D-peptide inhibitors of HIV assembly and maturation

描述(由申请人提供)：艾滋病毒组装和成熟的D-肽抑制剂背景：需要较少对耐药性敏感的新疗法来抗击全球艾滋病毒流行。病毒的组装和成熟仍然是一个重要的、但在很大程度上尚未开发的抗病毒目标。HIV的组装和成熟是通过HIV Gag多聚蛋白及其亚域(如基质蛋白(MA)和衣壳蛋白(CA))的自结合在蛋白质水平上介导的。来源于MA和CA的多肽或选自噬菌体文库的多肽已被证明能够在体外阻止病毒的组装和成熟。然而，这些基于多肽的组装/成熟抑制剂的抗病毒效果各不相同，这在很大程度上是因为它们在富含蛋白酶的活细胞环境中稳定性较差，这是多肽疗法的固有缺点。完全由D-氨基酸组成的多肽，即D-肽，对蛋白质的降解具有抵抗力，与传统的L多肽相比，生物利用度大大提高，免疫原性降低。因此，D-肽非常适合作为治疗开发的先导药物化合物。目的/假设：通过化学蛋白质合成和镜像噬菌体展示，我们试图开发基于D-肽的HIV-MA拮抗剂和CA的C-末端结构域(C-CA)，作为一类新型的治疗HIV-1感染的药物。具体目标：(1)利用固相肽合成和天然化学连接相结合的方法合成完全由D-氨基酸组成的HIV-1 MA和C-CA的D-对映体；(2)筛选抗DMA和DC-CA的噬菌体表达肽库，并确定与D-蛋白有效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HIV在外周血单核细胞中复制的假设。研究设计：我们将根据化学合成的DMA和DC-CA筛选噬菌体表达的多肽库，鉴定D-蛋白的高亲和力结合。通过肽合成将L反转为D，将噬菌体优化的L多肽配体转化为与天然LMA和LC-CA特异结合的镜像D-形式。我们将对D-肽与LMA或LC-CA之间的相互作用进行结构表征，并在体外评价它们的抗病毒活性。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来提高靶细胞对多肽的摄取。意义：这项拟议的研究试图通过开发D-肽抑制剂来填补这一明显的空白，以防止未成熟和成熟的艾滋病毒颗粒的组装。这项提案中概述的具体目标如果实现，将使我们能够设计用于临床前研究的D-肽抗病毒疗法。这类新型的多肽拮抗剂模拟已知的组装/成熟抑制剂的活性，与传统的小分子药物相比具有高度的特异性和较低的毒性，在生物利用度、药代动力学和免疫原性方面优于传统的L多肽类抗病毒药物。D-肽拮抗剂的鉴定可能有助于更好地了解病毒的组装和成熟过程，并有助于设计其他类别的拮抗分子来抑制HIV的复制。 公共卫生相关性：目前针对HIV-1感染患者的抗逆转录病毒疗法(ART)使用的是针对病毒酶、逆转录酶和蛋白酶的抑制剂组合。抗逆转录病毒疗法减少了病毒载量，减缓了艾滋病毒向艾滋病的发展，为世界艾滋病死亡人数的稳步下降做出了贡献。尽管ART取得了成功，但它并不能从感染细胞中根除艾滋病毒，而且，在ART的许多并发症中，出现了对目前的抗逆转录病毒疗法无效的抗药性艾滋病毒株。这项拟议的研究旨在开发一种新型的D-肽抑制剂，作为武器库中的额外武器，通过专门针对艾滋病毒的组装和成熟来对抗艾滋病毒感染。

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.