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包膜病毒的两个属。过去，由EBOV在中非人和非人类灵长类动物中暴发的暴发性出血热的死亡率高达90%，最近在西非爆发的死亡率为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，迫切需要开发有效的抗丝病毒感染的抗病毒疗法。瞄准 包膜病毒进入细胞作为一种抗病毒策略已被证明对广泛的病毒疾病有效。然而，丝状病毒融合机制的细胞内隔离使开发阻断EBOV糖蛋白(GP)介导的病毒进入的抗病毒药物具有挑战性。我们的目标是通过增加一个穿透细胞的多肽序列并将一个脂质部分连接到融合抑制多肽(C-肽)来克服这一挑战。我们已经证明，在小鼠体内注射后，我们的脂联型EBOV C肽的血浆水平可以持续保持。该C肽体外抑制EB病毒感染，IC_(50)为0.2M 并有效地保护小鼠免受致命的EBOV感染。我们建议利用这些初步结果来设计、合成和评估新型EBOV C肽类似物，这些类似物可以通过鼻腔或皮下给药，并能增强对多种丝状病毒的疗效。类似物将在感染性分析中评估抗病毒活性，对人类细胞的细胞毒性，以及GP2亚区结合的相互作用。有希望的类似物将在体外评估对不同丝状病毒的抗病毒活性和对小鼠的毒性。我们还将确定抗病毒耐药性的分子决定因素。选定的类似物将在小鼠模型的挑战实验中进行测试。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的给药途径的有效抗病毒药物来显著增强我们应对自然和故意流行病威胁的能力，以遏制急性丝状病毒暴发。我们提出了以下具体目标：目的1.利用结构导向突变和蛋白质工程来优化EBOV C肽抑制剂的抗病毒效力和生物利用度。A)设计和合成新的C-肽类似物；b)在细胞培养中表征对一系列丝状病毒的抗病毒活性；c)通过体外病毒进化实验表征病毒耐药性的决定因素。目的2.评价新型EBOV C肽抑制剂鼻腔或肠外给药对小鼠致死性EBOV感染的保护作用。A)分析改良的EBOV C肽类似物在体内的生物分布；b)评估小鼠的毒性；c)评估融合抑制剂在EBOV感染小鼠模型中的体内效力和活性广度。

 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.