Investigation Into Modes Of Action And Virtual Screening Of Natural Neuraminidase Inhibitors

Influenza (commonly referred to as flu) is a respiratory infectious disease caused by influenza virus, and it is highly infectious and spreads fast. Flu epidemics cause30-50deaths every year, and flu pandemics may cause hundreds of millions’infection and claim tens of millions’death. Influenza virus (IV) is a RNA virus from family Orthomyxoviridae, which is classified into3types:A, B and C. All the flu pandemics are caused by Influenza virus A. According to differences of hemagglutinin (HA) and neuraminidase (NA), influenza virus A is named HxNy.As NA plays a biologically vital role in the replication of IV and contains a conservative pocket among different subtypes, it is regarded as one of the most important targets for anti-IV drugs. Up to date, NA inhibitors are the mostly effective drugs to treat influenza clinically. After H7N9occurred in China in2013,3NAIs have been taken as powerful weapons to combat this war. These drugs include Oseltamivir, Zanamivir and Peramivir.Till now,9serotypes of NAs are identified. They are classified into two genetically distinct groups:group-1include Nl, N4, N5, N8, group-2include N2, N3, N6, N7and N9. The two groups have structural differences in ’150-loop" and "150-cavity". But strangely09N1(from2009H1N1) are found to have a more similar "150-loop" as group2NA. As influenza virus mutation and drug-resistance problems become severe, the steps for discovering new structural and anti-drug resistance NAIs should be accelerated. NAI discovery and mechanism investigation based on the structure of09N1may provide new ideas for new NAIs.During1981to2010,80%of new antiviral entities registered are natural products or derivatives with natural product pharmacophores. In recent years, researches on natural NAIs have outnumbered those on chemical synthetic NAIs. Traditional chinese medicine (TCM) are widely used in our country, and they have also played an important role in the anti-flu war. Accelerating the process of modernization of TCM is a must for TCM to become internationally applied, while how to illustrate the principal base and action mechanism of active components has always been a key and difficult point. As CADD has a unique function in investigate the interactions between moleculars, it can be used in discovering active components from the anti-influenza TCM and illustrating their functions.This research makes full use of the crystal structures of NA in the protein data bank (PDB), collects structural and activity information of natural NAIs and components from anti-flu TCM, and use different theories and techniques of CADD. First, evaluate the molecular docking effectiveness of softwares GOLD and Surflex-Dock on09N1, and explore the binding modes of natural09N1inhibitors (09NAI) to09N1through molecular docking; build pharmacophore models for09N1inhibitors; built Anti-influenza Chinese Medicine Components Database (AICMD), and search for new NAIs from Traditional Chinese Medicine Database (TCMD) and AICMD, on the basis of pharmacophore model, drug-likeness and molecular docking.Objectives:Explore the binding modes of09NAI to09N1on molecular level through molecular docking; build pharmacophore models for09N1inhibitors, based on the structural features of NAIs on the market and protein-ligand interactions in the09N1-NAI crystal complex; build AICMD, and screen new NAIs from TCMD and AICMD.Methods:1Using molecular docking softwares GOLD and Surflex-Dock, docked ligands from09N1-NAI crystal complex back to original protein09N1, checked reliability of the docking based on RMSD and Key H-bonds repetition rate (presented by KISS Score) of docking conformations.2Collected natural products with09N1inhibitory activity, then explore the binding modes of09NAI to09Nland compared their binding modes with NAIs on the market, with the use of molecular docking. 3Built pharmacophore models for09N1inhibitors, based on the structural features of NAIs on the market and protein-ligand interactions in the09N1-NAI crystal complex.4Screened TCMD on the basis of pharmacophore models, drug-likeness and molecular docking techniques, aiming at discovering potential NAIs.5According to the frequencies of anti-flu herbs appearing in the clinical and experimental researches which were concluded in literatures, collected components of these herbs in sequence, and built AICMD which was subsequently screened for new NAIs.Results:1RMSD between ligand docking conformations obtained by GOLD and Surflex-Dock and ligand conformations in original crystal complexes are less than2.5A, and all highest-score conformations have high KISS Scores. Using RMSD and KISS Score as evaluation criterion, compared GoldScore and ChemScore which are both scoring functions of GOLD, and found docking based on GoldScore preformed better.2Collected10natural products with09N1inhibitory activity. Based on molecular docking, all09NAIs interact with site S1in NA through H-bond or electrostatic interaction.5structural fragments are summarized to replace chemical groups in known NAIs, which include5-hydroxychromone,2-Hydroxyacetophenone, benzyl aliphatic ketones (carbon number between benzyl and carbonyl is1-3), pyrocatechol and1,3-propylene ketone. were summarized. Different from other09NAI, katsumadain A binds to NA mainly by hydrophobic force and Van der Waals force, rather than H-bond or electrostatic interaction. A pharmacophore with4features were built based on all09NAIs. Two H-bond acceptor can form H-bonds with site S1, one hydrophobic center represent the mother nucleus structures of09NAIs, and the other hydrophobic center, adjoining to H-bond acceptor, has hydrophobic interaction with "430-loop". 3Built4pharmacophore models based on similarities and differences of4NAIs on the market in their structural features and interactions modes. There is one pharmacophore model with3features, which include one H-Bond acceptor/positively charged centre, one H-Bond donor/negtively charged centre, and one H-Bond acceptor. There are two pharmacophore models with4features, which include all the features of the3-feature-pharmacophore model, and a hydrophobic centre or H-bond acceptor, respectively. There is one pharmacophore model with5features, which include all the features of the two4-feature-pharmacophore models.450potential NAIs were obtained from TCMD, which include glucosinolates (GSs), flavonoids, polyphenols, betaxanthins, alkaloid and anthrones. Among them, GSs got much higher GoldScores than ZMR. All of the50compounds have strong H-bond interactions with site S1, and the functioning fragments include sulfate group, carboxyl group, mother nucleus structures of flavonoids and phenol-like structure. What is noteworthy is that alkaloid AL2bind to NA mainly by hydrophobic force and Van der Waals force.5Collected components from the top4anti-flu herbs, which are Glycyrrhizae, Lonicera japonica, Scutellaria baicalensis and Forsythia suspensa, and built AICMD with620compounds. Molecular docking results showed, among the top25scored compounds,12compounds are from Glycyrrhizae,10are from Lonicera japonica,2compounds are from Scutellaria baicalensis, and1compound is from Forsythia suspensa.Conclusions:1Experiments validate the reliability of docking results by GOLD and Surflex-Dock when they are performed on09N1, and it’s better to choose GoldScore as scoring function of GOLD, which provide references for docking experiment on09N1.4pharmacophore models for09N1inhibitors are determined, and among them, model1-3are suited to search for09N1or classical Nl inhibitors. On the basis of pharmacophore models, drug-likeness and molecular docking, two virtual screening methods were used to search TCMD, and complementarily structurally different compounds obtained, which improves the screening efficiency and is instructive for virtual screening on large databases.2Anti-influenza Chinese Medicine Database (AICMD) is different from other databases. It is mainly for the screening of anti-flu active components, which can facilitate anti-flu drug design by CADD and accelerate the research of principal base and action mechanism of anti-flu TCM. It is of originality and practical value.3Flavonoids, polyphenols and compounds with carboxyl group, which are obtained from TCMD and AICMD, have the structural features of NAIs on the market or natural NAIs, and they are worth further reseach. Among them, vitexin, epicatechin-3-O-gallate, baicalin, luteolin and galuteolin were found to have NA inhibitory activity, and this research illuminated their interactions with NA. Flavonoid F2and lignan PP3were found to inhibit IV, this research suggested they may inhibit NA to perform anti-flu activity. Flavonoids with prenyl structures obtained from Glycyrrhizae are potential NAI with anti-drug resistance properties.4GSs from TCMD got much higher GoldScores than ZMR, lignans PP1and PP2from Lonicera japonica. Have very similar binding modes as ZMR, and they are potential new NAIs. Our research will provide new ideas for posterior pharmacological tests and clues for illustrating the principal base and action mechanism of anti-flu Chinese medicine.5Through molecular docking research, all09NAIs and potential NAIs from TCMD and AICMD interact with site S1through H-bond or electrostatic interactions, which provides an important hint for NAI design.6structural fragments have been summarized which can interact with S1, that is5-hydroxychromone,2-Hydroxyacetophenone, benzyl aliphatic ketones (carbon number between benzyl and carbonyl is1-3), pyrocatechol,1,3-propylene ketone and sulfate group. 6Different form NAIs on the market, katsumadain A with09N1inhibitory activity and alkaloid AL2from TCMD both bind to NA maily by hydrophobic force and Van der Waals force, and they both have hydrophobic interaction with "245-loop" and "430-loop". Pharmacophore built based on09NAIs has a hydrophobic center feature, which has hydrophobic interaction with "430-loop". Baicalin from Scutellaria baicalensis has hydrophobic interaction with "245-loop". Therefore,"245-loop" and "430-loop" may play important roles in NAI design, and natural products with abundant structural types are of tremendous research value, which can be used to discover new structural and action mechanism NAIs.