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It is urgent to find an effective antiviral drug against SARS-CoV-2. In this study, 96 virus-drug associations (VDAs) from 12 viruses including SARS-CoV-2 and similar viruses and 78 small molecules are selected. Complete genomic sequence similarity of viruses and chemical structure similarity of drugs are then computed. A KATZ-based VDA prediction method (VDA-KATZ) is developed to infer possible drugs associated with SARS-CoV-2. VDA-KATZ obtained the best AUCs of 0.8803 when the walking length is 2. The predicted top 3 antiviral drugs against SARS-CoV-2 are remdesivir, oseltamivir, and zanamivir. Molecular docking is conducted between the predicted top 10 drugs and the virus spike protein/human ACE2. The results showed that the above 3 chemical agents have higher molecular binding energies with ACE2. For the first time, we found that zidovudine may be effective clues of treatment of COVID-19. We hope that our predicted drugs could help to prevent the spreading of COVID., (Copyright © 2020. Published by Elsevier Inc.)

Neuraminidase (NA) of influenza viruses enables the virus to access the cell membrane. It degrades the sialic acid contained in extracellular mucin. Later, it is responsible for releasing newly formed virions from the membrane of infected cells. Both processes become key functions within the viral cycle. Therefore, it is a therapeutic target for research of the new antiviral agents. Structure-activity relationships studies have revealed which are the important functional groups for the receptor-ligand interaction. Influenza virus type A NA activity was inhibited by five scaffolds without structural resemblance to sialic acid. Intending small organic compound repositioning along with drug repurposing, this study combined in silico simulations of ligand docking into the known binding site of NA, along with in vitro bioassays. The five proposed scaffolds are N -acetylphenylalanylmethionine, propanoic 3-[(2,5-dimethylphenyl) carbamoyl]-2-(piperazin-1-yl) acid, 3-(propylaminosulfonyl)-4-chlorobenzoic acid, ascorbic acid (vitamin C), and 4-(dipropylsulfamoyl) benzoic acid (probenecid). Their half maximal inhibitory concentration (IC 50 ) was determined through fluorometry. An acidic reagent 2'- O -(4-methylumbelliferyl)-α-d N -acetylneuraminic acid (MUNANA) was used as substrate for viruses of human influenza H1N1 or avian influenza H5N2. Inhibition was observed in millimolar ranges in a concentration-dependent manner. The IC 50 values of the five proposed scaffolds ranged from 6.4 to 73 mM. The values reflect a significant affinity difference with respect to the reference drug zanamivir ( p < 0.001). Two compounds ( N -acetyl dipeptide and 4-substituted benzoic acid) clearly showed competitive mechanisms, whereas ascorbic acid reflected non-competitive kinetics. The five small organic molecules constitute five different scaffolds with moderate NA affinities. They are proposed as lead compounds for developing new NA inhibitors which are not analogous to sialic acid.

Simulating drug binding and unbinding is a challenge, as the rugged energy landscapes that separate bound and unbound states require extensive sampling that consumes significant computational resources. Here, we describe the use of interactive molecular dynamics in virtual reality (iMD-VR) as an accurate low-cost strategy for flexible protein-ligand docking. We outline an experimental protocol which enables expert iMD-VR users to guide ligands into and out of the binding pockets of trypsin, neuraminidase, and HIV-1 protease, and recreate their respective crystallographic protein-ligand binding poses within 5-10 minutes. Following a brief training phase, our studies shown that iMD-VR novices were able to generate unbinding and rebinding pathways on similar timescales as iMD-VR experts, with the majority able to recover binding poses within 2.15 Å RMSD of the crystallographic binding pose. These results indicate that iMD-VR affords sufficient control for users to carry out the detailed atomic manipulations required to dock flexible ligands into dynamic enzyme active sites and recover crystallographic poses, offering an interesting new approach for simulating drug docking and generating binding hypotheses., Competing Interests: DRG acknowledges cloud computing resources were provided from Oracle Corporation via a Bristol University Partnership Cloud award. This does not alter our adherence to PLOS ONE policies on sharing data and materials. There are no patents, products in development or marketed products to declare.

Neuraminidase (NA) is an important antiviral drug target. Zanamivir is one of the most potent NA inhibitors. In this paper, a series of zanamivir derivatives as potential NA inhibitors were studied by combination of molecular modeling techniques including 3D-QSAR, molecular docking, and molecular dynamics (MD) simulation. The results show that the best CoMFA (comparative molecular field analysis) model has q 2  = 0.728 and r 2  = 0.988, and the best CoMSIA (comparative molecular similarity indices analysis) model has q 2  = 0.750 and r 2  = 0.981, respectively. The built 3D-QSAR models show significant statistical quality and excellent predictive ability. Seven new NA inhibitors were designed and predicted. 20 ns of MD simulations were carried out and their binding free energies were calculated. Two designed compounds were selected to be synthesized and biologically evaluated by NA inhibition and virus inhibition assays. One compound (IC 50  = 0.670 µM, SI > 149) exhibits excellent antiviral activity against A/WSN/33 H1N1, which is superior to the reference drug zanamivir (IC 50  = 0.873 µM, SI > 115). The theoretical and experimental results may provide reference for development of new anti-influenza drugs., (Copyright © 2018. Published by Elsevier Ltd.)

Inhibitors of viral neuraminidase enzymes have been previously developed as therapeutics. Humans can express multiple forms of neuraminidase enzymes (NEU1, NEU2, NEU3, NEU4) that share a similar active site and enzymatic mechanism with their viral counterparts. Using a panel of purified human neuraminidase enzymes, we tested the inhibitory activity of 2-deoxy-2,3-dehydro-N-acetylneuraminic acid (DANA), zanamivir, oseltamivir, and peramivir against each of the human isoenzymes. We find that, with the exceptions of DANA and zanamivir, these compounds show generally poor activity against the human neuraminidase enzymes. To provide insight into the interactions of viral inhibitors with human neuraminidases, we conducted molecular dynamics simulations using homology models based on coordinates reported for NEU2. Simulations revealed that an organized water is displaced by zanamivir in binding to NEU2 and NEU3 and confirmed the critical importance of engaging the binding pocket of the C7-C9 glycerol sidechain. Our results suggest that compounds designed to target the human neuraminidases should provide more selective tools for interrogating these enzymes. Furthermore, they emphasize a need for additional structural data to enable structure-based drug design in these systems., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Neuraminidase (NA) inhibitors are used for the prevention and treatment of influenza A virus infections. Two subtypes of NA, N1 and N2, predominate in viruses that infect humans, but differential patterns of drug resistance have emerged in each subtype despite highly homologous active sites. To understand the molecular basis for the selection of these drug resistance mutations, structural and dynamic analyses on complexes of N1 and N2 NA with substrates and inhibitors were performed. Comparison of dynamic substrate and inhibitor envelopes and interactions at the active site revealed how differential patterns of drug resistance have emerged for specific drug resistance mutations, at residues I222, S246, and H274 in N1 and E119 in N2. Our results show that the differences in intermolecular interactions, especially van der Waals contacts, of the inhibitors versus substrates at the NA active site effectively explain the selection of resistance mutations in the two subtypes. Avoiding such contacts that render inhibitors vulnerable to resistance by better mimicking the dynamics and intermolecular interactions of substrates can lead to the development of novel inhibitors that avoid drug resistance in both subtypes.

The design of potent metabolically stable neuraminidase (NA) inhibitors represents an attractive approach for treating influenza virus infection. In this study, we describe the exploitation of the 150-cavity in the active site of group 1 NA for the design, synthesis, and in vitro evaluation of new triazole-containing N-acyl derivatives related to Zanamivir. Inhibition studies with influenza virus NAs of group 1 (H1N1) and group 2 (H3N2) revealed that several of them are good inhibitors, with IC50 values in the low nanomolar (2.3 nM-31 nM) range. Substituents that form stable van der Waals interaction with the 150-cavity residues play crucial roles in NA inhibition as demonstrated by the potency of 6a (H1N1 IC50 = 2.3 nM, and H3N2 IC50 = 2.9 nM). Docking studies indicated that the cyclohexane-substituted triazole ring extended toward the hydrophobic region in the active site of group 1 NA in open form. The high potency observed for inhibitor 6a may be attributable to the highly favorable hydrophobic interactions in this region., (Copyright © 2016 Elsevier Masson SAS. All rights reserved.)

Human parainfluenza type-3 virus (hPIV-3) is one of the principal aetiological agents of acute respiratory illness in infants worldwide and also shows high disease severity in the elderly and immunocompromised, but neither therapies nor vaccines are available to treat or prevent infection, respectively. Using a multidisciplinary approach we report herein that the approved drug suramin acts as a non-competitive in vitro inhibitor of the hPIV-3 haemagglutinin-neuraminidase (HN). Furthermore, the drug inhibits viral replication in mammalian epithelial cells with an IC50 of 30 μM, when applied post-adsorption. Significantly, we show in cell-based drug-combination studies using virus infection blockade assays, that suramin acts synergistically with the anti-influenza virus drug zanamivir. Our data suggests that lower concentrations of both drugs can be used to yield high levels of inhibition. Finally, using NMR spectroscopy and in silico docking simulations we confirmed that suramin binds HN simultaneously with zanamivir. This binding event occurs most likely in the vicinity of the protein primary binding site, resulting in an enhancement of the inhibitory potential of the N-acetylneuraminic acid-based inhibitor. This study offers a potentially exciting avenue for the treatment of parainfluenza infection by a combinatorial repurposing approach of well-established approved drugs.

Increased reports of oseltamivir (OTV)-resistant strains of the influenza virus, such as the H274Y mutation on its neuraminidase (NA), have created some cause for concern. Many studies have been conducted in the attempt to uncover the mechanism of OTV resistance in H274Y NA. However, most of the reported studies on H274Y focused only on the drug-bound system, so the direct effects of the mutation on NA itself prior to drug binding still remain unclear. Therefore, molecular dynamics simulations of NA in apo form, followed by principal component analysis and interaction energy calculations, were performed to investigate the structural changes of the NA binding site as a result of the H274Y mutation. It was observed that the disruption of the NA binding site due to the H274Y mutation was initiated by the repulsive effect of Y274 on the 250-loop, which in turn altered the hydrogen-bonding network around residue 274. The rotated W295 side chain caused the upward movement of the 340-loop. Consequently, sliding box docking results suggested that the binding pathway of OTV was compromised because of the disruption of this binding site. This study also highlighted the importance of the functional group at C6 of the sialic acid mimicry. It is hoped that these results will improve the understanding of OTV resistance and shed some light on the design of a novel anti-influenza drug.

Assessment of accurate drug binding affinity to a protein remains a challenge for in silico drug development. In this research, we used the smooth reaction path generation (SRPG) method to calculate binding free energies and determine potential of mean forces (PMFs) along the smoothed dissociation paths of influenza A neuraminidase and its variants with oseltamivir (Tamiflu) and zanamivir (Relenza) inhibitors. With the gained results, we found that the binding free energies of neuraminidase A/H5N1 in WT and two mutants (including H274Y and N294S) with oseltamivir and zanamivir show good agreement with experimental results. Additionally, the thermodynamic origin of the drug resistance of the mutants was also discussed from the PMF profiles.

The neuraminidase (NA) inhibitors oseltamivir and zanamivir are administered twice daily for 5days for treatment of influenza. Laninamivir is a 7-methoxy derivative of zanamivir, but a single dose is effective when taken as the laninamivir octanoate prodrug. We show here in IC50 kinetics assays and a solid phase reactivation assay that compared to zanamivir laninamivir also demonstrates slow binding to but slower dissociation from multiple wild type NAs. A D197E mutation in an influenza B and an E119G in an N9 neuraminidase which confer 15- and 150-fold resistance to laninamivir result in faster binding and dissociation. Despite similar IC50s our assays demonstrate more rapid dissociation of laninamivir from clade 1 compared to 2 H5N1 NAs., (Crown Copyright © 2014. Published by Elsevier B.V. All rights reserved.)

The influenza virus neuraminidase inhibitors are normally slow binding inhibitors, but many mutations leading to resistance, also result in the loss of the slow binding phenotype. Mutations can also affect the rate of dissociation of the inhibitors from the neuraminidase, but the assays to measure this require large amounts of virus and are time consuming. To more fully understand the impacts of mutations on the binding and dissociation of the neuraminidase inhibitors we have developed a solid phase reactivation assay, which can use small amounts of crude virus sample bound to an ELISA plate. Multiple viruses can be assayed simultaneously against multiple inhibitors. Using this assay we have demonstrated differences in the relative rates of dissociation of the inhibitors and reactivation of enzyme activity among different influenza A and B viruses for zanamivir, oseltamivir and peramivir. In general oseltamivir dissociated the fastest, and dissociation of peramivir was much slower than both the other inhibitors. Viruses with H274Y, E119V and E119G mutations demonstrated faster dissociation of the inhibitor to which they were resistant. Dissociation of zanamivir and oseltamivir were faster from the D197E mutant, but not of peramivir., (Crown Copyright © 2014. Published by Elsevier B.V. All rights reserved.)

Laninamivir octanoate (LO) is an octanoyl ester prodrug of the neuraminidase inhibitor laninamivir. After inhaled administration, LO exhibits clinical efficacy for both treatment and prophylaxis of influenza virus infection, resulting from hydrolytic bioactivation into its pharmacologically active metabolite laninamivir in the pulmonary tissue. In this study, we focused on the identification of LO-hydrolyzing enzymes from human pulmonary tissue extract using proteomic correlation profiling-a technology integration of traditional biochemistry and proteomics. In a single elution step by gel-filtration chromatography, LO-hydrolyzing activity was separated into two distinct peaks, designated as peak I and peak II. By mass spectrometry, 1160 and 1003 proteins were identified and quantitated for peak I and peak II, respectively, and enzyme candidates were ranked based on the correlation coefficient between the enzyme activity and the proteomic profiles. Among proteins with a high correlation value, S-formylglutathione hydrolase (esterase D; ESD) and acyl-protein thioesterase 1 (APT1) were selected as the most likely candidates for peak I and peak II, respectively, which was confirmed by LO-hydrolyzing activity of recombinant proteins. In the case of peak II, LO-hydrolyzing activity was completely inhibited by treatment with a specific APT1 inhibitor, palmostatin B. Moreover, immunohistochemical analysis revealed that both enzymes were mainly localized in the pulmonary epithelia, a primary site of influenza virus infection. These findings demonstrate that ESD and APT1 are key enzymes responsible for the bioactivation of LO in human pulmonary tissue.

Neuraminidase inhibitors (NAIs) play vital roles in controlling human influenza epidemics and pandemics. However, the emergence of new human influenza virus mutant strains resistant to existing antiviral drugs has been becoming a major challenge. Therefore, it is critical to uncover the mechanisms of drug resistance and seek alternative treatments to combat drug resistance. In this study, molecular dynamics (MD) simulations and Molecular Mechanics/Generalized Born Surface Area (MM/GBSA) were applied to investigate the different sensitivities of oseltamivir (OTV), zanamivir (ZNV), and peramivir (PRV) against the E119G mutant of 2009 A/H1N1 neuraminidase. The predicted binding free energies indicate that the E119G mutation in NA confers resistance to all of the three studied inhibitors. The ordering of the level of drug resistance predicted by the binding free energies for the three inhibitors is ZNV>PRV>OTV, which agrees well with the experimental data. Drug resistance arises primarily from the unfavorable shifts of the polar interactions between NA and the inhibitors. It comes as a surprise that the mutation of Glu119 that can form strong H-bonds with the inhibitors in the wild-type protein does not have direct impact on the binding affinities of both OTV and PRV due to the regulation of the strong unfavorable polar desolvation energies. The indirectly conformational variations of the inhibitors, which caused by the E119G mutation, are responsible for the loss of the binding free energies. However, for ZNV, the E119G mutation has both direct and indirect influences on the drug binding. The structural and quantitative viewpoint obtained from this study provides valuable information for the rational design of novel and effective drugs to combat drug resistance., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Laninamivir octanoate (LO) is a new neuraminidase inhibitor for inhalation. The objectives of this study were to model the population pharmacokinetics of LO and its active metabolite laninamivir after inhaled administration of LO using a pooled population of healthy subjects, and adult and pediatric patients with influenza virus infection from 8 clinical studies, and to evaluate covariate effects on pharmacokinetics. The pharmacokinetics of LO and laninamivir in plasma and urine are well-described by structural models that consist of a 2-compartment model for LO with instantaneous bolus input and first-order elimination; and a 1-compartment model for laninamivir with formation of laninamivir via the metabolic pathway from LO in systemic circulation, entry of laninamivir from the respiratory tract compartment, and linear elimination. Creatinine clearance was identified as a covariate of apparent total clearance for LO and renal clearances for LO and laninamivir, with the largest effect on laninamivir exposure. Body weight was identified to affect distribution volumes of LO and laninamivir and the metabolic clearance of LO; however there was no notable effect on exposures across the wide body weight range evaluated. The population pharmacokinetic model also provides insight into the likely kinetics of drug disposition in the respiratory tract following inhaled administration.

A single inhaled dose of laninamivir octanoate (LO), a long-acting neuraminidase inhibitor, exhibits efficacy in treating both adult and pediatric patients with influenza virus infection. The intrapulmonary pharmacokinetics (PK) of LO and laninamivir, a pharmacologically active metabolite, were investigated by a single-center, open-label study of healthy adult volunteers. Subgroups of five subjects each underwent bronchoalveolar lavage (BAL) 4, 8, 24, 48, 72, 168, and 240 h following a single inhaled administration of LO (40 mg). Plasma, BAL fluid, and alveolar macrophages (AM) were analyzed to determine LO and laninamivir concentrations, using validated liquid chromatography-tandem mass spectrometry methods. The concentrations in epithelial lining fluid (ELF) and AM from the first and subsequent BAL fluid samples were determined separately to explore the drug distribution in airways. Mean laninamivir concentrations in ELF, calculated using the first BAL fluids and BAL fluids collected 4 h after inhaled administration, were 8.57 and 2.40 μg/ml, respectively. The laninamivir concentration in ELF decreased with a longer half-life than that in plasma, and it exceeded the 50% inhibitory concentrations for viral neuraminidases at all time points examined for 240 h after the inhalation. Laninamivir exposure in ELF from the first BAL samples was 3.2 times higher than that in ELF from the subsequent BAL fluid samples. ELF concentration profiles of laninamivir support its long-lasting effect for treatment of patients with influenza virus infection by a single inhaled administration.

Zanamivir is the known potent anti-influenza agent targeting the key enzyme neuraminidase that cleaves sialic acid from cell receptors allowing release of newly formed virions. Molecular dynamics simulation was carried out to determine the dynamic behavior of Zanamivir upon its binding to flexible loops of neuraminidase and to analyse its interactions in the bioactive state. Neuraminidase exhibits wide range of affinity with structurally similar compounds. CoMFA study was used to determine quantitative structure-activity relationship for 36 carbocyclic Neuraminidase inhibitors (NIs). The CoMFA model was also successfully built using cross-validated r²cv = 0.580 and r²pred = 0.638.

We carried out full ab initio fragment molecular orbital (FMO) calculations for complexes comprising human neuraminidase-2 (hNEU2) and sialic acid analogues including anti-influenza drugs zanamivir (Relenza) and oseltamivir (Tamiflu) in order to examine the variation in the observed inhibitory activity toward hNEU2 at the atomic and electronic levels. We recently proposed the LERE (linear expression by representative energy terms)-QSAR (quantitative structure-activity relationship) procedure. LERE-QSAR analysis quantitatively revealed that the complex formation is driven by hydrogen-bonding and electrostatic interaction of hNEU2 with sialic acid analogues. The most potent inhibitory activity, that of zanamivir, is attributable to the strong electrostatic interaction of a positively charged guanidino group in zanamivir with negatively charged amino acid residues in hNEU2. After we confirmed that the variation in the observed inhibitory activity among sialic acid analogues is excellently reproducible with the LERE-QSAR equation, we examined the reason for the remarkable difference between the inhibitory potencies of oseltamivir as to hNEU2 and influenza A virus neuraminidase-1 (N1-NA). Several amino acid residues in close contact with a positively charged amino group in oseltamivir are different between hNEU2 and N1-NA. FMO-IFIE (interfragment interaction energy) analysis showed that the difference in amino acid residues causes a remarkably large difference between the overall interaction energies of oseltamivir with hNEU2 and N1-NA. The current results will be useful for the development of new anti-influenza drugs with high selectivity and without the risk of adverse side effects.

As one of the most important antiviral drugs against 2009 influenza A (H1N1), will zanamivir be effective for the possible drug resistant mutants? To answer this question, we combined multiple molecular dynamics simulations and molecular mechanics generalized Born surface area (MM-GBSA) calculations to study the efficiency of zanamivir over the most frequent drug-resistant strains of neuraminidase including R293K, R152K, E119A/D and H275Y mutants. The calculated results indicate that the modeled mutants of the 2009-H1N1 strains except H275Y will be significantly resistant to zanamivir. The resistance to zanamivir is mainly caused by the loss of polar interactions. The identified potential resistance sites in this study will be useful for the development of new effective anti-influenza drugs and to avoid the occurrence of the state without effective drugs to new mutant influenza strains.

There are 9 serotypes of neuraminidase (NA) from influenza A virus (N1 to N9), which are classified into two groups based on primary sequences (groups 1 and 2). The structural hallmark of the two groups is the presence or absence of an extra 150-cavity (formed by the 150-loop) in the active site. Thus far, structures of NAs from 6 out of the 9 serotypes have been solved. Here, we solved the N5 structure, the last unknown structure group 1 serotype with a unique Asn147 residue in its 150-loop, demonstrating that it has an extended 150-cavity that closes upon inhibitor binding.

Seasonal influenza epidemics cause illness and death each year, and the emergence of antigenically novel influenza A viruses are a continual pandemic threat. Disease and death can be averted by vaccination. The potency of killed virus vaccines is based on hemagglutinin (HA) content. However, antibodies that inhibit enzyme activity of the neuraminidase (NA) also reduce virus replication and protect against disease. Monoclonal NA-inhibiting (NI) antibodies recognize conformational epitopes, and it is anticipated that native tertiary structure is required for their induction. NA assembles as a tetramer and only this form has enzyme activity. Since small inhibitors of NA do not significantly alter conformation, we sought to determine whether neuraminidase-inhibiting (NI) antibodies would be induced by inhibitor-inactivated NA. We therefore evaluated responses of mice immunized with purified NA that was either inactivated by addition of zanamivir or denatured by heat treatment. NI antibodies were induced following immunization with NA from A/Wisconsin/67/2005 (H3N2) in which enzyme activity was inhibited by the former method but not the latter. Protection of mice against challenge with virus containing an antigenically matched NA correlated with the detection of NI antibodies in serum. Similar results were obtained when mice were immunized with whole H1N1 virus in which NA activity had been inhibited by the same two modalities. This demonstrates that native conformation of NA is necessary for induction of NI antibodies; enzyme activity provides a useful marker of intact structure, but absence of activity in NA that is correctly folded does not result in loss of immunogenicity. Thus any assay to assess potency of influenza vaccines with respect to NA content should consider the proportion of NA that is in a structurally native state., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

The influenza neuraminidase (NA) inhibitors zanamivir, oseltamivir and peramivir were all designed based on the knowledge that the transition state analogue of the cleaved sialic acid, 2-deoxy,2,3-dehydro N-acetyl neuraminic acid (DANA) was a weak inhibitor of NA. While DANA bound rapidly to the NA, modifications leading to the improved potency of these new inhibitors also conferred a time dependent or slow binding phenotype. Many mutations in the NA leading to decreased susceptibility result in loss of slow binding, hence this is a phenotypic marker of many but not all resistant NAs. We present here a simplified approach to determine whether an inhibitor is fast or slow binding by extending the endpoint fluorescent enzyme inhibition assay to a real time assay and monitoring the changes in IC(50)s with time. We carried out two reactions, one with a 30 min preincubation with inhibitor and the second without. The enzymatic reaction was started via addition of substrate and IC(50)s were calculated after each 10 min interval up to 60 min. Results showed that without preincubation IC(50)s for the wild type viruses started high and although they decreased continuously over the 60 min reaction time the final IC(50)s remained higher than for pre-incubated samples. These results indicate a slow equilibrium of association and dissociation and are consistent with slow binding of the inhibitors. In contrast, for viruses with decreased susceptibility, preincubation had minimal effect on the IC(50)s, consistent with fast binding. Therefore this modified assay provides additional phenotypic information about the rate of inhibitor binding in addition to the IC(50), and critically demonstrates the differential effect of incubation times on the IC(50) and K(i) values of wild type and mutant viruses for each of the inhibitors.

The outbreak and high speed global spread of the new strain of influenza A (H1N1) virus in 2009 poses a serious threat to the general population and governments. At present, the most effective drugs for the treatment of 2009 influenza A (H1N1) virus are neuraminidase inhibitors: mainly oseltamivir and zanamivir. The use of these two inhibitors will undoubtedly increase, and therefore it is more likely that drug-resistant influenza strains will arise. The identification of the potential resistance sites for these drugs in advance and the understanding of corresponding molecular basis to cause drug resistance are no doubt very important to fight against the new resistant influenza strains. In this study, first, the complexes of neuraminidase with the substrate sialic acid and two inhibitors oseltamivir and zanamivir were obtained by fitting them to the 3D structure of 2009 influenza A (H1N1) neuraminidase obtained by homology modeling. By using these complexes as the initial structures, molecular dynamics simulation and molecular mechanics generalized Born surface area (MM-GBSA) calculations were performed to identify the residues with significant contribution to the binding of substrate and inhibitors. By analyzing the difference of interaction profiles of substrate and inhibitors, the potential drug resistance sites for two inhibitors were identified. Parts of the identified sites have been verified to confer resistance to oseltamivir and zanamivir for influenza virus of the past flu epidemic. The identified potential resistance sites in this study will be useful for the development of new effective drugs against the drug resistance and avoid the situation of having no effective drugs to treat new mutant influenza strains.

The outbreak of avian influenza A subtype H5N1 virus has raised a global concern for both animal as well as human health. Recently, drug resistance in H5N1 infections has been widely reported due to neuraminidase mutations. Consequently, the understanding of inhibitor-neuraminidase interactions at the molecular level represents the main goal of our study. Molecular dynamics simulations were carried out for the neuraminidase N1 in complex with six inhibitors--oseltamivir, zanamivir, peramivir, and their phosphonate analogues. Molecular dynamics trajectories were extensively analyzed in terms of important interactions between inhibitors and the enzyme target. Results show that open and closed forms (defined by the relative position of the flexible 150-loop) of neuraminidase N1 interchange during the course of 20 ns molecular dynamics simulation of the protein-inhibitor complexes. Reported free energies of closing indicate that the carboxylate inhibitors prefer the closed form more than their phosphonate analogues. This can be understood in view of the negative total charge (-1 e0) of the phosphonate inhibitors, which repels the Asp151 residue of the loop away from the inhibitor and drives the complex into the open form. Obtained results constitute new valuable information to assist further drug development of inhibitors against the H5N1 avian influenza A virus and could also inspire similar studies for other systems of the influenza family such as the 2009 influenza A (H1N1) virus.

Influenza virus neuraminidase (NA) plays a crucial role in facilitating the spread of newly synthesized virus in the host and is an important target for controlling disease progression. The NA crystal structure from the 1918 "Spanish flu" (A/Brevig Mission/1/18 H1N1) and that of its complex with zanamivir (Relenza) at 1.65-A and 1.45-A resolutions, respectively, corroborated the successful expression of correctly folded NA tetramers in a baculovirus expression system. An additional cavity adjacent to the substrate-binding site is observed in N1, compared to N2 and N9 NAs, including H5N1. This cavity arises from an open conformation of the 150 loop (Gly147 to Asp151) and appears to be conserved among group 1 NAs (N1, N4, N5, and N8). It closes upon zanamivir binding. Three calcium sites were identified, including a novel site that may be conserved in N1 and N4. Thus, these high-resolution structures, combined with our recombinant expression system, provide new opportunities to augment the limited arsenal of therapeutics against influenza.

To provide detailed information and insight into the drug-target interaction, structure, solvation, and dynamic and thermodynamic properties, the three known-neuraminidase inhibitors-oseltamivir (OTV), zanamivir (ZNV), and peramivir (PRV)-embedded in the catalytic site of neuraminidase (NA) subtype N1 were studied using molecular dynamics simulations. In terms of ligand conformation, there were major differences in the structures of the guanidinium and the bulky groups. The atoms of the guanidinium group of PRV were observed to form many more hydrogen bonds with the surrounded residues and were much less solvated by water molecules, in comparison with the other two inhibitors. Consequently, D151 lying on the 150-loop (residues 147-152) of group-1 neuraminidase (N1, N4, N5, and N8) was considerably shifted to form direct hydrogen bonds with the --OH group of the PRV, which was located rather far from the 150-loop. For the bulky group, direct hydrogen bonds were detected only between the hydrophilic side chain of ZNV and residues R224, E276, and E277 of N1 with rather weak binding, 20-70% occupation. This is not the case for OTV and PRV, in which flexibility and steric effects due to the hydrophobic side chain lead to the rearrangement of the surrounded residues, that is, the negatively charged side chain of E276 was shifted and rotated to form hydrogen bonds with the positively charged moiety of R224. Taking into account all the ligand-enzyme interaction data, the gas phase MM interaction energy of -282.2 kcal/mol as well as the binding free energy (DeltaG(binding)) of -227.4 kcal/mol for the PRV-N1 are significantly lower than those of the other inhibitors. The ordering of DeltaG(binding) of PRV < ZNV < OTV agrees well with the ordering of experimental IC(50) value., ((c) 2008 Wiley-Liss, Inc.)