Trimerization of the N-terminal Domain of ACE2 for Bifunctional Trapping of Future SARS-CoV-2 Variants

摘要 由SARS-Cov-2引起的COVID-19大流行导致188个国家的3830万例感染病例 到2020年10月14日，死亡人数超过108万的国家和地区。虽然各种疫苗 在快速发展的情况下，人们严重担心SARS-COV-2的未来变种将演变， 在不久的将来感染人类。我们建议开发一类新的通用分子阻滞剂， SARS-CoV-2基于hACE 2的N-末端三螺旋束（3 HB，残基20-99），其被识别为 通过SARS-CoV和SARS-CoV-2作为细胞表面受体感染人细胞。基于我们之前的工作 在三聚化三螺旋束结构域时，我们假设hACE 2的N末端3 HB结构域可以被 三聚体化以与SARS-CoV-2的所有变体的可能的RBD高度有效和特异性结合。两 该项目将追求具体目标。在具体目标1中，我们将开发基于N-甲基-N- hACE 2的末端三螺旋束（3 HB），三聚体以高特异性结合SARS-CoV-2的RBD 和力量在具体目标2中，我们将使用定向分子进化来鉴定来自人的弗林蛋白酶抑制剂。 蛋白结构域文库，并将其与hACE 2的N-末端3 HB融合，以获得双功能SARS-CoV-2陷阱， 高度特异性地结合病毒RBD，同时抑制其弗林蛋白酶介导的预活化。由此产生的新型SARS-CoV- 2捕获分子将有可能普遍应用于阻断hACE 2介导的感染， 大多数，如果不是全部，SARS-CoV-2的未来变体。

Abstract The Covid-19 pandemic caused by SARS-Cov-2 has resulted in 38.3 million infection cases across 188 countries and territories with more than 1.08 million deaths by October 14 2020. While various vaccines are under expedite development, there is a serious concern that future variants of SARS-COV-2 will evolve and infect humans in the near future. We propose to develop a novel class of universal molecular blockers against SARS-CoV-2 based on the N-terminal three-helix bundle (3HB, residues 20-99) of hACE2, which is recognized by both SARS-CoV and SARS-CoV-2 as cell surface receptor to infect human cells. Based on our previous work in trimerizing three-helical bundle domains, we hypothesize that the N-terminal 3HB domain of hACE2 can be trimerized for highly potent and specific binding with presumably the RBD of all variants of SARS-CoV-2. Two specific aims will be pursued in this project. In specific aim 1, we will develop a trimeric trap based on the N- terminal three-helix bundle (3HB) of hACE2 that trivalently binds to the RBD of SARS-CoV-2 with high specificity and potency. In specific aim 2, we will use directed molecular evolution to identify a human furin inhibitor from a protein domain library and fuse it with the N-terminal 3HB of hACE2 for a bifunctional SARS-CoV-2 trap that highly specifically binds viral RBD while inhibits its furin-mediated preactivation. The resulting novel SARS-CoV- 2 trapping molecules will have the potential to be universally applied to block hACE2-mediated infection by vast majority of, if not all, future variants of SARS-CoV-2.