Modeling viral entry and its inhibition using SARS-CoV

描述(申请人提供)：严重急性呼吸综合征相关冠状病毒(SARS-CoV)是C类优先病原体，病死率高，仍有可能导致大流行。我们的目标是阐明这些被包裹的病毒在进入过程中介导膜融合的机制。将通过对化合物文库的有针对性和一般性的筛选来寻找进入的抑制剂。这项工作将对SARS-CoV治疗方法的开发以及了解SARS-CoV和其他具有类似特性的新出现的病毒感染(如埃博拉病毒)如何进入具有重要影响。此外，我们将为开发新的抗病毒药物确定新的靶点。我们先前已经确定，SARS-CoV的尖峰包膜糖蛋白(简称S)需要组织蛋白酶L以及可能的其他蛋白酶来处理，以促进其膜融合潜力。独特的是，在蛋白质降解之前，最初需要与专性受体(血管紧张素转换酶-2，ACE2)相互作用。我们假设，ACE2相互作用引起的构象变化不仅暴露了隐蔽的切割位点，而且还启动了S体内介导膜融合所必需的级联重排。然而，构象限制阻碍了这些重排的完成。因此，需要蛋白质分解来释放这些限制。我们基于以下假设：(1)能够阻断组织蛋白酶L的广谱和特异的蛋白水解酶抑制剂能有效地阻止SARS-CoV进入；(2)重组组织蛋白酶L以及一些丝氨酸蛋白酶如胰蛋白酶能成功地介导S的加工，从而导致膜融合；(3)在受体结合之后，但在蛋白降解之前需要一个温度依赖的步骤来实现高效的膜融合。观察到组织蛋白酶L缺陷细胞的SARS-CoV残留感染。因此，我们推测其他内体蛋白水解酶可以调节这些效应。此外，肺等组织中的细胞外或表面蛋白酶可能能够介导S的细胞表面激活。我们的目的是鉴定能够介导感染的蛋白酶及其实现机制。将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来寻找SARS-CoV介导的感染的抑制剂。其具体目的是：1.鉴定能够介导SARS冠状病毒感染的内体蛋白水解酶，并确定其切割位置和方式。这种蛋白分解活性的抑制剂将被描述为潜在的铅治疗药物。2.鉴定S介导的膜融合的触发因素，从而发现新的抑制剂靶点。3.鉴定S介导的SARS-CoV进入的小分子抑制剂，并表征其作用模式。这将确定针对SARS-CoV的两种抗病毒先导化合物，突出病毒进入抑制剂的目标，并为AIMS 1和2提供有用的试剂。亚洲蝙蝠中SARS相关病毒的高死亡率和庞大的动物储存库，加上缺乏已证实的治疗或预防试剂，引起了人们对SARS-CoV成为重大全球公共卫生危害的担忧。我们建议对广泛的小分子文库进行有针对性和一般性的筛选，以确定可以开发为SARS-CoV抗病毒药物的病毒进入抑制剂。候选抑制剂将被开发为预防和治疗这种严重呼吸道感染的先导化合物。

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.