STRUCTURE-GUIDED RECEPTOR/INHIBITOR TRIMERIZATION AND RELATED STRATEGIES AGAINST CORONAVIRUSES

项目摘要 目前的SARS-CoV-2大流行构成了迫在眉睫的全球公共卫生威胁。这是第三大专业 继SARS-CoV-1和MERS-CoV之后，人畜共患冠状病毒在过去20年中在人类中暴发。 因为主流的抗病毒方法，如疫苗和中和抗体，必须特别 针对每种病毒开发的药物，在有效的医疗补救措施方面总是滞后，这导致了严重的 死亡人数和经济混乱。由于病毒很少转换其受体特异性，因此我们在这里 提出一种利用三聚体受体作为诱饵来中和病毒的治疗策略。SARS冠状病毒- 1和SARS-CoV-2以血管紧张素转换酶2(ACE2)为靶点，获得细胞进入。“三张王牌” 诱饵以结构引导的方式设计，以匹配病毒尖峰的对称性和几何形状(S)， 其通过三聚体亲和力效应最大化结合亲和力。然后是附加的功能域 引入Tri-ACE2平台，达到更好的抗病毒活性。我们提供了强劲的初步数据 展示了这种设计的有效性。Tri-ACE2诱饵将病毒尖峰锁定在对称的“3-up”中 受体结合域(RBD)构象，以纳米分子中和多种嗜ACE2冠状病毒 浓度，约为单体ACE2的100倍。用AB最初设计的小型粘结剂取代ACE2 (微型-ACE2)通过实现超快的S结合，进一步将IC50提高到~20 PM。三网合一的优势 ACE2优于中和抗体在于其潜在的广谱抗所有嗜ACE2的活性 冠状病毒，以及预期的对病毒受体逃逸突变的抵抗力。因此，这条线 在未知的ACE2-2引起的未来爆发中，可能作为现成的治疗药物- 热带冠状病毒。我们建议探索结构导向受体多聚化的全部潜力。 作为一般的抗病毒策略。这些努力还将产生新的机械论观点，即 冠状病毒刺突蛋白的一般功能。具体目标包括：1)产生强有力的和规避的- 抗SARS-CoV-2的tri-ACE2抑制剂；2)产生具有皮摩尔抑制作用的三微型ACE2 3)Tri-ACE2抗SARS-CoV-2及相关基因的疗效测定 原代呼吸道细胞培养和动物模型中的病毒；4)确定预防和治疗 基于Tri-ACE2的抑制剂对金黄地鼠SARS-CoV-2感染的疗效。vt.在.的基础上 这个项目的完成，我们期待着我们将开发出安全、高效、广泛的-- 光谱抗冠状病毒药物，可以直接导致在非人类灵长类动物或人类身上进行试验。

Project Summary The current SARS-CoV-2 pandemic poses an immediate and global public health threat. This is the third major zoonotic-born coronavirus outbreak in humans in the past twenty years, after SARS-CoV-1 and MERS-CoV. Because mainstream antiviral approaches such as vaccines and neutralizing antibodies have to be specifically developed for each virus, there has always been a lag in effective medical remedy, which resulted in grave human death toll and economic disruption. Since viruses rarely switch their receptor specificity, here we propose a therapeutic strategy that utilizes the trimeric receptor as a decoy to neutralize the virus. SARS-CoV- 1 and SARS-CoV-2 target the angiotensin-converting enzyme 2 (ACE2) to gain cell entry. The “tri-ACE2” decoys are designed in a structure-guided fashion to match the symmetry and geometry of the viral spike (S), which maximizes the binding affinity through the trimer avidity effect. Additional functional domains are then introduced into the tri-ACE2 platform to achieve better antiviral activity. We present strong preliminary data demonstrating the effectiveness of such designs. Tri-ACE2 decoys lock the viral spikes in a symmetric “3-UP” receptor binding domain (RBD) conformation, neutralizing various ACE2-tropic coronaviruses with nanomolar concentrations, ~100-fold better than monomeric ACE2. Replacing ACE2 with ab initial designed minibinders (miniature-ACE2s) further improved the IC50 to ~20 pM by enabling ultrafast S-binding. The advantage of tri- ACE2 over neutralizing antibodies lies in its potential broad-spectrum activity against all ACE2-tropic coronaviruses, and in its expected resistance against evader mutations in the viral receptor. Therefore, this line of inhibitors can potentially serve as an off-the-shelf therapeutic in future outbreaks caused by unknown ACE2- tropic coronaviruses. We propose to explore the full potential of the structure-guided receptor multimerization as a general antiviral strategy. These efforts will also generate new mechanistic insight about how the coronavirus spike protein functions in general. The specific aims include: 1) Produce potent and evader- resistant tri-ACE2 inhibitors against SARS-CoV-2; 2) Produce tri-miniatureACE2 with picomolar inhibitory activity and novel antiviral mechanism; 3) Determine the efficacy of tri-ACE2 against SARS-CoV-2 and related viruses in primary airway cell cultures and animal models; 4) Determine the prophylactic and therapeutic efficacy of tri-ACE2 based inhibitors against SARS-CoV-2 infection in a Golden Syrian hamster model. Upon the completion of this project, we expect that we will have developed safe, highly effective, and broad- spectrum anti-coronavirus drugs that can directly lead to trials in nonhuman primates or humans.