Inhibitory Effect Of Calcein And Oseltamivir On Neuraminidase Of H7N9 Influenza Virus

Objective:To investigate the combined inhibitory effects of calcein and oseltamivir on the neuraminidase of H7N9 influenza virus,and explore the stability of the complex binding by means of molecular docking.Methods:(1)Virtual screening:The Autodock 4.2 simulation software was used to screen the 2 stable small molecules from the ZIM database of ZINC;(2)Enzymatic experiments:The inhibitor effect of the selected small molecule and its combination effect with oseltamivir on N9s were determined.The software CalcuSyn2.0 was employed to analyze the combination effect of the two drugs;(3)Molecular simulation:Autodock4.2 software was employed to evaluate the free energy of docking complexes of small molecules and N9s,to assess the stability of the formation of complexes,while Discovery Studio software was applied to analyze the hydrogen bond formation of the complexes to determine the stability of the complexes;(4)Molecular dynamics simulation:The RMSD and the RMSF values of the complexes were measured using the GROMACS software.Results:(1)Virtual screening:According to the virtual screening affinities,the two small molecules:dehydrocholic acid and calcein,were selected from ZIM database and conducted subsequent experimnets;(2)Enzymology experiment:Dehydrocholic acid has a weak inhibitory effect on N9s,while calcein has a good inhibitory effect on N9s.The IC50 value of calcein on N9WT was(12.6±2.2)?M and the IC50 on N9R294K was(30.1±1.6?M the IC50 value of oseltamivir N9WT was(1.7±0.1)nM and the IC50 for N9R294K was(26.5±1.0)?M.The CI values of ED50,ED75 and ED90 for N9WT were 0.71,0.48 and 0.33,respectively,while the CI values of ED50,ED75 and ED90 for N9R294K were 0.79,0.55 and 0.44,respectively.Therefore,the combined effect of calcein and oseltamivir against N9s was synergistic inhibition.(3)Molecular simulation:The evaluation result using Autodock 4.2 software showed that the the free energy of the two molecules with N9s was lower than that of the single molecule with N9s,indicating the stability of the former one is better than the latter one.The result from Discovery Studio software predicted that the hydrogen bond binding sites of the calcein-N9 complexes,showed that several hydrogen bonds were formed during the calcein-N9 binding,indicating the strong stability of the complexes.(4)Molecular dynamics simulation:The RMSD and RMSF values were also showed that the structure stability of the calcein-N9 complexes were high.Conclusions:(1)The combined effcet of calcein and oseltamivir with N9s is a synergistic inhibitory effect.(2)Computer simulations also confirm that calcinein and oseltamivir can form stable complexes with N9s.