Nie, Q.; Zhou, Y.; Yin, H.; Chen, K.; Tang, J.; Zhang, J. Z. H.; Zhan, J.; Qi, J.; Li, W.; Zhang, C.

One major strategy for the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) to evade antibody drugs or preventive vaccines is high mutation rate of the spike receptor binding domain (RBD). Because variable RBDs of different SARS-CoV-2 strains must bind to the same human receptor angiotensin-converting enzyme 2 (hACE2) for viral cell entry and infection, we hypothesize that designing a protein with the same or very similar hACE2 binding interface might have a broad-spectrum effect against various SARS-CoV-2 strains. The designed protein binds specifically to the WT-RBD (with micromolar affinity) but not to RBDs from other SARS-CoV-2 strains. However, two rounds of the E. coli display and Magnetic Cell Sorting (MACS) selection are sufficient to yield a protein named CYN1 with nanomolar binding affinities not only to the WT-RBD but also to those of Omicron BA.1, XBB.1.16, and JN.1. Molecular dynamics simulations and free-energy hotspot analysis revealed that CYN1\'s broader spectrum capability stems from its engagement of essentially all ACE2 hotspot residues critical for WT-RBD binding, unlike the designed protein. The discovery of CYN1, differing by only four mutations from the designed protein, confirms that targeting the small interface of human viral receptors, rather than the entire receptor, offers a viable strategy for developing broad-spectrum inhibitors. This approach minimizes potential off-target effects arising from receptor multifunctionality.