The Natural Products Withaferin A and Withanone from the Medicinal Herb <i>Withania somnifera</i> Are Covalent Inhibitors of the SARS-CoV‑2 Main Protease

The current COVID-19 pandemic caused by the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) created a global health crisis. The ability of vaccines to protect immunocompromised individuals and from emerging new strains are major concerns. Hence antiviral drugs against SARS-CoV-2 are essential. The SARS-CoV-2 main protease M^pro is vital for replication and an important target for antivirals. Using CMap analysis and docking studies, withaferin A (wifA) and withanone (win), two natural products from the medicinal herb <i>Withania somnifera</i> (ashwagandha), were identified as promising candidates that can covalently inhibit the viral protease M^pro. Cell culture, enzymatic, LC-MS/MS, computational, and equilibrium dialysis based assays were performed. DFT calculations indicated that wifA and win can form stable adducts with thiols. The cytotoxicity of M^pro was significantly reduced by wifA and win. Both wifA and win were found to irreversibly inhibit 0.5 μM M^pro with IC₅₀ values of 0.54 and 1.8 μM, respectively. LC-MS/MS analysis revealed covalent adduct formation with wifA at cysteines 145 and 300 of M^pro. The natural products wifA and win can irreversibly inhibit the SARS-CoV-2 main protease M^pro. Based on the work presented here we propose that both wifA and win have the potential to be safely used as preventative and therapeutic interventions for COVID-19.