Computational Investigations On The Active Conformations Of Neuraminidase Inhibitors As Well As Their Mechanisms Of Actions

With the development of separation and purification technologies as well as bioactivity researches,a lot of plant secondary metabolites have been isolated and validated with the obvious anti-influenza effects.From the roots of medicinal herb Scutellaria baicalensis Georgi,5,7,4'-trihydroxy-8-methoxyflavone(MF)was isolated and found to be antiviral to various subtype neuraminidases of influenza,even to the varied subtypes.Therefore,MF is a potent anti-influenza lead drug.In this work,docking,molecular dynamics and density functional theory methods were combined to analyze the conformations of neuraminidase inhibitors in gas phase and aqueous solutions,then to explore the active form of inhibitors in neuraminidase-receptors.The respective contributions of each core template and functional group during the binding processes were evaluated.On such basis,a series of trihydroxymethoxyflavone analogues were designed starting from MF,aiming to discover the novel antiviral agents from plants and contribute to the in-depth understanding on the interacting mechanisms between neuraminidases and plant inhibitors.With the aid of density functional calculations,it was revealed that the neuraminidase inhibitors exist exclusively in the neutral form.However,docking and molecular dynamics simulations indicated that the zwitterionic rather than neutral isomers are the active form in neuraminidase-receptors.The neutral and zwitterionic isomers of 4-(N-acetylamino)-5-guanidino-3-(3-pentyloxy)benzoic acid(BA)are quite different in structure,charge distributions and etc.,and therefore are expected to show distinct bioactivities.With the presence of four water molecules,the BA geometry of the neutral isomer(nBA4)is induced to resemble the zwitterions and remains rather stable throughout the proton transfer process(nBA4?zBA4);in addition,the energy difference between the zwitterionic vs.neutral isomer is lowered from 24.76 to 2.54 kcal·mol-1.The zwitterion is the predominant isomer in aqueous solutions,consistent with the conformation preference in neuraminidase-receptors.The proton transfer process of nBA4?zBA4 is divided into two steps.Step 1 rather than Step 2 plays a decisive role owing to the larger energy barrier;however,Step 1 is not assisted by water solvents,even if not water-suppressed.Accordingly,the proton transfer process that leads to the formation of zwitterions is not facilitated in aqueous solutions,albeit they may be more stable than the neutral isomers.It is thus suggested that the designed antiviral inhibitors should be facile to transform into the zwitterionic form in aqueous solutions,in order to improve the oral bioavailability of the antiviral drugs.The data of docking and molecular dynamics simulations indicated that the binding modes and binding strengths of neuraminidase with various inhibitors,which consist of different core template/functional groups,are structurally and energetically different.Compared with 1A(oseltamivir carboxylate),the changes of core template or/and functional groups in the other inhibitors cause the reductions of interaction energies and numbers of H-bonds with the neuraminidases.Nonetheless,all these inhibitors occupy the proximity space at the active site of neuraminidase and share some commonness in their binding modes.The fragment approach was then used to analyze and understand the binding specificities of these inhibitors.The contributions of each core template and functional group were evaluated.It was found that the core templates rather than functional groups play a larger role during the binding processes;in addition,the binding qualities are determined by the synergistic effects of the core templates and functional groups.Among the inhibitors used in this study,1A(oseltamivir carboxylate)has the largest synergistic energy and its functional groups fit perfectly with the neuraminidase active site,consistent with the largest interaction energy,numerous H-bonds with the neuraminidase active-site residues as well as experimentally lowest IC50 value.Owing to the poorer metabolizability than oseltamivir,large contribution of the benzene core template and fine synergistic effects of the functional groups,BA should be an ideal lead drug for optimizing neuraminidase inhibitors.Based on the above studies,a series of analogues were designed in order to overcome the current disadvantages of MF.It was found that the binding locations of derivatives at the neuraminidase active site will not be influenced much with the addition of functional groups(carboxyl and guanidino/N-acetylamino),and there are some connections between the structures of derivatives and their interaction energies.Compound 9,with the replacements of carboxyl and guanidino groups at C7 and C5 of MF,demonstrates the highest interaction energy among the covered derivatives,equals-297.49 kcal·mol-1,and is larger than the values of available lead drugs BA and MF(-160.64 and-83.51 kcal·mol-1).The space orientations of carboxyl and guanidino groups within the compound 9 at the neuraminidase active site are in good agreement with the current drugs.In addition,compound 9 has strong interactions with the conserved active-site residues Asp151 and Glu227,which is similar to the situation of MF.Therefore,compound 9 is a novel plant neuraminidase inhibitor with potential applications and will arouse the interest of experimental aspects to design novel anti-influenza drugs.