Novel Small Molecule Targeting the Hemagglutinin Stalk of Influenza Viruses

Abstract

Combating influenza is one of the perennial global public health issues to be managed. Antiviral drugs are useful for the treatment of influenza in the absence of an appropriate vaccine. However, the appearance of resistant strains necessitates a constant search for new drugs. In this study, we investigated novel anti-influenza drug candidates using in vitro and in vivo assays. We identified anti-influenza hit compounds using a high-throughput screening method with a green fluorescent protein-tagged recombinant influenza virus. Through subsequent analyses of their cytotoxicity and pharmacokinetic properties, one candidate (IY7640) was selected for further evaluation. In a replication kinetics analysis, IY7640 showed greater inhibitory effects during the early phase of viral infection than the viral neuraminidase inhibitor oseltamivir. In addition, we observed that hemagglutinin (HA)-mediated membrane fusion was inhibited by IY7640 treatment, indicating that the HA stalk region, which is highly conserved across various (sub)types of influenza viruses, may be the molecular target of IY7640. In an escape mutant analysis in cells, amino acid mutations were identified at the HA stalk region of the 2009 pandemic H1N1 (pH1N1) virus. Even though the in vivo efficacy of IY7640 did not reach complete protection in a lethal challenge study in mice, these results suggest that IY7640 has potential to be developed as a new type of anti-influenza drug. IMPORTANCE Anti-influenza drugs with broad-spectrum efficacy against antigenically diverse influenza viruses can be highly useful when no vaccines are available. To develop new anti-influenza drugs, we screened a number of small molecules and identified a strong candidate, IY7640. When added at the time of or after influenza virus infection, IY7640 was observed to successfully inhibit or reduce viral replication in cells. We subsequently discovered that IY7640 targets the stalk region of the influenza HA protein, which exhibits a relatively high degree of amino acid sequence conservation across various (sub)types of influenza viruses. Furthermore, IY7640 was observed to block HAmediated membrane fusion of H1N1, H3N2, and influenza B viruses in cells. Although it appears less effective against strains other than H1N1 subtype viruses in a challenge study in mice, we suggest that the small molecule IY7640 has potential to be optimized as a new anti-influenza drug.

Antiviral Efficacy of Pralatrexate against SARS-CoV-2

“…In our search of antiviral drug candidates ( Kim et al ., 2019 ), pralatrexate was initially found to be an oseltamivir-comparable candidate drug effective against seasonal influenza viruses ( Fig. 1 ).…”

Specific Detection of Influenza A and B Viruses by CRISPR-Cas12a-Based Assay

“…Viruses. Two types of influenza viruses, A/Puerto Rico/8/1934(H1N1) and B/Brisbane/60/2008, were propagated in MDCK cells, and viral titers were determined by plaque assay as previously described [ 15 ]. Experiments with viruses were performed in a biosafety level-2 (BSL-2) laboratory.…”

Morin Hydrate Inhibits Influenza Virus entry into Host Cells and Has Anti-inflammatory Effect in Influenza-infected Mice

“…Next, 100 µl of 0.5 M sodium acetate at different pHs was added to this mixture, followed by incubation at 37°C for 30 min. The mixtures were centrifuged at 1,200 rpm for 6 min to separate the intact erythrocytes, and the hemoglobin concentration in the supernatant was spectrophotometrically analyzed ( 2 18 ).…”

“…Influenza virus causes the flu, killing several thousand people every year worldwide, and in the 3 pandemic flu outbreaks occurred in the 20th century. The most recent influenza pandemic was of the H1N1 subtype, which began in Mexico in 2009 ( 1 2 3 ). Currently, for influenza prevention, vaccines against H1N1 and H3N2 subtypes of the influenza A virus, and 2 antigenically distinct virus lineages called the Yamagata and Victoria lineages are used as antigens, but these vaccines are not very effective.…”