Our research, guided by virtual screening, revealed a notable breakthrough in the quest for effective influenza A virus (IAV) inhibitors. The inhibition of neuraminidase activity was then evaluated with the Influenza Neuraminidase Assay Kit, using zanamivir as a positive control (Figure ). Two of the eleven compounds (compounds 1 and 7) selected through this innovative approach exhibited potential inhibition of NA activity. However, four compounds (compounds 4-6, and 11) yield higher values of readings than the virus-only control. We suspect that these compounds may exhibit unexpected stimulatory effects on the neuraminidase enzyme, either directly or indirectly, resulting in higher enzymatic activity levels compared to the virus-alone condition. However, we can't exclude the autofluorescence exhibited by these compounds. Moreover, compounds could potentially stabilize the neuraminidase enzyme, prolonging its activity and enhancing the signal generated by the substrate cleavage. Additionally, these compounds might inadvertently interact with assay components or viral particles in a manner that artificially amplifies the fluorescent signal generated by the cleavage of the MUNANA substrate. This may also explain why compound 9 did not exhibit clear inhibition of NA activity but demonstrated the ability to inhibit viruses in a cell-based assay with about 55% protection (Figure ). Compound 6 also showed a significant protection of about 30%. Nevertheless, it's important to consider that compounds 6 and 9, with no inhibition in the NA enzymatic activity, have off-target effects in the cell-based antiviral activity assay. The ineffectiveness of compounds 1 and 7 in the antiviral assay might be attributed to their membrane penetration ability.