Modeling viral entry and its inhibition using SARS-CoV

使用 SARS-CoV 模拟病毒进入及其抑制

• 批准号: 7740796
• 负责人: GRAHAM SIMMONS
• 金额: $ 43.01万
• 依托单位: VITALANT
• 依托单位国家: 美国
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-12-15 至 2012-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7740796
• 关键词: Acids; Address; Animals; Antiviral Agents; Asians; Biological Assay; Categories; Cathepsin L; Cell Line; Cell surface; Cells; Chiroptera; Clinical Trials; Complex; Containment; Coronavirus; Coronavirus Infections; Coupled; DNA Sequence Rearrangement; Development; Disease Outbreaks; Event; Future; Glycoproteins; Health Hazards; Human; Infection; Influenza; Lead; Libraries; Licensing; Lung; Mediating; Membrane Fusion; Membrane Lipids; Methods; Modeling; Molecular; Nature; Peptide Hydrolases; Peptidyl-Dipeptidase A; Pharmaceutical Preparations; Play; Pneumonia; Population; Process; Property; Protease Inhibitor; Proteolysis; Proteolytic Processing; Public Health; RNA Viruses; Reagent; Recombinants; Residual state; Respiratory Tract Infections; Role; Satellite Viruses; Screening procedure; Serine Protease; Severe Acute Respiratory Syndrome; Site; Staging; Sulfhydryl Compounds; Surface; Techniques; Temperature; Therapeutic; Time; Tissues; Trypsin; Vaccines; Viral; Virus; Virus Diseases; Virus Inhibitors; Work; base; cellular targeting; coronavirus receptor; design; experience; extracellular; high risk; high throughput screening; human disease; inhibitor/antagonist; mortality; novel; pandemic disease; pathogen; prevent; prophylactic; receptor; restraint; small molecule; small molecule libraries; therapy development; tool; transmission process

DESCRIPTION (provided by applicant): Severe Acute Respiratory Syndrome-associated Coronavirus (SARS-CoV) is a category C priority pathogen with a high fatality rate that retains the potential to cause a pandemic. We aim to elucidate the mechanisms by which such enveloped viruses mediate membrane fusion during the process of entry. Inhibitors of entry will be sought through targeted and general screening of compound libraries. This work will have important consequences on the development of treatments for SARS-CoV, as well as understanding how entry occurs for SARS-CoV and other emerging viral infections with similar properties, such as ebolavirus. Furthermore, we will identify novel targets for the development of new antivirals. We have previously determined that the spike envelope glycoprotein of SARS-CoV (termed S) requires proteolytic processing by cathepsin L, and likely other proteases, in order to facilitate its membrane fusion potential. Uniquely, interactions with obligate receptor (Angiotensin Converting Enzyme-2, ACE2) are initially required before proteolysis. We hypothesize that ACE2 interaction induces conformational changes that not only exposes cryptic cleavage sites, but also initiates the cascade of rearrangements within S necessary to mediate membrane fusion. However, conformational constraints prevent the completion of these rearrangements. Hence, proteolysis is required in order to release these restraints. We base these hypotheses on the observations that: (1) broad spectrum and specific protease inhibitors able to block cathepsin L efficiently inhibit SARS-CoV entry; (2) recombinant cathepsin L, as well as a number of serine proteases such as trypsin, are able to successfully mediate processing of S leading to membrane fusion; (3) an elevated temperature-dependent step subsequent to receptor engagement, but prior to proteolysis is required for efficient membrane fusion. Residual SARS-CoV infection of cathepsin L- deficient cells is observed. Thus, we postulate that other endosomal proteases can mediate these effects. Furthermore, extracellular or surface proteases in tissue such as the lung may be able to mediate cell surface activation of S. We aim to identify proteases able to mediate infection and the mechanisms by which this is achieved. Inhibitors of SARS-CoV mediated infection will be sought, by specifically targeting cellular proteases, as well as by conducting a broader screen of viral entry. The specific aims are to: 1. Identify endosomal proteases able to mediate SARS-CoV infection and characterize the sites and mode of cleavage. Inhibitors of this proteolytic activity will be characterized as potential lead therapeutics. 2. Characterize the triggers of S- mediated membrane fusion, and hence uncover new targets for inhibitors. 3. Identify small molecule inhibitors of SARS-CoV S-mediated entry, and characterize their mode of action. This will identify both lead compounds for antivirals directed against SARS-CoV, highlight targets for inhibitors of viral entry and provide useful reagents for aims 1 and 2. The high mortality rate and large animal reservoir of SARS-associated viruses in Asian bats, coupled with a lack of proven therapeutic or prophylactic reagents, raises concern about SARS-CoV becoming a significant worldwide public health hazard. We propose the targeted and general screening of extensive small molecule libraries in order to identify inhibitors of viral entry that can be developed as SARS-CoV antivirals. Candidate inhibitors will be developed as lead compounds for the prophylactic and therapeutic treatment of this serious respiratory infection.

描述（由申请人提供）：严重的急性呼吸道综合征相关的冠状病毒（SARS-COV）是一种C类优先病原体，其死亡率很高，它保留了引起大流行的潜力。我们旨在阐明这种包膜在进入过程中介导膜融合的机制。将通过对化合物库的有针对性和一般筛选来寻求进入的抑制剂。这项工作将对SARS-COV治疗的发展产生重要影响，并了解SARS-COV和其他具有相似特性（例如埃博拉病毒）的新兴病毒感染的进入。此外，我们将确定开发新抗病毒药的新目标。我们先前已经确定SARS-COV的尖峰包膜糖蛋白（称为S）需要组织蛋白酶L和可能的其他蛋白酶的蛋白水解加工，以促进其膜融合潜能。独特的是，在蛋白水解之前，最初需要与强制受体（血管紧张素转化酶-2，ACE2）的相互作用。我们假设ACE2相互作用会引起构象变化，不仅会暴露出隐性裂解位点，而且还引发了S介导膜融合所需的S内的重新排列。但是，构象约束阻止了这些重排的完成。因此，为了释放这些约束，需要蛋白水解。我们将这些假设基于：（1）能够有效阻断组织蛋白酶l有效抑制SARS-COV进入的广泛光谱和特定蛋白酶抑制剂的观察结果； （2）重组组织蛋白酶L以及许多丝氨酸蛋白酶（例如胰蛋白酶）能够成功介导S导致膜融合的S的加工； （3）受体参与后的温度依赖性步骤升高，但是在蛋白水解之前，需要有效的膜融合。观察到剩余的SARS-COV感染组织蛋白酶L缺乏细胞。因此，我们假设其他内体蛋白酶可以介导这些作用。此外，组织中的细胞外或表面蛋白酶（例如肺）可能能够介导S的细胞表面激活。我们旨在鉴定能够介导感染的蛋白酶以及实现这一目标的机制。通过专门针对细胞蛋白酶以及进行更广泛的病毒进入筛查，将寻求SARS-COV介导的感染的抑制剂。具体目的是：1。识别能够介导SARS-COV感染并表征切割的位点和模式的内体蛋白酶。该蛋白水解活性的抑制剂将被描述为潜在的铅疗法。 2。表征S介导的膜融合的触发器，因此发现了抑制剂的新靶标。 3。确定SARS-COV S介导的入口的小分子抑制剂，并表征其作用方式。 This will identify both lead compounds for antivirals directed against SARS-CoV, highlight targets for inhibitors of viral entry and provide useful reagents for aims 1 and 2. The high mortality rate and large animal reservoir of SARS-associated viruses in Asian bats, coupled with a lack of proven therapeutic or prophylactic reagents, raises concern about SARS-CoV becoming a significant worldwide public health hazard.我们提出了广泛的小分子文库的有针对性和一般筛查，以鉴定可以作为SARS-COV抗病毒药的病毒进入的抑制剂。候选抑制剂将被开发为对这种严重呼吸道感染的预防和治疗治疗的铅化合物。