Identification of neutralizing epitopes on SARS-CoV-2 spike for design of vaccines and small-molecule antivirals

冠状病毒是一种包膜、阳性、单链RNA病毒，分为甲型冠状病毒和乙型冠状病毒。冠状病毒感染哺乳动物和鸟类，通常会导致下呼吸道和/或上呼吸道疾病。冠状病毒对人类造成的疾病范围从普通感冒到全球流行/大流行，包括2003年的严重急性呼吸综合征(SARS-CoV)、2004年的人类CoV-NL63、2005年的人类CoV-HKU1、2012年的中东呼吸综合征冠状病毒(MERS-CoV)和2019年的SARS-CoV-2。目前还没有获得批准的疫苗或抗病毒药物来对抗冠状病毒感染，也缺乏 关于目前的SARSCoV-2疫情，经过测试和验证的治疗方法代表了一个巨大的全球关切。 2019年12月底，世界卫生组织注意到武汉市中国出现了异常大量的肺炎聚集性病例。在短短3个月的时间里，全球已确诊100多万例新冠肺炎病例，其中近5万人死亡。美国的这一数字现在正以惊人的速度增长(目前约占全球总数的25%)，而且 死亡的比例(目前约占总数的10%)。2002年，随着广东首例SARS-CoV大流行中国和2012年MERS-CoV暴发，人类冠状病毒感染的严重性和高死亡率非常明显。与SARS-CoV一样，目前的SARS-CoV-2也使用血管紧张素转换酶II(ACE2)作为细胞进入的宿主受体，其同源性为79%。冠状病毒表面暴露的刺突蛋白(S)负责识别和结合ACE2，是疫苗和抗病毒治疗的潜在靶点。重要的是，到目前为止从新冠肺炎患者中分离出来的抗体似乎结合了尖峰蛋白上的几个区域，这些区域随后成为小分子发现的理想靶点。 冠状病毒表面的刺突蛋白是一个糖基化的三聚体，由两个胞外结构域组成，即S1和S2。到目前为止，大多数NAB(中和抗体)对CoV的靶向是S1结构域，该结构域包含负责ACE2结合的受体结合结构域(RBD)。尽管如此，具有s2结构域表位的抗体，包括干细胞融合机制，在基于细胞和动物的感染模型中也具有中和潜力，并且通常比靶向抗体对其他冠状病毒有更广泛的反应。 S1。此外，来自人CoV-OC43(一种引起普通感冒的毒株)HR2结构域的螺旋肽EK1广泛与CoV的茎区域结合并抑制膜融合。我们这一补充的目的是确定SARS-CoV-2 S蛋白上的中和表位，如抗体靶向的表位、EK1肽和其他已报道的与RBD结合的多肽，以帮助基于结构的疫苗和小分子抗病毒设计。 具体地说，我们将利用我们在X射线结晶学(威尔逊)、电子显微镜(WARD)和小分子发现与药物化学(WURAN)方面的专业知识，从功能上表征中和表位和抗体结合基序，并将这些信息应用于高通量分析，以帮助疫苗设计、治疗性抗体的应用和使用，并发现SARS-CoV-2以及其他冠状病毒(包括SARS和MERS)S蛋白的特异性高亲和力小分子。我们预料到 我们从恢复期患者的B细胞中分离出的新型抗体的结构特征将提供有关表面热点的关键信息，这些热点可以被疫苗和小分子靶向。因此，抗体用于表位识别的共同特征将为化合物作为新冠肺炎抗病毒药物的主要候选药物的量身定制设计提供信息。重要的是，我们将对我们的小分子进行生物相关的假病毒空斑分析和基于细胞的感染模型，以及人类微粒体稳定性分析，以生成小分子概要，以便进入翻译研究。

Coronaviruses (CoVs) are enveloped, positive-sense, single-stranded RNA viruses and are divided into Alphaand Beta-coronaviruses. CoVs infect mammals and birds and typically result in lower and/or upper respiratory tract disease. The spectrum of illness in humans caused by CoVs range from common colds to worldwide epidemics/pandemics, including severe acute respiratory syndrome (SARS-CoV) in 2003, human CoV-NL63 in 2004, human CoV-HKU1 in 2005, Middle East respiratory syndrome coronavirus (MERS-CoV) in 2012, and SARS-CoV-2 in 2019. There are no approved vaccines or antiviral drugs to combat CoV infections and a lack of tested and validated therapeutics represents a tremendous global concern with respect to the current SARSCoV-2 outbreak. At the end of December 2019, the World Health Organization became aware of an abnormally large cluster of pneumonia cases localized in the city of Wuhan, China. Within a span of only 3 months, over one million confirmed cases of COVID-19 have been diagnosed worldwide with almost 50,0000 resulting in death. The numbers in the US are now growing at an alarming rate (currently ~25% of the global total) as well as the number of deaths (currently ~10% of total). The severity of human CoV infections and high mortality rates were strikingly apparent in 2002 with the first SARS-CoV pandemic in Guangdong, China, as well as the MERS-CoV outbreak in 2012. Like SARS-CoV, the current SARS-CoV-2, which is 79% identical, also employs angiotensin converting enzyme II (ACE2) as the host receptor for cellular entry. The CoV surface-exposed spike (S) protein is responsible for the recognition and binding of ACE2 and represents a potential target for development of vaccines and antiviral therapeutics. Importantly, antibodies (Abs) isolated to date from COVID-19 patients appear to bind several regions on the spike protein that then represent ideal targets for small molecule discovery. The spike protein on the CoV surface is a glycosylated trimer and consists of two extracellular domains, S1 and S2. The majority of nAbs (neutralizing Abs) to CoVs characterized to date target the S1 domain that contains the receptor-binding domain (RBD) responsible for ACE2 binding. Notwithstanding, Abs with epitopes on the S2 domain, consisting of the stem fusion machinery, also have neutralizing potential in both cell-based and animal models of infection and are generally more broadly reactive against other CoVs than those antibodies that target S1. Additionally, a helical peptide EK1 derived from the HR2 domain of human CoV-OC43 (a strain responsible for the common cold) broadly binds to the stem region of CoVs and inhibits membrane fusion. Our goal with this supplement is to define the neutralizing epitopes on the S protein of SARS-CoV-2, such as those targeted by antibodies, the EK1 peptide and other peptides reported to bind to the RBD, to aid in both structure-based vaccine and small molecule antiviral design. Specifically, we will leverage our combined expertise in x-ray crystallography (Wilson), electron microscopy (Ward), and small-molecule discovery and medicinal chemistry (Wolan) to functionally characterize neutralization epitopes and antibody binding motifs and apply this information into high-throughput assays to aid in vaccine design, applications and use of therapeutic antibodies, and for discovery of specific high affinity small molecules to the S protein of SARS-CoV-2, as well as other coronaviruses, including SARS and MERS. We anticipate that our structural characterization of novel Abs isolated from B cells of convalescent patients will provide critical information on surface hot spots, which can be targeted by vaccines and small molecules. As such, common features employed by antibodies for epitope recognition will inform on the tailored design of compounds as lead candidates for COVID-19 antivirals. Importantly, we will subject our small molecules to biologically relevant pseudovirus plaque assays and cell-based infection models, as well as human microsomal stability assays to generate a compendium of small molecules to move forward into translational studies.