Oseltamivir-resistant Strains Research Articles

Sodium Polyoxotungstate Inhibits the Replication of Influenza Virus by Blocking the Nuclear Import of vRNP.

Both pandemic and seasonal influenza are major health concerns, causing significant mortality and morbidity. Current influenza drugs primarily target viral neuraminidase and RNA polymerase, which are prone to drug resistance. Polyoxometalates (POMs) are metal cation clusters bridged by oxide anions. They have exhibited potent anti-tumor, antiviral, and antibacterial effects. They have remarkable activity against various DNA and RNA viruses, including human immunodeficiency virus, herpes simplex virus, hepatitis B and C viruses, dengue virus, and influenza virus. In this study, we have identified sodium polyoxotungstate (POM-1) from an ion channel inhibitor library. In vitro, POM-1 has been demonstrated to have potent antiviral activity against H1N1, H3N2, and oseltamivir-resistant H1N1 strains. POM-1 can cause virion aggregation during adsorption, as well as endocytosis. However, the aggregation is reversible; it does not interfere with virus adsorption and endocytosis. Our results suggest that POM-1 exerts its antiviral activity by inhibiting the nuclear import of viral ribonucleoprotein (vRNP). This distinct mechanism of action, combined with its wide range of efficacy, positions POM-1 as a promising therapeutic candidate for influenza treatment and warrants further investigation.

Bifunctional neuraminidase inhibitory and simultaneously anti-Mycoplasma pneumoniae of flavan-3-ols and flavanones: Combined molecular docking, virtual screening, ADMET, and synthesis prediction

After lifting COVID-19 restrictions, pneumonia caused by influenza A virus coinfection with Mycoplasma pneumoniae bacteria emerges as a matter of concern. Only a few reports have recently proposed natural compounds for treating the coinfection of influenza A virus and M. pneumoniae, especially resistant strains. Hence, this study demonstrated in silico investigation of two natural classes (flavan-3-ol and flavanone) that could become future inhibitors for bifunctional neuraminidase and anti-M. pneumoniae. Combining the molecular docking with based-virtual screening strategies for over 900 ligands with neuraminidase protein of oseltamivir-resistant virus strain at H274Y mutation (PDB ID: 3CL0), 03 flavan-3-ols and 46 flavanones interacted with both the active site and the adjacent 430-cavity, and showed lower binding energies than laninamivir (−8.1 kcal/mol). The four most suitable candidates (FL22, FN166, FN316, and ChEMBL1779463) also exhibited potential characteristics for inhibiting M. pneumoniae through some vital targets (30S Ribosome-binding factor A (PDB ID: 1PA4) and immunodominant protein P40/P90 complex (PDB ID: 6TLZ)). Furthermore, the total-synthesis strategy of ChEMBL1779463 was successfully proposed using commercial reagents and modern techniques. Our results will offer potential insights for future methods to identify biflavonoids, especially biflavanone, to simultaneously suppress the resistant strains of influenza A virus and M. pneumoniae.

Optimization of an allelic discrimination real-time RT-PCR assay for detection of H275Y oseltamivir resistance gene mutation among influenza A(H1N1)pdm09 patients from 2020 to 2022.

Influenza virus is known to cause mild to severe respiratory infections and is also prone to genetic mutations. Of all the mutations, neuraminidase (NA) gene mutations are a matter of concern, as most approved antivirals target this protein. During the 2020 influenza season, an emergence of mutation in the NA gene, affecting the binding of the World Health Organization (WHO)-recommended probes to the specific site of the NA gene, was reported by our group. As a result of this mutation, the WHO-recommended allelic discrimination real-time reverse transcriptase polymerase chain reaction (RT-PCR) assay was unable to detect wild-type (H275) or mutant oseltamivir-resistant (Y275) strains of influenza A(H1N1)pmd09 viruses. In the current study, the WHO-recommended probes were redesigned according to the mutation in the probe binding site. Fifty undetermined samples (2020-2021) from the previous study were retested with the newly designed probes and found to be positive for H275 and/or Y275. The results obtained were similar to the Sanger sequencing results from the previous study, suggesting that the redesigned probes were efficient in discriminating between wild-type and mutant-type viruses. Furthermore, 133 samples from 2022, making a total of 183 samples (2020-2022), were tested using improved allelic discrimination real-time RT-PCR, and the overall prevalence rate of oseltamivir resistance in 2020-2022 was found to be 0.54%.

Development of chalcone-like derivatives and their biological and mechanistic investigations as novel influenza nuclear export inhibitors

Concerning the emergence of resistance to current anti-influenza drugs, our previous phenotypic-based screening study identified the compound A9 as a promising lead compound. This chalcone analog, containing a 2,6-dimethoxyphenyl moiety, exhibited significant inhibitory activity against oseltamivir-resistant strains (H1N1 pdm09), with an EC50 value of 1.34 μM. However, it also displayed notable cytotoxicity, with a CC50 value of 41.46 μM. Therefore, compound A9 was selected as a prototype structure for further structural optimization in this study. Initially, it was confirmed that the substituting the α,β-unsaturated ketone with pent-1,4-diene-3-one as a linker group significantly reduced the cytotoxicity of the final compounds. Subsequently, the penta-1,4-dien-3-one group was utilized as a privileged fragment for further structural optimization. Following two subsequent rounds of optimizations, we identified compound IIB-2, which contains a 2,6-dimethoxyphenyl- and 1,4-pentadiene-3-one moieties. This compound exhibited inhibitory effects on oseltamivir-resistant strains comparable to its precursor (compound A9), while demonstrating reduced toxicity (CC50 > 100 μM). Furthermore, we investigated its mechanism of action against anti-influenza virus through immunofluorescence, Western blot, and surface plasmon resonance (SPR) experiments. The results revealed that compound IIB-2 can impede virus proliferation by blocking the export of influenza virus nucleoprotein. Thusly, our findings further emphasize influenza nuclear export as a viable target for designing novel chalcone-like derivatives with potential inhibitory properties that could be explored in future lead optimization studies.

Discovery of ligustrazine and chalcone derivatives as novel viral nucleoprotein nuclear export inhibitors against influenza viruses.

Influenza viruses pose a significant threat to human health worldwide due to seasonal epidemics and occasional global pandemics. These viruses can cause severe upper respiratory tract infections that contribute to high morbidity and mortality rates. The emergence of drug-resistant influenza viruses has created the need for the development of novel broad-spectrum antivirals. Here, we present a novel anti-influenza agent with new targets and mechanisms of action to address this problem. Our findings led to the discovery of a novel influenza virus inhibitor, a ligustrazine derivative known as A9. We have found that it exhibits broad-spectrum antiviral properties against influenza A and B viruses (IAV and IBV, respectively), including oseltamivir-resistant strain. Through multiple bioassays such as time-of-addition assay, indirect immunofluorescence assay, and nuclear-cytoplasmic fractionation assay, we demonstrated that A9 inhibits the nuclear export of the viral ribonucleoprotein (vRNP). Furthermore, escape mutant analyses and affinity studies determined by surface plasmon resonanceindicated that A9 specifically targets the nucleoprotein.In addition, four chalcone derivatives developed from A9 (B14, B29, B31, and B32), were found to effectively inhibit the replication of influenza virus through the same mechanism of action. In this manuscript we highlight A9 and its four derivatives as potential leads for the treatment of IAV and IBV infections, and their unique and novel mechanism of action probable benefit the field of anti-influenza drug discovery.

A five-day treatment course of zanamivir for the flu with a single, self-administered, painless microneedle array patch: Revolutionizing delivery of poorly membrane-permeable therapeutics

Seasonal influenza virus infections cause a substantial number of deaths each year. While zanamivir (ZAN) is efficacious against oseltamivir-resistant influenza strains, the efficacy of the drug is limited by its route of administration, oral inhalation. Herein, we present the development of a hydrogel-forming microneedle array (MA) in combination with ZAN reservoirs for treating seasonal influenza. The MA was fabricated from Gantrez® S-97 crosslinked with PEG 10,000. Various reservoir formulations included ZAN hydrate, ZAN hydrochloric acid (HCl), CarraDres™, gelatin, trehalose, and/or alginate. In vitro permeation studies with a lyophilized reservoir consisting of ZAN HCl, gelatin, and trehalose resulted in rapid and high delivery of up to 33 mg of ZAN across the skin with delivery efficiency of up to ≈75% by 24 h. Pharmacokinetics studies in rats and pigs demonstrated that a single administration of a MA in combination with a CarraDres™ ZAN HCl reservoir offered a simple and minimally invasive delivery of ZAN into the systemic circulation. In pigs, efficacious plasma and lung steady-state levels of ∼120 ng/mL were reached within 2 h and sustained between 50 and 250 ng/mL over 5 days. MA-enabled delivery of ZAN could enable a larger number of patients to be reached during an influenza outbreak.

Development of Digital Droplet PCR Targeting the Influenza H3N2 Oseltamivir-Resistant E119V Mutation and Its Performance through the Use of Reverse Genetics Mutants.

The monitoring of antiviral-resistant influenza virus strains is important for public health given the availability and use of neuraminidase inhibitors and other antivirals to treat infected patients. Naturally occurring oseltamivir-resistant seasonal H3N2 influenza virus strains often carry a glutamate-to-valine substitution at position 119 in the neuraminidase (E119V-NA). Early detection of resistant influenza viruses is important for patient management and for the rapid containment of antiviral resistance. The neuraminidase inhibition assay allows the phenotypical identification of resistant strains; however, this test often has limited sensitivity with high variability depending on the virus strain, drugs and assays. Once a mutation such as E119V-NA is known, highly sensitive PCR-based genotypic assays can be used to identify the prevalence of such mutant influenza viruses in clinical samples. In this study, based on an existing reverse transcriptase real-time PCR (RT-qPCR) assay, we developed a reverse transcriptase droplet digital PCR assay (RT-ddPCR) to detect and quantify the frequency of the E119V-NA mutation. Furthermore, reverse genetics viruses carrying this mutation were created to test the performance of the RT-ddPCR assay and compare it to the standard phenotypic NA assay. We also discuss the advantage of using an RT-ddPCR instead of qPCR method in the context of viral diagnostics and surveillance.

Preclinical Study of ZSP1273, a Potent Antiviral Inhibitor of Cap Binding to the PB2 Subunit of Influenza A Polymerase.

The influenza A virus is highly contagious and often causes global pandemics. The prevalence of strains of the influenza A virus that are resistant to approved drugs is a huge challenge for the current clinical treatment of influenza A. RNA polymerase is a pivotal enzyme in the replication of the influenza A virus, and it is a promising target for anti-influenza A therapies. In this paper, we report a novel and potent anti-influenza-A-virus inhibitor, ZSP1273, targeting the influenza A virus RNA polymerase, especially for multidrug-resistant strains. The inhibitory activity of ZSP1273 on RNA polymerase activity was 0.562 ± 0.116 nM (IC50 value), which was better than that of the clinical candidate compound VX-787 with the same target. In vitro, the EC50 values of ZSP1273 on normal influenza A virus strains (i.e., H1N1 and H3N2) varied from 0.01 nM to 0.063 nM, which were better than those of the licensed drug oseltamivir. Moreover, oseltamivir-resistant strains, baloxavir-resistant strains, and highly pathogenic avian influenza strains were also sensitive to ZSP1273. In vivo, ZSP1273 effectively reduced influenza A virus titers in a dose-dependent manner in a murine model and maintained a high survival rate in mice. In addition, the inhibitory activity of ZSP1273 on influenza A virus infection was also observed in a ferret model. Pharmacokinetic studies showed the favorable pharmacokinetic characteristics of ZSP1273 in mice, rats, and beagle dogs after single-dose and continuous multiple-dose administration. In conclusion, ZSP1273 is a highly effective anti-influenza A virus replication inhibitor, especially against multidrug-resistant strains. ZSP1273 is currently being studied in phase III clinical trials.

Rapid detection of traditional Chinese medicine with neuraminidase inhibitory activities based on high-throughput and virtual screening strategy

BackgroundOseltamivir, a neuraminidase inhibitor (NAI), is the primary and first-line anti-influenza drug. In recent years, more and more oseltamivir resistant strains appeared frequently. PurposeTo identify anti-influenza candidates to overcome oseltamivir resistance from traditional Chinese medicine. MethodsHigh-throughput screening platform was used to screen candidates with oseltamivir sensitive and resistant neuraminidase (NA) inhibitory activities from traditional Chinese medicine (TCM) and in validation of potential active components in vitro. Molecular docking is used for virtual screening of TCM compound libraries and analysis of the molecular mechanism of active compounds. ResultsSix out of 188 TCM formula granules were screened out with good inhibitory activities in oseltamivir sensitive (H5N1) and resistant NA (H274Y mutant). A compound library containing 679 candidates in the above six plants were docked into binding pockets of H5N1 NA and its H274Y mutant. The results indicated that diverse structural types, including flavonoids glycosides, alkaloids, caffeoylquinic acid derivatives, etc. showed good docking scores on the two NAs. Furthermore, 10 representative compounds tested in vitro showed inhibitory activities in both oseltamivir sensitive and resistant NA. The docking simulations revealed that natural TCM molecules bind with NA in an absolute different mode compared with oseltamivir, which occupy the extra cavities adjacent to the active site of NA, thus might make them non-susceptible to be influenced by mutation around the active sites. ConclusionsThe combination of high-throughput screening and molecular docking can efficiently screen out candidates that have the potential to overcome oseltamivir resistance from TCM.

Anti-influenza A virus activity of flavonoids in vitro: a structure-activity relationship.

Secondary plant metabolites from food extracts, namely daidzein, quercetin, and luteolin, exhibit anti-influenza virus effects, with IC50 values of 143.6, 274.8, and 8.0μM, respectively. The activities of these metabolites differ depending on the functional groups. Therefore, in this study, we focused on members of the flavonoid group, and investigated the anti-influenza viral effects of different flavonoid classes (flavone, isoflavone, flavonol, flavanone, and flavan-3-ol) in vitro. The IC50 values were 4.9-82.8μM, 143.6μM, 62.9-477.8μM, 290.4-881.1μM, and 22.9-6717.2μM, respectively, confirming their activity. The modifying group factors (number, position, type) in the flavonoid skeleton may be significantly related to the anti-influenza virus activity. Moreover, time-of-addition assay revealed that the mechanism of inhibition varied for the different classes; for example, flavonoids that inhibit virus adsorption or the early stage of viral growth. Interestingly, all the examined flavonoids inhibited the late stages of viral growth, suggesting that flavonoids mainly inhibit the late events in viral growth before the release of viral particles. Additionally, apigenin might be effective against oseltamivir-resistant strains. Our results may be important in the development of anti-influenza virus therapeutic strategies in the future.

An ultrasensitive, rapid and portable method for screening oseltamivir-resistant virus based on CRISPR/Cas12a combined with immunochromatographic strips.

Influenza is a significant public health challenge because of the emergence of antigenically shifted or highly virulent strains. The neuraminidase inhibitor oseltamivir is used as an antiviral drug in clinical treatment. However, its therapeutic effects can be greatly compromised by the emergence of drug-resistant mutant viruses. Thus, there is an urgent need to distinguish drug-resistant strains with a simple method. To address this, in the present study, we develop a rapid, sensitive and convenient molecular diagnosis method based on CRISPR/Cas12a technology and lateral flow detection (LFD). By targeting mutant sequences amplified by recombinase polymerase amplification (RPA) reaction, crRNA is designed to develop the CRISPR/Cas12a assay, and 2000 copies can be directly observed by the naked eye under blue light-emitting diode (LED) light. Combined with LFD, the limit of detection of RPA-CRISPR/Cas12a-LFD is about 20 copies of target sequence per reaction. Collectively, RPA-CRISPR/Cas12a-LFD method provides a novel alternative for the sensitive, specific and portable detection to diagnose oseltamivir-resistant mutant strains.

Urolithin M5 from the Leaves of Canarium album (Lour.) DC. Inhibits Influenza Virus by Targeting Neuraminidase.

Ganlanye (GLY), the leaf of Canarium album (Lour.) DC., is a traditional Chinese medicinal herb for warm disease treatment. We found that its aqueous extract could inhibit the influenza A virus. To find and characterize anti-influenza virus phytochemicals from GLY, we performed (1) bioassay-guided isolation, (2) a cell and animal assay, and (3) a mechanism study. Bioassay-guided isolation was used to identify the effective components. Influenza virus-infected MDCK cell and BALB/c mouse models were employed to evaluate the anti-influenza virus activities. A MUNANA assay was performed to find the NA inhibitory effect. As a result, urolithin M5 was obtained from the crude extract of GLY. It inhibited influenza virus activities in vitro and in vivo by suppressing the viral NA activity. In the MDCK cell model, urolithin M5 could inhibit an oseltamivir-resistant strain. In a PR8-infected mouse model, 200 mg/kg/d urolithin M5 protected 50% of mice from death and improved lung edema conditions. GLY was recorded as a major traditional herb for warm disease treatment. Our study identified GLY as a potent anti-influenza herb and showed urolithin M5 as the active component. We first report the in vivo activity of urolithin M5 and support the anti-influenza application of GLY.

Rosmarinic acid interferes with influenza virus A entry and replication by decreasing GSK3β and phosphorylated AKT expression levels.

The purpose of this study was to examine the invivo activity of rosmarinic acid (RA) - a phytochemical with antioxidant, anti-inflammatory, and antiviral properties - against influenza virus (IAV). An antibody-based kinase array and different invitro functional assays were also applied to identify the mechanistic underpinnings by which RA may exert its anti-IAV activity. We initially examined the potential efficacy of RA using an invivo mouse model. A time-of-addition assay and an antibody-based kinase array were subsequently applied to investigate mechanism-of-action targets for RA. The hemagglutination inhibition assay, neuraminidase inhibition assay, and cellular entry assay were also performed. RA increased survival and prevented body weight loss in IAV-infected mice. Invitro experiments revealed that RA inhibited different IAV viruses - including oseltamivir-resistant strains. From a mechanistic point of view, RA downregulated the GSK3β and Akt signaling pathways - which are known to facilitate IAV entry and replication into host cells. RA has promising preclinical efficacy against IAV, primarily by interfering with the GSK3β and Akt signaling pathways.

Binding mechanism of oseltamivir and influenza neuraminidase suggests perspectives for the design of new anti-influenza drugs.

Oseltamivir is a widely used influenza virus neuraminidase (NA) inhibitor that prevents the release of new virus particles from host cells. However, oseltamivir-resistant strains have emerged, but effective drugs against them have not yet been developed. Elucidating the binding mechanisms between NA and oseltamivir may provide valuable information for the design of new drugs against NA mutants resistant to oseltamivir. Here, we conducted large-scale (353.4 μs) free-binding molecular dynamics simulations, together with a Markov State Model and an importance-sampling algorithm, to reveal the binding process of oseltamivir and NA. Ten metastable states and five major binding pathways were identified that validated and complemented previously discovered binding pathways, including the hypothesis that oseltamivir can be transferred from the secondary sialic acid binding site to the catalytic site. The discovery of multiple new metastable states, especially the stable bound state containing a water-mediated hydrogen bond between Arg118 and oseltamivir, may provide new insights into the improvement of NA inhibitors. We anticipated the findings presented here will facilitate the development of drugs capable of combating NA mutations.

Insilico Identification of H5N1 Avian Influenza Neuraminidase Inhibitors

Background: H5N1 avian influenza virus is influenza-A virus subtype that is highly contagious and fatal among birds and humans. Although human-human transmission of H5N1 is rare, but once the virus has acquired this ability, a devastating pandemic may be inevitable. The virus is currently in WHO phase 3 pandemic alerts: A new influenza virus subtype causing disease in humans, but is yet to spread efficiently and sustainably among humans. Oseltamivir is a single most-win WHO recommended drug for the treatment and prophylaxis of H5N1 influenza virus infection. However, several oseltamivir resistant strains of H5N1 have been reported. Methods: In silico molecular docking algorithm and pharmacophore modelling simulations were used to identify a novel H5N1 avian influenza neuraminidase inhibitors. Results: Of the 518-neuraminidase analogue, five (eflornithine, eltrombopag, entecavir, aminosalicylic acid and pregabalin) demonstrated excellent binding affinities against viral neuraminidase similar to that of oseltamivir. Again, these drugs like oseltamivir interact mainly with arginine, tyrosine and glutamine at different positions of the neuraminidase. Similarly, oseltamivir and the five selected drugs superimpose to the active pocket of neuraminidase, further buttressing the previous findings. Furthermore, oseltamivir and the five drugs had common generated pharmacophores. Conclusion: This study was the first to demonstrate the anti-viral neuraminidase effect of eflornithine, eltrombopag, entecavir, aminosalicylic acid and pregabalin. Keywords: Neuraminidase, Oseltamivir, H5N1 avian influenza, Pharmacophore, molecular docking.

Discovery of oseltamivir-based novel PROTACs as degraders targeting neuraminidase to combat H1N1 influenza virus

Annual and sporadic influenza outbreaks pose a great threat to human health and the economy worldwide. Moreover, the frequent mutation of influenza viruses caused by antigen drift complicates the application of antiviral therapeutics. As such, there is an urgent need for novel antiviral agents to tackle the problem of insufficient efficacy of licensed drugs. Inspired by the success of the newly emerged PROTACs ( PRO teolysis TA rgeting C himeras) strategy, we report herein the design and synthesis of novel PROTAC molecules based on an oseltamivir scaffold to combat severe annual influenza outbreaks. Among these, several compounds showed good anti-H1N1 activity and efficient influenza neuraminidase (NA) degradation activity. The best compound, 8e , effectively induced influenza NA degradation in a dose-dependent manner and relied on the ubiquitin–proteasome pathway. Moreover, Compound 8e exhibited potent antiviral activity toward both wild-type H1N1 virus and an oseltamivir-resistant strain (H1N1, H274Y). A molecular docking study demonstrated that Compound 8e had good hydrogen-bonding and hydrophobic interactions with both the active sites of NA and Von Hippel-Lindau (VHL) proteins, which could effectively drive the favorable interaction of these two proteins. Thus, as the first report of a successful anti-influenza PROTAC, this proof of concept will greatly widen the application range of the PROTAC technique to antiviral drug discovery. • A series of oseltamivir derived PROTACs with diverse linkers and E3 ligands were synthesized. • The Compound 8e exhibited potent antiviral activity toward both wild-type H1N1 virus and an oseltamivir-resistant strain. • Mechanistic studies indicated that 8e degraded the NA protein through the ubiquitin-proteasome pathway. • This proof of concept work will greatly widen the application range of the PROTAC technique in antiviral drug discovery.

A Therapeutically Active Minibody Exhibits an Antiviral Activity in Oseltamivir-Resistant Influenza-Infected Mice via Direct Hydrolysis of Viral RNAs.

Emerging Oseltamivir-resistant influenza strains pose a critical public health threat due to antigenic shifts and drifts. We report an innovative strategy for controlling influenza A infections by use of a novel minibody of the 3D8 single chain variable fragment (scFv) showing intrinsic viral RNA hydrolyzing activity, cell penetration activity, and epidermal cell penetration ability. In this study, we examined 3D8 scFv’s antiviral activity in vitro on three different H1N1 influenza strains, one Oseltamivir-resistant (A/Korea/2785/2009pdm) strain, and two Oseltamivir-sensitive (A/PuertoRico/8/1934 and A/X-31) strains. Interestingly, the 3D8 scFv directly digested viral RNAs in the ribonucleoprotein complex. scFv’s reduction of influenza viral RNA including viral genomic RNA, complementary RNA, and messenger RNA during influenza A infection cycles indicated that this minibody targets all types of viral RNAs during the early, intermediate, and late stages of the virus’s life cycle. Moreover, we further addressed the antiviral effects of 3D8 scFv to investigate in vivo clinical outcomes of influenza-infected mice. Using both prophylactic and therapeutic treatments of intranasal administered 3D8 scFv, we found that Oseltamivir-resistant H1N1-infected mice showed 90% (prophylactic effects) and 40% (therapeutic effects) increased survival rates, respectively, compared to the control group. The pathological signs of influenza A in the lung tissues, and quantitative analyses of the virus proliferations supported the antiviral activity of the 3D8 single chain variable fragment. Taken together, these results demonstrate that 3D8 scFv has antiviral therapeutic potentials against a wide range of influenza A viruses via the direct viral RNA hydrolyzing activity.

Inhibition of Influenza Virus Replication by Oseltamivir Derivatives.

Characterized by the high morbidity and mortality and seasonal surge, the influenza virus (IV) remains a major public health challenge. Oseltamivir is commonly used as a first-line antiviral. As a neuraminidase inhibitor, it attenuates the penetration of viruses through the mucus on the respiratory tract and inhibits the release of virus progeny from infected cells. However, over the years, oseltamivir-resistant strains have been detected in the IV surveillance programs. Therefore, new antivirals that circumvent the resistant strains would be of great importance. In this study, two novel secondary amine derivatives of oseltamivir CUHK326 (6f) and CUHK392 (10i), which bear heteroaryl groups of M2-S31 proton channel inhibitors, were designed, synthesized and subjected to biological evaluation using plaque assay. Influenza A virus (A/Oklahoma/447/2008, H1N1), influenza B viruses (B/HongKong/CUHK33261/2012), an oseltamivir-resistant influenza A virus (A/HongKong/CUHK71923/2009, H1N1) and an oseltamivir-resistant influenza B virus (B/HongKong/CUHK33280/2012) were included in the antiviral effect assessment compared to oseltamivir carboxylate (OC). Both novel compounds significantly reduced the plaque size of seasonal IV A and B, and performed similarly to OC at their corresponding half-maximal inhibitory concentration (IC50). CUHK392 (10i) functioned more effectively than CUHK326 (6f). More importantly, these compounds showed an inhibitory effect on the oseltamivir-resistant strain under 10 nM with selective index (SI) of >200.

The Entry Blocker Peptide Produced in Chlamydomonas reinhardtii Inhibits Influenza Viral Replication in vitro.

This year, a respiratory virus caused an emergency pandemic alert in health services around the world, showing the need for biotechnological approaches to fight these diseases. The influenza virus is one of the main viral agents that generate pandemic outbreaks. Currently, the majority of co-circulating influenza A virus (IAV) strains are adamantine‐ and oseltamivir-resistant strains, and the challenge is to find new antivirals for more efficient treatments. The antiviral entry blocker (EB) peptide is a promising candidate for blocking the virus entry into cells. The aim of this research was to express the EB peptide in the microalgae Chlamydomonas reinhardtii and test its antiviral activity against IAV in vitro. The EB peptide nucleotide sequence was introduced into the nuclear genome of microalgae using Agrobacterium tumefaciens transformation. The EB peptide amount produced in transformed microalgae was 4.99 ± 0.067% of the total soluble protein. In hemagglutination inhibition assays using influenza A/H1N1 pdm and influenza A H1N1/Virginia/ATCC/2009 strains, we reported that the EB peptide extract from the microalgae showed 100-fold higher efficiency than the EB synthetic peptide. In addition, both the EB peptide extract and synthetic peptide inhibited viral replication in MDCK cells (IC50 = 20.7 nM and IC50 = 754.4 nM, respectively); however, the EB peptide extract showed a 32-fold higher antiviral effectiveness than the synthetic peptide against influenza A/H1N1 pdm. Extracts from untransformed and transformed microalgae and synthetic peptide did not show cytotoxic effect on MDCK cell monolayers. Thus, C. reinhardtii may be a fast, safe, and effective expression platform for production of peptides with significant antiviral activity and can be used as a prophylactic treatment to reduce viral propagation.

Inhibitory effects of aprotinin on influenza A and B viruses in vitro and in vivo

Influenza viruses cause significant morbidity and mortality worldwide. Long-term or frequent use of approved anti-influenza agents has resulted in drug-resistant strains, thereby necessitating the discovery of new drugs. In this study, we found aprotinin, a serine protease inhibitor, as an anti-influenza candidate through screening of compound libraries. Aprotinin has been previously reported to show inhibitory effects on a few influenza A virus (IAV) subtypes (e.g., seasonal H1N1 and H3N2). However, because there were no reports of its inhibitory effects on the other types of influenza viruses, we investigated the inhibitory effects of aprotinin in vitro on a wide range of influenza viruses, including avian and oseltamivir-resistant influenza virus strains. Our cell-based assay showed that aprotinin had inhibitory effects on seasonal human IAVs (H1N1 and H3N2 subtypes), avian IAVs (H5N2, H6N5, and H9N2 subtypes), an oseltamivir-resistant IAV, and a currently circulating influenza B virus. We have also confirmed its activity in mice infected with a lethal dose of influenza virus, showing a significant increase in survival rate. Our findings suggest that aprotinin has the capacity to inhibit a wide range of influenza virus subtypes and should be considered for development as a therapeutic agent against influenza.