Development of novel endosome-targeted Ebola virus entry inhibitors as antiviral agents

 描述：埃博拉病毒（EBOV）和马尔堡病毒（MARV）是构成丝状病毒科的两个包膜病毒属。在中非，由EBOV引起的人和非人灵长类动物暴发性出血热的死亡率在过去已达到90%，最近西非暴发的死亡率为60%。没有针对这些致命病原体的特定抗病毒治疗或疫苗被批准。因此，迫切需要开发针对丝状病毒感染的有效抗病毒疗法。靶向 作为抗病毒策略的包膜病毒的细胞进入已被证明对广泛的病毒性疾病有效。然而，丝状病毒融合机制的细胞内隔离使得开发阻断EBOV糖蛋白（GP）介导的病毒进入的抗病毒药具有挑战性。我们的目标是通过添加细胞穿透肽序列并将脂质部分与融合抑制肽（C肽）缀合来克服这一挑战。我们已经表明，在小鼠中肠胃外施用后，实现了我们的脂质缀合的EBOV C肽的持续血浆水平。该C肽抑制EBOV的体外感染，IC50为0.2 μ M 并有效地保护小鼠免受致死性EBOV感染。我们建议利用这些初步结果来设计、合成和评估新的EBOV C肽类似物，其可以鼻内或皮下递送，并且对广泛的丝状病毒具有增强的功效。将在感染性试验中评价类似物的抗病毒活性、对人细胞的细胞毒性以及GP2亚结构域结合相互作用。将评估有前景的类似物对不同丝状病毒的体外抗病毒活性和小鼠毒性。我们还将确定抗病毒药物耐药性的分子决定因素。将在小鼠模型中的激发实验中测试选定的类似物。我们预计，从我们拟议的研究中获得的知识将通过开发具有可行输送途径的强效抗病毒药物来遏制急性丝状病毒爆发，显着提高我们应对自然和故意流行病威胁的能力。我们提出以下具体目标：目标1。利用结构导向突变和蛋白质工程优化埃博拉病毒C肽抑制剂的抗病毒效力和生物利用度。a）新的C-肽类似物的设计和合成; B）在细胞培养物中对一系列丝状病毒的抗病毒活性的表征; c）通过体外病毒进化实验表征病毒抗性的决定因素。目标二。评价鼻内或胃肠外给药的新型EBOV C肽抑制剂对小鼠致死性EBOV感染的保护作用。a）分析改进的EBOV C-肽类似物的体内生物分布; B）评价小鼠中的毒性; c）评估融合抑制剂在EBOV感染的小鼠模型中的体内效力和活性宽度。

 DESCRIPTION: Ebola viruses (EBOV) and Marburg virus (MARV) are two genera of enveloped viruses that constitute the family of Filoviridae. Outbreaks of fulminant hemorrhagic fever in human and non-human primates in Central Africa caused by EBOV have reached mortality rates of up to 90% in the past and the mortality rate in the recent West Africa outbreak is 60%. No specific antiviral treatment or vaccine is approved for these deadly pathogens. There is thus an urgent need to develop effective antiviral therapies against infection by filoviruses. Targeting cell entry of enveloped viruses as an antiviral strategy has been proven effective against a wide range of viral diseases. However, the intracellular sequestration of filovirus fusion machinery makes it challenging to develop antivirals that block EBOV glycoprotein (GP)-mediated viral entry. We aim to overcome this challenge by adding a cell penetrating peptide sequence and conjugating a lipid moiety to fusion inhibitory peptides (C-peptides). We have shown that sustained plasma levels of our lipid-conjugated EBOV C-peptide are achieved after parenteral administration in mice. This C-peptide inhibits in vitro infection by EBOV with an IC50 of 0.2 M and efficiently protects mice from lethal EBOV infection. We propose to leverage these preliminary results to design, synthesize and evaluate novel EBOV C-peptide analogs that can be delivered intranasally or subcutaneously, and have enhanced efficacy against a broad range of filoviruses. Analogs will be evaluated for antiviral activity in infectivity assays, for cytotoxcity on human cells, and for GP2 subdomain-binding interactions. Promising analogs will be evaluated for in vitro antiviral activity against diverse filoviruses and for toxicity in mice. We ill also identify the molecular determinants of antiviral resistance. Selected analogs will be tested in challenge experiments in a mouse model. We anticipate that the knowledge gained from our proposed studies will significantly enhance our ability to address the threat of natural and intentional epidemics by developing potent antiviral drugs with feasible delivery routes for containing acute filovirus outbreaks. We propose the following specific aims: Aim 1. To use structure-guided mutagenesis and protein engineering to optimize the antiviral potency and bioavailability of EBOV C-peptide inhibitors. a) Design and synthesis of new C-peptide analogs; b) Characterization of antiviral activity against a range of filoviruses in cell culture; c) Characterization of determinants of viral resistance by in vitro virus evolution experiments. Aim 2. To evaluate the protection afforded by novel EBOV C-peptide inhibitors delivered intranasally or parenterally against lethal EBOV infection in mice. a) Analysis of in vivo biodistribution of improved EBOV C-peptide analogs; b) Evaluation of toxicity in mice; c) Assessment of in vivo potency and breadth of activity of fusion inhibitors in the mouse model of EBOV infection.