA的C-末端结构域（C-CA）的拮抗剂，作为一类用于治疗HIV-1感染的新型治疗剂。具体目标：（1）使用固相肽合成和天然化学连接的组合来合成完全由D-氨基酸组成的HIV-1 MA和C-CA的D-对映体;（2）针对DMA和DC-CA筛选噬菌体表达的肽文库，并鉴定与D-蛋白的生产性结合所需的最佳序列;（3）检验天然MA和C-CA的D肽拮抗剂抑制外周血单核细胞中HIV复制的假设。研究设计：我们将针对化学合成的DMA和DC-CA筛选噬菌体表达的肽库，鉴定D蛋白的高亲和力结合剂。通过肽合成从L到D的转化将噬菌体优化的L-肽配体转化为特异性结合天然LMA和LC-CA的镜像D-形式。我们将在结构上表征D-肽与LMA或LC-CA之间的相互作用，并在体外评估其抗病毒活性。此外，还将进行机制研究，以检查D肽如何破坏HIV Gag组装。最后，将设计新的递送载体以改善靶细胞对肽的摄取。重要性：这项拟议中的研究试图通过开发D肽抑制剂来填补这一明显的空白，以防止不成熟和成熟的HIV颗粒的组装。如果实现了本提案中概述的具体目标，将使我们能够设计用于临床前研究的D-肽抗病毒治疗剂。这种新型肽拮抗剂模仿已知的组装/成熟抑制剂的活性，与传统的小分子药物相比具有高特异性和低毒性，并且在生物利用度、药代动力学和免疫原性方面上级传统的基于L-肽的抗病毒剂。D-肽拮抗剂的鉴定可能有助于更好地理解病毒组装和成熟过程，并阐明设计其他类别的拮抗分子以抑制HIV复制的见解。 公共卫生相关性：目前用于HIV-1感染患者的抗逆转录病毒疗法（ART）利用靶向病毒酶逆转录酶和蛋白酶的抑制剂的组合。抗逆转录病毒疗法降低了病毒载量，减缓了艾滋病毒向艾滋病的发展，有助于世界上艾滋病死亡人数的稳步下降。尽管ART取得了成功，但它并没有从受感染的细胞中根除艾滋病毒，并且ART的许多并发症之一是出现对目前的抗逆转录病毒疗法没有反应的耐药艾滋病毒株。这项研究的目的是开发一种新型的D肽抑制剂，作为武器库中的额外武器，通过特异性靶向HIV组装和成熟来对抗HIV感染。

DESCRIPTION (provided by applicant): D-Peptide Inhibitors of HIV Assembly and Maturation Background: New therapies less susceptible to drug resistance are needed to combat the global HIV epidemic. Virus assembly and maturation remains a significant yet largely unexploited antiviral target. HIV assembly and maturation is mediated at the protein level by self-association of the HIV Gag polyprotein and of its sub-domains such as the matrix protein (MA) and capsid protein (CA). Peptides derived from MA and CA or selected from phage libraries have been shown to be able to block virus assembly and maturation in vitro. However, the antiviral effects of these peptide-based assembly/maturation inhibitors vary, due to a large extent to their poor stability in the protease-rich environment of the living cell - an inherent drawback of peptide therapeutics. Peptides composed entirely of D-amino acids, i.e., D-peptides, are resistant to proteolysis, which translates into much-improved bioavailability and reduced immunogenicity as compared with conventional L- peptides. Thus, D-peptides are ideally suited as lead drug compounds for therapeutic development. Objective/Hypothesis: We seek to develop, by the means of chemical protein synthesis and mirror-image phage display, D-peptide based antagonists of HIV MA and the C-terminal domain of CA (C-CA) as a novel class of therapeutic agents for the treatment of HIV-1 infection. Specific Aims: (1) Synthesize the D- enantiomers of HIV-1 MA and C-CA composed entirely of D-amino acids using a combination of solid phase peptide synthesis and native chemical ligation; (2) Screen phage-expressed peptide libraries against DMA and DC-CA and identify optimal sequences required for productive binding to the D-proteins; (3) Test the hypothesis that D-peptide antagonists of native MA and C-CA inhibit HIV replication in peripheral blood mononuclear cells. Study design: We will screen phage-expressed peptide libraries against chemically synthesized DMA and DC-CA, identifying high-affinity binders for the D-proteins. Inversion from L to D through peptide synthesis will convert phage-optimized L-peptide ligands to their mirror image D-forms that specifically bind native LMA and LC-CA. We will structurally characterize the interaction between the D-peptides and LMA or LC-CA, and evaluate their antiviral activity in vitro. In addition, mechanistic studies will be carried out to examine how the D-peptides disrupt HIV Gag assembly. Finally, novel delivery vehicles will be designed to improve peptide uptake by target cells. Significance: The proposed research seeks to fill the obvious gap by developing D-peptide inhibitors to prevent assembly of both immature and mature HIV particles. The specific aims outlined in this proposal, if achieved, will enable us to design D-peptide antiviral therapeutics for preclinical studies. This novel class of peptide antagonists, emulating the activity of known assembly/maturation inhibitors, enjoys high specificity and less toxicity compared with traditional small- molecule drugs, and is superior to conventional L-peptide-based antiviral agents with respect to bioavailability, pharmacokinetics, and immunogenicity. Identification of D-peptide antagonists may help better understand the virus assembly and maturation process and illuminate insight as well into designing other classes of antagonistic molecules to inhibit HIV replication. PUBLIC HEALTH RELEVANCE: Current antiretroviral therapy (ART) for HIV-1 infected patients utilizes a combination of inhibitors that target the viral enzymes reverse transcriptase and protease. ART reduces viral load and slows the progression of HIV to AIDS, contributing to a steady decrease in AIDS deaths in the world. Despite its success, ART does not eradicate HIV from infected cells, and, among many complications of ART is the emergence of drug-resistant HIV strains not responding to current antiretroviral regimens. The proposed research aims to develop a novel class of D-peptide inhibitors as additional weapons in the arsenal to fight HIV infection by specifically targeting HIV assembly and maturation.