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

鉴定 SARS-CoV-2 刺突上的中和表位，用于设计疫苗和小分子抗病毒药物

• 批准号: 10186653
• 负责人: Dennis R. Burton
• 金额: $ 81.69万
• 依托单位: SCRIPPS RESEARCH INSTITUTE, THE
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-05-01 至 2022-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10186653
• 关键词: 2019-nCoV; Affinity; Animal Model; Antibodies; Antiviral Agents; Awareness; B-Lymphocytes; Binding; Biological; Biological Assay; Birds; COVID-19; Cells; Cessation of life; China; Cities; Common Cold; Coronavirus; Coronavirus Infections; Cryoelectron Microscopy; Diagnosis; Disease Outbreaks; Electron Microscopy; Epidemic; Epitope Mapping; Epitopes; Extracellular Domain; Goals; Hot Spot; Human; Infection; Mammals; Membrane Fusion; Middle East Respiratory Syndrome; Middle East Respiratory Syndrome Coronavirus; Modeling; Negative Staining; Patients; Peptides; Peptidyl-Dipeptidase A; Pharmaceutical Chemistry; Plaque Assay; Pneumonia; Proteins; RNA Viruses; Reporting; Respiratory Tract Diseases; SARS coronavirus; Severe Acute Respiratory Syndrome; Severities; Structure; Surface; Testing; Therapeutic; Therapeutic Uses; Therapeutic antibodies; Upper respiratory tract; Vaccine Design; Vaccines; World Health Organization; X-Ray Crystallography; base; combat; design; high throughput screening; human coronavirus; lead candidate; mortality; novel; pandemic disease; receptor; receptor binding; small molecule; stem; translational study; vaccine development; ward

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.

冠状病毒（COVS）被包裹，阳性，单链RNA病毒，并分为α和β-核纳病毒。 COV感染哺乳动物和鸟类，通常导致下呼吸道下部和/或上呼吸道疾病。 COVS引起的人类疾病的范围从普通感冒到全球流行病/大流行病，包括2003年的严重急性呼吸综合症（SARS-COV），2004年的人类COV-NL63，2005年的Human Cov-HKU1，2005年的人类Cov-HKU1，中东呼吸道综合症（MERS-CORONAVIROS）在2012年或SARS-COV中没有2012年，SARS-COV，SARS-COV，SARS-COV，SARS-COV。抗病毒药物以对抗COV感染，缺乏 在当前的SARSCOV-2爆发方面，经过测试和验证的治疗学代表了全球的巨大关注。 在2019年12月底，世界卫生组织意识到了中国武汉市定位的一个异常大的肺炎病例。在仅3个月的时间内，全世界已诊断出超过一百万个已确认的Covid-19病例，造成近50,0000例死亡。现在，美国的数字正在以惊人的速度增长（目前约占全球总数的25％）以及数字 死亡人数（目前约占总数的10％）。在2002年，人类COV感染和高死亡率的严重程度显而易见，中国广东的首次SARS-COV大流行以及2012年的MERS-COV爆发。例如，当前的SARS-COV-2（当前的SARS-COV-2）（当前的SARS-COV-2），即79％相同的相同，同样相同，也使用Angiotensin trengein nezymein trension trementing enzyme ii（ACE2）作为Cell Ordoror的宿主。 COV表面暴露的尖峰蛋白负责ACE2的识别和结合，并代表了疫苗和抗病毒药疗法开发的潜在靶标。重要的是，迄今为止，与19个患者分离的抗体（ABS）似乎结合了峰值蛋白上的几个区域，然后代表了小分子发现的理想靶标。 COV表面上的尖峰蛋白是糖基化的三聚体，由两个细胞外域S1和S2组成。迄今为止，大多数NABS（中和ABS）靶向包含负责ACE2结合的受体结合域（RBD）的S1域。尽管如此，由S2结构域上的表位（由茎融合机械组成）的ABS在基于细胞的感染和动物模型中也具有中和潜力，并且通常对其他COV更广泛地反应，而不是针对那些靶向的抗体 S1。此外，源自人CoV-OC43的HR2结构域的螺旋肽EK1（负责普通感冒的菌株）广泛地与COV的茎区域结合并抑制膜融合。我们使用这种补充剂的目标是定义SARS-COV-2的S蛋白上和中和表位，例如抗体靶向的表位，EK1肽和其他报道与RBD结合的肽，以帮助基于结构的疫苗和小分子抗病毒抗病毒抗病毒。 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 SARS-COV-2的S蛋白以及其他冠状病毒（包括SARS和MER）的分子。我们预料到这一点 我们从康复患者B细胞中分离出的新型AB的结构表征将提供有关表面热点的关键信息，这些信息可以由疫苗和小分子靶向。因此，抗体用于表位识别的共同特征将告知化合物的量身定制设计，作为COVID-19抗病人的铅候选者。重要的是，我们将使小分子进行生物学相关的假病毒斑块测定法和基于细胞的感染模型，以及人类的微粒体稳定性测定法，以生成小分子的汇编，以向前转化。