Neuraminidase Active Site Research Articles

Neuraminidase inhibitor susceptibility testing of influenza type B viruses in China during 2010 and 2011 identifies viruses with reduced susceptibility to oseltamivir and zanamivir

Influenza type B viruses are responsible for substantial morbidity and mortality in humans. Antiviral drugs are an important supplement to vaccination for reducing the public health impact of influenza virus infections. Influenza B viruses are not sensitive to M2 inhibitors which limit the current therapeutic options to two neuraminidase inhibitors (NAIs), oseltamivir and zanamivir, which are licensed in many countries. Drug resistance is a public health concern which has necessitated monitoring of influenza virus drug susceptibilities through active global surveillance. Here, we report the results of drug susceptibility surveillance of influenza type B viruses (n=680) collected in mainland China during two calendar years, 2010 and 2011, assessed using functional neuraminidase (NA) inhibition (NI) assays. Four influenza B viruses exhibited reduced susceptibilities to oseltamivir, but not zanamivir, and shared the amino acid substitution I221T (ATC→ACC), at this conserved residue in the NA active site (I222T in N2 numbering). Additionally, a single virus with reduced susceptibility to both oseltamivir and zanamivir was identified and contained an amino acid substitution D197N (GAC→AAC) at another conserved residue in the NA active site (D198N in N2 numbering). This report underlies the importance of continued influenza antiviral susceptibility surveillance globally, even in countries where the use of NAIs has been low or non-existing.

Subtype- and antigenic site-specific differences in biophysical influences on evolution of influenza virus hemagglutinin

BackgroundInfluenza virus undergoes rapid evolution by both antigenic shift and antigenic drift. Antibodies, particularly those binding near the receptor-binding site of hemagglutinin (HA) or the neuraminidase (NA) active site, are thought to be the primary defense against influenza infection, and mutations in antibody binding sites can reduce or eliminate antibody binding. The binding of antibodies to their cognate antigens is governed by such biophysical properties of the interacting surfaces as shape, non-polar and polar surface area, and charge.MethodsTo understand forces shaping evolution of influenza virus, we have examined HA sequences of human influenza A and B viruses, assigning each amino acid values reflecting total accessible surface area, non-polar and polar surface area, and net charge due to the side chain. Changes in each of these values between neighboring sequences were calculated for each residue and mapped onto the crystal structures.ResultsAreas of HA showing the highest frequency of pairwise changes agreed well with previously identified antigenic sites in H3 and H1 HAs, and allowed us to propose more detailed antigenic maps and novel antigenic sites for H1 and influenza B HA. Changes in biophysical properties differed between HAs of different subtypes, and between different antigenic sites of the same HA. For H1, statistically significant differences in several biophysical quantities compared to residues lying outside antigenic sites were seen for some antigenic sites but not others. Influenza B antigenic sites all show statistically significant differences in biophysical quantities for all antigenic sites, whereas no statistically significant differences in biophysical quantities were seen for any antigenic site is seen for H3. In many cases, residues previously shown to be under positive selection at the genetic level also undergo rapid change in biophysical properties.ConclusionsThe biophysical consequences of amino acid changes introduced by antigenic drift vary from subtype to subtype, and between different antigenic sites. This suggests that the significance of antibody binding in selecting new variants may also be variable for different antigenic sites and influenza subtypes.

Recovery of Influenza B Virus with the H273Y Point Mutation in the Neuraminidase Active Site from a Human Patient

The H275Y oseltamivir resistance mutation confers high-level resistance to oseltamivir in isolates of human A(H1N1) influenza. We report the recovery and identification of an influenza B virus with the H273Y neuraminidase point mutation directly from a human patient. The H273Y influenza B isolate is resistant to oseltamivir and peramivir but sensitive to zanamivir.

Molecular dynamic and monte carlo study on nanoenergetic binding sites of neuraminidase in different media

Influenza panedemic affect 25 to 30% of the world's population. Neuraminidase (NA) is the most important surfase glycoprotein of the virus causing cleavage of the sialic acid moieties and releasing of newly formed viral particles. The active site of NA is highly conserved all subtype of influaenza virus, then Neuraminidase is the target of drug designs. Using molecular dynamic (MD) and Monte Carlo simulatory methods, the NA structure and its stability different dielcteric (vacuum, water and methanol) and different tempeartures (298, 310, 315, 329 and 333K) was assessed. Measurements of potential energy (kcal/mol) of binding sites NA in different dielectrics and in different temperaturesrevealed that at time step size 0 ps, drug binding sites have maximum energy level, and at time step size 100 ps, have minimum energy level and maximum stability. Key words: Neuraminidase, influenza, molecular dynamic, binding sit, free energy, dielectric

Influenza B Viruses with Mutation in the Neuraminidase Active Site, North Carolina, USA, 2010–11

Oseltamivir is 1 of 2 antiviral medications available for the treatment of influenza B virus infections. We describe and characterize a cluster of influenza B viruses circulating in North Carolina with a mutation in the neuraminidase active site that may reduce susceptibility to oseltamivir and the investigational drug peramivir but not to zanamivir.

Structural and Functional Analysis of Laninamivir and its Octanoate Prodrug Reveals Group Specific Mechanisms for Influenza NA Inhibition

The 2009 H1N1 influenza pandemic (pH1N1) led to record sales of neuraminidase (NA) inhibitors, which has contributed significantly to the recent increase in oseltamivir-resistant viruses. Therefore, development and careful evaluation of novel NA inhibitors is of great interest. Recently, a highly potent NA inhibitor, laninamivir, has been approved for use in Japan. Laninamivir is effective using a single inhaled dose via its octanoate prodrug (CS-8958) and has been demonstrated to be effective against oseltamivir-resistant NA in vitro. However, effectiveness of laninamivir octanoate prodrug against oseltamivir-resistant influenza infection in adults has not been demonstrated. NA is classified into 2 groups based upon phylogenetic analysis and it is becoming clear that each group has some distinct structural features. Recently, we found that pH1N1 N1 NA (p09N1) is an atypical group 1 NA with some group 2-like features in its active site (lack of a 150-cavity). Furthermore, it has been reported that certain oseltamivir-resistant substitutions in the NA active site are group 1 specific. In order to comprehensively evaluate the effectiveness of laninamivir, we utilized recombinant N5 (typical group 1), p09N1 (atypical group 1) and N2 from the 1957 pandemic H2N2 (p57N2) (typical group 2) to carry out in vitro inhibition assays. We found that laninamivir and its octanoate prodrug display group specific preferences to different influenza NAs and provide the structural basis of their specific action based upon their novel complex crystal structures. Our results indicate that laninamivir and zanamivir are more effective against group 1 NA with a 150-cavity than group 2 NA with no 150-cavity. Furthermore, we have found that the laninamivir octanoate prodrug has a unique binding mode in p09N1 that is different from that of group 2 p57N2, but with some similarities to NA-oseltamivir binding, which provides additional insight into group specific differences of oseltamivir binding and resistance.

Molecular Docking, 3D‐QSAR Studies, and In Silico ADME Prediction of p‐Aminosalicylic Acid Derivatives as Neuraminidase Inhibitors

Neuraminidase (NA) is a major glycoprotein of influenza virus which is essential for viral infection. It offers a potential target for antiviral drug design and discovery. To develop novel potent neuraminidase inhibitors (NAI), Surflex-Dock was employed to dock 40 hydrophobic p-aminosalicylic acid derivatives into the active site of NA. The 3D-quantitative structure-activity relationship studies involving comparative molecular field analysis (CoMFA) and comparative molecular similarity indices analysis (CoMSIA) were carried out on 40 molecules. Both CoMFA (q(2) = 0.628, r(2) = 0.697) and CoMSIA (q(2) = 0.746, r(2) = 0.816) gave reasonable results. A preliminary pharmacokinetic profile of these NAI was also performed on the basis of Volsurf predictions. The results obtained from this study will be useful in the design of novel potent NAI.

Replication inhibition activity of carbocycles related to oseltamivir on influenza A virus in vitro

We have recently demonstrated that newly synthesized oseltamivir derivatives that contain a substituted triazole ring at the C-5 amino group interact with the 150 cavity found specifically in the group-1 neuraminidase (NA) subtypes of influenza A virus. These compounds exhibited in vitro inhibition activity of a group-1 NA enzyme incorporated in virus-like particles (VLPs). In the current study, we tested these nine triazole-containing carbocycles as well as an amino- and a guanidino-substituted derivative in virus replication inhibitory assays in vitro. None of the triazole-containing carbocycles significantly inhibited influenza A virus replication in MDCK cells with either a virus strain containing a group-1 or a group-2 subtype NA. In contrast, the amino- and guanidino-substituted derivatives clearly inhibited the cytopathic effect or spread of virus infection detected by immunostaining in MDCK monolayers as well as progeny virus release; these compounds were also reported to have shown the highest inhibition of group-1 NA in the context of VLPs. These results, together with the structures of these compounds, suggest that hydrogen-bonding interactions between the polar amino or guanidino functions and complementary groups in the neuraminidase active site ( e.g. Asp151, Glu 119) may be essential for strong inhibition of the neuraminidase enzyme and, in turn, the inhibition of influenza A virus replication.

Identification of critical residues of influenza neuraminidase in viral particle release

BackgroundInfluenza neuraminidase (NA) is essential for virus release from its host cells and it is one of the targets for structure-based antiviral drug design.ResultsIn this report, we established a pseudoviral particle release assay to study NA function, which is based on lentiviral particles pseudotyped with influenza glycoproteins HA and NA as a surrogate system. Through an extensive molecular analysis, we sought to characterize important residues governing NA function. We identified five residues of NA, 234, 241, 257, 286 and 345, four of which (except 345) map away from the active site of NA when projected onto the three-dimensional structure of avian influenza H5N1 NA, and substitutions of these residues adversely affected the NA-mediated viral particle release, suggesting that these residues are critical for NA enzymatic activity.ConclusionThrough extensive chimeric and mutational analyses, we have identified several residues, which map away from the active site and are critical for NA function. These findings provide new insights into NA-mediated pseudoviral particle release and may have important implications in drug design and therapeutics against influenza infection.

Computer-Based De Novo Designs of Tripeptides as Novel Neuraminidase Inhibitors

The latest influenza A (H1N1) pandemic attracted worldwide attention and called for the urgent development of novel antiviral drugs. Here, seven tripeptides are designed and explored as neuraminidase (NA) inhibitors on the structural basis of known inhibitors. Their interactions with NA are studied and compared with each other, using flexible docking and molecular dynamics simulations. The various composed tripeptides have respective binding specificities and their interaction energies with NA decrease in the order of FRI > FRV > FRT > FHV > FRS > FRG > YRV (letters corresponding to amino acid code). The Arg and Phe portions of the tripeptides play important roles during the binding process: Arg has strong electrostatic interactions with the key residues Asp151, Glu119, Glu227 and Glu277, whereas Phe fits well in the hydrophobic cave within the NA active site. Owing to the introduction of hydrophobic property, the interaction energies of FRV and FRI are larger; in particular, FRI demonstrates the best binding quality and shows potential as a lead compound. In addition, the influence of the chemical states of the terminal amino acids are clarified: it is revealed that the charged states of the N-terminus (NH3+) and C-terminus (COO−) are crucial for the tripeptide inhibitory activities and longer peptides may not be appropriate. In addition, the medium inhibiting activity by acetylation of the N-terminus indicates the possible chemical modifications of FRI. Experimental efforts are expected in order to actualize the tripeptides as potent NA inhibitors in the near future.

Using Selectively Applied Accelerated Molecular Dynamics to Enhance Free Energy Calculations.

Accelerated molecular dynamics (aMD) has been shown to enhance conformational space sampling relative to classical molecular dynamics; however, the exponential reweighting of aMD trajectories, which is necessary for the calculation of free energies relating to the classical system, is oftentimes problematic, especially for systems larger than small poly peptides. Here, we propose a method of accelerating only the degrees of freedom most pertinent to sampling, thereby reducing the total acceleration added to the system and improving the convergence of calculated ensemble averages, which we term selective aMD. Its application is highlighted in two biomolecular cases. First, the model system alanine dipeptide is simulated with classical MD, all-dihedral aMD, and selective aMD, and these results are compared to the infinite sampling limit as calculated with metadynamics. We show that both forms of aMD enhance the convergence of the underlying free energy landscape by 5-fold relative to classical MD; however, selective aMD can produce improved statistics over all-dihedral aMD due to the improved reweighting. Then we focus on the pharmaceutically relevant case of computing the free energy of the decoupling of oseltamivir in the active site of neuraminidase. Results show that selective aMD greatly reduces the cost of this alchemical free energy transformation, whereas all-dihedral aMD produces unreliable free energy estimates.

Synergistic effects in the designs of neuraminidase ligands: Analysis from docking and molecular dynamics studies

Docking and molecular dynamics were used to study the nine ligands (see Scheme 1) at the neuraminidase (NA) active sites. Their binding modes are structurally and energetically different, with details given in the text. Compared with 1A (oseltamivir carboxylate), the changes of core template or/and functional groups in the other ligands cause the reductions of interaction energies and numbers of H-bonds with the NA proteins. Nonetheless, all these ligands occupy the proximity space at the NA active sites and share some commonness in their binding modes. The fragment approach was then used to analyze and understand the binding specificities of the nine ligands. 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 nine ligands, 1A (oseltamivir carboxylate) has the largest synergistic energy and its functional groups fit perfectly with the NA active site, consistent with the largest interaction energy, numerous H-bonds with the NA active-site residues as well as experimentally lowest IC 50 value. Owing to the poorer metabolizability than oseltamivir, large contribution of the benzene core template and fine synergistic effects of the functional groups, the 4-(N-acetylamino)-5-guanidino-3-(3-pentyloxy)benzoic acid should be an ideal lead compound for optimizing NA drugs.

Effect of neuraminidase inhibitor-resistant mutations on pathogenicity of clade 2.2 A/Turkey/15/06 (H5N1) influenza virus in ferrets.

The acquisition of neuraminidase (NA) inhibitor resistance by H5N1 influenza viruses has serious clinical implications, as this class of drugs can be an essential component of pandemic control measures. The continuous evolution of the highly pathogenic H5N1 influenza viruses results in the emergence of natural NA gene variations whose impact on viral fitness and NA inhibitor susceptibility are poorly defined. We generated seven genetically stable recombinant clade 2.2 A/Turkey/15/06-like (H5N1) influenza viruses carrying NA mutations located either in the framework residues (E119A, H274Y, N294S) or in close proximity to the NA enzyme active site (V116A, I117V, K150N, Y252H). NA enzyme inhibition assays showed that NA mutations at positions 116, 117, 274, and 294 reduced susceptibility to oseltamivir carboxylate (IC50s increased 5- to 940-fold). Importantly, the E119A NA mutation (previously reported to confer resistance in the N2 NA subtype) was stable in the clade 2.2 H5N1 virus background and induced cross-resistance to oseltamivir carboxylate and zanamivir. We demonstrated that Y252H NA mutation contributed for decreased susceptibility of clade 2.2 H5N1 viruses to oseltamivir carboxylate as compared to clade 1 viruses. The enzyme kinetic parameters (Vmax, Km and Ki) of the avian-like N1 NA glycoproteins were highly consistent with their IC50 values. None of the recombinant H5N1 viruses had attenuated virulence in ferrets inoculated with 106 EID50 dose. Most infected ferrets showed mild clinical disease signs that differed in duration. However, H5N1 viruses carrying the E119A or the N294S NA mutation were lethal to 1 of 3 inoculated animals and were associated with significantly higher virus titers (P<0.01) and inflammation in the lungs compared to the wild-type virus. Our results suggest that highly pathogenic H5N1 variants carrying mutations within the NA active site that decrease susceptibility to NA inhibitors may possess increased virulence in mammalian hosts compared to drug-sensitive viruses. There is a need for novel anti-influenza drugs that target different virus/host factors and can limit the emergence of resistance.

Potential drug-like inhibitors of Group 1 influenza neuraminidase identified through computer-aided drug design

Pandemic (H1N1) influenza poses an imminent threat. Nations have stockpiled inhibitors of the influenza protein neuraminidase in hopes of protecting their citizens, but drug-resistant strains have already emerged, and novel therapeutics are urgently needed. In the current work, the computer program AutoGrow is used to generate novel predicted neuraminidase inhibitors. Given the great flexibility of the neuraminidase active site, protein dynamics are also incorporated into the computer-aided drug-design process. Several potential inhibitors are identified that are predicted to bind to neuraminidase better than currently approved drugs.

Modeling gene sequences over time in 2009 H1N1 Influenza A Virus populations

BackgroundA sudden emergence of Influenza A Virus (IAV) infections with a new pandemic H1N1 IAV is taking place since April of 2009. In order to gain insight into the mode of evolution of these new H1N1 strains, we performed a Bayesian coalescent Markov chain Monte Carlo (MCMC) analysis of full-length neuraminidase (NA) gene sequences of 62 H1N1 IAV strains (isolated from March 30th to by July 28th, 2009).ResultsThe results of these studies revealed that the expansion population growth model was the best to fit the sequence data. A mean of evolutionary change of 7.84 × 10-3 nucleotide substitutions per site per year (s/s/y) was obtained for the NA gene. A significant contribution of first codon position to this mean rate was observed. Maximum clade credibility trees revealed a rapid diversification of NA genes in different genetic lineages, all of them containing Oseltamivir-resistant viruses of very recent emergence. Mapping of naturally occurring amino acid substitutions in the NA protein from 2009 H1N1 IAV circulating in 62 different patients revealed that substitutions are distributed all around the surface of the molecule, leaving the hydrophobic core and the catalytic site essentially untouched.ConclusionHigh evolutionary rates and fast population growth have contributed to the initial transmission dynamics of 2009 H1N1 IAV. Naturally occurring substitutions are preferentially located at the protein surface and do not interfere with the NA active site. Antigenic regions relevant for vaccine development can differ from previous vaccine strains and vary among patients.

Characterization of the Influenza A H5N1 Viruses of the 2008-09 Outbreaks in India Reveals a Third Introduction and Possible Endemicity

Widespread infection of highly pathogenic avian influenza A H5N1 was reported from backyard and commercial poultry in West Bengal (WB), an eastern state of India in early 2008. Infection gradually spread to Tripura, Assam and Sikkim, the northeastern states, with 70 outbreaks reported between January 2008 and May 2009. Whole genome sequence analysis of three isolates from WB, one isolate from Tripura along with the analysis of hemagglutinin (HA) and neuraminidase (NA) genes of 17 other isolates was performed during this study. In the HA gene phylogenetic tree, all the 2008-09 Indian isolates belonged to EMA3 sublineage of clade 2.2. The closest phylogenetic relationship was found to be with the 2007-09 isolates from Bangladesh and not with the earlier 2006 and 2007 Indian isolates implying a third introduction into the country. The receptor-binding pocket of HA1 of two isolates from WB showed S221P mutation, one of the markers predicted to be associated with human receptor specificity. Two substitutions E119A (2 isolates of WB) and N294S (2 other isolates of WB) known to confer resistance to NA inhibitors were observed in the active site of neuraminidase. Several additional mutations were observed within the 2008-09 Indian isolates indicating genetic diversification. Overall, the study is indicative of a possible endemicity in the eastern and northeastern parts of the country, demanding active surveillance specifically in view of the critical mutations that have been observed in the influenza A H5N1 viruses.

P.066 Consequences of combinations of mutations in the neuraminidase active site on the cell surface expression, the activity and the sensibility of the neuraminidase to the neuraminidase inhibitors

P.066 Consequences of combinations of mutations in the neuraminidase active site on the cell surface expression, the activity and the sensibility of the neuraminidase to the neuraminidase inhibitors

Alanine-Scanning Mutagenesis for Revealing the Role of Highly Conserved Regions of Influenza A Virus Neuraminidase

Background: Neuraminidase (NA), a surface glycoprotein of influenza A virus, is an important molecular target for antiviral drugs. Recent reported NA-inhibition drug resistant cases, such as mutations in H274Y in N1 and R292K and E119G/A/D in N9 and N2, however, has raised concerns about the needs for better understanding NA functional role towards developing a new anti-influenza drug. Methods: Twenty-eight highly conserved amino acid residues in influenza A viral NA protein were identified through in silico analysis based on 2,827 NA sequences deposited in GenBank, NCBI. To understand the role of conserved residues on viral viability, we have introduced series of mutations (alanine substitutions) into NA by reverse genetics using A/WSN/33(H1N1) as a backbone. Results: Seven out of 28 mutants were rescued, indicating that the other 21 positions in NA are essential to viral viability. Among those 21 lethal mutants, 5 were rescued by exogenously adding NA from C. perfringens, suggesting that these 5 positions may reside on the NA active site. This assumption was reinforced by structural modeling by SWISS-MODEL server. By simulation, we also found 9/21 mutants located on the side-surface of NA protein. When being substituted, they remarkably reduced the virus survival by losing its biological function, possibly due to the associated structural alteration. Conclusion: This study identified several amino acid residues important for viral viability. Via structural simulation they are found located at NA active sites or oligomerization sites. We believe the results obtained herein provide valuable information in antiviral drug design targeting influenza NA protein. Mok, C.K., et al. 2008 Chang Gung University 2 Table of

Rapid molecular detection of the H275Y oseltamivir resistance gene mutation in circulating influenza A (H1N1) viruses

In early 2008, drug susceptibility surveillance of influenza viruses in Europe revealed that some influenza A viruses (subtype H1N1) circulating during the winter season of 2007 and 2008 were resistant to the neuraminidase inhibitor, oseltamivir. This resistance arises due to a histidine to tyrosine substitution in the neuraminidase active site (H275Y in N1 nomenclature). Current methods to detect this mutation involve an end-point reverse transcription polymerase chain reaction followed by nucleotide sequencing. While accurate, this approach has the limitation of being time-consuming, labour-intensive and expensive. Herein we describe a one-step allelic discrimination assay which rapidly (2 h) detects this resistance mutation. The sensitivity of the assay was as low as 10 copies per reaction and is capable of detecting the antiviral resistance mutation in a mixture of wild type H275 and mutant H275Y targets.

Impact of influenza A virus neuraminidase mutations on the stability, activity, and sensibility of the neuraminidase to neuraminidase inhibitors

Background The influenza neuraminidase plays a critical role in the spread of the influenza A and B viruses. Through the cleavage of terminal sialic acid from glycoconjugates, it facilitates the elution of progeny virions from infected cells and prevents their self-aggregation. Objectives Our objective was to study the impact of mutations at framework sites not under direct selective pressure in the neuraminidase active site. Study design In the A/Moscow/10/99 (H3N2) virus background, viruses containing mutations in NA framework residues (E119D, R156K, W178L, S179A, D198N, I222L, E227G, H274Y, E277G, N294D, and E425G) were constructed by reverse genetics. After several passages on MDCK cells, fluorimetric assays were conducted to assess the neuraminidase activity and sensibility to the neuraminidase inhibitors (IC50). Results Among the viruses detectable through the phenotypic tests, R156K, I222L, H274Y, N294D and E425G viruses presented a NA activity between 70% and 100% of the A/Moscow/10/99 wild type one. The D198N and the E119D mutations decreased seriously in NA activity compared to the wild-type (>10-fold). The I222L mutation reduced susceptibility to oseltamivir (18-fold). Conclusion With the exception of one mutation, framework mutations on N2 background do not induce resistance. Nevertheless they tend to decrease slowly the sensitivity to one or the other inhibitors.