The His41 site of 3CLpro is a new target for designing broad-spectrum antiviral agents against coronaviruses via a non-covalent pan-binding mode

<title>Abstract</title> <p> Ongoing mutations of coronaviruses make control extremely difficult, urgently demanding broad-spectrum antivirals to complement vaccines. In this study, we screened and identified antiviral drugs targeting the coronavirus 3CL protease (3CLpro) and further investigated their broad-spectrum antiviral activity. The results indicated that Isoliquiritigenin (ISL) interacts with the conserved His41 site of 3CLpro through a non-covalent pan-binding mode and exhibits broad-spectrum antiviral activity against six coronaviruses from four genera. ISL exhibits high affinity for the coronavirus 3CLpro (K _D = 10 ^− 6 –10 ^− 7 M), which is abolished upon the His41 mutation (K _D &gt; 10 ^− 3 M). Its enzyme-inhibitory activity stems from anchoring to 3CLpro His41 via a π-π stacking network. By rationally fortifying this π-π stacking, we developed the optimized derivative ISL-221, achieving an order-of-magnitude leap in pan-3CLpro affinity (K _D = 10 ^− 7 –10 ^− 8 M). <italic>In vivo</italic> , ISL-221 demonstrated 83.33%, 100%, and 100% protection against coronavirus PEDV, TGEV, and PDCoV, respectively; ISL-221 markedly reduced lung viral loads to 1.65 ± 0.68 Log ₁₀ copies/g against SARS-CoV-2 infection in mice, exhibiting efficacy comparable to Paxlovid. This study establishes the highly conserved 3CLpro His41 site as a tractable broad-spectrum design site against four genera of coronaviruses, reveals the unique non-covalent π-π stacking mechanism, and validates mechanism-guided optimization for developing potent pan-coronavirus therapeutics. </p>