Molecular Modeling On The Interacting Mechanisms Between Neuraminidases And The Plant Inhibitors

Scutellaria baicalensis Georgi,commonly used in Chinese medicine,has flavonoids as the main chemical constituents.It has been proved with antiviral,anti-allergic,anti-platelet, anti-inflammatory,antioxidant and antitumor activities.From the roots of Scutellaria baicalensis Georgi,5,7,4′-trihydroxy-8-methoxyflavone(MF) was isolated and identified to be antiviral to the neuraminidases of influenza;however,the interactions between MF and neuraminidases have not been clear yet.In this work,molecular mechanics,molecular docking and density functional theory methods were combined to study the roles of heteroatoms(Ca²⁺ and water molecules near the active site) on the neuraminidase structures,the bioactivity conformers of neuraminidase inhibitors and the interaction modes between MF and neuraminidases.The results contribute to the in-depth understanding on the interacting mechanisms between neuraminidases and the plant inhibitors.The structural analyses implicated that,with the absence of the calcium ion near the active site,the neuraminidase active site shrinks remarkably so that the inhibitor can not be docked with,consistent with the binding-site-searching result.With the inclusion of the calcium ion, the shapes and sizes of the neuraminidase active site are steady and can combine with the plant inhibitors,close to those in the crystal structures.The calcium ion is crucial to the maintenance of the active sites while the conserved water molecules exert relatively slight influences. Moreover,larger movements were observed in the side chains of the key residues rather than in the backbone atoms.It was also observed that the heteroatoms can cause the secondary structural transitions in the active sites as well as the charge,hydrophilicity vs.hydrophobicity and polarity vs.nonpolarity redistributions in the sub-regions of the active sites.The structural and property analyses indicated that sub-regions 1 and 3 are responsible for the locations of substrates whereas the orientations of substrates can be tuned according to the different properties of sub-regions 2 and 4.The predictions of the binding modes were supported by the molecular docking results.With the aid of density functional theory,it was found that,in gas phase the lowest-energy structure of the neuraminidase inhibitor,4-(N-acetylamino)-5-guanidino-3-(3-pentyloxy) benzoic acid(BA) is neutral,while in solution the zwitterionic conformer of BA is the most stabilized.The atom positions and charges distributions in the neutral and zwitterionic conformers of BA are different;for example,in the zwitterionic conformer,the distance between the N-acetyl group and the positive charged guanidinium group is smaller,which matches better with the sub-regions 2 and 3 in the neuraminidase active pocket.The data of flexible molecular docking and molecular dynamics simulations showed that the inter-energies and absolute binding free energy between the zwitterionic conformer and the neuraminidase are larger than those of the neutral conformer.These have proved that bioactive conformer of BA is zwitterionic.The interactions between the zwitterionic BA and neuraminidase are centralized on 8 key residues,while in the case of the neutral conformer on 13 residues.With the flexible molecular docking program Affinity,an in-depth and systemetic investaigation was carried out on the interacting mechanisms between 5,7,4'-trihydroxy-8-methoxyflavone(MF) and N1 and N9 subtype neuraminidases.It was found that there exist one and two binding modes in the cases of N1 and N9 subtype neuraminidases,and their corresponding largest binding energies were calculated to be -70.26 and -83.51 kcal·mol^-1, respectively.The further analyses revealed that great distinctions are present in the bindings of MF to N1 and N9 subtype neuraminidases such as the interacting forces,hydrogen bonding and the interacted key amino acid residues.In contrast with the monotonous binding mode of the current oseltamivir drug,MF can be tightly bound with various subtypes of neuraminidases or with the varied ones.Therefore,MF is a novel anti-influenza virus drug with potential applications.Combined with the previous results,we pointed out how to effectively modify the functional groups of MF.