Singh A.Xu S.H.Tsai Y.D.Yang Z.S.Tang H.J.Tsai Y.J.Tsai H.Y.Yuan C.W.Chen C.C.Thitithanyanont A.Wang S.F.Chiu H.T.Mahidol University2025-06-132025-06-132025-08-01Bioorganic Chemistry Vol.163 (2025)00452068https://repository.li.mahidol.ac.th/handle/123456789/110682Severe diseases like severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) outbreak urge efficient discovery of drugs for emergent needs and precision medicine. This study demonstrates an interdisciplinary solution by identification of a chief anti-SARS-CoV-2 small molecule, 2,3,5,8-tetrahydroxy-6-methylnaphthalene-1,4-dione (TMD) from a Taiwanese traditional medicine, Antrodia cinnamomea. TMD was found to inhibit the key viral replication enzymes, 3-chymotrypsin-like protease (3CLpro) and papain-like protease (PLpro), by structure-based prediction analysis using the Explore program of BT&D² drug targeting system. Subsequently, TMD was total-synthesized and shown to inhibit Omicron BA.2 and BA.5 variants. The enzymatic inhibition kinetics studies revealed the competitive inhibition and dual-target nature of TMD and its potential capability to target the proteases in mixed-type mode, consistent with the in silico mechanistic analysis. The predicted less off-target side effects of TMD also agreed with the animal toxicity test result of no apparent toxicity. This study provides new insight into anti-viral mechanism of Antrodia cinnamomea and a new lead drug with improved pharmacokinetics to combat SARS-CoV-2 infection.Pharmacology, Toxicology and PharmaceuticsChemistryBiochemistry, Genetics and Molecular BiologyArticleSCOPUS10.1016/j.bioorg.2025.1086462-s2.0-10500745686410902120