Computational study on new natural polycyclic compounds of H1N1 influenza virus neuraminidase

A new strain of influenza A (H1N1) virus is a major cause of morbidity and mortality around the world. The neuraminidase of the influenza virus has been the most potential target for the anti-influenza drugs such as oseltamivir and zanamivir. However, the emergence of drug-resistant variants of these drugs makes a pressing need for the development of new neuraminidase inhibitors for controlling illness and transmission. Here a 3D structure model of H1N1 avian influenza virus neuraminidase type 1 (N1) was constructed based on the structure of the template H5N1 avian influenza virus N1. Upon application of virtual screening technique for N1 inhibitors, two novel compounds (ZINC database ID: ZINC02128091, ZINC02098378) were found as the most favorable interaction energy with N1. Docking results showed that the compounds bound not only in the active pocket, but also in a new hydrophobic cave which contains Arg368, Trp399, Ile427, Pro431 and Lys432 of N1. Our result suggested that both of the screened compounds containing the hydrophobic group bring a strong conjugation effect with Arg293, Arg368 Lys432 of N1 by pi-pi interaction. However, the control inhibitors zanamivir and oseltamivir do not have this effect. The details of N1-compound binding structure obtained will be valuable for the development of a new anti-influenza virus agent.     

In silico prediction of drug resistance due to S247R mutation of Influenza H1N1 neuraminidase protein

We present here in silico studies on antiviral drug resistance due to a novel mutation of influenza A/H1N1 neuraminidase (NA) protein. Influenza A/H1N1 virus was responsible for a recent pandemic and is currently circulating among the seasonal influenza strains. M2 and NA are the two major viral proteins related to pathogenesis in humans and have been targeted for drug designing. Among them, NA is preferred because the ligand-binding site of NA is highly conserved between different strains of influenza virus. Different mutations of the NA active site residues leading to drug resistance or susceptibility of the virus were studied earlier. We report here a novel mutation (S247R) in the NA protein that was sequenced earlier from the nasopharyngeal swab from Sri Lanka and Thailand in the year 2009 and 2011, respectively. Another mutation (S247N) was already known to confer resistance to oseltamivir. We did a comparative study of these two mutations vis-a-vis the drug-sensitive wild type NA to understand the mechanism of drug resistance of S247N and to predict the probability of the novel S247R mutation to become resistant to the currently available drugs, oseltamivir and zanamivir. We performed molecular docking- and molecular dynamics-based analysis of both the mutant proteins and showed that mutation of S247R affects drug binding to the protein by positional displacement due to altered active site cavity architecture, which in turn reduces the affinity of the drug molecules to the NA active site. Our analysis shows that S247R may have high probability of being resistant.     

Insights from investigating the interaction of oseltamivir (Tamiflu) with neuraminidase of the 2009 H1N1 swine flu virus

The neuraminidase (NA) of influenza virus is the target of anti-flu drugs oseltamivir and zanamivir. Clinical practices showed that oseltamivir was effective to treat the 2009-H1N1 influenza but failed to the 2006-H5N1 avian influenza. To perform an in-depth analysis on such a drug-resistance problem, the 2009-H1N1-NA structure was developed. To compare it with the crystal 2006-H5N1-NA structure as well as the 1918 influenza virus H1N1-NA structure, the multiple sequential and structural alignments were performed. It has been revealed that the hydrophobic residue Try347 in H5N1-NA does not match with the hydrophilic carboxyl group of oseltamivir as in the case of H1N1-NA. This may be the reason why H5N1 avian influenza virus is drug-resistant to oseltamivir. The finding provides useful insights for how to modify the existing drugs, such as oseltamivir and zanamivir, making them not only become more effective against H1N1 virus but also effective against H5N1 virus.     

Crystal structure of the drug‐resistant S31N influenza M2 proton channel

The M2 protein is a small proton channel found in the influenza A virus that is necessary for viral replication. The M2 channel is the target of a class of drugs called the adamantanes, which block the channel pore and prevent the virus from replicating. In recent decades mutations have arisen in M2 that prevent the adamantanes from binding to the channel pore, with the most prevalent of these mutations being S31N. Here we report the first crystal structure of the S31N mutant crystallized using lipidic cubic phase crystallization techniques and solved to 1.59 Å resolution. The Asn31 residues point directly into the center of the channel pore and form a hydrogen‐bonded network that disrupts the drug‐binding site. Ordered waters in the channel pore form a continuous hydrogen bonding network from Gly34 to His37.     

In Silico Design of an Oseltamivir Derivative with Increased Affinity against Wild-Type and Mutant Variants of Neuraminidase and Hemagglutinin of Influenza A H1N1 Virus

Antiviral resistance has turned into a world concern nowadays. Influenza A H1N1 emerged as a problem at the world level due to the neuraminidase (NA) mutations. The NA mutants conferred resistance to oseltamivir and zanamivir. Several efforts were conducted to develop better anti-influenza A H1N1 drugs. Our research group combined in silico methods to create a compound derived from oseltamivir to be tested in vitro against influenza A H1N1. Here we show the results of a new compound derived from oseltamivir but with specific chemical modifications, with significant affinity either on NA (in silico and in vitro assays) or HA (in silico) from influenza A H1N1 strain. We include docking and molecular dynamics (MD) simulations of the oseltamivir derivative at the binding site onto NA and HA of influenza A H1N1. Additionally, the biological experimental results show that oseltamivir derivative decreases the lytic-plaque formation on viral susceptibility assays, and it does not show cytotoxicity. Finally, oseltamivir derivative assayed on viral NA showed a concentration-dependent inhibition behavior at nM, depicting a high affinity of the compound for the enzyme, corroborated with the MD simulations results, placing our designed oseltamivir derivative as a potential antiviral against influenza A H1N1.     

Analogue inhibitors by modifying oseltamivir based on the crystal neuraminidase structure for treating drug-resistant H5N1 virus

The worldwide spread of H5N1 avian influenza and the increasing reports about its resistance to the existing drugs have made a priority for the development of the new anti-influenza molecules. The crystal structure of H5N1 avian influenza neuraminidase reported recently by Russell et al. [R.J. Russell, L.F. Haire, D.J. Stevens, P.J. Collins, Y. P. Lin, G.M. Blackburn, A.J. Hay, S.J. Gamblin, J.J. Skehel, The structure of H5N1 avian influenza neuraminidase suggests new opportunities for drug design, Nature 443 (2006) 45-49] have provided new opportunities for drug design in this regard. It is revealed through the structure that the active sites of the group-1 neuraminidases, which contain the N1 subtype, have a very different three-dimensional structure from those of group-2 neuraminidases. The key difference is in the 150-loop cavity adjacent to the conserved active site in neuraminidase. Based on these findings and by modifying oseltamivir, six analog inhibitors were proposed as candidates for developing inhibitors against H5N1 virus, particularly against the oseltamivir-resistant H5N1 virus strain.     

H1N1 2009 Pandemic Influenza Virus: Resistance of the I223R Neuraminidase Mutant Explained by Kinetic and Structural Analysis

Two classes of antiviral drugs, neuraminidase inhibitors and adamantanes, are approved for prophylaxis and therapy against influenza virus infections. A major concern is that antiviral resistant viruses emerge and spread in the human population. The 2009 pandemic H1N1 virus is already resistant to adamantanes. Recently, a novel neuraminidase inhibitor resistance mutation I223R was identified in the neuraminidase of this subtype. To understand the resistance mechanism of this mutation, the enzymatic properties of the I223R mutant, together with the most frequently observed resistance mutation, H275Y, and the double mutant I223R/H275Y were compared. Relative to wild type, K_M values for MUNANA increased only 2-fold for the single I223R mutant and up to 8-fold for the double mutant. Oseltamivir inhibition constants (K_I) increased 48-fold in the single I223R mutant and 7500-fold in the double mutant. In both cases the change was largely accounted for by an increased dissociation rate constant for oseltamivir, but the inhibition constants for zanamivir were less increased. We have used X-ray crystallography to better understand the effect of mutation I223R on drug binding. We find that there is shrinkage of a hydrophobic pocket in the active site as a result of the I223R change. Furthermore, R223 interacts with S247 which changes the rotamer it adopts and, consequently, binding of the pentoxyl substituent of oseltamivir is not as favorable as in the wild type. However, the polar glycerol substituent present in zanamivir, which mimics the natural substrate, is accommodated in the I223R mutant structure in a similar way to wild type, thus explaining the kinetic data. Our structural data also show that, in contrast to a recently reported structure, the active site of 2009 pandemic neuraminidase can adopt an open conformation.     

奥司他韦、利巴韦林和盐酸金刚乙胺对甲型H1N1流感病毒的抑制作用

目的细胞水平测试奥司他韦、利巴韦林和盐酸金刚乙胺对甲型流感H1N1病毒的抑制或杀伤作用。方法通过在MDCK细胞系和甲型H1N1病毒株间建立药物剂量-效应关系确定导致细胞死亡的效力与抑制病毒复制的效力的比值（治疗指数）,测试药物的抗病毒效果。结果奥司他韦、利巴韦林和盐酸金刚乙胺对MDCK细胞的半数中毒浓度分别为（1134.7±186.8）μg/mL、（742.0±76.9）μg/mL、（94.6±1.9）μg/mL,对甲型H1N1病毒的治疗指数（TI）分别为71.19、24.9和3.12。结论奥司他韦对甲型H1N1病毒抑制作用最强,利巴韦林其次,盐酸金刚乙胺对甲型H1N1病毒抑制效果较弱。     

Screening from the world's largest TCM database against H1N1 virus

The swine influenza virus (H1N1) 2009 pandemic highlights the importance of having effective anti-viral strategies. Recently, oseltamivir (Tamiflu) resistant influenza viruses are identified; which further emphasizes the urgency in developing new antiviral agents. In influenza virus replication cycle, viral surface glycoprotein, hemagglutinin, is responsible for viral entry into host cells. Hence, a potentially effective antiviral strategy is to inhibit viral entry mechanism. To develop novel antiviral agent that inhibits viral entry, we analyzed 20,000 traditional Chinese medicine (TCM) ingredients in hemagglutinin subtype H1 sialic acid binding site found on H1N1 virus. We then performed molecular dynamics simulations to investigate receptor-ligand interaction of the candidates obtained from docking. Here, we report three TCM derivatives that have high binding affinities to H1 sialic acid binding site residues based on structure-based calculations. The top three derivatives, xylopine_2, rosmaricine_14 and rosmaricine_15, all have an amine group that interact with Glu83 and a pyridinium group that interact with Asp103. Molecular dynamics simulations show that these derivatives form strong hydrogen bonding with Glu83 but interact transiently with Asp103. We therefore suggest that an enhanced hemagglutinin inhibitor, based on our scaffold, should be designed to bind both Glu83 and Asp103 with high affinity.     

Insights from investigating the interactions of adamantane-based drugs with the M2 proton channel from the H1N1 swine virus

The M2 proton channel is one of indispensable components for the influenza A virus that plays a vital role in its life cycle and hence is an important target for drug design against the virus. In view of this, the three-dimensional structure of the H1N1-M2 channel was developed based on the primary sequence taken from a patient recently infected by the H1N1 (swine flu) virus. With an explicit water-membrane environment, molecular docking studies were performed for amantadine and rimantadine, the two commercial drugs generally used to treat influenza A infection. It was found that their binding affinity to the H1N1-M2 channel is significantly lower than that to the H5N1-M2 channel, fully consistent with the recent report that the H1N1 swine virus was resistant to the two drugs. The findings and the relevant analysis reported here might provide useful structural insights for developing effective drugs against the new swine flu virus.     

Multidrug resistant 2009 A/H1N1 influenza clinical isolate with a neuraminidase I223R mutation retains its virulence and transmissibility in ferrets

Only two classes of antiviral drugs, neuraminidase inhibitors and adamantanes, are approved for prophylaxis and therapy against influenza virus infections. A major concern is that influenza virus becomes resistant to these antiviral drugs and spreads in the human population. The 2009 pandemic A/H1N1 influenza virus is naturally resistant to adamantanes. Recently a novel neuraminidase I223R mutation was identified in an A/H1N1 virus showing cross-resistance to the neuraminidase inhibitors oseltamivir, zanamivir and peramivir. However, the ability of this virus to cause disease and spread in the human population is unknown. Therefore, this clinical isolate (NL/2631-R223) was compared with a well-characterized reference virus (NL/602). In vitro experiments showed that NL/2631-I223R replicated as well as NL/602 in MDCK cells. In a ferret pathogenesis model, body weight loss was similar in animals inoculated with NL/2631-R223 or NL/602. In addition, pulmonary lesions were similar at day 4 post inoculation. However, at day 7 post inoculation, NL/2631-R223 caused milder pulmonary lesions and degree of alveolitis than NL/602. This indicated that the mutant virus was less pathogenic. Both NL/2631-R223 and a recombinant virus with a single I223R change (recNL/602-I223R), transmitted among ferrets by aerosols, despite observed attenuation of recNL/602-I223R in vitro. In conclusion, the I223R mutated virus isolate has comparable replicative ability and transmissibility, but lower pathogenicity than the reference virus based on these in vivo studies. This implies that the 2009 pandemic influenza A/H1N1 virus subtype with an isoleucine to arginine change at position 223 in the neuraminidase has the potential to spread in the human population. It is important to be vigilant for this mutation in influenza surveillance and to continue efforts to increase the arsenal of antiviral drugs to combat influenza.     

Triple Combination of Amantadine,Ribavirin, and Oseltamivir Is Highly Active and Synergistic against Drug Resistant Influenza Virus Strains In Vitro

The rapid emergence and subsequent spread of the novel 2009 Influenza A/H1N1 virus (2009 H1N1) has prompted the World Health Organization to declare the first pandemic of the 21^st century, highlighting the threat of influenza to public health and healthcare systems. Widespread resistance to both classes of influenza antivirals (adamantanes and neuraminidase inhibitors) occurs in both pandemic and seasonal viruses, rendering these drugs to be of marginal utility in the treatment modality. Worldwide, virtually all 2009 H1N1 and seasonal H3N2 strains are resistant to the adamantanes (rimantadine and amantadine), and the majority of seasonal H1N1 strains are resistant to oseltamivir, the most widely prescribed neuraminidase inhibitor (NAI). To address the need for more effective therapy, we evaluated the in vitro activity of a triple combination antiviral drug (TCAD) regimen composed of drugs with different mechanisms of action against drug-resistant seasonal and 2009 H1N1 influenza viruses. Amantadine, ribavirin, and oseltamivir, alone and in combination, were tested against amantadine- and oseltamivir-resistant influenza A viruses using an in vitro infection model in MDCK cells. Our data show that the triple combination was highly synergistic against drug-resistant viruses, and the synergy of the triple combination was significantly greater than the synergy of any double combination tested (P<0.05), including the combination of two NAIs. Surprisingly, amantadine and oseltamivir contributed to the antiviral activity of the TCAD regimen against amantadine- and oseltamivir-resistant viruses, respectively, at concentrations where they had no activity as single agents, and at concentrations that were clinically achievable. Our data demonstrate that the TCAD regimen composed of amantadine, ribavirin, and oseltamivir is highly synergistic against resistant viruses, including 2009 H1N1. The TCAD regimen overcomes baseline drug resistance to both classes of approved influenza antivirals, and thus may represent a highly active antiviral therapy for seasonal and pandemic influenza.     

Molecular Dynamics Simulations Suggest that Electrostatic Funnel Directs Binding of Tamiflu to Influenza N1 Neuraminidases

Oseltamivir (Tamiflu) is currently the frontline antiviral drug employed to fight the flu virus in infected individuals by inhibiting neuraminidase, a flu protein responsible for the release of newly synthesized virions. However, oseltamivir resistance has become a critical problem due to rapid mutation of the flu virus. Unfortunately, how mutations actually confer drug resistance is not well understood. In this study, we employ molecular dynamics (MD) and steered molecular dynamics (SMD) simulations, as well as graphics processing unit (GPU)-accelerated electrostatic mapping, to uncover the mechanism behind point mutation induced oseltamivir-resistance in both H5N1 “avian” and H1N1pdm “swine” flu N1-subtype neuraminidases. The simulations reveal an electrostatic binding funnel that plays a key role in directing oseltamivir into and out of its binding site on N1 neuraminidase. The binding pathway for oseltamivir suggests how mutations disrupt drug binding and how new drugs may circumvent the resistance mechanisms.     

Oseltamivir boosts 2009 H1N1 virus infectivity in vitro

The neuraminidase inhibitor oseltamivir has been identified to have significant anti-influenza activity in clinical practice. However, its efficacy has not been verified in enough subtypes of influenza A virus, particularly, the current pandemic virus, H1N1. In vitro, using our influenza pseudotyped particle system, oseltamivir displayed significant inhibitory effects on viral NA activity and pp release. Conversely, a boosting effect on viral infection was observed, particularly with the 2009 H1N1 pp at oseltamivir concentrations above 0.025 μM. Further testing on two wild 2009 H1N1 virus strains, A/California/07/09 and A/Sichuan/1/09, as well as a seasonal flu virus, A/Baoan/51/2008, confirmed these findings.     

禽流感病毒H5N1全基因组克隆与分析

为明确广东地区分离的一株禽流感病毒H5N1的遗传背景,建立流感病毒反向遗传的平台。对该株禽流感病毒进行了空斑纯化与组织细胞培养,检测其在MDCK细胞中的增殖特性;利用H5N1病毒通用引物,通过RT-PCR对该病毒全基因组的8条片段进行全长克隆及测序分析;将H5N1的8条全长基因组片段分别插入反向遗传通用载体中,构建禽流感病毒H5N1的感染性克隆。结果表明,该H5N1毒株在MDCK细胞中可不依赖胰酶进行有效增殖与复制,可使MDCK细胞出现典型细胞病变,具有高致病性禽流感病毒的细胞增殖特征。RT-PCR克隆得到该H5N1毒株的PB2、PB1、PA、HA、NP、NA、M和NS八条全长片段,经测序分析确认该毒株的基因序列,其内部编码序列出现多处突变,其中HA连接肽为多个连续碱性氨基酸,表明该毒株可不依赖胰酶进行有效复制,与细胞培养结果一致,未出现抗药性的遗传突变。PCR与测序证明,插入H5N1八个全长基因组片段的载体序列完全正确,表明成功构建了该毒株的感染性克隆。为明确病毒遗传信息,建立流感病毒反向遗传的平台,为进一步研究禽流感病毒相关疫苗提供了研究基础。     

Pseudovirus-based neuraminidase inhibition assays reveal potential H5N1 drug-resistant mutations

The use of antiviral drugs such as influenza neuraminidase (NA) inhibitors is a critical strategy to prevent and control flu pandemic, but this strategy faces the challenge of emerging drug-resistant strains. F or a highly pathogenic avian influenza (HPAI) H5N1 virus, biosafety restrictions have significantly limited the efforts to monitor its drug responses and mechanisms involved. In this study, a rapid and biosafe assay based on NA pseudovirus was developed to study the resistance of HPAI H5N1 virus to NA inhibitor drugs. The H5N1 NA pseudovirus was comprehensively tested using oseltamivir-sensitive strains and their resistant mutants. Results were consistent with those in previous studies, in which live H5N1 viruses were used. Several oseltamivir-resistant mutations reported in human H1N1 were also identifi ed to cause decreased oseltamivir sensitivity in H5N1 NA by using the H5N1 NA pseudovirus. Thus, H5N1 NA pseudoviruses could be used to monitor HPAI H5N1 drug resistance rapidly and safely.     

Insights from modeling the 3D structure of H5N1 influenza virus neuraminidase and its binding interactions with ligands

The highly pathogenic H5N1 influenza virus, which is rapidly mutating and becoming increasingly drug-resistant, was investigated by means of structure-activity relationship between NA (neuraminidase) and three inhibitors, i.e., DANA (2,3-didehydro-2-deoxy-N-acetylneuraminic acid), zanamivir, and oseltamivir. A homology model of the H5N1-NA from the highly pathogenic chicken H5N1 A viruses isolated during the 2003-2004 influenza outbreaks in Japan was built based on the crystal structure of N9-NA complexed with DANA (PDB code: 1F8B). It was found that the traditional constituent residues around the active site of NA family are highly conserved in the H5N1-NA. However, a partially lipophilic pocket composed by Ala248 and Thr249 in N9-NA becomes a hydrophilic pocket because the two residues in the H5N1-NA are replaced by hydrophilic residues Ser227 and Asn228, respectively. On the other hand, two hydrophilic residues Asn347 and Asn348 in the N9-NA are replaced by two lipophilic residues Ala323 and Tyr324 in the H5N1-NA, respectively, leading to the formation of a new lipophilic pocket. This kind of subtle variation not only destroys the original lipophilic environment but also changes the complement interaction between the H5N1-NA and DANA. Such a finding might provide insights into the secret why some of H5N1 strains bear high resistance for existing NA inhibitors, and stimulate new strategies for designing new drugs against these viruses.     

Designing Inhibitors of M2 Proton Channel against H1N1 Swine Influenza Virus

Background

M2 proton channel of H1N1 influenza A virus is the target protein of anti-flu drugs amantadine and rimantadine. However, the two once powerful adamantane-based drugs lost their 90% bioactivity because of mutations of virus in recent twenty years. The NMR structure of the M2 channel protein determined by Schnell and Chou (Nature, 2008, 451, 591–595) may help people to solve the drug-resistant problem and develop more powerful new drugs against H1N1 influenza virus.

Methodology

Docking calculation is performed to build the complex structure between receptor M2 proton channel and ligands, including existing drugs amantadine and rimantadine, and two newly designed inhibitors. The computer-aided drug design methods are used to calculate the binding free energies, with the computational biology techniques to analyze the interactions between M2 proton channel and adamantine-based inhibitors.

Conclusions

1) The NMR structure of M2 proton channel provides a reliable structural basis for rational drug design against influenza virus. 2) The channel gating mechanism and the inhibiting mechanism of M2 proton channel, revealed by the NMR structure of M2 proton channel, provides the new ideas for channel inhibitor design. 3) The newly designed adamantane-based inhibitors based on the modeled structure of H1N1-M2 proton channel have two pharmacophore groups, which act like a “barrel hoop”, holding two adjacent helices of the H1N1-M2 tetramer through the two pharmacophore groups outside the channel. 4) The inhibitors with such binding mechanism may overcome the drug resistance problem of influenza A virus to the adamantane-based drugs.     

Emergence of Eurasian Avian-Like Swine Influenza A (H1N1) Virus from an Adult Case in Fujian Province,China

正Dear Editor,As we known,pigs play a vital role as genetic mixing vessels for human and avian influenza viruses as their tracheal epitheliums possess both sialic acid a-2,6-Gal and a-2,3-Gal receptors(Ma et al.2008),and swine influenza viruses occasionally infect humans(Shinde et al.2009).The Eurasian avian-like swine influenza A(H1N1)virus     

In silico analysis of drug-resistant mutant of neuraminidase (N294S) against oseltamivir

The recent H1N1 influenza pandemic has attracted worldwide attention due to the high infection rate. Oseltamivir is a new class of anti-viral agent approved for the treatment and prevention of influenza infections. The principal target for this drug is a virus surface glycoprotein, neuraminidase (NA), which facilitates the release of nascent virus and thus spreads infection. Until recently, only a low prevalence of neuraminidase inhibitor (NAI) resistance (<1 %) had been detected in circulating viruses. However, there have been reports of significant numbers of A (H1N1) influenza strains with a N294S neuraminidase mutation that was highly resistant to the NAI, oseltamivir. Hence, in the present study, we highlight the effect of point mutation-induced oseltamivir resistance in H1N1 subtype neuraminidases by molecular simulation approach. The docking analysis reveals that mutation (N294S) significantly affects the binding affinity of oseltamivir with mutant type NA. This is mainly due to the decrease in the flexibility of binding site residues and the difference in prevalence of hydrogen bonds in the wild and mutant structures. This study throws light on the possible effects of drug-resistant mutations on the large functionally important collective motions in biological systems.