Abstract
The recent global pandemic was a spillover from the SARS-CoV-2 virus. Viral entry involves the receptor binding domain (RBD) of the viral spike protein interacting with the protease domain (PD) of the cellular receptor, ACE2. We hereby present a comprehensive mutational landscape of the effects of ACE2-PD point mutations on RBD-ACE2 binding using a saturation mutagenesis approach based on microarray-based oligo synthesis and a single-cell screening assay. We observed that changes in glycosylation sites and directly interacting sites of ACE2-PD significantly influenced ACE2-RBD binding. We further engineered an ACE2 decoy receptor with critical point mutations, D30I, L79W, T92N, N322V, and K475F, named C4-1. C4-1 shows a 200-fold increase in neutralization for the SARS-CoV-2 D614G pseudotyped virus compared to wild-type soluble ACE2 and a sevenfold increase in binding affinity to wild-type spike compared to the C-terminal Ig-Fc fused wild-type soluble ACE2. Moreover, C4-1 efficiently neutralized prevalent variants, especially the omicron variant (EC 50 = 16 ng/mL), and rescued monoclonal antibodies, vaccine, and convalescent sera neutralization from viral immune-escaping. We hope to next investigate translating the therapeutic potential of C4-1 for the treatment of SARS-CoV-2.
Keywords: ACE2; SARS-CoV-2; decoy receptor; high-throughput screening; protein engineering.
【저자키워드】 SARS-CoV-2, ACE2, High-throughput screening, decoy receptor, protein engineering., 【초록키워드】 Treatment, Vaccine, neutralization, mutations, SARS-CoV-2 virus, protease, monoclonal antibodies, viral entry, variants, binding affinity, Spike protein, Receptor binding domain, global pandemic, Protein, Pseudotyped virus, RBD, Microarray, D614G, Omicron variant, receptor, Critical, single-cell, convalescent sera, binding, soluble ACE2, Mutagenesis, Point mutation, ACE2 binding, critical point, viral spike protein, domain, therapeutic potential, ACE2-RBD binding, RBD binding, wild-type, cellular receptor, Effect, approach, prevalent, neutralized, glycosylation site, RBD-ACE2, significantly, changes in, increase in, C-terminal, rescued, fused, the SARS-CoV-2, the SARS-CoV-2 virus, 【제목키워드】 variant, Mutagenesis, prevalent, neutralize,