Neuraminidase from Influenza A and B Viruses is Susceptible to the Compound 4-(4-Phenyl-1H-1,2,3-Triazol-1-yl)-2,2,6,6-Tetramethylpiperidine-1- Oxyl.

BACKGROUND: Since the influenza virus is the main cause of acute seasonal respiratory infections and pandemic outbreaks, antiviral drugs are critical to mitigate infections and impair chain of transmission. Neuraminidase inhibitors (NAIs) are the main class of anti-influenza drugs in clinical use. Nevertheless, resistance to oseltamivir (OST), the most used NAI, has been detected in circulating strains of the influenza virus. Therefore, novel compounds with anti-influenza activity are necessary. OBJECTIVE: To verify whether the NA from influenza A and B virus is susceptible to the compound 4-(4- phenyl-1H-1,2,3-triazol-1-yl)-2,2,6,6-tetramethylpiperidine-1-oxyl (Tritempo). METHODS: Cell-free neuraminidase inhibition assays were performed with Tritempo, using wild-type (WT) and OST-resistant influenza strains. Cell-based assays in MDCKs were performed to confirm Tritempo`s antiviral activity and cytotoxicity. Multiple passages of the influenza virus in increasing concentrations of our compound, followed by the sequencing of NA gene and molecular docking, were used to identify our Tritempo's target. RESULTS AND DISCUSSION: Indeed, Tritempo inhibited the neuraminidase activity of WT and OSTresistant strains of influenza A and B, at the nanomolar range. Tritempo bound to WT and OST-resistant influenza NA isoforms at the sialic acid binding site with low free binding energies. Cell-free assays were confirmed using a prototypic influenza A infection assay in MDCK cells, in which we found an EC50 of 0.38 µM, along with very low cytotoxicity, CC50 > 2,000 µM. When we passaged the influenza A virus in the presence of Tritempo, a mutant virus with the G248P change in the NA was detected. This mutant was resistant to Tritempo but remained sensitive to OST, indicating no cross-resistance between the studied and reference drugs. CONCLUSION: Our results suggest that Tritempo's chemical structure is a promising one for the development of novel antivirals against influenza.

Herpes simplex type 1 activates glycolysis through engagement of the enzyme 6-phosphofructo-1-kinase (PFK-1).

UNLABELLED: Viruses such as HIV, HCV, Mayaro and HCMV affect cellular metabolic pathways, including glycolysis. Although some studies have suggested that the inhibition of glycolysis affects HSV-1 replication and that HSV-1-infected eyes have increased lactate production, the mechanisms by which HSV-1 induces glycolysis have never been investigated in detail. In this study, we observed an increase in glucose uptake, lactate efflux and ATP content in HSV-1-infected cells. HSV-1 triggered a MOI-dependent increase in the activity of phosphofructokinase-1 (PFK-1), a key rate-limiting enzyme of the glycolytic pathway. After HSV-1 infection, we observed increased PFK-1 expression, which increased PFK-1 total activity, and the phosphorylation of this enzyme at serine residues. HSV-1-induced glycolysis was associated with increased ATP content, and these events were critical for viral replication. In summary, our results suggest that HSV-1 triggers glycolysis through a different mechanism than other herpesviruses, such as HCMV. Thus, this study contributes to a better understanding of HSV-1 pathogenesis and provides insights into novel targets for antiviral therapy. HIGHLIGHTS: ►HSV-1 activates glycolysis by PFK-1 activation. ►In HSV-1-infected cells PFK-1 synthesis is up-regulated and phosphorylated at serine residues. ►PFK-1 knockdown impairs HSV-1 replication. ►HSV-1-mediated glycolysis activation increases ATP content.