In Silico and In Vitro Identification of Pan-Coronaviral Main Protease Inhibitors from a Large Natural Product Library

The main protease (M(pro) or 3CL(pro)) in coronaviruses represents a promising specific drug target as it is essential for the cleavage of the virus polypeptide and has a unique cleavage site that does not exist in human host proteases. In this study, we explored potential natural pan-coronavirus drugs using in vitro and in silico approaches and three coronavirus main proteases as treatment targets. The PyRx program was used to screen 39,442 natural-product-like compounds from the ZINC database and 121 preselected phytochemicals from medicinal plants with known antiviral activity. After assessment with Lipinski’s rule of five, molecular docking was performed for the top 33 compounds of both libraries. Enzymatic assays were applied for the top candidates from both in silico approaches to test their ability to inhibit SARS-CoV-2 M(pro). The four compounds (hypericin, rosmarinic acid, isorhamnetin, and luteolin) that most efficiently inhibited SARS-CoV-2 M(pro) in vitro were further tested for their efficacy in inhibiting M(pro) of SARS-CoV-1 and MERS-CoV. Microscale thermophoresis was performed to determine dissociation constant (Kd) values to validate the binding of these active compounds to recombinant M(pro) proteins of SARS-CoV-2, SARS-CoV-1, and MERS-CoV. The cytotoxicity of hypericin, rosmarinic acid, isorhamnetin, and luteolin was assessed in human diploid MRC-5 lung fibroblasts using the resazurin cell viability assay to determine their therapeutic indices. Sequence alignment of M(pro) of SARS-CoV-2 demonstrated 96.08%, 50.83%, 49.17%, 48.51%, 44.04%, and 41.06% similarity to M(pro) of other human-pathogenic coronaviruses (SARS-CoV-1, MERS-CoV, HCoV-NL63, HCoV-OC43, HCoV-HKU1, and HCoV-229E, respectively). Molecular docking showed that 12 out of 121 compounds were bound to SARS-CoV-2 M(pro) at the same binding site as the control inhibitor, GC376. Enzyme inhibition assays revealed that hypericin, rosmarinic acid, isorhamnetin, and luteolin inhibited M(pro) of SARS-CoV-2, while hypericin and isorhamnetin inhibited M(pro) of SARS-CoV-1; hypericin showed inhibitory effects toward M(pro) of MERS-CoV. Microscale thermophoresis confirmed the binding of these compounds to M(pro) with high affinity. Resazurin assays showed that rosmarinic acid and luteolin were not cytotoxic toward MRC-5 cells, whereas hypericin and isorhamnetin were slightly cytotoxic. We demonstrated that hypericin represents a potential novel pan-anti-coronaviral agent by binding to and inhibiting M(pro) of several human-pathogenic coronaviruses. Moreover, isorhamnetin showed inhibitory effects toward SARS-CoV-2 and SARS-CoV-1 M(pro), indicating that this compound may have some pan-coronaviral potential. Luteolin had inhibitory effects against SARS-CoV-2 M(pro).