An engineered chimeric ACE2-HR2 peptide exhibits potent and broad-spectrum activity against SARS-CoV-2 variants

The persistent circulation of SARS-CoV-2 and limited efficacy of current vaccines in preventing viral transmission underscore the critical need for broad-spectrum antivirals. Here, we report a novel engineering strategy to develop A3M6L35HR2 (FL), a cholesterol-free recombinant peptide derived from our previously reported A1L35HR2, by incorporating an optimized angiotensin-converting enzyme 2 (ACE2)-mimetic peptide A3M6 with six residue mutations. A3M6L35HR2 (FL) exhibited superior inhibitory potency against diverse SARS-CoV-2 variants with IC₅₀ from 1.7 to 16.2 nM, including antigenically distinct Omicron sublineages, achieving 2- to 29-fold enhanced activity compared to A1L35HR2. A3M6L35HR2 (FL) exhibits a higher α-helicity (70 %) and stronger binding affinity (K_D = 0.03 nM) with the viral HR1 domain, thereby effectively suppressing spike-mediated membrane fusion at nanomolar concentrations. Combining A3M6L35HR2 (FL) with RBD-targeting protein T-ACE2 exhibited antagonistic effect, attributed to competitive RBD binding by both agents. These findings establish A3M6L35HR2 (FL) as a promising antiviral candidate against current and emerging SARS-CoV-2 variants.