Neuraminidase Inhibitors Research Articles

Misclassification of Both Influenza Infection and Oseltamivir Exposure Status in Administrative Data

This cross-sectional study examines documented hospitalization for influenza and administration of neuraminidase inhibitors in US children.

Screening for Q-markers of Antiviral Granules based on neuraminidase inhibitors and Effect-constituent index

BackgroundAntiviral granules (AG) is a classic traditional Chinese medicine commonly employed for influenza treatment. However, there is an urgent need to improve its quality control standards. PurposeThe quality markers (Q-markers), guided by the Effect-constituent Index (ECI), were screened through in vitro and in vivo component identification, molecular docking, and neuraminidase (NA) inhibition experiments. MethodsThe chemical components in AG underwent analysis using UPLC-Q-Orbitrap HRMS and GC–MS, and serum pharmacochemistry was employed to analyze the components absorbed into the bloodstream. Molecular docking was used to evaluate the affinity of the prototype components to NA, and the inhibitory activity of the candidate components was later confirmed through NA inhibitor experiments. Following this, a UHPLC-MS/MS method was developed to screen and quantify the Q-markers in 20 sample batches. Lastly, the ECI was formulated using a multi-index comprehensive evaluation method that combines composition, content, and activity. ResultsA total of 246 compounds were identified in AG, and 89 prototype components and 19 metabolic components were detected in the serum of the rats. Molecular docking revealed that the binding energy of 35 candidate active ingredients was better than that of the original ligand and the target protein. Among these candidate active ingredients, 12 candidate Q-markers demonstrated significant inhibitory effects in NA inhibition experiments, with IC50 values ranging from 0.954 to 3.408 mM. A UHPLC-MS/MS content determination method was established for the quantification of 11 Q-markers, including Forsythiaside A, Pogostone, Verbascoside, Mangiferin, 5-methoxy-8-hydroxypsoralen, Curcumenol, Luteolin-7-glucuronide, Kaempferol-3-O-rutinoside, Baicalin, Rutin, and Bergaptol, in 20 batches of samples. The ECI was calculated based on activity and content of these Q-markers. Pearson's correlation analysis of the NA inhibitory effect of the samples and the ECI resulted in an R value of 0.859, indicating a good correlation. Furthermore, this correlation coefficient surpassed those observed between the content of each Q-marker and the NA inhibitory effect. ConclusionThe Q-markers for treating influenza with AG have been successfully screened, and the ECI of AG has been constructed. These findings will provide beneficial strategies for improving the quality control level of AG.

Facile synthesis of C5-azido derivatives of thiosialosides and 2,3-dehydro-5-N-acetylneuraminic acid (DANA)

Neuraminic acid (Neu5Ac, also known as sialic acid) is an important monosaccharide found in glycoproteins and glycolipids which plays a vital role in regulation of physiological functions and pathological conditions. The study of sialoglycans has benefitted from the development of glycomimetic probes and inhibitors for proteins and enzymes that interact with and modify neuraminic acid in glycan chains. Methods to access sialoside intermediates with high yield are needed to facilitate the design of new targets. Here, we report the synthesis of C5-azido thiosialosides using a mild method to deprotect the C5-acetamido functional group followed by the use of a diazo-transfer reagent. We examined two diazo-transfer strategies and compared their yields and tolerance of acetate protecting groups. The same methods and comparisons were also performed for the 2,3-dehydro-5-N-acetylneuraminic acid (DANA) scaffold which is commonly used to generate inhibitors of neuraminidase (sialidase) enzymes. We found that C5-azido derivatives of both thiosialosides and DANA could be produced in five or six steps with yields up to 76 % and 83 %, respectively. Diazo-transfer reagents compared in this study were trifluoromethanesulfonyl azide (TfN3) and imidazole-1-sulfonyl azide (ImzSO2N3). We found that both reagents were compatible with this method and showed comparable yields. Finally, we show that C5-azido derivatives can help to avoid O, N-acyl protecting group migration which was observed in C5-NHAc analogs.

An Investigation of Severe Influenza Cases in Russia during the 2022-2023 Epidemic Season and an Analysis of HA-D222G/N Polymorphism in Newly Emerged and Dominant Clade 6B.1A.5a.2a A(H1N1)pdm09 Viruses.

In Russia, during the COVID-19 pandemic, a decrease in influenza circulation was initially observed. Influenza circulation re-emerged with the dominance of new clades of A(H3N2) viruses in 2021-2022 and A(H1N1)pdm09 viruses in 2022-2023. In this study, we aimed to characterize influenza viruses during the 2022-2023 season in Russia, as well as investigate A(H1N1)pdm09 HA-D222G/N polymorphism associated with increased disease severity. PCR testing of 780 clinical specimens showed 72.2% of them to be positive for A(H1N1)pdm09, 2.8% for A(H3N2), and 25% for influenza B viruses. The majority of A(H1N1)pdm09 viruses analyzed belonged to the newly emerged 6B.1A.5a.2a clade. The intra-sample predominance of HA-D222G/N virus variants was observed in 29% of the specimens from A(H1N1)pdm09 fatal cases. The D222N polymorphic variant was registered more frequently than D222G. All the B/Victoria viruses analyzed belonged to the V1A.3a.2 clade. Several identified A(H3N2) viruses belonged to one of the four subclades (2a.1b, 2a.3a.1, 2a.3b, 2b) within the 3C.2a1b.2a.2 group. The majority of antigenically characterized viruses bore similarities to the corresponding 2022-2023 NH vaccine strains. Only one influenza A(H1N1)pdm09 virus showed reduced inhibition by neuraminidase inhibitors. None of the influenza viruses analyzed had genetic markers of reduced susceptibility to baloxavir.

In-silico design of novel 2-((4-chloro-6-methoxy-1H-indol-3-yl)thio)-N-(2-ethoxyphenyl)acetamide derivatives as potential inhibitors of influenza neuraminidase protein receptor

Influenza virus transmission is largely mediated by its mutation and genome reassortment from distinct strains resulting in drug-resistances and pandemics. This necessitates the need for the discovery of more potential influenza inhibitors to prevent future epidemics. An in-silico approach was utilized here to design six new (21a-f) potential inhibitors of influenza neuraminidase (NA) using a hit compound 21 with good binding affinity, predicted activity, and pharmacokinetic properties in our previous work. The modeled activities (pEC50) of the newly designed compounds (ranging between 8.188 and 7.600) were better than that of the hit compound 21 with predicted activity (pEC50) of 6.0101 and zanamivir (pEC50 of 5.6755) as the standard reference control used. The MolDock scores (ranging between −189.67 and −142.47 ​kcal/mol) of these newly designed compounds in the NA binding cavity were also better than the hit template 21 with a MolDock score of −125.33 ​kcal/mol and zanamivir standard drug (−136.36 ​kcal/mol). In addition, the conformational stability of the best-designed compound 21a in the NA binding cavity was further studied through the MD simulation of 100 ​ns. Moreover, the drug-likeness and ADMET predictions of these designed compounds showed their good oral bioavailability and pharmacokinetic profiling respectively. More so, the DFT calculations also revealed the relevance of these designed compounds in view of their smaller band energy gaps from the frontier molecular orbital calculations. This study could serve as a reliable in-silico perspective for the search and discovery of potential anti-influenza agents.

Naturally Occurring Isorhamnetin Glycosides as Potential Agents Against Influenza Viruses: Antiviral and Molecular Docking Studies.

Influenza remains one of the most widespread infections, causing an annual illness in adults and children. Therefore, the search for new antiviral drugs is one of the priorities of practical health care. Eight isorhamnetin glycosides were purified from Persicaria species, characterized by nuclear magnetic resonance spectroscopy and mass spectrometry and then evaluated as potential agents against influenza virus. A comprehensive in vitro and in vivo assessment of the compounds revealed that compound 5 displayed the most potent inhibitory activity with an EC50 value of 1.2-1.3 μM, better than standard drugs (isorhamnetin 28.0-56.0 μM and oseltamivir 1.3-9.1 μM). Molecular docking results also revealed that compound 5 has the lowest binding energy (-10.7 kcal/mol) among the tested compounds and isorhamnetin (-8.1 kcal/mol). The ability of the isorhamnetin glycosides to suppress the reproduction of the influenza virus was studied on a model of a cell culture and chicken embryos. The ability of active compounds to influence the structure of the virion, as well as the activity of hemagglutinin and neuraminidase, has been demonstrated. Compound 1, 5, and 6 demonstrated the most effective inhibition of virus replication for all tested viruses. Molecular dynamics simulation techniques were run for 100 ns for compound 5 with two protein receptors Hem (1RUY) and Neu (3BEQ). These results revealed that the Hem-complex system acquired a relatively more stable conformation and even better descriptors than the other Neu-complex studied systems, suggesting that it can be an effective inhibiting drug toward hemagglutinin than neuraminidase inhibition. Based on the reported results, compound 5 can be a good candidate to be evaluated for effectiveness in preclinical testing.

Amphiphilic Sialic Acid Derivatives as Potential Dual-Specific Inhibitors of Influenza Hemagglutinin and Neuraminidase.

In the shadow of SARS-CoV-2, influenza seems to be an innocent virus, although new zoonotic influenza viruses evolved by mutations may lead to severe pandemics. According to WHO, there is an urgent need for better antiviral drugs. Blocking viral hemagglutinin with multivalent N-acetylneuraminic acid derivatives is a promising approach to prevent influenza infection. Moreover, dual inhibition of both hemagglutinin and neuraminidase may result in a more powerful effect. Since both viral glycoproteins can bind to neuraminic acid, we have prepared three series of amphiphilic self-assembling 2-thio-neuraminic acid derivatives constituting aggregates in aqueous medium to take advantage of their multivalent effect. One of the series was prepared by the azide-alkyne click reaction, and the other two by the thio-click reaction to yield neuraminic acid derivatives containing lipophilic tails of different sizes and an enzymatically stable thioglycosidic bond. Two of the three bis-octyl derivatives produced proved to be active against influenza viruses, while all three octyl derivatives bound to hemagglutinin and neuraminidase from H1N1 and H3N2 influenza types.

Recombinant A(H6N1)-H274Y avian influenza virus with dual drug resistance does not require permissive mutations to retain the replicative fitness in vitro and in ovo

The possible emergence of drug-resistant avian flu raises concerns over the limited effectiveness of currently approved antivirals (neuraminidase inhibitors - NAIs) in the hypothetical event of a zoonotic spillover. Our study demonstrated that the recombinant avian A(H6N1) viruses showed reduced inhibition (RI) by multiple NAI drugs following the introduction of point mutations found predominantly in the neuraminidase gene (NA) of NAI-resistant human influenza strains (E119V, R292K and H274Y; N2 numbering). Moreover, A(H6N1)-H274Y showed increased replication efficiency in vitro, and a fitness advantage over wild-type (WT) when co-inoculated into embryonated hen’s eggs. The results presented in our study together with the zoonotic potential of the A(H6N1) virus as evidenced by the human infection from 2013, highlight the need for enhanced monitoring of NAI resistance-associated signatures in circulating LPAI (low pathogenic avian influenza) globally.

Impact of antiviral therapy on short- and long-term outcomes of patients with chronic obstructive pulmonary disease after influenza infection.

Respiratory complications often accompany influenza in patients with chronic obstructive pulmonary disease (COPD). In this retrospective study, we quantified the impact of antiviral therapy on exacerbations, healthcare resource utilization (HRU), and costs in patients with COPD across 5 influenza seasons. Using claims data from US MarketScan® databases, we identified patients with COPD who had an influenza diagnosis during the 2012-2016 influenza seasons. Patients who received a neuraminidase inhibitor within 48 h of diagnosis (N= 4134) were identified and propensity score-matched 1:1 to a comparator cohort of untreated patients. We determined COPD- and pneumonia-related HRU and costs during month 1, each subsequent quarter, and months 2-13. Antiviral-treated patients had a significantly lower frequency of COPD-related outcomes than untreated patients during all periods (exacerbations: 10.4% vs 18.2% [month 1] and 17.7% vs 24.2% [months 2-13]; inpatient visit: 2.5% vs 7.9% [month 1] and 3.8% vs 6.7% [months 2-13]; P< 0.0001, all comparisons). Treated patients also had significantly lower outpatient and emergency department (ED) visits beyond month 1. Pneumonia-related inpatient, ED, and outpatient visits were significantly lower in antiviral-treated patients than in untreated patients over all periods (P< 0.0001, all comparisons). In all HRU categories, COPD- and pneumonia-related costs were significantly lower in treated patients over all periods (month-1 ED visit costs were higher). Antiviral treatment in patients with COPD and influenza is associated with significantly lower HRU and costs in the postinfection month and for an entire year following infection compared with untreated patients.

Antiviral Activity of Probenecid and Oseltamivir on Influenza Virus Replication.

Influenza can cause respiratory infections, leading to significant morbidity and mortality in humans. While current influenza vaccines offer varying levels of protection, there remains a pressing need for effective antiviral drugs to supplement vaccine efforts. Currently, the FDA-approved antiviral drugs for influenza include oseltamivir, zanamivir, peramivir, and baloxavir marboxil. These antivirals primarily target the virus, making them vulnerable to drug resistance. In this study, we evaluated the efficacy of the neuraminidase inhibitor, oseltamivir, against probenecid, which targets the host cells and is less likely to engender resistance. Our results show that probenecid has superior antiviral efficacy compared to oseltamivir in both in vitro replication assays and in vivo mouse models of influenza infection.

2116. Efficacy of CD388, a Novel Drug Fc-Conjugate (DFC), is Driven by the Small Molecule Neuraminidase Inhibitor (NAI)

Abstract Background Influenza prevention remains a significant public health concern that is still not adequately addressed by vaccines or current therapeutic options. Cidara has developed CD388, a multivalent conjugate of a dimeric NAI with a proprietary hIgG1 Fc domain engineered for extended half-life. CD388 is in clinical development (NCT05285137 and NCT05523089) for the prevention of seasonal influenza A and B. Herein we analyze the contribution of Fc-mediated immune effector function to CD388 efficacy. Methods CD388, Fc engineered to extend PK, and closely related Fc modified analogues (immune-active ‘IA’ or immune-silent ‘IS’) were generated. CD388-IA can engage Fc gamma receptors (FcγRs) whereas IS fails to engage FcγRs required to trigger Fc-mediated immune effector functions (e.g. antibody-dependent cellular cytotoxicity). Efficacy studies were conducted in BALB/c WT or Fcer1g-/- mice. The Fcer1g-/- mice are deficient in activating FcγRs thereby excluding contribution of Fc-mediated immunity to efficacy. After lethal challenge with influenza A virus, CD388 or analogues were administered subcutaneously or intramuscularly two hours post-infection. Animals were monitored daily for 14 days or longer for survival (&amp;lt; 20% BW loss). For viral burden quantification, lungs were harvested on day 4 post-infection and viral titers were determined by plaque assay. Results CD388-IA and -IS at comparable drug to antibody ratios (DARs) of 4.5±0.5 (used in the clinical candidate) demonstrated comparable protection (Figure 1) and comparable dose-dependent viral burden and cytokine reduction in a lethal mouse model (Table 1). Additionally, the CD388-IA analogue at DAR 4.7 was protective in Fcer1g-/- mice at the same doses required for protection in WT mice. However, a CD388-IA analogue at low DAR of 1, that has reduced antiviral activity, demonstrated improved efficacy in WT mice as compared to KO mice (Figure 2).Figure 1.Efficacy of CD388-IA and CD388-IS against A/PR/8/1934 (H1N1) in a lethal mouse model in BALB/c WT mice showing survival (A) and body weight (B)Figure 2.Efficacy of CD388-IA at DAR 1 or 4.7 against A/PR/8/1934 (H1N1) in a lethal mouse model in BALB/c WT (B, D) or KO (A, C) miceTable 1.Viral burden reduction and cytokine levels in fold-change versus uninfected control for IL-6, MIP-1□ and MCP-1 on day 4 post-infection in a lethal influenza A/H1N1 mouse model. Conclusion These data combined demonstrate that the efficacy of CD388 at DAR 4.5±0.5 is driven predominantly by the intrinsic antiviral activity of the small molecule NAI and is independent of the contribution of Fc-mediated effector functions. Disclosures Simon Döhrmann, PhD, Cidara Therapeutics: Stocks/Bonds James Levin, PhD, Cidara Therapeutics: Stocks/Bonds Elizabeth Abelovski, B.S., Cidara Therapeutics: Ownership Interest Amanda Almaguer, Bachelors, Cidara Therapeutics: Stocks/Bonds Rajvir Grewal, n/a, Cidara Therapeutics: Ownership Interest Karin Amundson, BSc, Cidara Therapeutics: Stocks/Bonds Joanne Fortier, BSc, Cidara Therapeutics: Stocks/Bonds Thanh Lam, PhD, Cidara Therapeutics: Stocks/Bonds Thomas P. Brady, Ph.D., Cidara Therapeutics: Stocks/Bonds Allen Borchardt, PhD, Cidara Therapeutics: Stocks/Bonds Jason N. Cole, Ph.D., Cidara Therapeutics: Stocks/Bonds Leslie W. Tari, Ph.D., Cidara Therapeutics: Stocks/Bonds

Bioactive Pyrrolo[2,1-f][1,2,4]triazines: Synthesis, Molecular Docking, In Vitro Cytotoxicity Assay and Antiviral Studies

A series of 2,4-disubstituted pyrrolo[2,1-f][1,2,4]triazines containing both aryl and thienyl substituents were synthesized by exploiting the 1,3-cycloaddition reaction of N(1)-ethyl-1,2,4-triazinium tetrafluoroborates with dimethyl acetylenedicarboxylate. The antiviral activity of the synthesized compounds against influenza virus strain A/Puerto Rico/8/34 (H1N1) was studied in experiments on Madin-Darby canine kidney (MDCK) cell culture. Among the pyrrolo[2,1-f][1,2,4]triazine derivatives, compounds with low toxicity and high antiviral activity were identified. Dimethyl 4-(4-methoxyphenyl)-7-methyl-2-p-tolylpyrrolo[2,1-f][1,2,4]triazine-5,6-dicarboxylate was found to demonstrate the best antiviral activity (IC50 4 µg/mL and selectivity index 188). Based on the results of in vitro tests and molecular docking studies performed, a plausible mechanism of action for these compounds was suggested to involve inhibition of neuraminidase.

Molecular identification of neuraminidase gene mutations in influenza A/H1N1 and A/H3N2 isolates of Mazandaran province, north of Iran

ObjectivesThe neuraminidase (NA) mutations causing resistance to NA inhibitors (NAIs) mostly compromise the fitness of influenza viruses. Considering the importance of these mutations, constant monitoring of the effectiveness of available drugs is critical. This study aimed to identify NA mutations in the influenza A/H1N1 and A/H3N2 subtypes in the samples of Mazandaran, Iran from 2016 to 2020. MethodsIn this cross-sectional study, 20 influenza A/H1N1 and 20 influenza A/H3N2 samples were included in the study. After design of appropriate primers for NA gene, all samples subjected to RT-PCR and electrophoresis. Then the PCR product was sequenced to determine the mutations. ResultsIn the present study, no oseltamivir resistance-related mutations were detected. Still, NA gene showed variations compared to the vaccine strains. In A/H1N1, a total of 43 mutations were detected. Similarly, in A/H3N2, a total of 66 mutations were observed. In all isolates of H1N1, N200S, N248D and I321V mutations were detected in the antigenic site of NA protein, which can affect vaccine incompatibility and virus escape from the host's immune system. Also, H150R mutation was observed in the NA active site of H3N2, which is the cause of agglutination by NA protein. Also, S245N mutation was identified as a new N-Glycosylation site of H3N2 subtype. ConclusionsThe study of NA gene sequences revealed no oseltamivir resistance mutations. In H1N1 isolates, ca. 97% identities and in the H3N2 subtype, 96% identities were observed compared to reference isolate of 2009, which indicates the importance of constant monitoring of the emergence of the drug resistance mutations.

Longitudinal Analysis of Neuraminidase and Hemagglutinin Antibodies to Influenza A Viruses after Immunization with Seasonal Inactivated Influenza Vaccines.

Neuraminidase (NA)-based immunity could reduce the harmful impact of novel antigenic variants of influenza viruses. The detection of neuraminidase-inhibiting (NI) antibodies in parallel with anti-hemagglutinin (HA) antibodies may enhance research on the immunogenicity and duration of antibody responses to influenza vaccines. To assess anti-NA antibodies after vaccination with seasonal inactivated influenza vaccines, we used the enzyme-linked lectin assay, and anti-HA antibodies were detected in the hemagglutination inhibition assay. The dynamics of the anti-NA antibody response differed depending on the virus subtype: antibodies to A/H3N2 virus neuraminidase increased later than antibodies to A/H1N1pdm09 subtype neuraminidase and persisted longer. In contrast to HA antibodies, the fold increase in antibody titers to NA after vaccination poorly depended on the preexisting level. At the same time, NA antibody levels after vaccination directly correlated with titers before vaccination. A difference was found in response to NA antigen between split and subunit-adjuvanted vaccines and in NA functional activity in the vaccine formulations.

BİR İNFLUENZA VİRÜSÜ NÖRAMİNİDAZ İNHİBİTÖRÜ OLAN PERAMIVİR’İN SAPTANMASI İÇİN MOLEKÜLER BASKI POLİMER İLE ELEKTROKİMYASAL SENSÖRÜN GELİŞTİRİLMESİ

Objective: Influenza viruses are the most common cause of influenza. Antiviral drugs prevent the spread of the virus through the infected cells. Peramivir is one of the antiviral drugs that is an inhibitor of influenza virus neuraminidase. In our study, we aimed to develop a MIP-based electrochemical sensor to determine Peramivir. This study is the first to create an electrochemical sensor for Peramivir. MIP(PERA)/GCE was fabricated with the electropolymerization of 4-aminophenol (4-AP) and ortophenilendiamine (o-PD) in the presence of Peramivir. The developed MIP(PERA)/GCE was applied to the commercial serum sample for analysis of Peramivir. Material and Method: PERA is supplied by Tobio Novelpharma pharmaceutical company (İstanbul, Türkiye). Potassium ferricyanide ([K3Fe(CN)6]), potassium ferrocyanide (K4[Fe(CN)6].3H2O), and potassium chloride (KCl), 4-aminophenol (4-AP) and ortophenilendiamine (o-PD), commercial human serum sample, dopamine, ascorbic acid, uric acid, paracetamol, KNO3, Na2SO4 ve MgCl2 were obtained from Sigma-Aldrich (St. Louis, Missouri, USA). Methanol, acetic acid, oxalic acid, hydrochloric acid, acetonitrile, sodium hydroxide, and ethanol were purchased by Merck. The redox process was investigated using cyclic voltammetry (CV) and differential pulse voltammetry (DPV) by the Dropsens μStat 400 Bipotantiostat/ Galvanostat (Metrohm, Herisau, Switzerland). Three-electrode systems consisted of a glassy carbon working electrode (GCE, 3mm2, BASi, USA), a saturated Ag/AgCl reference electrode, and a Pt wire. Result and Discussion: The sensor exhibits a linear range of 1-10 pM with a detection limit of 0.158 pM. The MIP(PERA)/GCE sensor could detect PERA from commercial serum samples with a high recovery of 101.81%.

Screening and identification of neuraminidase inhibitors from Baphicacanthus cusia by a combination of affinity ultrafiltration, HPLC-MS/MS, molecular docking, and fluorescent techniques

Natural products provide a new opportunity for the discovery of neuraminidase (NA)inhibitors. In this study, an affinity ultrafiltration (AUF) coupled with HPLC-MS/MS method was firstly developed and optimized for screening of NA inhibitors from natural products. The critical factors influencing the interaction of enzyme-ligand (including sample concentration, enzyme concentration, incubation time and temperature, pH of the buffer, and dissociation solvents and time) were investigated and optimized by a one-factor-at-a-time design. The method was then applied to discover NA inhibitory compounds in stems and leaves of Baphicacanthus cusia. As a result, five active alkaloids were screened out and identifiedas 2,4(1H,3H)-quinazolinedione (1), 4(3H)-quinazolinone (2), 2(3H)-benzoxazolone (3), tryptanthrin (4), and indirubin (5) through analysis of their DAD profiles, MS/MS fragments, and comparison with reference substances. These active compounds were further evaluated for their NA inhibitory activity using a fluorescence-based NA inhibition assay. The result from the fluorescent assay revealed that all the five compounds(1–5) showed pronounced NA inhibitory activities with IC50values of 98.98, 64.69, 40.16, 69.44, and 144.73 μM, respectively. Finally, molecular docking of these five alkaloids with NA showed that hydrogen bond and π-cation interactions dominated within the binding sites with binding energies ranging between −5.7 to −7.9 kcal/mol, which was supported by the results of the AUF and the fluorescence-based enzyme assay. The developed AUF method is simple and efficient for screening potential NA inhibitors from stems and leaves of B. cusia.

A novel N-heterocycles substituted oseltamivir derivatives as potent inhibitors of influenza virus neuraminidase: discovery, synthesis and biological evaluation

Our previous studies have shown that the introduction of structurally diverse benzyl side chains at the C5-NH2 position of oseltamivir to occupy 150-cavity contributes to the binding affinity with neuraminidase and anti-influenza activity. To obtain broad-spectrum neuraminidase inhibitors, we designed and synthesised a series of novel oseltamivir derivatives bearing different N-heterocycles substituents that have been proved to induce opening of the 150-loop of group-2 neuraminidases. Among them, compound 6k bearing 4-((r)-2-methylpyrrolidin-1-yl) benzyl group exhibited antiviral activities similar to or weaker than those of oseltamivir carboxylate against H1N1, H3N2, H5N1, H5N6 and H5N1-H274Y mutant neuraminidases. More encouragingly, 6k displayed nearly 3-fold activity enhancement against H3N2 virus over oseltamivir carboxylate and 2-fold activity enhancement over zanamivir. Molecular docking studies provided insights into the explanation of its broad-spectrum potency against wild-type neuraminidases. Overall, as a promising lead compound, 6k deserves further optimisation by fully considering the ligand induced flexibility of the 150-loop.

Drift and shape-new insights into human immunity against influenza virus neuraminidase.

Influenza virus hemagglutinin mediates infection by binding sialic acids, whereas neuraminidase cleaves sialic acids to release progeny virions. Both are targets of protective antibodies, but influenza vaccine strain selection and antigen dose are based on hemagglutinin alone. Virus characterization using first infection ferret sera indicates that escape from hemagglutination inhibiting (HI) antibodies occurs more frequently and is not coordinated with escape from neuraminidase inhibiting (NI) antibodies. A key question addressed by Daulagala et al. (P. Daulagala, B. R. Mann, K. Leung, E. H. Y. Lau, et al., mBio 14:e00084-23, 2023, https://doi.org/10.1128/mbio.00084-23) is how this translates to humans who encounter multiple influenza viruses throughout life. Their cross-sectional study, using sera from a wide age range of participants and H1N1 viruses spanning 1977-2015, indicates that NI antibodies are more broadly cross-reactive than HI antibodies. Both HI and NI titers were highest against strains encountered in childhood indicating that both are shaped by priming exposures. The study further supports the development of NA-optimized vaccines.

Structural Basis for Interactions between Influenza A Virus M2 Proton Channel and Adamantane-Based Antiviral Drugs

Influenza A virus pandemics still remain a threat to global health. One class of antiviral drugs, namely, inhibitors of the specific viral enzyme neuraminidase, is predominantly used in the fight against these pandemics. These antivirals include zanamivir (Relenza™) and oseltamivir (Tamiflu™). The viral resistance to this class of compounds steadily increases. The M2 proton channel of influenza A virus is an alternative clinically proven target for antiviral therapy. However, many circulating virus strains bear amino acid mutations in the M2 protein, causing resistance to drugs of the adamantane series, M2 blockers, such as rimantadine and amantadine. Consequently, inhibitors targeting mutants of the M2 channel are urgently needed for public biosafety and health. This review is devoted to structural-functional interactions used in practice and mediated by the action of experimental drugs on the protein target, the transmembrane domain of the influenza virus M2 proton channel. An analysis of the experimental and model structural data available in open access is presented.

Quadrivalent neuraminidase RNA particle vaccine protects pigs against homologous and heterologous strains of swine influenza virus infection

Influenza A virus in swine (IAV-S) continues to cause significant negative impact to both sows and growing pigs. The viral hemagglutinin (HA) and neuraminidase (NA) genes continue to evolve with HA diversifying at a faster rate than NA. Depending on country, whole inactivated virus (WIV) commercial and autogenous vaccines, as well as veterinary prescription vaccines targeting HA, are currently available. The use of these vaccines is focused on reducing virus and clinical signs in sows and to provide HA-specific maternally derived antibodies (MDA) to their suckling pigs. The deficiency in this strategy is that HA-MDA does not persist long enough to protect pigs through their growing phase from infection, and HA-MDA can interfere with effective pig immunization. This study evaluated the immunogenicity and efficacy of an adjuvanted, quadrivalent RNA Particle vaccine (Sequivity NA), currently licensed as Sequivity® IAV-S NA. This vaccine was formulated based on four NA antigens representing the major NA clades of IAV subtypes H1N1, H1N2 and H3N2 circulating in swine herds in the United States. In a series of trials, pigs were vaccinated twice, at three days and three weeks of age (WOA), followed by challenge with either homologous or heterologous IAV strains at 8 or 15 WOA. The Sequivity NA vaccine induced robust serum NA inhibition (NI) antibody and protected against IAV-S strains with homologous and heterologous NA to that of the vaccine. The magnitude and duration of nasal shedding was reduced in vaccinated-pigs challenged with either homologous or heterologous virus within the same NA clade. This NA-based RNA Particle vaccine avoids the known impact of HA-MDA on pig vaccination and provides a new tool to successfully reduce IAV-induced disease in the pig population.