Targeting SARS-Related Coronaviruses with a D-peptide Entry Inhibitor

项目摘要 21世纪世纪出现了多种致命的人类冠状病毒（SARS-CoV，MERS-CoV， 现在是SARS-CoV-2）。迫切需要治疗方案，以打击当前和不可避免的 类似SARS的流行病冠状病毒利用病毒共有的保守进入机制感染细胞 在多个家族（包括艾滋病毒、埃博拉病毒和流感病毒）中，三聚体病毒的两个区域 蛋白质（HR 1和HR 2）折叠形成高度稳定的六螺旋束结构， 细胞膜在一起，诱导膜融合。与HR 1结合的抑制剂阻断六螺旋束 形成并阻止病毒进入，防止感染。我们实验室专门从事镜像噬菌体展示（MIPD）， 一种创新的方法，以确定新的合成蛋白酶抗性D-肽候选药物，具有特殊的 专注于抑制病毒进入（我们的HIV-1药物CPT 31将开始临床试验）。D肽 （由镜像D-氨基酸组成的肽）不能被体内的蛋白酶消化， 因此，具有显著的治疗优势，包括延长的半衰期、较低的剂量、降低的 免疫原性（不消化MHC呈递），以及在富含蛋白酶的环境中的持久性，如 呼吸道为了解决当前的健康危机，我们正在加快药物发现过程， 靶向SARS相关冠状病毒保守HR 1的D肽进入抑制剂。我们设计了， 合成并表征了我们的HR 1模拟药物靶点，并将其用于MIPD以鉴定D-肽 6-螺旋束形成和病毒进入的抑制剂。 在这个提议中，我们将化学合成由MIPD鉴定的D-肽，并对其进行表征。 对SARS-CoV和SARS-CoV-2靶向亲和力（使用表面等离子体共振）和抗病毒活性 假病毒有希望的D肽将使用第二轮MIPD进行亲和力成熟，以优化 力量使用我们定制设计的PEG支架（CPT 31的骨架），我们将三聚化最高的 亲和力D-肽候选物，以提高对三聚体刺突靶标的亲合力并连接膜定位的 一组，如胆固醇，这将丰富的D-肽在细胞网站的病毒进入和改善体内 半衰期这些领先的D-肽将针对真实病毒进行测试（与USU研究所合作 抗病毒研究）。我们的目标是获得一种体外EC 90 ≤100 nM的D-肽候选物， SARS-CoV-2和SARS以及良好的治疗指数（EC 50/CC 50>100），以推进体内PK和疗效 研究，使用USU的SARS-CoV-2感染仓鼠模型。在资助期结束时，我们预计 有一个D-肽先导化合物，已证明在体内动物中有效，准备用于IND的临床前研究 和开发作为SARS相关冠状病毒治疗和/或预防。

Project Summary The 21st century has seen the emergence of multiple lethal human coronaviruses (SARS-CoV, MERS-CoV, and now SARS-CoV-2). There is an urgent need for therapeutic options to combat the current and inevitable future SARS-like pandemics. Coronaviruses infect cells using a conserved entry mechanism shared by viruses across multiple families (including HIV, Ebola, and influenza) in which two regions of the trimeric viral spike protein (HR1 and HR2) collapse to form a highly stable six-helix bundle structure that forces the viral and cellular membranes together, inducing membrane fusion. Inhibitor binding to HR1 blocks six-helix bundle formation and stops viral entry, preventing infection. Our lab specializes in mirror-image phage display (MIPD), an innovative approach to identify novel synthetic protease-resistant D-peptide drug candidates, with a special focus on the inhibition of viral entry (with our HIV-1 drug, CPT31, set to begin clinical trials). D-peptides (peptides composed of mirror-image D-amino acids) cannot be digested by proteases in the body and, therefore, possess significant therapeutic advantages including extended half-life, lower dosing, reduced immunogenicity (not digested for MHC presentation), and durability in protease-rich environments such as the respiratory tract. To address the current health crisis, we are expediting our drug discovery process to identify D-peptide entry inhibitors that target the conserved HR1 of SARS-related coronaviruses. We have designed, synthesized, and characterized our HR1 mimic drug targets and are using them in MIPD to identify D-peptide inhibitors of 6-helix bundle formation and viral entry. In this proposal, we will chemically synthesize the D-peptides identified by MIPD and characterize their target affinity (using surface plasmon resonance) and antiviral activity against SARS-CoV and SARS-CoV-2 pseudoviruses. Promising D-peptides will be affinity-matured using a second round of MIPD to optimize potency. Using our custom-designed PEG scaffold (the backbone of CPT31), we will trimerize the highest affinity D-peptide candidates to improve avidity for the trimeric spike target and attach a membrane-localizing group, such as cholesterol, that will enrich the D-peptide at the cellular site of viral entry and improve in vivo half-life. These leading D-peptides will be tested against authentic virus (in collaboration with USU's Institute for Antiviral Research). Our objective is to have one D-peptide candidate with ≤100 nM in vitro EC90 against SARS-CoV-2 and SARS and a good therapeutic index (EC50/CC50 >100) to advance to in vivo PK and efficacy studies, using USU's hamster model of SARS-CoV-2 infection. At the end of the grant period, we expect to have one D-peptide lead with demonstrated in vivo animal efficacy, poised for IND-enabling preclinical studies and development as a SARS-related coronavirus treatment and/or preventative.