SARS-CoV-2 and its emerging variants continue to pose a significant global public health threat. The SARS-CoV-2 main protease (Mpro) is a critical target for the development of antiviral agents that can inhibit viral replication and transcription. In this study, we identified chebulagic acid (CHLA), isolated from Terminalia chebula Retz., as a potent non-peptidomimetic and non-covalent Mpro inhibitor. CHLA exhibited intermolecular interactions and provided significant protection to Vero E6 cells against a range of SARS-CoV-2 variants, including the wild-type, Delta, Omicron BA.1.1, BA.2.3, BA.4, and BA.5, with EC₅₀ values below 2 μmol/L. Moreover, in vivo studies confirmed the antiviral efficacy of CHLA in K18-hACE2 mice. Notably, CHLA bound to a unique groove at the interface between Mpro domains I and II, which was revealed by the high-resolution crystal structure (1.4 Å) of the Mpro–CHLA complex, shrinking the substrate binding pocket of Mpro and inducing Mpro aggregation. CHLA was proposed to act as an allosteric inhibitor. Pharmacokinetic profiling and safety assessments underscore CHLA's potential as a promising broad-spectrum antiviral candidate. These findings report a novel binding site on Mpro and identify antiviral activity of CHLA, providing a robust framework for lead compounds discovery and elucidating the underlying molecular mechanisms of inhibition.

COVID-19  /  SARS-CoV-2  /  Main protease (M^pro)  /  Broad-spectrum antiviral activity  /  Chebulagic acid (CHLA)  /  Allosteric inhibitor  /  X-ray crystallography  /  Molecular interaction