Inhibition of Ebola Virus Infection with Cathepsin L Inhibitors

组织蛋白酶 L 抑制剂抑制埃博拉病毒感染

• 批准号: 7475277
• 负责人: Norton P Peet
• 金额: $ 30万
• 依托单位: MICROBIOTIX, INC
• 依托单位国家: 美国
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-08-01 至 2010-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7475277
• 关键词: Address; Affinity; Africa; Antidotes; Antiviral Therapy; Biological Assay; Bioterrorism; Calpain; Categories; Cathepsin L; Cathepsins B; Cells; Cellular Assay; Centers for Disease Control and Prevention (U.S.); Containment; Cysteine Protease; Democratic Republic of the Congo; Disease; Disease Outbreaks; Dose; Drug Design; Drug Kinetics; Ebola virus; Environmental air flow; Enzyme Inhibitor Drugs; Enzyme Inhibitors; Enzymes; Exhibits; Fluorescence; Glycoproteins; Goals; Human; In Vitro; Infection; Inhibitory Concentration 50; Ivory Coast; Lead; Liver Microsomes; Lung; Measures; Mechanics; Methods; National Institute of Allergy and Infectious Disease; Nature; Oral; Permeability; Phase; Plaque Assay; Preparation; Process; Property; Protein Isoforms; Rain; Rate; Recombinants; Renal dialysis; Reporting; Research; Reston; SARS coronavirus; Structure; Sudan; Symptoms; Testing; Therapeutic; Therapeutic Intervention; Time; TimeLine; Toxicology; Vaccination; Vaccines; Viral; Viral Hemorrhagic Fevers; Viral Physiology; Virulent; Virus; Virus Diseases; Virus Inhibitors; base; biodefense; biosafety level 4 facility; concept; cytotoxicity; day; design; forest; in vivo; indexing; inhibitor/antagonist; innovation; mortality; nonhuman primate; novel; pathogen; pre-clinical; prevent; small molecule

DESCRIPTION (provided by applicant): Ebola virus is one of the most lethal infectious threats to mankind. These infections cause severe hemorrhagic fevers in humans and non-human primates and produce mortality rates of up to 90%. Ebola virus is classified in the NIAID Biodefense Research Agenda for CDC as a Category A Agent (USDHHS-NIH) and is a biosafety level 4 (BSL4) pathogen. There are four species of the Ebola virus, and there is currently no vaccine or antiviral therapy against Ebola virus infections for humans. The elusive nature of the natural reservoir makes eradication of the agent impossible and elevates the control of this virus and bioterrorist threat through vaccination or therapeutic means to a level of paramount importance. Currently, only heroic, symptomatic measures are available for the treatment of Ebola infections. The overall goal of this research is to address this critical need by developing small molecule, orally active antidotes to Ebola virus infections, which can be used prophylactically or in the 4-16 day post-infection period when fever and hemorrhagic symptoms of disease are present. The cysteine proteinase cathepsin L, a lysosomal enzyme, has been reported recently to be a new target for inhibition of SARS virus infections. Furthermore, inhibitors of cathepsin L can prevent SARS coronavirus entry into target cells. Ebola virus and SARS virus both employ cathepsin L activity for viral entry and cathepsin L is strongly implicated in the processing of the Ebola virus glycoprotein. Our strategy is to take advantage of this discovery to develop inhibitors of this new anti-Ebola target, cathepsin L, which is required for virus entry. This innovative approach involves targeting a host enzyme rather than a viral component. In preliminary studies, we built a pseudovirus carrying the Ebola glycoprotein, which can be studied under BSL2 containment, and demonstrated the capability of the pseudovirus to infect 293T cells. With an initial set of cathepsin L inhibitors, we demonstrated (i) potent and selective cathepsin L inhibitory activity versus recombinant enzyme and (ii) cell-contained enzyme; (iii) lack of cytotoxicity; and (iv) inhibition of pseudotype Ebola virus entry into 293T cells. These results validate this host enzyme at the cellular level as a target for anti-Ebola therapy for the first time. In Phase I, we will design and build new potent and selective inhibitors of cathepsin L by rational drug design and structure-based drug design methods, employing a rapid sensitive fluorescence assay for cathepsin L inhibition and inhibitor-enzyme co-crystal structures. We will prioritize the most potent inhibitors for their selectivity (eg, minimal affinity for cathepsin B and calpain), minimal cytotoxicity, cell permeability, favorable ADME properties and efficacy in an Ebola pseudotype assay as well as a virulent wild-type Ebola virus tested in a BSL4 facility. We will establish Proof of Concept by demonstrating the activity of pseudotype virus inhibitors in an infectious Ebola virus plaque assay in a BSL4 containment facility at USMARIID. In Phase II, these leads will be developed into preclinical candidates and in vivo IND enabling efficacy and pharmacokinetic and toxicology studies will be performed. Ebola virus infections constitute one of the most severe threats of infection to mankind. The natural reservoir for the Ebola virus is unknown, which increases the need for therapeutic intervention. This proposal describes a rational approach for the preparation of small molecule inhibitors of Ebola virus infection.

描述（由申请人提供）：埃博拉病毒是对人类最致命的传染性威胁之一。这些感染引起人类和非人类灵长类动物的严重出血狂，并产生高达90％的死亡率。埃博拉病毒被分类为CDC的Niaid Biodefense研究议程，为A类药物（USDHHS-NIH），是生物安全水平4（BSL4）病原体。埃博拉病毒有四种，目前尚无针对人类埃博拉病毒感染的疫苗或抗病毒疗法。自然储层的难以捉摸的性质使根除对代理的不可能，并通过疫苗接种或治疗手段提高了对这种病毒和生物恐怖主义威胁的控制，至关重要。目前，只有英雄，有症状的措施可用于治疗埃博拉病毒感染。这项研究的总体目的是通过开发小分子，口服活性的埃博拉病毒感染，可以预防性地使用，或在感染后4-16天使用疾病的发烧和出血性症状。据报道，半胱氨酸蛋白酶组织蛋白酶组织蛋白酶L（一种溶酶体酶）最近是抑制SARS病毒感染的新靶标。此外，组织蛋白酶L的抑制剂可以防止SARS冠状病毒进入靶细胞。埃博拉病毒和SARS病毒均采用组织蛋白酶L活性进行病毒进入，而组织蛋白酶L强烈与埃博拉病毒糖蛋白的加工有关。我们的策略是利用这一发现来开发这种新的抗欧博拉靶标的抑制剂，这是病毒进入所必需的。这种创新的方法涉及靶向宿主酶，而不是病毒成分。在初步研究中，我们建立了一个携带埃博拉糖蛋白的假病毒，可以在BSL2遏制下进行研究，并证明了假病毒感染293T细胞的能力。使用初始的组织蛋白酶L抑制剂，我们证明了（i）有效的和选择性的组织蛋白酶L抑制活性与重组酶和（ii）含细胞的酶； （iii）缺乏细胞毒性； （iv）抑制假埃博拉病毒进入293T细胞。这些结果首次证实了该宿主酶在细胞水平上作为抗欧博拉治疗的靶标。在第一阶段，我们将通过合理的药物设计和基于结构的药物设计方法设计和建立组织蛋白酶L的新有效抑制剂，采用快速敏感的荧光测定法对组织蛋白酶l抑制作用和抑制剂 - 酶共晶结构。我们将优先考虑其选择性最有效的抑制剂（例如，对组织蛋白酶B和钙蛋白酶的亲和力最小），最小的细胞毒性，细胞渗透性，良好的ADME特性和功效性的性质和功效，对ebola pseudotype分析以及一种有毒的野生型埃博拉犬Virus对bsl4 bsl4 bsl4 bsl4 besl4 bsl4 bsl4 bsl4 bsl4 bsl4 bsl4 besl4。我们将通过证明在USMARIID的BSL4遏制设施中在感染性埃博拉病毒斑块测定中证明伪型病毒抑制剂的活性来建立概念证明。在第二阶段，将将这些潜在客户发展为临床前候选者，并将在体内启用功效和药代动力学和毒理学研究。 埃博拉病毒感染构成了对人类感染最严重的威胁之一。埃博拉病毒的天然储层尚不清楚，这增加了对治疗干预的需求。该提案描述了一种制备埃博拉病毒感染的小分子抑制剂的合理方法。