Influenza Virus Neuraminidase Research Articles

Structural and functional characterization of avian influenza H9N2 virus neuraminidase with a combination of five novel mutations

The influenza virus neuraminidase (NA) protein is responsible for actively cleaving the sialic acid (SA) bound to the viral hemagglutinin. In the present study we identified a combination of five novel amino acid substitutions in the NA, conferring increased substrate binding and altered surface characteristics to a low pathogenic avian influenza (LPAI) H9N2 strain. The H9N2 virus strain reported from India, namely, A/Environmental/India/1726265/2017 (H9N2-1726265) showed the combination of amino acid substitutions T149I, R249W, G346A, W403R and G435R, which were in the vicinity of the enzyme active site cavity. The strain A/chicken/India/99321/2009 (H9N2-99321) did not show these substitutions and was used for comparison. Virus elution was studied using turkey red blood cells (tRBCs). NA enzyme kinetics assays were carried out using the MUNANA substrate, which is a SA analogue. Homology modelling and molecular docking were performed to determine alterations in the surface characteristics and substrate binding. H9N2-1726265 showed enhanced elution from turkey red blood cells. Enzyme kinetics revealed a lower KM of H9N2-1726265 (111.5 μM) as compared to H9N2-99321 (135.2 μM), indicating higher substrate binding affinity of H9N2-1726265 due to which the NA enzyme cleaved the SA more efficiently, leading to faster elution. Molecular docking revealed a greater number of binding interactions of H9N2-1726265 to SA as compared to H9N2-99321 which explained the greater substrate binding affinity. Changes in the surface charge, hydrophobicity, and contour, were observed in H9N2-1726265 NA due to the substitutions. Thus, the novel combination of five amino acids near the sialic acid binding site of NA, resulted in altered surface characteristics, higher substrate binding affinity and virus elution.

Protective human monoclonal antibodies target conserved sites of vulnerability on the underside of influenza virus neuraminidase

Continuously evolving influenza viruses cause seasonal epidemics and pose global pandemic threats. Although viral neuraminidase (NA) is an effective drug and vaccine target, our understanding of the NA antigenic landscape still remains incomplete. Here, we describe NA-specific human antibodies that target the underside of the NA globular head domain, inhibit viral propagation of a wide range of human H3N2, swine-origin variant H3N2, and H2N2 viruses, and confer both pre- and post-exposure protection against lethalH3N2 infection in mice. Cryo-EM structures of two such antibodies in complex with NA reveal non-overlapping epitopes covering the underside of the NA head. These sites are highly conserved among N2 NAs yet inaccessible unless the NA head tilts or dissociates. Our findings help guide the development of effective countermeasures against ever-changing influenza viruses by identifying hidden conserved sites of vulnerability on the NA underside.

Applied Research Note: Development and validation of a highly specific polyclonal antibody targeting neuraminidase of novel H3N8 avian influenza virus

Applied Research Note: Development and validation of a highly specific polyclonal antibody targeting neuraminidase of novel H3N8 avian influenza virus

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%.

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.

Immunity induced by vaccination with recombinant influenza B virus neuraminidase protein breaks viral transmission chains in guinea pigs in an exposure intensity-dependent manner.

Vaccines that can slow respiratory virus transmission in the population are urgently needed for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and influenza virus. Here, we describe how a recombinant neuraminidase-based influenza virus vaccine reduces transmission in vaccinated guinea pigs in an exposure intensity-based manner.

New insights into the neuraminidase-mediated hemagglutination activity of influenza A(H3N2) viruses

Influenza virus neuraminidase (NA) can act as a receptor-binding protein, a role commonly attributed to hemagglutinin (HA). In influenza A(H3N2) viruses, three NA amino acid residues have previously been associated with NA-mediated hemagglutination: T148, D151, and more recently, H150. These residues are part of the 150-loop of the NA monomer. Substitutions at 148 and 151 arise from virus propagation in laboratory cell cultures, whereas changes at 150 occurred during virus evolution in the human host. In this study, we examined the effect of natural amino acid polymorphism at position 150 on NA-mediated hemagglutination. Using the A/Puerto Rico/8/34 backbone, we generated a comprehensive panel of recombinant A(H3N2) viruses that have different NAs but shared an HA that displays poor binding to red blood cells (RBCs). None of the tested substitutions at 150 (C, H, L, R, and S) promoted NA-binding. However, we identified two new determinants of NA-binding, Q136K and T439R, that emerged during virus culturing. Similar to T148I, both Q136K and T439R reduced NA enzyme activity by 48–86% and inhibition (14- to 173-fold) by the NA inhibitor zanamivir. NA-binding was observed when a virus preparation contained approximately 10% of NA variants with either T148I or T439R, highlighting the benefit of using deep sequencing in virus characterization. Taken together, our findings provide new insights into the molecular mechanisms underlying the ability of NA to function as a binding protein. Information gained may aid in the design of new and improved NA-targeting antivirals.

Novel mono- and multivalent N-acetylneuraminic acid glycoclusters as potential broad-spectrum entry inhibitors for influenza and coronavirus infection

N-acetylneuraminic acid (Neu5Ac) is a glycan receptor of viruses spread in many eukaryotic cells. The present work aimed to design, synthesis and biological evaluation of a panel of Neu5Ac derivatives based on a cyclodextrin (CD) scaffold for targeting influenza and coronavirus membrane proteins. The multivalent Neu5Ac glycoclusters efficiently inhibited chicken erythrocyte agglutination induced by intact influenza virus in a Neu5Ac density–dependent fashion. Compared with inhibition by Neu5Ac, the multivalent inhibitor with 21 Neu5Ac residues on the primary face of the β-CD scaffold afforded 1788-fold higher binding affinity inhibition for influenza virus hemagglutinin with a dissociation constant (KD) of 3.87 × 10−7 M. It showed moderate binding affinity to influenza virus neuraminidase, but with only about one-thirtieth the potency of that with the HA protein. It also exhibited strong binding affinity to the spike protein of three human coronaviruses (severe acute respiratory syndrome coronavirus, Middle East respiratory syndrome coronavirus, and severe acute respiratory syndrome coronavirus 2), with KD values in the low micromolar range, which is about 10-time weaker than that of HA. Therefore, these multivalent sialylated CD derivatives have possible therapeutic application as broad-spectrum antiviral entry inhibitors for many viruses by targeting the Neu5Ac of host cells.

Identification of Anti-Influenza A Compounds Inhibiting the Viral Non-Structural Protein 1 (NS1) Using a Type I Interferon-Driven Screening Strategy.

There is an urgent need to identify efficient antiviral compounds to combat existing and emerging RNA virus infections, particularly those related to seasonal and pandemic influenza outbreaks. While inhibitors of the influenza viral integral membrane proton channel protein (M2), neuraminidase (NA), and cap-dependent endonuclease are available, circulating influenza viruses acquire resistance over time. Thus, the need for the development of additional anti-influenza drugs with novel mechanisms of action exists. In the present study, a cell-based screening assay and a small molecule library were used to screen for activities that antagonized influenza A non-structural protein 1 (NS1), a highly conserved, multifunctional accessory protein that inhibits the type I interferon response against influenza. Two potential anti-influenza agents, compounds 157 and 164, were identified with anti-NS1 activity, resulting in the reduction of A/PR/8/34(H1N1) influenza A virus replication and the restoration of IFN-β expression in human lung epithelial A549 cells. A 3D pharmacophore modeling study of the active compounds provided a glimpse of the structural motifs that may contribute to anti-influenza virus activity. This screening approach is amenable to a broader analysis of small molecule compounds to inhibit other viral targets.

Highly Cross-Reactive and Protective Influenza A Virus H3N2 Hemagglutinin- and Neuraminidase-Specific Human Monoclonal Antibodies.

Due to antigenic drift and shift of influenza A viruses (IAV) and the tendency to elicit predominantly strain-specific antibodies, humanity remains susceptible to new strains of seasonal IAV and is at risk from viruses with pandemic potential for which limited or no immunity may exist. The genetic drift of H3N2 IAV is specifically pronounced, resulting in two distinct clades since 2014. Here, we demonstrate that immunization with a seasonal inactivated influenza vaccine (IIV) results in increased levels of H3N2 IAV-specific serum antibodies against hemagglutinin (HA) and neuraminidase (NA). Detailed analysis of the H3N2 B cell response indicated expansion of H3N2-specific peripheral blood plasmablasts 7 days after IIV immunization which expressed monoclonal antibodies (MAbs) with broad and potent antiviral activity against many H3N2 IAV strains as well as prophylactic and therapeutic activity in mice. These H3N2-specific B cell clonal lineages persisted in CD138+ long-lived bone marrow plasma cells. These results demonstrate that IIV-induced H3N2 human MAbs can protect and treat influenza virus infection in vivo and suggest that IIV can induce a subset of IAV H3N2-specific B cells with broad protective potential, a feature that warrants further study for universal influenza vaccine development. IMPORTANCE Influenza A virus (IAV) infections continue to cause substantial morbidity and mortality despite the availability of seasonal vaccines. The extensive genetic variability in seasonal and potentially pandemic influenza strains necessitates new vaccine strategies that can induce universal protection by focusing the immune response on generating protective antibodies against conserved targets within the influenza virus hemagglutinin and neuraminidase proteins. We have demonstrated that seasonal immunization with inactivated influenza vaccine (IIV) stimulates H3N2-specific monoclonal antibodies in humans that are broad and potent in their neutralization of virus in vitro. These antibodies also provide protection from H3N2 IAV in a mouse model of infection. Furthermore, they persist in the bone marrow, where they are expressed by long-lived antibody-producing plasma cells. This significantly demonstrates that seasonal IIV can induce a subset of H3N2-specific B cells with broad protective potential, a process that if further studied and enhanced could aid in the development of a universal influenza vaccine.

Neuraminidase 1 promotes renal fibrosis development in male mice

The functions of the influenza virus neuraminidase has been well documented but those of the mammalian neuraminidases remain less explored. Here, we characterize the role of neuraminidase 1 (NEU1) in unilateral ureteral obstruction (UUO) and folic acid (FA)-induced renal fibrosis mouse models. We find that NEU1 is significantly upregulated in the fibrotic kidneys of patients and mice. Functionally, tubular epithelial cell-specific NEU1 knockout inhibits epithelial-to-mesenchymal transition, inflammatory cytokines production, and collagen deposition in mice. Conversely, NEU1 overexpression exacerbates progressive renal fibrosis. Mechanistically, NEU1 interacts with TGFβ type I receptor ALK5 at the 160-200aa region and stabilizes ALK5 leading to SMAD2/3 activation. Salvianolic acid B, a component of Salvia miltiorrhiza, is found to strongly bind to NEU1 and effectively protect mice from renal fibrosis in a NEU1-dependent manner. Collectively, this study characterizes a promotor role for NEU1 in renal fibrosis and suggests a potential avenue of targeting NEU1 to treat kidney diseases.

Influenza virus and pneumococcal neuraminidases enhance catalysis by similar yet distinct sialic acid–binding strategies

Influenza A viruses and the bacterium Streptococcus pneumoniae (pneumococci) both express neuraminidases that catalyze release of sialic acid residues from oligosaccharides and glycoproteins. Although these respiratory pathogen neuraminidases function in a similar environment, it remains unclear if these enzymes use similar mechanisms for sialic acid cleavage. Here, we compared the enzymatic properties of neuraminidases from two influenza A subtypes (N1 and N2) and the pneumococcal strain TIGR4 (NanA, NanB, and NanC). Insect cell-produced N1 and N2 tetramers exhibited calcium-dependent activities and stabilities that varied with pH. In contrast, E.coli-produced NanA, NanB, and NanC were isolated as calcium insensitive monomers with stabilities that were more resistant to pH changes. Using a synthetic substrate (MUNANA), all neuraminidases showed similar pH optimums (pH 6-7) that were primarily defined by changes in catalytic rate rather than substrate binding affinity. Upon using a multivalent substrate (fetuin sialoglycans), much higher specific activities were observed for pneumococcal neuraminidases that contain an additional lectin domain. In virions, N1 and especially N2 also showed enhanced specific activity toward fetuin that was lost upon the addition of detergent, indicating the sialic acid-binding capacity of neighboring hemagglutinin molecules likely contributes to catalysis of natural multivalent substrates. These results demonstrate that influenza and pneumococcal neuraminidases have evolved similar yet distinct strategies to optimize their catalytic activity.

A practical and rapid screening method for influenza virus neuraminidase inhibitors based on fluorescence detection.

A new analytical method for rapid screening of influenza virus neuraminidase inhibitors was established. The method is based on the principle that, given a certain amount of neuraminidase, the sample and the neuraminidase act in the microplate for a period of time, and the active neuraminidase that is not inhibited by the sample can generate a fluorescence value at a specific wavelength after binding to the substrate, and the rate of inhibition of neuraminidase by the sample can be calculated based on the actual detected fluorescence value. This newly developed method was used to screen and evaluate the in vitro anti-neuraminidase activity of 39 high-purity compounds contained in three traditional Chinese herbal medicines, and finally 25 compounds with strong activity were obtained. The newly established neuraminidase inhibitor analytical method has these advantages of practicality, rapidity, high sensitivity and low cost, and has a good value for promotion and application. This article newly establishes a rapid, sensitive, simple and practical screening method for influenza virus neuraminidase inhibitors, which is a great complement to the existing methods and has a good promotion and application value.

Essential oil from Prunella vulgaris L. as a valuable source of bioactive constituents: In vitro anti-bacterial, anti-viral, immunoregulatory, anti-inflammatory, and chemical profiles

• We identified 56 chemical components of PVO and fatty acids are the most. • PVO had an antibacterial effect on gram-negative bacteria and gram-positive bacteria. • PVO had an antiviral effect. • PVO had a significant anti-inflammatory effect. • PVO has dual immunomodulatory effects. Our preliminary results suggested a favorable therapeutical effect of essential oil from Prunella vulgaris L. (PVO) on pelvic inflammation. In this study, we further analyzed the chemical components of PVO through gas chromatography-mass spectrometry (GC-MS) and liquid chromatography-tandem mass spectrometry (LC-MS), and its anti-bacterial, anti-inflammatory, and immunoregulatory properties. Fifty-six chemical components were identified with GC-MS, most of which were fatty acids. The positive and negative ion modes of LC-MS were used to detect 48 and 37 compounds with content above 0.1%, respectively, most of which were terpenoids and lipids. The anti-bacterial experiment revealed that PVO could strongly inhibit both gram-negative and gram-positive bacteria. It had a significant inhibiting effect on influenza virus neuraminidase, with an inhibition ratio of over 50%. Besides, PVO dramatically inhibited the NO release and the levels of IL-6 in RAW264.7 cells induced by LPS. Moreover, immunological results indicated that PVO could synergistically facilitate concanavalin A (ConA) to stimulate the secretion of Th1 and Th2 cytokines from the splenic lymphocytes of mice but also regulate the Th1/Th2 balance to exert dual immunoregulatory effects. Generally, these results proved the anti-bacterial, anti-inflammatory, and immunoregulatory effects of PVO, thus providing a reference for the application of PVO as a food or drug.

Identification and anti-viral activities of butanolide skeleton from Cinnamomum kotoense and Cinnamomum subavenium by inhibiting H5N1 neuraminidase

The human influenza virus and avian influenza virus represents a serious threat to human health. Although zanamivir and oseltamivir against influenza virus neuraminidase and thus the viral replication is not available. The oseltamivir-resistant H5N1 virus has been isolated from patient. Moreover, several cases have been reported where patients developed adverse symptoms due to a long-term use of oseltamivir. Effective antiviral drugs are essential for early control of an influenza pandemic. Natural products have been used as important resources for medicine and healthy food to improve the quality of life. Cinnamon bark is widely used as a spice and herb medicine. In this study, we have isolated and identified three butanolide derivatives, kotomolide A, isolinderanolide B and linderanolide B from Cinnamomum kotoense and Cinnamomum subavenium. Further, enzyme-based screening and anti-viral activity were also examined. Our study provides a starting point for developing new therapeutic or preventive agents from natural products for avian influenza virus.

Structural and inhibitor sensitivity analysis of influenza B–like viral neuraminidases derived from Asiatic toad and spiny eel

Influenza virus neuraminidase (NA) is an important target for antiviral development because it plays a crucial role in releasing newly assembled viruses. Two unique influenza-like virus genomes were recently reported in the Wuhan Asiatic toad and Wuhan spiny eel. Their NA genes appear to be highly divergent from all known influenza NAs, raising key questions as to whether the Asiatic toad influenza-like virus NA (tNA) and spiny eel NA (eNA) have canonical NA activities and structures and whether they show sensitivity to NA inhibitors (NAIs). Here, we found that both tNA and eNA have neuraminidase activities. A detailed structural analysis revealed that tNA and eNA present similar overall structures to currently known NAs, with a conserved calcium binding site. Inhibition assays indicated that tNA is resistant to NAIs, while eNA is still sensitive to NAIs. E119 is conserved in canonical NAs. The P119E substitution in tNA can restore sensitivity to NAIs, and, in contrast, the E119P substitution in eNA decreased its sensitivity to NAIs. The structures of NA-inhibitor complexes further provide a detailed insight into NA-inhibitor interactions at the atomic level. Moreover, tNA and eNA have unique N-glycosylation sites compared with canonical NAs. Collectively, the structural features, NA activities, and sensitivities to NAIs suggest that fish- and amphibian-derived influenza-like viruses may circulate in these vertebrates. More attention should be paid to these influenza-like viruses because their NA molecules may play roles in the emergence of NAI resistance.

Analysis and Control of Viral Infection Mechanisms by Glycobiology

Glycans are present in all living organisms, including viruses, bacteria, and animals, and perform various biological functions. The author has been studying influenza viruses' glycan usage mechanisms, particularly the functional analysis of neuraminidase (NA), a viral sialidase. The authors recently focused on influenza virus NAs' high sialidase activity with the aim of using sialidase activity detection as a virus detection technology. Using the probe BTP3-Neu5Ac, allows fluorescent imaging of sialidase activity, we created a new technique for easy, rapid, and high sensitivity fluorescent imaging of virus-infected cells. The detection of viruses using BTP3-Neu5Ac does not require specific antibodies and can be performed by simply adding reagents. Furthermore, fluorescence imaging of sialidase as a virus detection technology has many advantages, including isolating viruses from fluorescently imaged infected cells. This detection technique is easy to use in basic research and hygiene testing, where viral culture is conducted and is expected to be widely used.

Discovery of Novel Boron-Containing N-Substituted Oseltamivir Derivatives as Anti-Influenza A Virus Agents for Overcoming N1-H274Y Oseltamivir-Resistant.

To address drug resistance to influenza virus neuraminidase inhibitors (NAIs), a series of novel boron-containing N-substituted oseltamivir derivatives were designed and synthesized to target the 150-cavity of neuraminidase (NA). In NA inhibitory assays, it was found that most of the new compounds exhibited moderate inhibitory potency against the wild-type NAs. Among them, compound 2c bearing 4-(3-boronic acid benzyloxy)benzyl group displayed weaker or slightly improved activities against group-1 NAs (H1N1, H5N1, H5N8 and H5N1-H274Y) compared to that of oseltamivir carboxylate (OSC). Encouragingly, 2c showed 4.6 times greater activity than OSC toward H5N1-H274Y NA. Moreover, 2c exerted equivalent or more potent antiviral activities than OSC against H1N1, H5N1 and H5N8. Additionally, 2c demonstrated low cytotoxicity in vitro and no acute toxicity at the dose of 1000 mg/kg in mice. Molecular docking of 2c was employed to provide a possible explanation for the improved anti-H274Y NA activity, which may be due to the formation of key additional hydrogen bonds with surrounding amino acid residues, such as Arg152, Gln136 and Val149. Taken together, 2c appeared to be a promising lead compound for further optimization.

Iterative Optimization and Structure-Activity Relationship Studies of Oseltamivir Amino Derivatives as Potent and Selective Neuraminidase Inhibitors via Targeting 150-Cavity.

With our continuous endeavors in seeking neuraminidase (NA) inhibitors, we reported herein three series of novel oseltamivir amino derivatives with the goal of exploring the druggable chemical space inside the 150-cavity of influenza virus NAs. Among them, around half of the compounds in series C were demonstrated to be better inhibitors against both wild-type and oseltamivir-resistant group-1 NAs than oseltamivir carboxylate (OSC). Notably, compounds 12d, 12e, 15e, and 15i showed more potent or equipotent antiviral activity against H1N1, H5N1, and H5N8 viruses compared to OSC in cellular assays. Furthermore, compounds 12e and 15e exhibited high metabolic stability in human liver microsomes (HLMs) and low inhibitory effect on main cytochrome P450 (CYP) enzymes, as well as low acute/subacute toxicity and certain antiviral efficacy in vivo. Also, pharmacokinetic (PK) and molecular docking studies were performed. Overall, 12e and 15e possess great potential to serve as anti-influenza candidates and are worthy of further investigation.