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-
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.