Engineered Trimeric ACE2 Binds and Locks “Three-up” Spike Protein to Potently Inhibit SARS-CoVs and Mutants

Guo, Liang; Bi, Wenwen; Wang, Xinling; Xu, Wei; Yan, Renhong; Zhang, Yuanyuan; Zhao, Kai; Li, Yaning; Zhang, Mingfeng; Bao, Xingyue; Cai, Xia; Li, Yutang; Qu, Di; Jiang, Shibo; Xie, Youhua; Zhou, Qiang; Lu, Lu; Dang, Bobo

SARS-CoV-2 enters cells via ACE-2, which binds the spike protein with moderate affinity. Despite a constant background mutational rate, the virus must retain binding with ACE2 for infectivity, providing a conserved constraint for SARS-CoV-2 inhibitors. To prevent mutational escape of SARS-CoV-2 and to prepare for future related coronavirus outbreaks, we engineered a de novo trimeric ACE2 (T-ACE2) protein scaffold that binds the trimeric spike protein with extremely high affinity (KD < 1 pM), while retaining ACE2 native sequence. T-ACE2 potently inhibits all tested pseudotyped viruses including SARS-CoV-2, SARS-CoV, eight naturally occurring SARS-CoV-2 mutants, two SARSr-CoVs as well as authentic SARS-CoV-2. The cryo-EM structure reveals that T-ACE2 can induce the transit of spike protein to “three-up” RBD conformation upon binding. T-ACE2 thus represents a promising class of broadly neutralizing proteins against SARS-CoVs and mutants.