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

项目摘要 目前的SARS-CoV-2大流行对全球公共卫生构成了直接威胁。这是第三大 在SARS-CoV-1和MERS-CoV之后，在过去的二十年里，人畜共患冠状病毒在人类中爆发。 因为主流的抗病毒方法，如疫苗和中和抗体， 针对每一种病毒的发展，有效的医疗补救措施总是滞后，这导致了严重的 造成人员死亡和经济混乱。由于病毒很少改变它们的受体特异性， 提出了一种治疗策略，利用三聚体受体作为诱饵来中和病毒。SARS冠状病毒 1和SARS-CoV-2靶向血管紧张素转换酶2（ACE 2）以获得细胞进入。“三ACE 2” 以结构导向的方式设计诱饵以匹配病毒刺突（S）的对称性和几何形状， 其通过三聚体亲合力效应使结合亲和力最大化。然后， 引入tri-ACE 2平台以实现更好的抗病毒活性。我们提供了强有力的初步数据 证明了这种设计的有效性。Tri-ACE 2诱饵将病毒尖峰锁定在对称的“3-UP”中 受体结合结构域（RBD）构象，用纳摩尔浓度中和各种ACE 2嗜性冠状病毒 浓度，比单体ACE 2好约100倍。用ab初始设计的微型活页夹替代ACE 2 （miniature-ACE 2s）通过实现超快S-结合进一步将IC 50提高至约20 pM。三位一体的优势- ACE 2过度中和抗体在于其对所有ACE 2嗜性的潜在广谱活性， 冠状病毒，并在其预期的抵抗病毒受体中的逃避突变。因此，这条线 的抑制剂可能作为一种现成的治疗方法，在未来由未知的ACE 2- 热带冠状病毒我们建议探索结构引导的受体多聚化的全部潜力 作为一般的抗病毒策略。这些努力也将产生新的机械洞察力， 冠状病毒刺突蛋白的功能一般。具体目标包括：1）生产有效的和逃避的- 抗SARS-CoV-2的三-ACE 2抑制剂; 2）产生具有皮摩尔抑制性的三-miniatureACE 2 活性和新的抗病毒机制; 3）确定tri-ACE 2对SARS-CoV-2和相关病毒的效力 原代气道细胞培养物和动物模型中的病毒; 4）确定预防和治疗 在金黄色叙利亚仓鼠模型中，基于三-ACE 2的抑制剂对SARS-CoV-2感染的功效。后 这个项目的完成，我们预计将开发出安全、高效、广泛的- 可以直接导致在非人类灵长类动物或人类中进行试验的抗冠状病毒药物。

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.