Isoliquiritin for COVID-19

Background The global outbreak of coronavirus disease (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has raised significant public health concerns. Qingyan Dropping Pills (QDP), as a recommended drug, is issued by the National Health Commission of the People’s Republic of China for the treatment of COVID-19. However, its bioactive compounds and their mechanisms of action remain largely unidentified. In this study, the integration of computational and experimental approaches was performed to identify the bioactive compounds in QDP and elucidate its mechanisms against COVID-19. Methods Utilizing UPLC-Q/TOF-MS, the chemical compounds of QDP were delineated, followed by network pharmacology analysis and molecular docking targeting SARS-CoV-2 spike protein (S pro ), main protease (M pro ), and papain-like protease (PL pro ). To validate the inhibitory activity of these compounds, fluorescence resonance energy transfer (FRET) and surface plasmon resonance (SPR) assays were employed. The antivival efficacy was tested in Vero E6 cells infected with SARS-CoV-2 Omicron BA.5 variant. Moreover, anti-inflammatory potential was evaluated via the measurement of inflammatory markers, including nitric oxide (NO), interleukin-6 (IL-6), interleukin-1 beta (IL-1 β ), and tumor necrosis factor-alpha (TNF- α ). Results Among the 48 identified compounds, 33 demonstrated potential antiviral activity against COVID-19. Notably, Hamamelitannin (HAM), corilagin (COR), and rhoifolin (RHO) effectively interacted with S pro , M pro and PL pro in silico . In SPR assays, the equilibrium dissociation constant ( K D ) for COR and RHO ranged from 4.515 × 10 −8 M to 7.718 × 10 −6 M, while HAM showed strong binding affinity to S pro ( K D = 9.33 × 10 −8 M) but weaker affinity for M pro and PL pro . In FRET assays, COR and RHO inhibited..

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.