A combinatorial and computational Tandem approach towards a universal therapeutics against ACE2-mediated coronavirus infections

site-saturation mutagenesis to predict key ACE2-RBD interacting residues. Subsequent generation of ACE2 mutants and high-throughput screening identified specific ACE2 residue substitutions that enhanced binding to both SARS-CoV and SARS-CoV-2 RBDs. The triple mutant ACE2-YHA demonstrated significantly enhanced binding affinity to SARS-CoV, SARS-CoV-2, and bat SARSr-CoVs' RBDs. It effectively neutralized SARS-CoV and numerous SARS-CoV-2 variants with picomolar IC50s in pseudotyped virus assays. Notably, ACE2-YHA displayed potent neutralization against major variants of concern, including Delta and Omicron, in human airway epithelia, positioning it as a promising universal decoy for current and future ACE2-binding coronavirus outbreaks.