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

Abstract

The main protease (Mpro or 3CLpro) 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 Mpro. The four compounds (hypericin, rosmarinic acid, isorhamnetin, and luteolin) that most efficiently inhibited SARS-CoV-2 Mpro in vitro were further tested for their efficacy in inhibiting Mpro 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 Mpro 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 Mpro of SARS-CoV-2 demonstrated 96.08%, 50.83%, 49.17%, 48.51%, 44.04%, and 41.06% similarity to Mpro 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 Mpro at the same binding site as the control inhibitor, GC376. Enzyme inhibition assays revealed that hypericin, rosmarinic acid, isorhamnetin, and luteolin inhibited Mpro of SARS-CoV-2, while hypericin and isorhamnetin inhibited Mpro of SARS-CoV-1; hypericin showed inhibitory effects toward Mpro of MERS-CoV. Microscale thermophoresis confirmed the binding of these compounds to Mpro 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 Mpro of several human-pathogenic coronaviruses. Moreover, isorhamnetin showed inhibitory effects toward SARS-CoV-2 and SARS-CoV-1 Mpro, indicating that this compound may have some pan-coronaviral potential. Luteolin had inhibitory effects against SARS-CoV-2 Mpro.

Eryngium creticum L.: Chemical Characterization, SARS-CoV-2 Inhibitory Activity, and In Silico Study

“…These results are in accordance with the literature because Z -rosmarinic acid is reported to be not stable enough to be isolated in a pure form. 13 …”

“…Recently, E -rosmarinic acid has been reported to inhibit SARS-CoV-2 replication and SARS-CoV-2 3CL pro 13 However, the activity of the Z -isomer has not been reported yet. This prompted us to investigate and compare the 3CL pro inhibitory potential of compounds 1 and 2 ( 2 Z and 2 E ) ( Table 3 ).…”

“… 16 According to recent studies, rosmarinic acid showed medium inhibitory activity against SARS-CoV-2 replication 17 and SARS-CoV-2 -3CL pro . 13 This prompted us to investigate the inhibitory potential of E -rosmarinic acid 1 versus ( E / Z )-rosmarinic acid mixture 2 against the main protease of coronavirus.…”

SARS-CoV-2 omicron variants succumbin vitrotoArtemisia annuahot water extracts

“…Because we and others [12,14] showed that ART is not the most likely anti-SARS-CoV-2 therapeutic phytochemical in A. annua extracts, questions remain regarding the identity of these non-ART phytochemicals. To resolve that question, several groups have used in silico approaches [22,23]. Tang et al screened the Traditional Chinese Medicines for systems Pharmacology Databased and Analysis Platform to identify all phytochemicals reportedly in A. annua then ranked them according to oral bioavailability and drug likeness (OB and DL, respectively).…”

Microscale Thermophoresis as a Tool to Study Protein Interactions and Their Implication in Human Diseases

“…The lack of vaccines and antivirals is partly due to a poor understanding of the structural characteristics of the viral proteins that are involved in viral infection and replication [ 89 ]. Teams all over the world are using MST to study CoV since fast screening on large libraries can be performed [ 90 ]. Here we will highlight specific cases to illustrate the benefit of the methodology.…”