Salinomycin Inhibits Influenza Virus Infection by Disrupting Endosomal Acidification and Viral Matrix Protein 2 Function.

Screening of chemical libraries with 2,000 synthetic compounds identified salinomycin as a hit against influenza A and B viruses, with 50% effective concentrations ranging from 0.4 to 4.3 µM in cells. This compound is a carboxylic polyether ionophore that exchanges monovalent ions for protons across lipid bilayer membranes. Monitoring the time course of viral infection showed that salinomycin blocked nuclear migration of viral nuclear protein (NP), the most abundant component of the viral ribonucleoprotein (vRNP) complex. It caused cytoplasmic accumulation of NP, particularly within perinuclear endosomes, during virus entry. This was primarily associated with failure to acidify the endosomal-lysosomal compartments. Similar to the case with amantadine (AMT), proton channel activity of viral matrix protein 2 (M2) was blocked by salinomycin. Using purified retroviral Gag-based virus-like particles (VLPs) with M2, it was proved that salinomycin directly affects the kinetics of a proton influx into the particles but in a manner different from that of AMT. Notably, oral administration of salinomycin together with the neuraminidase inhibitor oseltamivir phosphate (OSV-P) led to enhanced antiviral effect over that with either compound used alone in influenza A virus-infected mouse models. These results provide a new paradigm for developing antivirals and their combination therapy that control both host and viral factors.IMPORTANCE Influenza virus is a main cause of viral respiratory infection in humans as well as animals, occasionally with high mortality. Circulation of influenza viruses resistant to the matrix protein 2 (M2) inhibitor, amantadine, is highly prevalent. Moreover, the frequency of detection of viruses resistant to the neuraminidase inhibitors, including oseltamivir phosphate (OSV-P) or zanamivir, is also increasing. These issues highlight the need for discovery of new antiviral agents with different mechanisms. Salinomycin as the monovalent cation-proton antiporter exhibited consistent inhibitory effects against influenza A and B viruses. It plays multifunctional roles by blocking endosomal acidification and by inactivating the proton transport function of M2, the key steps for influenza virus uncoating. Notably, salinomycin resulted in marked therapeutic effects in influenza virus-infected mice when combined with OSV-P, suggesting that its chemical derivatives could be developed as an adjuvant antiviral therapy to treat influenza infections resistant or less sensitive to existing drugs.

Characterization and mechanisms of anti-influenza virus metabolites isolated from the Vietnamese medicinal plant Polygonum chinense.

BACKGROUND: Polygonum chinense Linn. is a common medicinal plant in Southeast Asia and has been used in traditional medicine in Vietnam. The plant contains phytochemicals with various biological properties; however, its antiviral effect has not yet been demonstrated. This study was aimed to evaluate the anti-influenza virus activity of crude extracts of P. chinense, to characterize antiviral metabolites therefrom and to investigate their mechanisms of antiviral action. METHODS: The methanol (MeOH) extract and organic solvent layers of P. chinense were prepared by extraction and partition with relevant solvents. The ethyl acetate (EtOAc) layer showing antiviral activity was chromatographed repeatedly on SiO2 and Sephadex LH-20 columns to give eight pure metabolites. Their chemical structures were determined by NMR and MS spectral data. Anti-influenza virus activity of the eight metabolites against virus strains A/Puerto Rico/8/34 (H1N1, PR8), A/Hong Kong/8/68 (H3N2, HK) and B/Lee/40 (Lee) was evaluated on the basis of cytopathic effect (CPE) and plaque inhibition assays. Time-of-addition, confocal microscopy and neuraminidase inhibition assay were performed for mode-of-action studies of active ingredients. RESULTS: The MeOH extract of P. chinense showed anti-influenza virus activity with EC50 values ranging from 38.4 to 55.5 µg/mL in a CPE inhibition assay. Among the eight pure metabolites isolated from P. chinense, ellagic acid (PC5), methyl gallate (PC7) and caffeic acid (PC8) significantly inhibited viral replication in a dose-dependent manner in both plaque inhibition and CPE inhibition assays with EC50 values ranging from 14.7 to 81.1 µg/mL and CC50 values higher than 300 µg/mL. Mode-of-action studies suggested that PC5 and PC7 suppress virus entry into or replication in cells, while PC8 targets influenza viral neuraminidase, even oseltamivir-resistant one. CONCLUSION: These results demonstrated that P. chinense and its metabolites possess effective anti-influenza virus activities. The botanical materials of P. chinense could be a promising multitargeted inhibitor of influenza A and B viruses and applied to development of a novel herbal medicine.