Modeling viral entry and its inhibition using SARS-CoV

描述（由申请方提供）：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，具有高致死率，仍有可能引起大流行。我们的目的是阐明这种包膜病毒介导的膜融合过程中的入境的机制。将通过化合物文库的靶向和一般筛选来寻找进入抑制剂。这项工作将对SARS-CoV治疗方法的发展产生重要影响，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。此外，我们将确定新的目标，为新的抗病毒药物的发展。我们先前已经确定，SARS-CoV的刺突包膜糖蛋白（称为S）需要通过组织蛋白酶L和可能的其他蛋白酶进行蛋白水解加工，以促进其膜融合潜力。独特的是，与专性受体（血管紧张素转换酶-2，ACE 2）的相互作用最初需要在蛋白水解之前。我们假设ACE 2相互作用诱导构象变化，不仅暴露隐藏的切割位点，而且还启动了介导膜融合所必需的S内重排级联反应。然而，构象限制阻止这些重排的完成。因此，需要蛋白水解以释放这些限制。我们基于以下观察结果提出了这些假设：（1）广谱特异性蛋白酶抑制剂能够有效地阻断组织蛋白酶L抑制SARS-CoV进入;（2）重组组织蛋白酶L以及许多丝氨酸蛋白酶如胰蛋白酶能够成功地介导S的加工，导致膜融合;（3）在受体接合之后但在蛋白水解之前的升高的温度依赖性步骤是有效的膜融合所必需的。观察到组织蛋白酶L缺陷细胞的残余SARS-CoV感染。因此，我们假设其他内体蛋白酶可以介导这些作用。此外，组织如肺中的细胞外或表面蛋白酶可能能够介导S.我们的目标是确定能够介导感染的蛋白酶和实现这一目标的机制。将通过特异性靶向细胞蛋白酶以及通过对病毒进入进行更广泛的筛选来寻找SARS-CoV介导的感染的抑制剂。具体目标是：1.鉴定能够介导SARS-CoV感染的内体蛋白酶，并表征切割的位点和模式。这种蛋白水解活性的抑制剂将被表征为潜在的先导治疗剂。2.表征S介导的膜融合的触发因素，从而发现抑制剂的新靶点。3.识别SARS-CoV S介导的进入的小分子抑制剂，并表征其作用模式。这将确定两种针对SARS冠状病毒的抗病毒药物的先导化合物，突出病毒进入抑制剂的靶点，并为目标1和2提供有用的试剂。亚洲蝙蝠的高死亡率和SARS相关病毒的大型动物储库，加上缺乏经过验证的治疗或预防试剂，引起了人们对SARS冠状病毒成为一个重大的全球公共卫生危害的关注。我们建议对广泛的小分子文库进行靶向和一般性筛选，以确定可开发为SARS-CoV抗病毒药物的病毒进入抑制剂。候选抑制剂将作为先导化合物开发，用于预防和治疗这种严重的呼吸道感染。

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.