Exploring the Therapeutic Potential of <i>Cordyceps</i> Mushroom on SARS-CoV-2 Using Virtual Screening against M^pro and In Vitro Validation of Cordycepin

Pathogenic coronavirus, including COVID-19, threatens human health, and there have been strong demands for efficient therapeutics. <i>Cordyceps militaris</i> is a medicinal mushroom that has long been used for immune enhancement, anticancer, and antiviral effects. Therefore, the inhibitory potentials of constituents of <i>C. militaris</i> against COVID-19 were analyzed using various virtual screening analyses. Among ten constituents of <i>C. militaris</i>, cordycepin, the major component, and 3'-deoxyuridine and 2'-<i>O</i>-methyl-adenosine showed strong binding affinity to M^pro, a potential target for COVID-19 therapeutics. Considering the structure-activity relationship, nucleosides having deoxyribose and methoxyribose moiety are important for the affinity to M^pro. Cordycepin is also bound to M^pro mutants, and the binding mechanisms between cordycepin and M^pro were investigated further by MD simulation and MM/PBSA analysis. Principal component analysis also confirmed the conformational change of M^pro by cordycepin, which inhibits the function of M^pro. <i>In vitro</i>, the efficacy of cordycepin was measured using Vero cells infected with SARS-CoV-2, which showed excellent inhibition with an IC₅₀ value of 29 μM. Conclusively, the constituents of <i>C. militaris</i> are expected to inhibit SARS-CoV-2 replication through binding to M^pro. Therefore, <i>C. militaris</i> can be an essential therapeutic for coronavirus through the synergistic effect of its constituents.