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Influenza viruses contribute significantly to the global health burden, necessitating the development of strategies against transmission as well as effective antiviral treatments. The present study reports a biomimetic strategy inspired by the natural antiviral properties of mucins. A bovine serum albumin (BSA) conjugate decorated with the multivalent neuraminidase inhibitor Zanamivir (ZA-BSA) was synthesized using copper-free click chemistry. This synthetic pseudo-mucin exhibited potent neuraminidase inhibitory activity against several influenza strains. Virus capture and growth inhibition assays demonstrated its effective absorption of virion particles and ability to prevent viral infection in nanomolar concentrations. Investigation of the underlying antiviral mechanism of ZA-BSA revealed a dual mode of action, involving disruption of the initial stages of host-cell binding and fusion by inducing viral aggregation, followed by blocking the release of newly assembled virions by targeting neuraminidase activity. Notably, the conjugate also exhibited potent inhibitory activity against Oseltamivir-resistant neuraminidase variant comparable to the monomeric Zanamivir. These findings highlight the application of multivalent drug presentation on protein scaffold to mimic mucin adsorption of viruses, together with counteracting drug resistance. This innovative approach has potential for the creation of antiviral agents against influenza and other viral infections., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Sialosides containing C8-modified sialic acids are challenging synthetic targets but potentially useful probes for diagnostic substrate profiling of sialidases and elucidating the binding specificity of sialic acid-interacting proteins. Here, we demonstrate efficient chemoenzymatic methods for synthesizing para-nitrophenol-tagged α2-3- and α2-6-linked sialyl galactosides containing C8-acetamido, C8-azido, or C8-amino derivatized N-acetylneuraminic acid (Neu5Ac). High-throughput substrate specificity studies showed that the C8-modification of sialic acid significantly changes its recognition by sialidases from humans, various bacteria, and different influenza A and B viruses. Sialosides carrying Neu5Ac with a C8-azido modification were generally well tolerated by all the sialidases we tested, whereas sialosides containing C8-acetamido-modified Neu5Ac were only cleaved by selective bacterial sialidases. In contrast, sialosides with C8-amino-modified Neu5Ac were cleaved by a combination of selective bacterial and influenza A virus sialidases. These results indicate that sialosides terminated with a C8-amino or C8-acetamido-modified sialic acid can be used with other sialosides for diagnostic profiling of disease-causing sialidase-producing pathogens., (© 2024 Wiley-VCH GmbH.)

Influenza virus neuraminidase (NA) is a major antiviral drug target and has recently reemerged as a key target of antibody-mediated protective immunity. Here we show that recombinant NAs across non-bat subtypes adopt various tetrameric conformations, including an "open" state that may help explain poorly understood variations in NA stability across viral strains and subtypes. We use homology-directed protein design to uncover the structural principles underlying these distinct tetrameric conformations and stabilize multiple recombinant NAs in the "closed" state, yielding two near-atomic resolution structures of NA by cryo-EM. In addition to enhancing thermal stability, conformational stabilization improves affinity to protective antibodies elicited by viral infection, including antibodies targeting a quaternary epitope and the broadly conserved catalytic site. Stabilized NAs can also be integrated into viruses without affecting fitness. Our findings provide a deeper understanding of NA structure, stability, and antigenicity, and establish design strategies for reinforcing the conformational integrity of recombinant NA proteins., (© 2022. This is a U.S. Government work and not under copyright protection in the US; foreign copyright protection may apply.)

The growing resistance of the influenza virus to widely used competitive neuraminidase inhibitors occupying the active site of the enzyme requires the development of bifunctional compounds that can simultaneously interact with other regulatory sites on the protein surface. When developing such an inhibitor and combining structural fragments that could be located in the sialic acid cavity of the active site and the adjacent 430-cavity, it is necessary to select a suitable linker not only for connecting the fragments, but also to ensure effective interactions with the unique arginine triad Arg118-Arg292-Arg371 of neuraminidase. Using molecular modeling, we have demonstrated the usefulness of the sulfonamide group in the linker design and the potential advantage of this functional group over other isosteric analogues.

Encouraged by our earlier discovery of N1-selective inhibitors, the 150-cavity of influenza virus neuraminidases (NAs) could be further exploited to yield more potent oseltamivir derivatives. Herein, we report the design, synthesis and biological evaluation of a series of novel oseltamivir derivatives via the structural modifications at C 5 -NH 2 of oseltamivir targeting 150-cavity. Among them, compound 5c bearing 4-(3-methoxybenzyloxy)benzyl group exhibited the most potent activity, which was lower or modestly improved activities than oseltamivir carboxylate (OSC) against N1 (H1N1), N1 (H5N1) and N1 (H5N1-H274Y). Specifically, there was 30-fold loss of activity against the wild-type strain H1N1. However, 5c displayed 4.85-fold more potent activity than OSC against H5N1-H274Y NA. Also, 5c demonstrated low cytotoxicity in vitro and no acute toxicity in mice. Molecular docking studies provided insights into the high potency of 5c against N1 and N1-H274Y mutant NAs. Besides, the in silico prediction of physicochemical properties and CYP enzymatic inhibitory ability of representative compounds were conducted to evaluate their drug-like properties., Competing Interests: Declaration of competing interest The authors declare no conflict of interest., (Copyright © 2020 Elsevier Masson SAS. All rights reserved.)

Altered sialylation patterns play a role in chronic autoimmune diseases such as rheumatoid arthritis (RA). Recent studies have shown the pro-inflammatory activities of immunoglobulins (Igs) with desialylated sugar moieties. The role of neuraminidases (NEUs), enzymes which are responsible for the cleavage of terminal sialic acids (SA) from sialoglycoconjugates, is not fully understood in RA. We investigated the impact of zanamivir, an inhibitor of the influenza virus neuraminidase, and mammalian NEU2/3 on clinical outcomes in experimental arthritides studies. The severity of arthritis was monitored and IgG titers were measured by ELISA. (2,6)-linked SA was determined on IgG by ELISA and on cell surfaces by flow cytometry. Zanamivir at a dose of 100 mg/kg (zana-100) significantly ameliorated collagen-induced arthritis (CIA), whereas zana-100 was ineffective in serum transfer-induced arthritis. Systemic zana-100 treatment reduced the number of splenic CD138 + /TACI + plasma cells and CD19 + B cells, which was associated with lower IgG levels and an increased sialylation status of IgG compared to controls. Our data reveal the contribution of NEU2/3 in CIA. Zanamivir down-modulated the T and B cell-dependent humoral immune response and induced an anti-inflammatory milieu by inhibiting sialic acid degradation. We suggest that neuraminidases might represent a promising therapeutic target for RA and possibly also for other antibody-mediated autoimmune diseases.

Visualization of virus-infected cells is usually performed by immunostaining with an antiviral antibody. On the other hand, we established an easy method for fluorescence (FL) imaging of cells infected with influenza A and B viruses and some paramyxoviruses without the need for cell fixation and an antiviral antibody. These viruses and the cells they have infected express the viral surface enzyme "neuraminidase" or "hemagglutinin-neuraminidase" that shows sialidase activity. Sialidase activity is fluorescently visualized by using a sialidase fluorogenic probe developed in our previous study. The probe enables histochemical FL imaging of the virus-infected cells and is applicable to virus isolation and detection of an influenza virus resistant to antiinfluenza drugs of sialidase inhibitors.

Neuraminidase (NA) inhibitors are one of the two major classes of antivirals available for the treatment and prevention of influenza. X-ray crystal structure determination of NA head domains and their complexes with various inhibitors are of importance for the design and optimization of anti-influenza drugs. However, the globular tetrameric properties of NA head domains may produce crystals with pathological imperfections, lattice-translocation defects, making structure determination no longer straightforward. In this report, using a crystal of the NA head domain from the Wuhan Asiatic toad influenza virus as an example, the identification and solution of this type of crystal pathology are presented. Furthermore, its underlying mechanism of formation is explored.

The recently discovered 340-cavity in influenza neuraminidase (NA) N6 and N7 subtypes has introduced new possibilities for rational structure-based drug design. However, the plasticity of the 340-loop (residues 342-347) and the role of the 340-loop in NA activity and substrate binding have not been deeply exploited. Here, we investigate the mechanism of 340-cavity formation and demonstrate for the first time that seven of nine NA subtypes are able to adopt an open 340-cavity over 1.8 μs total molecular dynamics simulation time. The finding that the 340-loop plays a role in the sialic acid binding pathway suggests that the 340-cavity can function as a druggable pocket. Comparing the open and closed conformations of the 340-loop, the side chain orientation of residue 344 was found to govern the formation of the 340-cavity. Additionally, the conserved calcium ion was found to substantially influence the stability of the 340-loop. Our study provides dynamical evidence supporting the 340-cavity as a druggable hotspot at the atomic level and offers new structural insight in designing antiviral drugs.

Neuraminidase (NA) is one of the targets for the development of new antivirals against the influenza virus. The recombinant Escherichia coli cells, namely the strains BL21(DE3)pLysS and ArcticExpress(DE3) were used to produce the influenza virus neuraminidase. Although the different conditions of induction were tested, the accumulation of over-expressed NA in insoluble fraction occurred independently of these conditions. The level of over-expressed protein represents 26.15 % of the total cellular proteins. Therefore, the aim of these study was to design the procedure for isolation of recombinant neuraminidase from IBs and subsequently its solubilization and refolding to its native and active form. The highest purity of IBs (86 %) was achieved after repeatedly washing for at least five times with 2 M urea. The best solubilizing agent for releasing NA from IBs was the solution of 8 M urea at pH 8.0 with 94.8 ± 0.4 mg/L released proteins. The most appropriate buffer for refolding of solubilized NA was found to be 50 mM Tris-HCl at pH 7.5 (102 ± 24.2 mg proteins) and the addition of glycerol or arginine had no stimulating effect on protein recovery. The determination of non-glycosylated activity of refolded NA monomer (K m = 0.51 g/L; V max = 9.73 U/mg; k cat = 8.76 s -1 ) using fetuin as a substrate in the coupled enzyme reaction system was the highlight of this work. This procedure provides a way to produce active form of NA monomer by recombinant E. coli cells., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier B.V. All rights reserved.)

To characterize bat influenza H18N11 virus, we propagated a reverse genetics-generated H18N11 virus in Madin-Darby canine kidney subclone II cells and detected two mammal-adapting mutations in the neuraminidase (NA)-like protein (NA-F144C and NA-T342A, N2 numbering) that increased the virus titers in three mammalian cell lines (i.e., Madin-Darby canine kidney, Madin-Darby canine kidney subclone II, and human lung adenocarcinoma [Calu-3] cells). In mice, wild-type H18N11 virus replicated only in the lungs of the infected animals, whereas the NA-T342A and NA-F144C/T342A mutant viruses were detected in the nasal turbinates, in addition to the lungs. Bat influenza viruses have not been tested for their virulence or organ tropism in ferrets. We detected wild-type and single mutant viruses each possessing NA-F144C or NA-T342A in the nasal turbinates of one or several infected ferrets, respectively. A mutant virus possessing both the NA-F144C and NA-T342A mutations was isolated from both the lung and the trachea, suggesting that it has a broader organ tropism than the wild-type virus. However, none of the H18N11 viruses caused symptoms in mice or ferrets. The NA-F144C/T342A double mutation did not substantially affect virion morphology or the release of virions from cells. Collectively, our data demonstrate that the propagation of bat influenza H18N11 virus in mammalian cells can result in mammal-adapting mutations that may increase the replicative ability and/or organ tropism of the virus; overall, however, these viruses did not replicate to high titers throughout the respiratory tract of mice and ferrets. IMPORTANCE Bats are reservoirs for several severe zoonotic pathogens. The genomes of influenza A viruses of the H17N10 and H18N11 subtypes have been identified in bats, but no live virus has been isolated. The characterization of artificially generated bat influenza H18N11 virus in mammalian cell lines and animal models revealed that this virus can acquire mammal-adapting mutations that may increase its zoonotic potential; however, the wild-type and mutant viruses did not replicate to high titers in all infected animals., (Copyright © 2020 American Society for Microbiology.)

Background: Neuraminidase inhibitors (NAIs) are the only class of antivirals in clinical use against influenza virus approved worldwide. However, approximately 1-3% of circulating strains present resistance mutations to oseltamivir (OST), the most used NAI. Therefore, it is important to catalogue new molecules to inhibit influenza virus, especially OST-resistant strains. Natural products from tropical plants used for human consumption represent a worthy class of substances. Their use could be stimulated in resource-limited setting where the access to expensive antiviral therapies is restricted., Methods: We evaluated the anti-influenza virus activity of agathisflavone derived from Anacardium occidentale L., Results: The neuraminidase (NA) activity of wild-type and OST-resistant influenza virus was inhibited by agathisflavone, with IC50 values ranging from 20 to 2.0 µM, respectively. Agathisflavone inhibited influenza virus replication with EC50 of 1.3 µM. Sequential passages of the virus in the presence of agathisflavone revealed the emergence of mutation R249S, A250S and R253Q in the NA gene. These changes are outside the OST binding region, meaning that agathisflavone targets this viral enzyme at a region different than conventional NAIs., Conclusion: Altogether our data suggest that agathisflavone has a promising chemical structure for the development of anti-influenza drugs., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)

The discovery of novel potent neuraminidase (NA) inhibitors remains an attractive approach for treating infectious diseases caused by influenza. In this study, we describe the design and synthesis of novel N-substituted oseltamivir derivatives for probing the 150-cavity which is nascent to the activity site of NA. NA inhibitory studies showed that new derivatives demonstrated the inhibitory activity with IC 50 values at nM level against NA of a clinical influenza virus strain. Moreover, the in silico ADME predictions showed that the selected compounds had comparable properties with oseltamivir carboxylate, which demonstrated the druggablity of these derivatives. Furthermore, molecular docking studies showed that the most potent compound 6f and 10i could adopt different modes of binding interaction with NA, which may provide novel solutions for treating oseltamivir-resistant influenza. Based on the research results, we consider that compounds 6f and 10i have the potential for further studies as novel antiviral agents., (Copyright © 2019 Elsevier Masson SAS. All rights reserved.)

Neuraminidase (NA) plays a crucial role in the replication and transmission of influenza virus. NA inhibitors have been developed as effective treatments for influenza A and B infections. In this paper, a new lead neuraminidase inhibitor 6a (IC 50  = 7.10 ± 0.2 μM) was discovered by ligand-based virtual screening, receptor-based virtual screening, molecular dynamics simulation (MD), and bioassay validation. MD simulation indicates that the morpholinyl group of 6a could be embedded in 430-loop of NA. To exploit the 430-loop in the active site, a series of novel acylhydrazone NA inhibitors 6b-6g were designed and synthesized based on the lead compound 6a. Compound 6e exerts the most potency, with IC 50 value of 2.37 ± 0.5 μM against NA, which is lower than that of oseltamivir carboxylate (OC) (IC 50  = 3.84 μM). Overall, this work provided unique insights in the discovery of potent inhibitors against NA., (Copyright © 2019 Elsevier Masson SAS. All rights reserved.)

A panel of divalent oseltamivir and guanidino oseltamivir analogues with esterification on the carboxyl acid group as potent inhibitors of influenza virus neuraminidase was prepared via click reaction. The primary structure activity relationship study demonstrated that appropriate distance between two oseltamivir monomers around 30 Å can crosslink two adjacent neuraminidase tetramers on the virion surface and result in highly effective NA inhibitors against three strains of influenza virus and H7N9 virus like particle. This strategy also provides a basis for the multivalent modification on oseltamivir., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

In these studies, we analyzed substituted piperazine based berberine analogs conjugated through a pentyloxy side chain for their in vitro and in silico biological effects. All the final analogs were screened for their in vitro antiviral action against a collection of different influenza virus strains using the CPE assay and SRB assay. Moreover, their cytotoxicity towards non-cancer cell lines was examined employing Madin-Darby canine kidney (MDCK) cell lines. The anti-influenza activities of berberine-piperazine derivatives (BPD) were evaluated in the range from 35.16 μg/mL to 90.25 μg/mL of the IC 50 s along with cytotoxicity level which was observed in the range 44.8 μg/mL to 3890.6 μg/mL of CC 50 s towards MDCK cells. In an effort to know the mechanism of action of BPD1-BPD23, results of Neuraminidase inhibition assay and Molecular docking studies carried out against neuraminidase as the target enzyme revealed that titled compounds are potential neuraminidase inhibitors that merge to the active site of neuraminidase, with moderate to high binding energy., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Reagents to visualize and localize neuraminidase activity would be valuable probes to study the role of neuraminidases in normal cellular processes as well as during viral infections or cancer development. Herein, a new class of neuraminidase-imaging probes that function as proximity ligation reagents by releasing a highly reactive fluorophore that tags nearby cellular material is described. It is further demonstrated that it is possible to create an influenza virus-specific reagent, which can specifically detect influenza virus infections in mammalian cells. These reagents have potential use as specific histological probes independent of viral antigenicity and, therefore, offer some advantages over commonly used anti-neuraminidase antibodies., (© 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

A total of 13672 viruses, collected by World Health Organization recognised National Influenza Centres between May 2016 and May 2017, were assessed for neuraminidase inhibitor susceptibility by four WHO Collaborating Centres for Reference and Research on Influenza and one WHO Collaborating Centre for the Surveillance Epidemiology and Control of Influenza. The 50% inhibitory concentration (IC 50 ) was determined for oseltamivir and zanamivir for all viruses, and for peramivir and laninamivir in a subset (n = 8457). Of the viruses tested, 94% were obtained from the Western Pacific, Americas and European WHO regions, while limited viruses were available from the Eastern Mediterranean, African and South East Asian regions. Reduced inhibition (RI) by one or more neuraminidase inhibitor was exhibited by 0.2% of viruses tested (n = 32). The frequency of viruses with RI has remained low since this global analysis began (2015/16: 0.8%, 2014/15: 0.5%; 2013/14: 1.9%; 2012/13: 0.6%) but 2016/17 has the lowest frequency observed to date. Analysis of 13581 neuraminidase sequences retrieved from public databases, of which 5243 sequences were from viruses not included in the phenotypic analyses, identified 58 further viruses (29 without phenotypic analyses) with amino acid substitutions associated with RI by at least one neuraminidase inhibitor. Bringing the total proportion to 0.5% (90/18915). This 2016/17 analysis demonstrates that neuraminidase inhibitors remain suitable for treatment and prophylaxis of influenza virus infections, but continued monitoring is important. An expansion of surveillance testing is paramount since several novel influenza antivirals are in late stage clinical trials with some resistance already having been identified., (Copyright © 2018 The Authors. Published by Elsevier B.V. All rights reserved.)

Neuraminidase has been considered as an important target for designing agents against influenza viruses. In a discovery of anti-influenza agents with epigoitrin as the initial lead compound, a series of 1-amino-2-alkanols were synthesized and biologically evaluated. The in vitro evaluation indicated that (E)-1-amino-4-phenylbut-3-en-2-ol (C1) had better inhibitory activities than 2-amino-1-arylethan-1-ol derivatives. To our surprise, sulfonation of C1 with 4-methoxybenzenesulfonyl chloride afforded more active inhibitor II with up to 6.4 μM IC 50 value against neuraminidase. Furthermore, docking of inhibitor II into the active site of NA found that the H atoms in both NH 2 and OH groups of inhibitor II were the key factors for potency. Molecular docking research did not explained very well the observed structure-activity relationship (SAR) from amino acid residue level, but also aided the discovery of (E)-1-amino-4-phenylbut-3-en-2-ol derivatives as novel and potent NA inhibitors., (Copyright © 2018. Published by Elsevier Ltd.)

Several subtypes of avian influenza viruses (AIVs) are emerging as novel human pathogens, and the frequency of related infections has increased in recent years. Although neuraminidase (NA) inhibitors (NAIs) are the only class of antiviral drugs available for therapeutic intervention for AIV-infected patients, studies on NAI resistance among AIVs have been limited, and markers of resistance are poorly understood. Previously, we identified unique NAI resistance substitutions in AIVs of the N3, N7, and N9 NA subtypes. Here, we report profiles of NA substitutions that confer NAI resistance in AIVs of the N4, N5, N6, and N8 NA subtypes using gene-fragmented random mutagenesis. We generated libraries of mutant influenza viruses using reverse genetics (RG) and selected resistant variants in the presence of the NAIs oseltamivir carboxylate and zanamivir in MDCK cells. In addition, two substitutions, H274Y and R292K (N2 numbering), were introduced into each NA gene for comparison. We identified 37 amino acid substitutions within the NA gene, 16 of which (4 in N4, 4 in N5, 4 in N6, and 4 in N8) conferred resistance to NAIs (oseltamivir carboxylate, zanamivir, or peramivir) as determined using a fluorescence-based NA inhibition assay. Substitutions conferring NAI resistance were mainly categorized as either novel NA subtype specific (G/N147V/I, A246V, and I427L) or previously reported in other subtypes (E119A/D/V, Q136K, E276D, R292K, and R371K). Our results demonstrate that each NA subtype possesses unique NAI resistance markers, and knowledge of these substitutions in AIVs is important in facilitating antiviral susceptibility monitoring of NAI resistance in AIVs. IMPORTANCE The frequency of human infections with avian influenza viruses (AIVs) has increased in recent years. Despite the availability of vaccines, neuraminidase inhibitors (NAIs), as the only available class of drugs for AIVs in humans, have been constantly used for treatment, leading to the inevitable emergence of drug-resistant variants. To screen for substitutions conferring NAI resistance in AIVs of N4, N5, N6, and N8 NA subtypes, random mutations within the target gene were generated, and resistant viruses were selected from mutant libraries in the presence of individual drugs. We identified 16 NA substitutions conferring NAI resistance in the tested AIV subtypes; some are novel and subtype specific, and others have been previously reported in other subtypes. Our findings will contribute to an increased and more comprehensive understanding of the mechanisms of NAI-induced inhibition of influenza virus and help lead to the development of drugs that bind to alternative interaction motifs., (Copyright © 2017 American Society for Microbiology.)

Peramivir is an effective anti-influenza drug in the clinical treatment of influenza, but its efficacy toward the H275Y mutant is reduced. The previously reported cocrystal structures of inhibitors in the mutant neuraminidase (NA) suggest that the hydrophobic side chain should be at the origin of reduced binding affinity. In contrast, zanamivir having a hydrophilic glycerol side chain still possesses high affinity toward the H275Y NA. We thus designed five peramivir analogues (5-9) carrying hydrophilic glycol or glycerol side chains, and evaluated their roles in anti-influenza activity, especially for the H275Y mutant. The synthetic sequence involves a key step of (3 + 2) cycloaddition reactions between alkenes and nitrile oxides to construct the scaffold of peramivir carrying the desired hydrophilic side chains and other appropriate functional groups. The molecular docking experiments reveal that the hydrophilic side chain can provide extra hydrogen bonding with the translocated Glu-276 residue in the H275Y NA active site. Thus, the H275Y mutant may be even more sensitive than wild-type virus toward the peramivir analogues bearing hydrophilic side chains. Notably, the peramivir analogue bearing a glycerol side chain inhibits the H275Y mutant with an IC 50 value of 35 nM, which is better than the WSN virus by 9 fold.

In this study, we investigated the antiviral activity of newly synthesized berberine derivatives (BD) against influenza virus infection using several strains in in vitro and in silico. The CPE reduction, pre-incubation, NA activity inhibition and molecular docking assays were used for antiviral evaluation. The anti-influenza activities of BDs were stronger than plant-derived pure commercial berberine, and some of the BDs were more potent than control drug Oseltamivir. The cytotoxicity level was observed in the range 63.16-1639μg/mL for synthesized BDs. Additionally, BDs were detected as able to block influenza viral particles. We targeted neuraminidase one of the influenza surface protein for further probing. Moreover, BDs registered competitive NA inhibition activity comparing with Oseltamivir. The active site of viral NA subunit was fully blocked by BD as the same location as Oseltamivir. The binding energies between influenza NA subunit and BD-5 were higher than Oseltamivir. More H-bonds and NA residues were occupied by BD for stronger binding ability than Oseltamivir. These results indicated that BD inhibits various strains of influenza virus by blocking of viral NA subunit., (Copyright © 2017. Published by Elsevier Ltd.)

Influenza A and B viruses possess an enzyme "sialidase" that cleavages terminal sialic acid from glycochains. These viral sialidase proteins are highly expressed on the virus infected cells. We developed sialidase imaging probe "BTP3-Neu5Ac" that enables histochemical fluorescence staining of sialidase activity. BTP3-Neu5Ac was able to perform speedy and easy fluorescence imaging of these virus infected cells, with no needs of specific antibody and cell fixation. In addition, combination use of anti-influenza drugs (sialidase inhibitors) and BTP3-Neu5Ac resulted in selective fluorescence imaging for detection and high-efficiency isolation of drug-resistant virus. Fluorescence imaging of drug-resistant virus will be a powerful method for study of the drug-resistance mechanism, for monitoring of drug-resistant viruses. A novel tool for fluorescence imaging of viral sialidase activity is described in this review.

Despite being a common viral disease, influenza has very negative consequences, causing the death of around half a million people each year. A neuraminidase located on the surface of the virus plays an important role in viral reproduction by contributing to the release of viruses from infected host cells. The treatment of influenza is mainly based on the administration of neuraminidase inhibitors. The neuraminidase inhibitors zanamivir, laninamivir, oseltamivir and peramivir have been commercialized and have been demonstrated to be potent influenza viral neuraminidase inhibitors against most influenza strains. In order to create more potent neuraminidase inhibitors and fight against the surge in resistance resulting from naturally-occurring mutations, these anti-influenza drugs have been used as templates for the development of new neuraminidase inhibitors through structure-activity relationship studies. Here, we review the synthetic routes to these commercial drugs, the modifications which have been performed on these structures and the effects of these modifications on their inhibitory activity.

Influenza virus remains an emerging virus and causes pandemics with high levels of fatality. After screening different plant extracts with potential anti-influenza activity, a water extract of Taxodium distichum stems (TDSWex) showed excellent activity against influenza viruses. The EC 50 of TDSWex was 0.051 ± 0.024 mg/mL against influenza virus A/WSN/33. TDSWex had excellent antiviral efficacy against various strains of human influenza A and B viruses, particularly oseltamivir-resistant clinical isolates and a swine-origin influenza strain. We observed that the synthesis of viral RNA and protein were inhibited in the presence of TDSWex. The results of the time-of-addition assay suggested that TDSWex inhibited viral entry and budding. In the hemagglutination inhibition assay, TDSWex inhibited the hemagglutination of red blood cells, implying that the extract targeted hemagglutin-related functions such as viral entry. In the attachment and penetration assay, TDSWex showed antiviral activity with EC 50 s of 0.045 ± 0.026 and 0.012 ± 0.003 mg/mL, respectively. In addition, TDSWex blocked neuraminidase activity. We conclude that TDSWex has bimodal activities against both hemagglutinin and neuraminidase during viral replication.

Background: Laboratory correlates of influenza vaccine protection can best be identified by examining people who are infected despite vaccination. While the importance of antibody to viral hemagglutinin (HA) has long been recognized, the level of protection contributed independently by antibody to viral neuraminidase (NA) has not been determined., Methods: Sera from a controlled trial of the efficacies of inactivated influenza vaccine (IIV) and live attenuated influenza vaccine (LAIV) were tested by hemagglutination inhibition (HAI) assay, microneutralization (MN) assay, and a newly standardized lectin-based neuraminidase inhibition (NAI) assay., Results: The NAI assay detected a vaccine response in 37% of IIV recipients, compared with 77% and 67% of participants in whom responses were detected by the HAI and MN assays, respectively. For LAIV recipients, the NAI, HAI, and MN assays detected responses in 6%, 21%, and 17%, respectively. In IIV recipients, as NAI assay titers rose, the frequency of infection fell, similar to patterns seen with HAI and MN assays. HAI and MN assay titers were highly correlated, but NAI assay titers exhibited less of a correlation. Analyses suggested an independent role for NAI antibody in protection, which was similar in the IIV, LAIV, and placebo groups., Conclusions: While NAI antibody is not produced to a large extent in response to current IIV, it appears to have an independent role in protection. As new influenza vaccines are developed, NA content should be considered., Clinical Trials Registration: NCT00538512., (© The Author 2015. Published by Oxford University Press on behalf of the Infectious Diseases Society of America. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.)

Influenza is a major cause of severe respiratory infections leading to excessive hospitalizations and deaths globally; annual epidemics, pandemics, and sporadic/endemic avian virus infections occur as a result of rapid, continuous evolution of influenza viruses. Emergence of antiviral resistance is of great clinical and public health concern. Currently available antiviral treatments include four neuraminidase inhibitors (oseltamivir, zanamivir, peramivir, laninamivir), M2-inibitors (amantadine, rimantadine), and a polymerase inhibitor (favipiravir). In this review, we focus on resistance issues related to the use of neuraminidase inhibitors (NAIs). Data on primary resistance, as well as secondary resistance related to NAI exposure will be presented. Their clinical implications, detection, and novel therapeutic options undergoing clinical trials are discussed.

A population drug-disease model was developed to describe the time course of influenza virus with and without oseltamivir treatment and to investigate opportunities for antiviral combination therapy. Data included viral titers from 208 subjects, across 4 studies, receiving placebo and oseltamivir at 20 to 200 mg twice daily for 5 days. A 3-compartment mathematical model, comprising target cells infected at rate β, free virus produced at rate p and cleared at rate c, and infected cells cleared at rate δ, was implemented in NONMEM with an inhibitory Hill function on virus production (p), accounting for the oseltamivir effect. In congruence with clinical data, the model predicts that the standard 75-mg regimen initiated 2 days after infection decreased viral shedding duration by 1.5 days versus placebo; the 150-mg regimen decreased shedding by an additional average 0.25 day. The model also predicts that initiation of oseltamivir sooner postinfection, specifically at day 0.5 or 1, results in proportionally greater decreases in viral shedding duration of 5 and 3.5 days, respectively. Furthermore, the model suggests that combining oseltamivir (acting to subdue virus production rate) with an antiviral whose activity decreases viral infectivity (β) results in a moderate additive effect dependent on therapy initiation time. In contrast, the combination of oseltamivir with an antiviral whose activity increases viral clearance (c) shows significant additive effects independent of therapy initiation time. The utility of the model for investigating the pharmacodynamic effects of novel antivirals alone or in combination on emergent influenza virus strains warrants further investigation., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

Mechanism-based inhibition of influenza neuraminidases by difluorosialic acids (DFSA) is not only rendered highly specific by incorporation of 4-amino or 4-guanidine substituents but also the half-life for reactivation is greatly increased. Measurement of rate constants for spontaneous hydrolysis of a series of such substituted DFSAs reveals, surprisingly, that inherent inductive effects play very little role in this rate reduction and that interactions with the enzyme are more important.

The applicability of combined quantum mechanics/molecular mechanics (QM/MM) methods for the calculation of absolute binding free energies of conserved water molecules in protein/ligand complexes is demonstrated. Here, we apply QM/MM Monte Carlo simulations to investigate binding of water molecules to influenza neuraminidase. We investigate five different complexes, including those with the drugs oseltamivir and peramivir. We investigate water molecules in two different environments, one more hydrophobic and one hydrophilic. We calculate the free-energy change for perturbation of a QM to MM representation of the bound water molecule. The calculations are performed at the BLYP/aVDZ (QM) and TIP4P (MM) levels of theory, which we have previously demonstrated to be consistent with one another for QM/MM modeling. The results show that the QM to MM perturbation is significant in both environments (greater than 1 kcal mol(-1)) and larger in the more hydrophilic site. Comparison with the same perturbation in bulk water shows that this makes a contribution to binding. The results quantify how electronic polarization differences in different environments affect binding affinity and also demonstrate that extensive, converged QM/MM free-energy simulations, with good levels of QM theory, are now practical for protein/ligand complexes.

Neuraminidase (NA) is the second most abundant influenza surface glycoprotein and contributes to virus replication in several ways, most notably by removing sialic acids from the host and viral glycoproteins, releasing newly formed virus particles from infected cells. Antibodies that block this enzyme activity restrict virus replication in vitro. This chapter describes foundational epidemiologic and human influenza challenge studies that provide evidence of an association between NA inhibiting antibodies and resistance to disease. Mouse challenge studies show that while NA immunity is infection-permissive, NA-specific antibodies attenuate infection and prevent severe disease. NA immunity is most effective against homologous viruses but there is substantial protection against viruses with a heterologous NA (different lineage within a NA subtype). Monoclonal antibodies specific for conserved antigenic domains of subtype N1 protect against seasonal and pandemic H1N1 as well as H5N1 virus challenge. Clinical studies demonstrate that licensed seasonal vaccines contain immunogenic amounts of NA, but the contribution of this immunity to vaccine efficacy is currently not known. New types of influenza vaccines could be designed to elicit NA immunity. Because NA induces heterologous immunity, it could be an important constituent of universal influenza vaccines that aim to protect against unexpected emerging viruses.

Vaccination is the best measure to prevent influenza pandemics. Here, we studied the protective effect against heterologous influenza viruses, including A/reassortant/NYMC X-179A (pH1N1), A/Chicken/Henan/12/2004 (H5N1), A/Chicken/Jiangsu/7/2002 (H9N2) and A/Guizhou/54/89×A/PR/8/34 (A/Guizhou-X) (H3N2), in mice first vaccinated with a DNA vaccine of haemagglutinin (HA) or neuraminidase (NA) of A/PR/8/34 (PR8) and then infected with the homologous virus. We showed that PR8 HA or NA vaccination both protected mice against a lethal dose of the homologous virus; PR8 HA or NA DNA vaccination and then PR8 infection in mice offered poor or excellent protection, respectively, against a second, heterologous influenza virus challenge. In addition, before the second heterologous influenza infection, the highest antibody level against nucleoprotein (NP) and matrix (M1 and M2) proteins was found in the PR8 NA-vaccinated and PR8-infected group. The level of induced cellular immunity against NP and M1 showed a trend consistent with that seen in antibody levels. However, PR8 HA+NA vaccination and then PR8 infection resulted in limited protection against heterologous influenza virus challenge. Results of the present study demonstrated that infection of the homologous influenza virus in mice already immunized with a NA vaccine could provide excellent protection against subsequent infection of a heterologous influenza virus. These findings suggested that NA, a major antigen of influenza virus, could be an important candidate antigen for universal influenza vaccines., (© 2014 The Authors.)

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.)

Water molecules are commonplace in protein binding pockets, where they can typically form a complex between the protein and a ligand or become displaced upon ligand binding. As a result, it is often of great interest to establish both the binding free energy and location of such molecules. Several approaches to predicting the location and affinity of water molecules to proteins have been proposed and utilized in the literature, although it is often unclear which method should be used under what circumstances. We report here a comparison between three such methodologies, Just Add Water Molecules (JAWS), Grand Canonical Monte Carlo (GCMC), and double-decoupling, in the hope of understanding the advantages and limitations of each method when applied to enclosed binding sites. As a result, we have adapted the JAWS scoring procedure, allowing the binding free energies of strongly bound water molecules to be calculated to a high degree of accuracy, requiring significantly less computational effort than more rigorous approaches. The combination of JAWS and GCMC offers a route to a rapid scheme capable of both locating and scoring water molecules for rational drug design.

Influenza virus is rich in variation and mutations. It would be very convenient for virus detection and isolation to histochemically detect viral infection regardless of variation and mutations. Here, we established a histochemical imaging assay for influenza virus sialidase activity in living cells by using a new fluorescent sialidase substrate, 2-(benzothiazol-2-yl)-4-bromophenyl 5-acetamido-3,5-dideoxy-α-D-glycero-D-galacto-2-nonulopyranosidonic acid (BTP3-Neu5Ac). The BTP3-Neu5Ac assay histochemically visualized influenza virus-infected cells regardless of viral hosts and subtypes. Influenza virus neuraminidase-expressed cells, viral focus formation, and virus-infected locations in mice lung tissues were easily, rapidly, and sensitively detected by the BTP3-Neu5Ac assay. Histochemical visualization with the BTP3-Neu5Ac assay is extremely useful for detection of influenza viruses without the need for fixation or a specific antibody. This novel assay should greatly improve the efficiency of detection, titration, and isolation of influenza viruses and might contribute to research on viral sialidase.

For the treatment of seasonal flu and possible pandemic infections the development of new anti-influenza drugs that have good bioavailability against a broad spectrum of influenza viruses including the resistant strains is needed. In this review, we summarize previous methods for the structural modification of zanamivir, a potent neuraminidase inhibitor that has rare drug resistance, in order to develop effective anti-influenza drugs. We also report recent research into the design of multivalent zanamivir drugs and bifunctional zanamivir conjugates, some of which have shown better efficacy in animal experiments. As a step towards developing improved antivirals, conjugating anti-influenza drugs with anti-inflammatory agents can improve oral bioavailability and also exert synergistic effect in influenza therapy.

Background: Evidence of the effectiveness of influenza vaccination in children and elderly adults is limited, although this population has the highest risk for influenza infection., Materials and Methods: We enrolled 4443 participants, aged 3-97 years, who had influenza-kit-positive results during seasons 2007-12, including 2135 with influenza A, 534 with A/H1N1, and 1643 with influenza B. Eligible subjects completed a questionnaire to identify past influenza infection and vaccination history. For the diagnosis of current influenza infection, subjects were examined, and pharyngeal swabs were collected and tested using the Capilia flu rapid diagnosis kit to confirm influenza infection. An interim analysis was performed using clinician-based surveillance data for the entire four seasons of influenza infection in Japan., Results: In 3035 adults aged 14-64 years, administration of the influenza vaccine significantly reduced the frequency of infection (P<0.01) in the 2008 and 2010 seasons, but not in the 2009 and 2011 seasons. Moreover, the vaccine did not reduce the frequency of infection in children (aged <13 years) and older adults (aged >65 years) significantly. Laninamivir, oseltamivir phosphate, zanamivir hydrate, and amantadine hydrochloride were administered to 1381, 2432, 1044, and 100 patients, respectively. They were effective in >97% of patients, with no significant differences being found. Adverse effects were few. However, the recurrence rate of influenza infection after treatment was significantly reduced in patients who received laninamivir compared with that in those who received oseltamivir and zanamivir (P<0.01). The effectiveness of laninamivirdid not decrease., Conclusions: The vaccines administered had limited efficacy in reducing the frequency of influenza infection in young adults. Laninamivir significantly reduced the recurrence of influenza infection when compared with other neuraminidase inhibitors.

The live attenuated influenza vaccine (LAIV) consists of reassortant viruses with hemagglutinin (HA) and neuraminidase (NA) gene segments inherited from the circulating wild-type (WT) parental and the 6 internal protein-encoding gene segments from the cold-adapted attenuated master donor viruses (genome composition 6:2). In this study, we describe the obstacles to developing LAIV vaccine strains depending on the phenotypic peculiarities of the WT viruses used for reassortment. The genomic composition analysis of 849 reassortants revealed that over 80% of the reassortants based on the inhibitor-resistant WT viruses inherited WT NA as compared to 26% of reassortants based on the inhibitor-sensitive WT viruses. In addition, the highest percentage of the vaccine genotype reassortants was achieved when WT parental viruses were resistant to the non-specific serum inhibitors. We demonstrate that NA may play a role in the influenza virus sensitivity to the non-specific serum inhibitors. Replacing NA of the inhibitor-sensitive WT virus with the NA of the inhibitor-resistant master donor virus significantly decreased the sensitivity of the resulting reassortant virus to the non-specific inhibitors.

The correlation between precise interactions of influenza A virus hemagglutinins with host cell surface glycans having terminal sialic acids and host range specificity has provoked the development of a high-throughput viral-receptor specificity assay. Here, we describe the use of the virus itself as a specific antibody coupled to enzymes (virus with neuraminidase spikes) for determining its binding specificity to glycans, a strategy that reduces not only the cost but also the tedious steps of adding primary and secondary antibodies and washing between each step. All of the steps, including coating the glycopolymers onto microtiter plates, virus binding, and visual and quantitative detection of fluorescence products that correlate well with the amount of glycan-bound viruses, can be done within 3 h. This simple, rapid, sensitive, and reliable strategy is an ideal method for detection of high-throughput influenza virus receptor-binding preference not only for studies on viral evolution and transmission but also for viral surveillance in pandemic preparedness, leading to efficient prevention and control of the disease.

Silver-catalyzed endo-selective and copper-catalyzed exo-selective asymmetric [3 + 2] cycloadditions of acrylates to chiral iminoesters derived from D-glyceraldehyde have been investigated. The reaction diastereoselectively provides highly functionalized pyrrolidines. This approach was used to develop the first asymmetric synthesis of a key intermediate in the synthesis of pyrrolidine influenza neuramidinase inhibitors.

Novel 3,4-disubstituted-Neu5Ac2en derivatives have been synthesised to probe the open 150-loop conformation of influenza virus sialidases. Both equatorially and axially (epi) substituted C4 amino and guanidino 3-(p-tolyl)allyl-Neu5Ac2en derivatives were prepared, via the 4-epi-hydroxy derivative. The equatorially-substituted 4-amino derivative showed low micromolar inhibition of both group-1 (pdm09 H1N1) and group-2 (pdm57 H2N2) sialidases, and provides the first in vitro evidence that a group-2 sialidase may exhibit 150-loop flexibility., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Natural killer (NK) cells play an essential role in the defense against influenza virus, one of the deadliest respiratory viruses known today. The NKp46 receptor, expressed by NK cells, is critical for controlling influenza infections, as influenza-virus-infected cells are eliminated through the recognition of the viral hemagglutinin (HA) protein by NKp46. Here, we describe an immune-evasion mechanism of influenza viruses that is mediated by the neuraminidase (NA) protein. By using various NA blockers, we show that NA removes sialic acid residues from NKp46 and that this leads to reduced recognition of HA. Furthermore, we provide in vivo and in vitro evidence for the existence of this NA-mediated, NKp46-dependent immune-evasion mechanism and demonstrate that NA inhibitors, which are commonly used for the treatment of influenza infections, are useful not only as blockers of virus budding but also as boosters of NKp46 recognition., (Copyright © 2013 The Authors. Published by Elsevier Inc. All rights reserved.)

Influenza antiviral agents play important roles in modulating disease severity and in controlling pandemics while vaccines are prepared, but the development of resistance to agents like the commonly used neuraminidase inhibitor oseltamivir may limit their future utility. We report here on a new class of specific, mechanism-based anti-influenza drugs that function through the formation of a stabilized covalent intermediate in the influenza neuraminidase enzyme, and we confirm this mode of action with structural and mechanistic studies. These compounds function in cell-based assays and in animal models, with efficacies comparable to that of the neuraminidase inhibitor zanamivir and with broad-spectrum activity against drug-resistant strains in vitro. The similarity of their structure to that of the natural substrate and their mechanism-based design make these attractive antiviral candidates.

Introduction: At present, the key public health concern is to define the way in which the next influenza pandemic should be controlled. While influenza vaccines are available, their effectiveness could be significantly reduced if new strains differ significantly from those of the vaccines. Therefore, antiviral drugs play an important role in the prevention and management of influenza. The influenza neuraminidase (NA), a surface-glycoprotein enzyme involved in releasing the virus from the host cell, has been considered as an essential therapeutic target for treatment and prophylaxis of influenza infection. It is a highly conserved feature of the active site across all influenza A and B viruses and is of particular interest because compounds NA inhibitors (NAIs) can be cross-reactive against multiple types and subtypes of influenza. Currently, there are two NAI drugs which are licensed worldwide: oseltamivir and zanamivir, and two more drugs which have received recent approval in Japan: peramivir and laninamivir. Sudden changes in NAI susceptibility have stressed the urgent need in searching for novel inhibitors., Areas Covered: In this review, a potential pitfall in NA-based assays and the progress in the chemical synthesis of all patented NAIs from February 2006 to July 2012 are discussed., Expert Opinion: Both NA enzyme inhibition and X-ray crystallography data have suggested that the strategy of designing NAIs binding to the highly conserved region of NA can lead to inhibitors that are effective against all influenza NA subtypes. A number of new synthetic entries having unique structural frameworks that were designed based on computational study of X-ray structures of NA. They strongly exhibited the activity of NA in the low nanomolar range such as phosphonate congeners of zanamivir and oseltarmivir. Screening strategies based on the chemical diversity of natural products have revealed that flavonoids are the most prominent scaffolds possessing NA inhibitory activity. However, these substituted phenyl-benzopyrane compounds have been reported to exert a considerable quenching effect, causing false-positive results in the commonly used method of enzyme-based NA inhibition assays, and thus, reliability of the flavonoid-based NAIs reported in the literature.

Influenza A virus poses a great threat to global health, and oseltamivir (trade marked as Tamiflu), which targets influenza surface glycoprotein neuraminidase (NA), is used clinically as a major anti-influenza treatment. However, certain substitutions in NA can render an influenza virus resistant to this drug. In this study, using a lentiviral pseudotyping system, which alleviates the safety concerns of studying highly pathogenic influenza viruses such as avian influenza H5N1, that utilizes influenza surface glycoproteins (hemagglutinin or HA, and NA) and an HIV-core combined with a luciferase reporter gene as a surrogate assay, we first assessed the functionality of NA by measuring pseudovirion release in the absence or presence of oseltamivir. We demonstrated that oseltamivir displays a dose-dependent inhibition on NA activity. In contrast, a mutant NA (H274Y) is more resistant to oseltamivir treatment. In addition, the effects of several previously reported substitution NA mutants were examined as well. Our results demonstrate that this lentivirus-based pseudotyping system provides a quick, safe, and effective way to assess resistance to neuraminidase inhibitors. And we believe that as new mutations appear in influenza isolates, their impact on the effectiveness of current and future anti-NA can be quickly and reliably evaluated by this assay.

A macrocyclic mechanism-based inhibitor for neuraminidases (NAs) bearing a 2-difluoromethylphenyl aglycone and a linker between the aglycone and C-9 positions of sialic acid was synthesized and evaluated. The macrocyclic structure was designed to keep the aglycone moiety in the active site of the neuraminidase after cleavage of the glycoside bond. When Vibrio chorelae neuraminidase (VCNA) was treated with a similar acyclic derivative in the presence of detergent, the irreversible inhibition property was disabled. In contrast, this macrocyclic compound acted as an irreversible inhibitor for VCNA in the presence of detergent. Inhibition assay for various NAs using this macrocyclic compound revealed that the irreversible inhibition property depends on the k(cat) of the neuraminidase treated. NAs having small k(cat) values, such as Influenza viruses, Clostridium, Trypanosoma cruzi, and Human, were also inhibited irreversibly. However, Salmonella typhimurium NA, which has an extremely high k(cat) , was not affected irreversibly by the inhibitor. Interestingly, in contrast to common k(cat) inhibitors, the irreversibility of inhibition by this macrocyclic compound is inversely proportional to the k(cat) of the target neuraminidase., (Copyright © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Development of novel influenza neuraminidase inhibitors is critical for preparedness against influenza outbreaks. Knowledge of the neuraminidase enzymatic mechanism and transition-state analogue, 2-deoxy-2,3-didehydro-N-acetylneuraminic acid, contributed to the development of the first generation anti-neuraminidase drugs, zanamivir and oseltamivir. However, lack of evidence regarding influenza neuraminidase key catalytic residues has limited strategies for novel neuraminidase inhibitor design. Here, we confirm that influenza neuraminidase conserved Tyr406 is the key catalytic residue that may function as a nucleophile; thus, mechanism-based covalent inhibition of influenza neuraminidase was conceived. Crystallographic studies reveal that 2α,3ax-difluoro-N-acetylneuraminic acid forms a covalent bond with influenza neuraminidase Tyr406 and the compound was found to possess potent anti-influenza activity against both influenza A and B viruses. Our results address many unanswered questions about the influenza neuraminidase catalytic mechanism and demonstrate that covalent inhibition of influenza neuraminidase is a promising and novel strategy for the development of next-generation influenza drugs.

A series of C3 O-functionalized 2-acetamido-2-deoxy-Δ⁴-β-D-glucuronides were synthesized to explore noncharge interactions in subsite 2 of the influenza virus sialidase active site. In complex with A/N8 sialidase, the parent compound (C3 OH) inverts its solution conformation to bind with all substituents well positioned in the active site. The parent compound inhibits influenza virus sialidase at a sub-μM level; the introduction of small alkyl substituents or an acetyl group at C3 is also tolerated.

Influenza viruses are highly contagious for human population and result in acute respiratory infectious diseases ranging from mild to severe. Neuraminidase (NA) inhibitors (NAIs) (oseltamivir, zanamivir, peramivir and laninamivir), which target the NA glycoproteins of influenza A and B viruses are widely used in the prophylaxis and treatment of influenza virus infection. However, the substitutions of amino acids in NA or HA gene may lead to resistances to NAIs. NAI-resistance-related substitutions are typically specific to certain NA type or subtype. The sensitivity for NAI-resistance detection is affected by different assays used.