D-peptide inhibitors of HIV assembly and maturation

背景：为了对抗全球范围内的艾滋病流行，需要一种不易产生耐药性的新疗法。病毒的组装和成熟仍然是一个重要的抗病毒靶点，但在很大程度上尚未被利用。HIV的组装和成熟是通过HIV Gag多蛋白及其子结构域（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。从MA和CA衍生的肽或从噬菌体文库中选择的肽已被证明能够在体外阻断病毒的组装和成熟。然而，这些基于肽的组装/成熟抑制剂的抗病毒作用各不相同，这在很大程度上是由于它们在富含蛋白酶的活细胞环境中稳定性差——这是肽疗法的固有缺点。完全由d -氨基酸组成的肽，即d -肽，对蛋白质水解具有抗性，与传统的L-肽相比，这意味着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：我们寻求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端结构域（C-CA）拮抗剂，作为治疗HIV-1感染的新型治疗剂。具体目的：(1)采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)筛选针对DMA和DC-CA的噬菌体表达肽文库，确定与d蛋白有效结合所需的最佳序列；(3)验证天然MA和C-CA的d肽拮抗剂抑制HIV在外周血单核细胞复制的假设。研究设计：我们将筛选噬菌体表达的肽库，以对抗化学合成的DMA和DC-CA，确定d蛋白的高亲和力结合物。通过肽合成从L到D的转化将噬菌体优化的L肽配体转化为镜像的D形式，特异性结合天然LMA和LC-CA。我们将从结构上表征d肽与LMA或LC-CA之间的相互作用，并在体外评估它们的抗病毒活性。此外，机制研究将进行检查如何d肽破坏HIV Gag组装。最后，将设计新的递送载体来改善靶细胞对肽的摄取。意义：本研究旨在通过开发d肽抑制剂来阻止未成熟和成熟HIV颗粒的组装，从而填补这一明显的空白。本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。这种新型的肽拮抗剂，模拟了已知的组装/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优于传统的基于l肽的抗病毒药物。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，并阐明设计其他类拮抗剂分子来抑制HIV复制的见解。

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.