Virtual screening pipeline and ligand modelling for H5N1 neuraminidase

The H5N1 virus neuraminidase structure was solved in two different conformations depending on the inhibitor concentration. In the absence of oseltamivir or at a low concentration, the neuraminidase structure assumes an open form that closes at a high oseltamivir concentration due to the shift of the so-called 150-loop near the active site. Although the close conformation is similar to all the other structurally known neuraminidase types, it doesn’t appear to be the most likely physiological condition for N1.To investigate the specific ligand binding properties of the open form, we screened by docking simulation, a large dataset of ligands and compared the results with closed form. The virtual screening procedure was implemented in a docking pipeline that also performs a step-by-step, target specific, filtering approach for data reduction. The selected ligands display binding ability involving multiple sites of interaction including the active site and an adjacent cavity made available by the 150-loop shift. Two ligands are especially interesting and are proposed as substituents to design oseltamivir derivatives specifically suited for the open conformation.     

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.     

Probing molecular level interaction of oseltamivir with H5N1-NA and model membranes by molecular docking, multinuclear NMR and DSC methods

Structure-based drug design has led to the introduction of three drugs — oseltamivir (GS-4104), zanamivir (GG-167) and peramivir (RWJ-270201) which target the enzyme neuraminidase, for treatment of influenza infections. Using comparative docking studies we propose that more potent molecules against neuraminidase can be obtained by appending extra positively charged substituents at the C5 position of the oseltamivir skeleton. This provides an additional interaction with the enzyme and may overcome the problem of resistance encountered with these drugs. To get an insight into the transport and absorption of oseltamivir — the ethyl ester prodrug (GS-4104) as well as its mechanism of action, we have carried out ¹H, ¹³C, ³¹P NMR, DSC and TEM studies on GS-4104 with model membranes prepared from DMPC/DPPC/POPC. These studies reveal that interactions between GS-4104 and the membrane are both electrostatic (involving H-bonding) and hydrophobic (involving the hydrophobic chain and cyclohexene ring of GS-4104) in nature. The prodrug is seen to increase the fluidity as well as stabilize the bilayer phase of the membrane. This property may be responsible for preventing viral entry into the cells by preventing fusion of the virus outer coat with the cell membrane.     

The derivatives of oseltamivir design passing through the important cleft of neuraminidase against influenza virus by de novo design

Owing to its unique function to release the progeny virus particles from the surface of an infected cell, neuraminidase has drawn special attention for developing new drugs to treat influenza viruses. The 150-cavity that is adjacent to the active pocket of the group-1 neuraminidase (N1) renders the conformational change from ‘open’ form to ‘closed’ form when enzyme is binding with a ligand. Consequently, it would be a better strategy to design multi-binding-site inhibitors including X and R groups with proper shapes, sizes and electronic charges fitting into the active site. The NCI and ZINC fragment databases were screened for finding the optimal fragments with de novo design technique. By doing so, 24 derivatives of oseltamivir were obtained by linking the fragments at two different sites of the scaffold of oseltamivir. Molecular docking and dynamics showed that these compounds not only adopt more favourable conformation but also have stronger binding interaction with receptor. Most importantly, all compounds skilfully pass through the cleft (formed by Glu119 and Arg156) and fit into 150-cavity. Therefore, the selected 24 derivatives may become promising candidates for treating influenza virus; in addition, the findings reported here may at least provide useful insights and stimulate new strategy in this area.     

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.     

A computational-experimental approach identifies mutations that enhance surface expression of an oseltamivir-resistant influenza neuraminidase

The His274→Tyr (H274Y) oseltamivir (Tamiflu) resistance mutation causes a substantial decrease in the total levels of surface-expressed neuraminidase protein and activity in early isolates of human seasonal H1N1 influenza, and in the swine-origin pandemic H1N1. In seasonal H1N1, H274Y only became widespread after the occurrence of secondary mutations that counteracted this decrease. H274Y is currently rare in pandemic H1N1, and it remains unclear whether secondary mutations exist that might similarly counteract the decreased neuraminidase surface expression associated with this resistance mutation in pandemic H1N1. Here we investigate the possibility of predicting such secondary mutations. We first test the ability of several computational approaches to retrospectively identify the secondary mutations that enhanced levels of surface-expressed neuraminidase protein and activity in seasonal H1N1 shortly before the emergence of oseltamivir resistance. We then use the most successful computational approach to predict a set of candidate secondary mutations to the pandemic H1N1 neuraminidase. We experimentally screen these mutations, and find that several of them do indeed partially counteract the decrease in neuraminidase surface expression caused by H274Y. Two of the secondary mutations together restore surface-expressed neuraminidase activity to wildtype levels, and also eliminate the very slight decrease in viral growth in tissue-culture caused by H274Y. Our work therefore demonstrates a combined computational-experimental approach for identifying mutations that enhance neuraminidase surface expression, and describes several specific mutations with the potential to be of relevance to the spread of oseltamivir resistance in pandemic H1N1.     

Three new powerful oseltamivir derivatives for inhibiting the neuraminidase of influenza virus

Owing to its unique function in assisting the release of newly formed virus particles from the surface of an infected cell, neuraminidase, an antigenic glycoprotein enzyme, is a main target for drug design against influenza viruses. The group-1 neuraminidase of influenza virus possesses a 150-cavity, which is adjacent to the active pocket, and which renders conformational change from the ‘open’ form to the ‘closed’ form when the enzyme is binding with a ligand. Using AutoGrow evolutionary algorithm, one very unique fragment is screened out from the fragment databases by exploiting additional interactions with the 150-cavity. Subsequently, three derivatives were constructed by linking the unique fragment to oseltamivir at its three different sites. The three derivatives thus formed show much stronger inhibition power than oseltamivir, and hence may become excellent candidates for developing new and more powerful drugs for treating influenza. Or at the very least, the findings may stimulate new strategy or provide useful insights for working on the target vitally important to the health of human beings.     

New stilbenoid with inhibitory activity on viral neuraminidases from Erythrina addisoniae

Influenza occurs with seasonal variations and reaches the peak prevalence in winter causing the death of many people worldwide. A few inhibitors of viral neuraminidase, including amantadine, rimantadine, zanamivir, and oseltamivir, have been used as influenza therapy. However, as drug-resistant influenza viruses are generated rapidly, there is a need to identify new agents for chemotherapy against influenza. Therefore, research on more effective drugs has been given high priority. During the course of an anti-influenza screening program on natural products, two new compounds (1 and 2) along with seven known flavonoid derivatives (3-9) were isolated as active principles from an EtOAc-soluble extract of the root bark of Erythrina addisoniae. The stilbenoid (2) and chalcone (3, 4, and 6) compounds of the isolates exhibited stronger activity than the isoflavone ones. Compound 2, which is a formylated stilbenoid derivative, exhibited strong inhibition of both influenza H1N1 and H9N2 neuraminidases with IC(50) values of 8.80±0.34 μg/mL and 7.19±0.40 μg/mL, respectively.     

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.     

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.     

Combating influenza: natural products as neuraminidase inhibitors

The ever increasing threat of influenza pandemic outbreaks represents a serious concern for public health. Various therapeutic and prophylactic means are available which helps to counter viral infections including vaccines and curative such as zanamivir and oseltamivir. However, with the inception of unfamiliar strains which show resistance to the available drugs manifests the rapid demand for discovery of rational drug as antiviral agents. Neuraminidase, a crucial enzyme for viral replication is the most promising target for new drugs because of its highly conserved residues. Nature provides with a myriad of natural bioactive compounds constituting a plethora of chemical entities that can be useful in drug discovery against influenza. This review is an update on neuraminidase enzyme highlighting its structure, function, catalytic mechanism and its inhibition by natural products. Various approved neuraminidase inhibitors and neuraminidase inhibition assays along with their susceptibility have been described. A discussion on published reports about 267 plant secondary metabolites tested in the past 7 years (2011–2017) for their neuraminidase inhibition activity is presented. Moreover, the recent techniques using QSAR to develop third generation neuraminidase inhibitors have been described. This work summarizes the recent development in experimental and theoretical research based on neuraminidase inhibitors that will help in the discovery of antiviral agents in coming future.

     

Environmental levels of the antiviral oseltamivir induce development of resistance mutation H274Y in influenza A/H1N1 virus in mallards

Oseltamivir (Tamiflu®) is the most widely used drug against influenza infections and is extensively stockpiled worldwide as part of pandemic preparedness plans. However, resistance is a growing problem and in 2008–2009, seasonal human influenza A/H1N1 virus strains in most parts of the world carried the mutation H274Y in the neuraminidase gene which causes resistance to the drug. The active metabolite of oseltamivir, oseltamivir carboxylate (OC), is poorly degraded in sewage treatment plants and surface water and has been detected in aquatic environments where the natural influenza reservoir, dabbling ducks, can be exposed to the substance. To assess if resistance can develop under these circumstances, we infected mallards with influenza A/H1N1 virus and exposed the birds to 80 ng/L, 1 µg/L and 80 µg/L of OC through their sole water source. By sequencing the neuraminidase gene from fecal samples, we found that H274Y occurred at 1 µg/L of OC and rapidly dominated the viral population at 80 µg/L. IC₅₀ for OC was increased from 2–4 nM in wild-type viruses to 400–700 nM in H274Y mutants as measured by a neuraminidase inhibition assay. This is consistent with the decrease in sensitivity to OC that has been noted among human clinical isolates carrying H274Y. Environmental OC levels have been measured to 58–293 ng/L during seasonal outbreaks and are expected to reach µg/L-levels during pandemics. Thus, resistance could be induced in influenza viruses circulating among wild ducks. As influenza viruses can cross species barriers, oseltamivir resistance could spread to human-adapted strains with pandemic potential disabling oseltamivir, a cornerstone in pandemic preparedness planning. We propose surveillance in wild birds as a measure to understand the resistance situation in nature and to monitor it over time. Strategies to lower environmental levels of OC include improved sewage treatment and, more importantly, a prudent use of antivirals.     

Inhibitory potency of flavonoid derivatives on influenza virus neuraminidase

The constant risk of emerging new influenza virus strains that are resistant to established inhibitors like oseltamivir leaves influenza neuraminidase (NA) a prominent target for drug design. The inhibitory activity of several flavonoid derivatives was experimentally tested in comparison to oseltamivir for the NA expressed by the seasonal influenza virus strains A/California/7/09 (A(H1N1)pdm09), A/Perth/16/09 (A(H3N2)), and B/Brisbane/60/08. IC₅₀ values of polyphenols confirmed moderate inhibition in the μM range. Structurally, the amount and site of glycosylation of tested flavonoids have no significant influence on their inhibitory potency. In a pharmacophore-based docking approach the structure–activity relationship was evaluated. Molecular dynamics simulations revealed highly flexible parts of the enzyme and the contribution of salt bridges to the structural stability of NA. The findings of this study elucidate the impact of flavonoids on viral neuraminidase activity and the analysis of their modes of action provide valuable information about the mechanism of NA inhibition.     

Highly potent and long-acting trimeric and tetrameric inhibitors of influenza virus neuraminidase

A set of trimeric and tetrameric derivatives 6-11 of the influenza virus neuraminidase inhibitor zanamivir 1 have been synthesized by coupling a common monomeric zanamivir derivative 3 onto various multimeric carboxylic acid core groups. These discrete multimeric compounds are all significantly more antiviral than zanamivir and also show outstanding long-lasting protective activity when tested in mouse influenza infectivity experiments.     

Anti-virals for influenza virus infection

Dramatic advances in the diagnosis and treatment of influenza in Japan has been made in recent years. Rapid diagnosis tests for influenza are routinely performed in Japanese hospitals. Both zanamivir and oseltamivir have been approved for the treatment of influenza since 2001, in addition to amantadine. Japan has the highest figure of neuraminidase inhibitor-use in the world because the treatment of influenza with neuraminidase inhibitors is covered by Japan's National Health Insurance program. Therefore, we should carefully observe the appearance of resistance strains and side effects to neuraminidase inhibitors.     

Synthesis and in vitro study of novel neuraminidase inhibitors against avian influenza virus

Evidences of oseltamivir resistant influenza patients raised the need of novel neuraminidase inhibitors. In this study, five oseltamivir analogs PMC-31-PMC-36, synthesised according to the outcomes of a rational design analysis aimed to investigate the effects of substitution at the 5-amino and 4-amido groups of oseltamivir on its antiviral activity, were screened for their inhibition against neuraminidase N1 and N3. The enzymes used as models were from the avian influenza A H7N1 and H7N3 viruses. The neuraminidase inhibition assay was carried out by using recombinant species obtained from a baculovirus expression system and the fluorogenic substrate MUNANA. The assay was validated by using oseltamivir carboxylate as a reference inhibitor. Among the tested compounds, PMC-36 showed the highest inhibition on N1 with an IC(50) of 14.6±3.0nM (oseltamivir 25±4nM), while PMC-35 showed a significant inhibitory effect on N3 with an IC(50) of 0.1±0.03nM (oseltamivir 0.2±0.02nM). The analysis of the inhibitory properties of this panel of compounds allowed a preliminary assessment of a structure-activity relationship for the modification of the 4-amido and 5-amino groups of oseltamivir carboxylate. The substitution of the acetamido group in the oseltamivir structure with a 2-butenylamido moiety reduced the observed activity, while the introduction of a propenylamido group was well tolerated. Substitution of the free 5-amino group of oseltamivir carboxylate with an azide, decreased the activity against both N1 and N3. When these structural changes were both introduced, a dramatic reduction of activity was observed for both N1 and N3. The alkylation of the free 5-amino group in oseltamivir carboxylate introducing an isopropyl group seemed to increase the inhibitory effect for both N1 and N3 neuraminidases, displaying a more pronounced effect against N1.     

Design, synthesis, and antiviral properties of 4'-substituted ribonucleosides as inhibitors of hepatitis C virus replication: the discovery of R1479

A series of 4'-substituted ribonucleoside derivatives has been prepared and evaluated for inhibition of hepatitis C virus (HCV) RNA replication in cell culture. The most potent and non-cytotoxic derivative was compound 28 (4'-azidocytidine, R1479) with an IC(50) of 1.28 microM in the HCV replicon system. The triphosphate of compound 28 was prepared and shown to be an inhibitor of RNA synthesis mediated by NS5B (IC(50)=320 nM), the RNA polymerase encoded by HCV. Data on related analogues have been used to generate some preliminary requirements for activity within this series of nucleosides.     

Characterization of two distinct neuraminidases from avian-origin human-infecting H7N9 influenza viruses

An epidemic of an avian-origin H7N9 influenza virus has recently emerged in China, infecting 134 patients of which 45 have died. This is the first time that an influenza virus harboring an N9 serotype neuraminidase (NA) has been known to infect humans. H7N9 viruses are divergent and at least two distinct NAs and hemagglutinins (HAs) have been found, respectively, from clinical isolates. The prototypes of these viruses are A/Anhui/1/2013 and A/Shanghai/1/2013. NAs from these two viruses are distinct as the A/Shanghai/1/2013 NA has an R294K substitution that can confer NA inhibitor oseltamivir resistance. Oseltamivir is by far the most commonly used anti-influenza drug due to its potency and high bioavailability. In this study, we show that an R294K substitution results in multidrug resistance with extreme oseltamivir resistance (over 100 000-fold) using protein- and virus-based assays. To determine the molecular basis for the inhibitor resistance, we solved high-resolution crystal structures of NAs from A/Anhui/1/2013 N9 (R294-containing) and A/Shanghai/1/2013 N9 (K294-containing). R294K substitution results in an unfavorable E276 conformation for oseltamivir binding, and consequently loss of inhibitor carboxylate interactions, which compromises the binding of all classical NA ligands/inhibitors. Moreover, we found that R294K substitution results in reduced NA catalytic efficiency along with lower viral fitness. This helps to explain why K294 has predominantly been found in clinical cases of H7N9 infection under the selective pressure of oseltamivir treatment and not in the dominant human-infecting viruses. This implies that oseltamivir can still be efficiently used in the treatment of H7N9 infections.     

Susceptibility of antiviral drugs against 2009 influenza A (H1N1) virus

The recent outbreak of the novel strain of influenza A (H1N1) virus has raised a global concern of the future risk of a pandemic. To understand at the molecular level how this new H1N1 virus can be inhibited by the current anti-influenza drugs and which of these drugs it is likely to already be resistant to, homology modeling and MD simulations have been applied on the H1N1 neuraminidase complexed with oseltamivir, and the M2-channel with adamantanes bound. The H1N1 virus was predicted to be susceptible to oseltamivir, with all important interactions with the binding residues being well conserved. In contrast, adamantanes are not predicted to be able to inhibit the M2 function and have completely lost their binding with the M2 residues. This is mainly due to the fact that the M2 transmembrane of the new H1N1 strain contains the S31N mutation which is known to confer resistance to adamantanes.     

The inhibitory performance of flavonoid cyanidin-3-sambubiocide against H274Y mutation in H1N1 influenza virus

Oseltamivir (Tamiflu) is the most accepted antiviral drug that targets the neuraminidase (NA) protein to inhibit the viral release from the host cell. Few H1N1 influenza strains with the H274Y mutation creates drug resistance to oseltamivir. In this study, we report that flavonoid cyanidin-3-sambubiocide (C3S) compound acts as a potential inhibitor against H274Y mutation. The drug resistance mechanism and inhibitory activity of C3S and oseltamivir against wild-type (WT) and H274Y mutant-type (MT) have been studied and compared based on the results of molecular docking, molecular dynamics, and quantum chemical methods. Oseltamivir has been found less binding affinity with MT. C3S has more binding affinity with WT and MT proteins. From the dynamical study, the 150th loop of the MT protein has found more deformation than WT. A single H274Y mutation induces the conformational changes in the 150th loop which leads to produce more resistance to oseltamivir. The 150th cavity is more attractive target for C3S to stop the conformational changes in the MT, than 430th cavity of NA protein. The C3S is stabilized with MT by more number of hydrogen bonds than oseltamivir. The electrostatic interaction energy shows a stronger C3S binding with MT and this compound may be more effective against oseltamivir-resistant virus strains.