Neuraminidase inhibition of Dietary chlorogenic acids and derivatives - potential antivirals from dietary sources.

Plants rich in chlorogenic acids (CGAs), caffeic acids and their derivatives have been found to exert antiviral effects against influenza virus neuroaminidase. In this study several dietary naturally occurring chlorogenic acids, phenolic acids and derivatives were screened for their inhibitory activity against neuroaminidases (NAs) from C. perfringens, H5N1 and recombinant H5N1 (N-His)-Tag using a fluorometric assay. There was no significant difference in inhibition between the different NA enzymes. The enzyme inhibition results indicated that chlorogenic acids and selected derivatives, exhibited high activities against NAs. It seems that the catechol group from caffeic acid was important for the activity. Dietary CGA therefore show promise as potential antiviral agents. However, caffeoyl quinic acids show low bioavailibility and are intensly metabolized by the gut micro flora, only low nM concentrations are observed in plasma and urine, therefore a systemic antiviral effect of these compounds is unlikely. Nevertheless, gut floral metabolites with a catechol moiety or structurally related dietary phenolics with a catechol moiety might serve as interesting compounds for future investigations.

Anti-influenza activities of polyphenols from the medicinal mushroom Phellinus baumii.

Five polyphenols were isolated from the ethanolic extract of the fruiting bodies of Phellinus baumii. These compounds were identified by various spectroscopic methods as hispidin, hypholomine B, inoscavin A, davallialactone, and phelligridin D. All compounds inhibited noncompetitively H1N1, H5N1, and H3N2 neuraminidase activity and reduced the amount of virally-induced cytopathic effect (CPE) according to an MDCK cell-based assay.

A virtual screening approach for identifying plants with anti H5N1 neuraminidase activity.

Recent outbreaks of highly pathogenic and occasional drug-resistant influenza strains have highlighted the need to develop novel anti-influenza therapeutics. Here, we report computational and experimental efforts to identify influenza neuraminidase inhibitors from among the 3000 natural compounds in the Malaysian-Plants Natural-Product (NADI) database. These 3000 compounds were first docked into the neuraminidase active site. The five plants with the largest number of top predicted ligands were selected for experimental evaluation. Twelve specific compounds isolated from these five plants were shown to inhibit neuraminidase, including two compounds with IC50 values less than 92 µM. Furthermore, four of the 12 isolated compounds had also been identified in the top 100 compounds from the virtual screen. Together, these results suggest an effective new approach for identifying bioactive plant species that will further the identification of new pharmacologically active compounds from diverse natural-product resources.

Identification of potential inhibitors of H5N1 influenza A virus neuraminidase by ligand-based virtual screening approach.

The neuraminidase (NA) of the influenza virus is the target of antiviral drug, oseltamivir. Recently, cases were reported that influenza virus becoming resistant to oseltamivir, necessitating the development of new long-acting antiviral compounds. In this report, a novel class of lead molecule with potential NA inhibitory activity was identified using a combination of virtual screening (VS), molecular docking, and molecular dynamic approach. The PubChem database was used to perform the VS analysis by employing oseltamivir as query. Subsequently, the data reduction was carried out by employing molecular docking study. Furthermore, the screened lead molecules were analyzed with respect to the Lipinski rule of five, drug-likeness, toxicity profiles, and other physico-chemical properties of drugs by suitable software program. Final screening was carried out by normal mode analysis and molecular dynamic simulation approach. The result indicates that CID 25145634, deuterium-enriched oseltamivir, become a promising lead compound and be effective in treating oseltamivir sensitive as well as resistant influenza virus strains.

Screen anti-influenza lead compounds that target the PA(C) subunit of H5N1 viral RNA polymerase.

The avian influenza (H5N1) viral RNA polymerase protein PA(C) was used as a target to screen nine chlorogenic acid derivatives for their polymerase inhibitor activity. Among them, seven compounds were PA(C) ligands, and four inhibited influenza RNA polymerase activity. These results aid in the design of anti-influenza agents based on caffeoylquinic acid.

Consensus QSAR model for identifying novel H5N1 inhibitors.

Due to the importance of neuraminidase in the pathogenesis of influenza virus infection, it has been regarded as the most important drug target for the treatment of influenza. Resistance to currently available drugs and new findings related to structure of the protein requires novel neuraminidase 1 (N1) inhibitors. In this study, a consensus QSAR model with defined applicability domain (AD) was developed using published N1 inhibitors. The consensus model was validated using an external validation set. The model achieved high sensitivity, specificity, and overall accuracy along with low false positive rate (FPR) and false discovery rate (FDR). The performance of model on the external validation set and training set were comparable, thus it was unlikely to be overfitted. The low FPR and low FDR will increase its accuracy in screening large chemical libraries. Screening of ZINC library resulted in 64,772 compounds as probable N1 inhibitors, while 173,674 compounds were defined to be outside the AD of the consensus model. The advantage of the current model is that it was developed using a large and diverse dataset and has a defined AD which prevents its use on compounds that it is not capable of predicting. The consensus model developed in this study is made available via the free software, PaDEL-DDPredictor.

A novel small-molecule inhibitor of the avian influenza H5N1 virus determined through computational screening against the neuraminidase.

Computational molecular docking provides an efficient and innovative approach to examine small molecule and protein interactions. We have utilized this method to identify potential inhibitors of the H5N1 neuraminidase protein. Of the 20 compounds tested, 4-(4-((3-(2-amino-4-hydroxy-6-methyl-5-pyrimidinyl)propyl)amino)phenyl)-1-chloro-3-buten-2-one (1) (NSC89853) demonstrated the ability to inhibit viral replication at a level comparable to the known neuraminidase inhibitor oseltamivir. Compound 1 demonstrated efficacy across a number of cell-lines assays and in both the H1N1 and H5N1 viruses. The predicted binding of 1 to the known H5N1 neuraminidase structure indicates a binding interface largely nonoverlapping with that of oseltamivir or another neuraminidase inhibitor zanamivir. These results indicate that 1 or similar molecules would remain effective in the presence of virus mutations conferring resistance to either oseltamivir or zanamivir and also vice versa.

Binding mechanism of H5N1 influenza virus neuraminidase with ligands and its implication for drug design.

To simulate new strategies for designing effective drugs against bird flu, we have carried out extensive studies by using various computer-aided drug design tools. Molecule AG7088 was first docked to the active site of H5N1 avian influenza neuraminidase (PBD code: 2HTY). The results thus obtained were compared with those by docking zanamivir (Relenza) and oseltamivir (Tamiflu) to the same receptor, respectively. It has been found that the compound AG7088 has better binding energy than zanamivir and oseltamivir. Thus, it was adopted as a template to perform the similarity search of 392,698 druggable compounds in order to find the leading candidates for the next step of modeling studies. Nine analogs of AG7088 were singled out through a series of docking studies. Finally, the molecular dynamics simulation technique was utilized to investigate into the binding interactions between the H5N1 receptor and the nine analogs, with a focus on the binding pocket, intermolecular surfaces and hydrogen bonds. This study may be used as a guide for mutagenesis studies for designing new inhibitors against H5N1.

Design of oseltamivir analogs inhibiting neuraminidase of avian influenza virus H5N1.

Neuraminidase is an important target for design of antiviral agents in the prophylaxis and treatment of avian influenza virus infections. We have shown the applicability of computer-assisted combinatorial techniques in the design, focusing and in silico screening of a virtual library of analogs of oseltamivir (Tamiflu) with the goal to find potent inhibitors of influenza A neuraminidase N1 that fill the cavity found adjacent to the active site. Crystal structure of oseltamivir-N1 complex was used in the structure-based focusing and virtual screening of the designed library. A target-specific Piecewise Linear Potential type 1 scoring function fitted for a training set of 14 carbocyclic inhibitors and validated for three other inhibitors was used to select virtual hits with predicted inhibitory activities in the subnanomolar range. The results of this computational study are useful as a rational guide for synthetic and medicinal chemists who are developing new drugs against the avian influenza virus H5N1.

Crystal structure of an avian influenza polymerase PA(N) reveals an endonuclease active site.

The heterotrimeric influenza virus polymerase, containing the PA, PB1 and PB2 proteins, catalyses viral RNA replication and transcription in the nucleus of infected cells. PB1 holds the polymerase active site and reportedly harbours endonuclease activity, whereas PB2 is responsible for cap binding. The PA amino terminus is understood to be the major functional part of the PA protein and has been implicated in several roles, including endonuclease and protease activities as well as viral RNA/complementary RNA promoter binding. Here we report the 2.2 ångström (A) crystal structure of the N-terminal 197 residues of PA, termed PA(N), from an avian influenza H5N1 virus. The PA(N) structure has an alpha/beta architecture and reveals a bound magnesium ion coordinated by a motif similar to the (P)DX(N)(D/E)XK motif characteristic of many endonucleases. Structural comparisons and mutagenesis analysis of the motif identified in PA(N) provide further evidence that PA(N) holds an endonuclease active site. Furthermore, functional analysis with in vivo ribonucleoprotein reconstitution and direct in vitro endonuclease assays strongly suggest that PA(N) holds the endonuclease active site and has critical roles in endonuclease activity of the influenza virus polymerase, rather than PB1. The high conservation of this endonuclease active site among influenza strains indicates that PA(N) is an important target for the design of new anti-influenza therapeutics.

Design of multi-binding-site inhibitors, ligand efficiency, and consensus screening of avian influenza H5N1 wild-type neuraminidase and of the oseltamivir-resistant H274Y variant.

The binding sites of wild-type avian influenza A H5N1 neuraminidase, as well as those of the Tamiflu (oseltamivir)-resistant H274Y variant, were explored computationally to design inhibitors that target simultaneously several adjacent binding sites of the open conformation of the virus protein. The compounds with the best computed free energies of binding, in agreement by two docking methods, consensus scoring, and ligand efficiency values, suggest that mimicking a polysaccharide, beta-lactam, and other structures, including known drugs, could be routes for multibinding site inhibitor design. This new virtual screening method based on consensus scoring and ligand efficiency indices is introduced, which allows the combination of pharmacodynamic and pharmacokinetic properties into unique measures.

Ensemble-based virtual screening reveals potential novel antiviral compounds for avian influenza neuraminidase.

Avian influenza virus subtype H5N1 is a potential pandemic threat with human-adapted strains resistant to antiviral drugs. Although virtual screening (VS) against a crystal or relaxed receptor structure is an established method to identify potential inhibitors, the more dynamic changes within binding sites are neglected. To accommodate full receptor flexibility, we use AutoDock4 to screen the NCI diversity set against representative receptor ensembles extracted from explicitly solvated molecular dynamics simulations of the neuraminidase system. The top hits are redocked to the entire nonredundant receptor ensemble and rescored using the relaxed complex scheme (RCS). Of the 27 top hits reported, half ranked very poorly if only crystal structures are used. These compounds target the catalytic cavity as well as the newly identified 150- and 430-cavities, which exhibit dynamic properties in electrostatic surface and geometric shape. This ensemble-based VS and RCS approach may offer improvement over existing strategies for structure-based drug discovery.