ABSTRACT
Two classes of antivirals targeting the viral neuraminidase (NA) and endonuclease are currently the only clinically useful drugs for the treatment of influenza. However, resistance to both antivirals has been observed in clinical isolates, and there was widespread resistance to oseltamivir (an NA inhibitor) among H1N1 viruses prior to 2009. This potential for resistance and lack of diversity for antiviral targets highlights the need for new influenza antivirals with a higher barrier to resistance. In this study, we identified an antiviral compound, M85, that targets host kinases, epidermal growth factor receptor (EGFR), and phosphoinositide 3 class II ß (PIK3C2ß) and is not susceptible to resistance by viral mutations. M85 blocks endocytosis of influenza viruses and inhibits a broad-spectrum of viruses with minimal cytotoxicity. In vitro, we found that combinations of M85 and oseltamivir have strong synergism. In the mouse model for influenza, treatment with the combination therapy was more protective against a lethal viral challenge than oseltamivir alone, indicating that development of M85 could lead to combination therapies for influenza. Finally, through this discovery of M85 and its antiviral mechanism, we present the first description of PIK3C2ß as a necessary host factor for influenza virus entry.
Subject(s)
Antiviral Agents/pharmacology , Enzyme Inhibitors/pharmacology , Orthomyxoviridae/drug effects , Oseltamivir/pharmacology , Phosphatidylinositol 3-Kinases/drug effects , Virus Internalization/drug effects , Animals , Cell Line , Chlorocebus aethiops , Class II Phosphatidylinositol 3-Kinases/drug effects , Disease Models, Animal , Drug Combinations , Drug Evaluation, Preclinical , Drug Resistance, Viral/drug effects , Drug Synergism , ErbB Receptors , Female , Humans , Influenza A Virus, H1N1 Subtype/drug effects , Male , Mice , Mice, Inbred BALB C , Vero CellsABSTRACT
For the first time in nearly 20 years there is a new class of antiviral drug for influenza. The latest approved antiviral is baloxavir marboxil (trade name, Xofluza) which targets the endonuclease function of the viral PA polymerase subunit and prevents the transcription of viral mRNA. The most promising aspect of this new drug is its pharmacology which allows for effective treatment of influenza A or B virus infection with just a single dose. A clinical trial showed greater reductions in viral loads with baloxavir marboxil treatment compared with oseltamivir, although no difference in the time to alleviation of symptoms between these two drugs. With this new class of influenza drug comes exciting prospects for combination therapy with the neuraminidase inhibitors which may help to abate concerns about the development of resistance.
Subject(s)
Antiviral Agents/pharmacology , Influenza, Human/drug therapy , Orthomyxoviridae/drug effects , Oxazines/pharmacology , Pyridines/pharmacology , Thiepins/pharmacology , Triazines/pharmacology , Animals , Clinical Trials as Topic , Dibenzothiepins , Drug Resistance, Viral , Humans , Influenza A virus/drug effects , Influenza B virus/drug effects , Morpholines , Orthomyxoviridae Infections/drug therapy , Pyridones , Viral Load/drug effectsABSTRACT
The discovery of the green fluorescent protein from Aequorea victoria has revolutionized the field of cell and molecular biology. Since its discovery a growing panel of fluorescent proteins, fluorophores and fluorescent-coupled staining methodologies, have expanded the analytical capabilities of flow cytometry. Here, we exploit the power of genetic engineering to barcode individual cells with genes encoding fluorescent proteins. For genetic engineering, we utilize retroviral technology, which allows for the expression of ectopic genetic information in a stable manner in mammalian cells. We have genetically barcoded both adherent and nonadherent cells with different fluorescent proteins. Multiplexing power was increased by combining both the number of distinct fluorescent proteins, and the fluorescence intensity in each channel. Moreover, retroviral expression has proven to be stable for at least a 6-month period, which is critical for applications such as biological screens. We have shown the applicability of fluorescent barcoded multiplexing to cell-based assays that rely themselves on genetic barcoding, or on classical staining protocols. Fluorescent genetic barcoding gives the cell an inherited characteristic that distinguishes it from its counterpart. Once cell lines are developed, no further manipulation or staining is required, decreasing time, nonspecific background associated with staining protocols, and cost. The increasing number of discovered and/or engineered fluorescent proteins with unique absorbance/emission spectra, combined with the growing number of detection devices and lasers, increases multiplexing versatility, making fluorescent genetic barcoding a powerful tool for flow cytometry-based analysis.
