Cardamonin as a p38 MAPK Signaling Pathway Activator Inhibits Human Coronavirus OC43 Infection in Human Lung Cells.

A natural chalcone, cardamonin (2',4'-dihydroxy-6'-methoxychalcone; CDN) was isolated from the seeds of Alpinia katsumadai Hayata, which has been traditionally used to treat stomach aches. CDN has been reported to possess various pharmacological properties, including anticancer and anti-inflammatory effects. This study evaluated the antiviral activity of CDN against human coronavirus HCoV-OC43 and determined the mode of action in HCoV-OC43-infected human lung cell lines (MRC-5 and A549 cells). CDN significantly inhibited HCoV-OC43-induced cytopathic effects with an IC50 of 3.62 µM and a CC50 of >50 µM, resulting in a selectivity index of >13.81. CDN treatment reduced the level of viral RNA and the expression of spike and nucleocapsid proteins in HCoV-OC43-infected cells as determine through qRT-PCR and Western blot analysis. Additionally, the activation of p38 mitogen-activated protein kinase (MAPK) by anisomycin decreased viral protein expression, whereas an inhibitor of p38 MAPK signaling, SB202190, increased viral protein expression. CDN also amplified and extended the p38 MAPK signaling pathway in HCoV-OC43-infected cells. In conclusion, CDN inhibited HCoV-OC43 infection by activating the p38 MAPK signaling pathway and has potential as a therapeutic agent against human coronavirus.

Natural Polyphenols, 1,2,3,4,6-O-Pentagalloyglucose and Proanthocyanidins, as Broad-Spectrum Anticoronaviral Inhibitors Targeting Mpro and RdRp of SARS-CoV-2.

The natural plant dietary polyphenols 1,2,3,4,6-O-Pentagalloylglucose (PGG) and proanthocyanidin (PAC) have potent antioxidant activity and a variety of pharmacological activities, including antiviral activity. In this study, we examined the inhibitory effect of PGG and PAC on SARS-CoV-2 virus infection, and elucidated its mode of action. PGG and PAC have dose-dependent inhibitory activity against SARS-CoV-2 infection in Vero cells. PGG has a lower IC50 (15.02 ± 0.75 µM) than PAC (25.90 ± 0.81 µM), suggesting that PGG has better inhibitory activity against SARS-CoV-2 than PAC. The PGG and PAC inhibit similar Mpro activities in a protease activity assay, with IC50 values of 25-26 µM. The effects of PGG and PAC on the activity of the other essential SARS-CoV-2 viral protein, RdRp, were analyzed using a cell-based activity assay system. The activity of RdRp is inhibited by PGG and PAC, and PGG has a lower IC50 (5.098 ± 1.089 µM) than PAC (21.022 ± 1.202 µM), which is consistent with their inhibitory capacity of SARS-CoV-2 infection. PGG and PAC also inhibit infection by SARS-CoV and MERS-CoV. These data indicate that PGG and PAC may be candidate broad-spectrum anticoronaviral therapeutic agents, simultaneously targeting the Mpro and RdRp proteins of SARS-CoV-2.

Natural Polyphenols, 1,2,3,4,6-O-Pentagalloyglucose and Proanthocyanidins, as Broad-Spectrum Anticoronaviral Inhibitors Targeting Mpro and RdRp of SARS-CoV-2

The natural plant dietary polyphenols 1,2,3,4,6-O-Pentagalloylglucose (PGG) and proanthocyanidin (PAC) have potent antioxidant activity and a variety of pharmacological activities, including antiviral activity. In this study, we examined the inhibitory effect of PGG and PAC on SARS-CoV-2 virus infection, and elucidated its mode of action. PGG and PAC have dose-dependent inhibitory activity against SARS-CoV-2 infection in Vero cells. PGG has a lower IC50 (15.02 ±0.75 μM) than PAC (25.90 ±0.81 μM), suggesting that PGG has better inhibitory activity against SARS-CoV-2 than PAC. The PGG and PAC inhibit similar Mpro activities in a protease activity assay, with IC50 values of 25–26 μM. The effects of PGG and PAC on the activity of the other essential SARS-CoV-2 viral protein, RdRp, were analyzed using a cell-based activity assay system. The activity of RdRp is inhibited by PGG and PAC, and PGG has a lower IC50 (5.098 ±1.089 μM) than PAC (21.022 ±1.202 μM), which is consistent with their inhibitory capacity of SARS-CoV-2 infection. PGG and PAC also inhibit infection by SARS-CoV and MERS-CoV. These data indicate that PGG and PAC may be candidate broad-spectrum anticoronaviral therapeutic agents, simultaneously targeting the Mpro and RdRp proteins of SARS-CoV-2.

SARS-CoV-2 RdRp Inhibitors Selected from a Cell-Based SARS-CoV-2 RdRp Activity Assay System.

The COVID-19 pandemic, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), urgently needs effective prophylactic and therapeutic drugs. RNA-dependent RNA polymerase (RdRp), essential for replicating and transcribing a viral RNA genome, is highly conserved in coronaviruses; thus, it is a potential target for inhibiting coronavirus infection. In this study, we generated the cell-based SARS-CoV-2 RdRp activity assay system by modifying a previously reported cell-based MERS-CoV RdRp activity assay system to screen for SARS-CoV-2 RdRp inhibitors. The assay system consisted of an expression plasmid encoding SARS-CoV-2 RdRp and an RdRp activity reporter plasmid. RdRp activity in the cells could be conveniently detected by luminescence after transfection. We confirmed that SARS-CoV-2 RdRp replicated double-stranded RNA using immunofluorescence staining and the inhibition of RdRp activity by remdesivir and lycorine using this system. Moreover, the Z-factor of this system was calculated to be 0.798, suggesting the reproducibility and reliability of the high-throughput screening system. Finally, we screened nucleoside and nucleotide analogs and identified adefovir dipivoxil, emtricitabine, telbivudine, entecavir hydrate, moroxydine and rifampin as novel SARS-CoV-2 RdRp inhibitors and therapeutic candidates for COVID-19 This system provides an effective high-throughput screening system platform for developing potential prophylactic and therapeutic drugs for COVID-19 and emerging coronavirus infections.

Lycorine, a non-nucleoside RNA dependent RNA polymerase inhibitor, as potential treatment for emerging coronavirus infections.

BACKGROUND: Highly effective novel treatments need to be developed to suppress emerging coronavirus (CoV) infections such as COVID-19. The RNA dependent RNA polymerase (RdRp) among the viral proteins is known as an effective antiviral target. Lycorine is a phenanthridine Amaryllidaceae alkaloid isolated from the bulbs of Lycoris radiata (L'Hér.) Herb. and has various pharmacological bioactivities including antiviral function. PURPOSE: We investigated the direct-inhibiting action of lycorine on CoV's RdRp, as potential treatment for emerging CoV infections. METHODS: We examined the inhibitory effect of lycorine on MERS-CoV, SARS-CoV, and SARS-CoV-2 infections, and then quantitatively measured the inhibitory effect of lycorine on MERS-CoV RdRp activity using a cell-based reporter assay. Finally, we performed the docking simulation with lycorine and SARS-CoV-2 RdRp. RESULTS: Lycorine efficiently inhibited these CoVs with IC50 values of 2.123 ± 0.053, 1.021 ± 0.025, and 0.878 ± 0.022 µM, respectively, comparable with anti-CoV effects of remdesivir. Lycorine directly inhibited MERS-CoV RdRp activity with an IC50 of 1.406 ± 0.260 µM, compared with remdesivir's IC50 value of 6.335 ± 0.731 µM. In addition, docking simulation showed that lycorine interacts with SARS-CoV-2 RdRp at the Asp623, Asn691, and Ser759 residues through hydrogen bonding, at which the binding affinities of lycorine (-6.2 kcal/mol) were higher than those of remdesivir (-4.7 kcal/mol). CONCLUSIONS: Lycorine is a potent non-nucleoside direct-acting antiviral against emerging coronavirus infections and acts by inhibiting viral RdRp activity; therefore, lycorine may be a candidate against the current COVID-19 pandemic.

A Cell-Based Reporter Assay for Screening Inhibitors of MERS Coronavirus RNA-Dependent RNA Polymerase Activity.

Severe acute respiratory syndrome (SARS), Middle East respiratory syndrome (MERS), and coronavirus disease 2019 (COVID-19) are emerging zoonotic diseases caused by coronavirus (CoV) infections. The viral RNA-dependent RNA polymerase (RdRp) has been suggested as a valuable target for antiviral therapeutics because the sequence homology of CoV RdRp is highly conserved. We established a cell-based reporter assay for MERS-CoV RdRp activity to test viral polymerase inhibitors. The cell-based reporter system was composed of the bicistronic reporter construct and the MERS-CoV nsp12 plasmid construct. Among the tested nine viral polymerase inhibitors, ribavirin, sofosbuvir, favipiravir, lamivudine, zidovudine, valacyclovir, vidarabine, dasabuvir, and remdesivir, only remdesivir exhibited a dose-dependent inhibition. Meanwhile, the Z-factor and Z'-factor of this assay for screening inhibitors of MERS-CoV RdRp activity were 0.778 and 0.782, respectively. Ribavirin and favipiravir did not inhibit the MERS-CoV RdRp activity, and non-nucleoside HCV RdRp inhibitor, dasabuvir, partially inhibited MERS-CoV RdRp activity. Taken together, the cell-based reporter assay for MERS-CoV RdRp activity confirmed remdesivir as a direct inhibitor of MERS-CoV RdRp in cells. A cell-based MERS-CoV RdRp activity reporter assay is reliable and accurate for screening MERS-CoV RdRp-specific inhibitors. It may provide a valuable platform for developing antiviral drugs for emerging CoV infections.

Kurarinone Inhibits HCoV-OC43 Infection by Impairing the Virus-Induced Autophagic Flux in MRC-5 Human Lung Cells.

Kurarinone is a prenylated flavonone isolated from the roots of Sophora flavescens. Among its known functions, kurarinone has both anti-apoptotic and anti-inflammatory properties. Coronaviruses (CoVs), including HCoV-OC43, SARS-CoV, MERS-CoV, and SARS-CoV-2, are the causative agents of respiratory virus infections that range in severity from the common cold to severe pneumonia. There are currently no effective treatments for coronavirus-associated diseases. In this report, we examined the anti-viral impact of kurarinone against infection with the human coronavirus, HCoV-OC43. We found that kurarinone inhibited HCoV-OC43 infection in human lung fibroblast MRC-5 cells in a dose-dependent manner with an IC50 of 3.458 ± 0.101 µM. Kurarinone inhibited the virus-induced cytopathic effect, as well as extracellular and intracellular viral RNA and viral protein expression. Time-of-addition experiments suggested that kurarinone acted at an early stage of virus infection. Finally, we found that HCoV-OC43 infection increased the autophagic flux in MRC-5 cells; kurarinone inhibited viral replication via its capacity to impair the virus-induced autophagic flux. As such, we suggest that kurarinone may be a useful therapeutic for the treatment of diseases associated with coronavirus infection.