Structure‐Based Development of Ultra‐Broad‐Spectrum 3C‐Like Protease Inhibitors

Recurrence of coronavirus outbreaks and zoonotic origins of human coronaviruses underscore the importance of developing pan-coronavirus antivirals. The highly conserved 3C-like protease (3CL^pro) in coronaviruses, together with the well-established druggability, makes it an ideal target for broad-spectrum antiviral therapeutics. Here, the inhibitory activity of approved 3CL^pro inhibitors, including nirmatrelvir, ensitrelvir, and simnotrelvir, against fifteen 3CL^pros is first reported by enzymatic assays. Despite their potent inhibition toward 3CL^pros of β-CoVs, these inhibitors show reduced potency against 3CL^pros from the other three genera, particularly against two newly identified human coronaviruses (α-CCoV-HuPn-2018 and δ-PDCoV). In this context, continued efforts in structure-based optimization of nirmatrelvir lead to the identification of compound 8 that potently inhibits a panel of 32 3CL^pros across all subgenera (IC₅₀s: 19-146 nm), with an IC₅₀ value of 61 and 81 nm against α-CCoV-HuPn-2018 and δ-PDCoV 3CL^pros, respectively. Moreover, it effectively inhibits nirmatrelvir-resistant 3CL^pro mutants and demonstrates broad-spectrum antiviral efficacy in cells. These findings suggest an important rule that a small, non-cyclic P2 segment and a P4 segment with a suitable size are preferred by the design of ultra-broad-spectrum 3CL^pro inhibitors, and provide a proof-of-concept guide for developing broad-spectrum antivirals as potential pan-CoV therapeutics.