M2 Ion Channel Blockers Research Articles

Harnessing PROTACs to combat H5N1 influenza: A new frontier in viral destruction.

H5N1, a highly pathogenic avian influenza virus, poses an ongoing and significant threat to global public health, primarily due to its potential to cause severe respiratory illness and high mortality rates in humans. Despite extensive efforts in vaccination and antiviral therapy, H5N1 continues to exhibit high mutation rates, resulting in recurrent outbreaks and the emergence of drug-resistant strains. Traditional antiviral therapies, such as neuraminidase inhibitors and M2 ion channel blockers, have demonstrated limited efficacy, necessitating the exploration of innovative therapeutic strategies. Proteolysis-targeting chimeras (PROTACs) emerge as a novel and promising approach, leveraging the ubiquitin-proteasome system to selectively degrade pathogenic proteins. Unlike conventional inhibitors that only block protein function, PROTACs eliminate the target protein, providing a sustained therapeutic effect and potentially reducing the development of resistance. This paper offers a comprehensive examination of the current landscape of H5N1 infections, detailing the pathogenesis and challenges associated with existing treatments. It further explores the mechanism of action, design, and therapeutic potential of PROTACs in inhibiting H5N1. By targeting essential viral proteins, such as hemagglutinin and the RNA-dependent RNA polymerase complex, PROTACs hold the potential to revolutionize the treatment of H5N1 infections, offering a new frontier in antiviral therapy.

Antiviral Effects of ABMA and DABMA against Influenza Virus In Vitro and In Vivo via Regulating the Endolysosomal Pathway and Autophagy.

Influenza virus is an acute and highly contagious respiratory pathogen that causes great concern to public health and for which there is a need for extensive drug discovery. The small chemical compound ABMA and its analog DABMA, containing an adamantane or a dimethyl-adamantane group, respectively, have been demonstrated to inhibit multiple toxins (diphtheria toxin, Clostridium difficile toxin B, Clostridium sordellii lethal toxin) and viruses (Ebola, rabies virus, HSV-2) by acting on the host’s vesicle trafficking. Here, we showed that ABMA and DABMA have antiviral effects against both amantadine-sensitive influenza virus subtypes (H1N1 and H3N2), amantadine-resistant subtypes (H3N2), and influenza B virus with EC50 values ranging from 2.83 to 7.36 µM (ABMA) and 1.82 to 6.73 µM (DABMA), respectively. ABMA and DABMA inhibited the replication of influenza virus genomic RNA and protein synthesis by interfering with the entry stage of the virus. Molecular docking evaluation together with activity against amantadine-resistant influenza virus strains suggested that ABMA and DABMA were not acting as M2 ion channel blockers. Subsequently, we found that early internalized H1N1 virions were retained in accumulated late endosome compartments after ABMA treatment. Additionally, ABMA disrupted the early stages of the H1N1 life cycle or viral RNA synthesis by interfering with autophagy. ABMA and DABMA protected mice from an intranasal H1N1 challenge with an improved survival rate of 67%. The present study suggests that ABMA and DABMA are potential antiviral leads for the development of a host-directed treatment against influenza virus infection.

Advancements in Host-Based Interventions for Influenza Treatment.

Influenza is a major acute respiratory infection that causes mortality and morbidity worldwide. Two classes of conventional antivirals, M2 ion channel blockers and neuraminidase inhibitors, are mainstays in managing influenza disease to lessen symptoms while minimizing hospitalization and death in patients with severe influenza. However, the development of viral resistance to both drug classes has become a major public health concern. Vaccines are prophylaxis mainstays but are limited in efficacy due to the difficulty in matching predicted dominant viral strains to circulating strains. As such, other potential interventions are being explored. Since viruses rely on host cellular functions to replicate, recent therapeutic developments focus on targeting host factors involved in virus replication. Besides controlling virus replication, potential targets for drug development include controlling virus-induced host immune responses such as the recently suggested involvement of innate lymphoid cells and NADPH oxidases in influenza virus pathogenesis and immune cell metabolism. In this review, we will discuss the advancements in novel host-based interventions for treating influenza disease.

Second-Generation Nitazoxanide Derivatives: Thiazolides are Effective Inhibitors of the Influenza a Virus

The only small molecule drugs currently available for treatment of influenza A virus (IAV) are M2 ion channel blockers and sialidase inhibitors. The prototype thiazolide, nitazoxanide, has successfully completed Phase III clinical trials against acute uncomplicated influenza. We report the activity of seventeen thiazolide analogs against A/PuertoRico/8/1934(H1N1), a laboratory-adapted strain of the H1N1 subtype of IAV, in a cell culture-based assay. A total of eight analogs showed IC50s in the range of 0.14-5.0μM. Additionally a quantitative structure-property relationship study showed high correlation between experimental and predicted activity based on a molecular descriptor set. A range of thiazolides show useful activity against an H1N1 strain of IAV. Further evaluation of these molecules as potential new small molecule therapies is justified.

Synthesis and Anti-Influenza Activity of Pyridine, Pyridazine, and Pyrimidine C-Nucleosides as Favipiravir (T-705) Analogues.

Influenza viruses are responsible for seasonal epidemics and occasional pandemics which cause significant morbidity and mortality. Despite available vaccines, only partial protection is achieved. Currently, there are two classes of widely approved anti-influenza drugs: M2 ion channel blockers and neuraminidase inhibitors. However, the worldwide spread of drug-resistant influenza strains poses an urgent need for novel antiviral drugs, particularly with a different mechanism of action. Favipiravir (T-705), a broad-spectrum antiviral agent, has shown potent anti-influenza activity in cell-based assays, and its riboside (2) triphosphate inhibited influenza polymerase. In one of our approaches to treat influenza infection, we designed, prepared, and tested a series of C-nucleoside analogues, which have an analogy to 2 and were expected to act by a similar antiviral mechanism as favipiravir. Compound 3c of this report exhibited potent inhibition of influenza virus replication in MDCK cells, and its triphosphate was a substrate of and demonstrated inhibitory activity against influenza A polymerase. Metabolites of 3c are also presented.

Mutations in influenza A virus during amantadine-oseltamivir combination therapy

Although two classes of antiviral drugs, M2 ion channel blockers and neuraminidase (NA) inhibitors, are available to combat seasonal H1N1 and H3N2, and highly pathogenic avian (e.g., H5N1) influenza, the emergence and spread of drug-resistant viruses is of great concern. Animal studies suggest that combination chemotherapy with M2 and NA inhibitors may be an effective option to reduce the emergence of drug-resistant viruses. In this study, we evaluated the antiviral susceptibility of clinical isolates from immunocompetent children infected with a seasonal influenza A virus and treated with a combination of amantadine and oseltamivir. We found that amantadine-resistant viruses emerged during this combination treatment. While viruses with mutations known to confer oseltamivir resistance were not detected, we found viruses with mutations in NA that reduced sialidase activity and viruses with hemagglutinin mutations. These findings suggest that while the amantadine-oseltamivir combination is an effective therapeutic option for influenza, drug-resistant viruses do appear with this combination therapy.

Screening and identification of inhibitors against influenza A virus from a US drug collection of 1280 drugs

Infection with influenza A virus is still a global concern since it causes significant mortality, morbidity and economic loss. New burst pandemics and rapid emergence of drug-resistance strains in recent years call for novel antiviral therapies. One promising way to overcome this problem is searching new inhibitors among thousands of drugs approved in the clinic for the treatment of different diseases or approved to be safe by clinical trials. In the present work, a collection of 1280 compounds, most of which have been clinically used in human or animal, were screened for anti-influenza activity and 41 hits (SI>4.0) were obtained. Next the 18 hit compounds with SI >10.0 were tested for antiviral activity against 7 other influenza virus strains in canine-originated MDCK cells, 9 compounds exhibited broad antiviral spectrum. The antiviral effects of the 9 compounds were also confirmed in human-originated A549 cells and chicken-originated DF1cells, by infectious virus yield reduction assay and indirect immunofluorescent assay. Results from the time of addition assay showed that the 9 candidates impaired different stages of influenza virus life cycle, indicating they are novel inhibitors with different mechanisms compared with the existing M2 ion-channel blockers or neuraminidase (NA) inhibitors. Taken together, our findings provide 9 novel drug candidates for the treatment of influenza virus infection. Further mechanism of action study of these inhibitors may lead to the discovery of new anti-influenza targets and structure–activity relationship (SAR) study can be initiated to improve the efficacy of these new classes of influenza inhibitors.

Antiviral strategies against influenza virus: towards new therapeutic approaches.

Influenza viruses are major human pathogens responsible for respiratory diseases affecting millions of people worldwide and characterized by high morbidity and significant mortality. Influenza infections can be controlled by vaccination and antiviral drugs. However, vaccines need annual updating and give limited protection. Only two classes of drugs are currently approved for the treatment of influenza: M2 ion channel blockers and neuraminidase inhibitors. However, they are often associated with limited efficacy and adverse side effects. In addition, the currently available drugs suffer from rapid and extensive emergence of drug resistance. All this highlights the urgent need for developing new antiviral strategies with novel mechanisms of action and with reduced drug resistance potential. Several new classes of antiviral agents targeting viral replication mechanisms or cellular proteins/processes are under development. This review gives an overview of novel strategies targeting the virus and/or the host cell for counteracting influenza virus infection.

Emergence of a novel drug resistant H7N9 influenza virus: evidence based clinical potential of a natural IFN-α for infection control and treatment

The novel avian H7N9 influenza virus has caused more than 130 human infections with 43 deaths (as of September, 2013) in China. Because of the lack of existing immunity against H7 subtype influenza viruses in the human population and the absence of a licensed commercial vaccine, antiviral drugs are critical tools for the treatment of infection with this novel H7N9. Both M2-ion channel blockers and neuraminidase inhibitors are used as antiviral drugs for influenza infections of humans. The emerging H7N9 viruses are resistant to the M2-ion channel blockers because of a S31N mutation in the M2 protein; additionally, some H7N9 isolates have gained neuraminidase R292K substitution resulting in broad resistance to neuraminidase inhibitors. In this study we report that Alferon N can inhibit wild type and 292K H7N9 viruses replication in vitro. Since Alferon N is approved for clinical use, this would allow a rapid regulatory approval process for this drug under pandemic threat.

Inhibitory and combinatorial effect of diphyllin, a v-ATPase blocker, on influenza viruses

An influenza pandemic poses a serious threat to humans and animals. Conventional treatments against influenza include two classes of pathogen-targeting antivirals: M2 ion channel blockers (such as amantadine) and neuraminidase inhibitors (such as oseltamivir). Examination of the mechanism of influenza viral infection has shown that endosomal acidification plays a major role in facilitating the fusion between viral and endosomal membranes. This pathway has led to investigations on vacuolar ATPase (v-ATPase) activity, whose role as a regulating factor on influenza virus replication has been verified in extensive genome-wide screenings. Blocking v-ATPase activity thus presents the opportunity to interfere with influenza viral infection by preventing the pH-dependent membrane fusion between endosomes and virions. This study aims to apply diphyllin, a natural compound shown to be as a novel v-ATPase inhibitor, as a potential antiviral for various influenza virus strains using cell-based assays. The results show that diphyllin alters cellular susceptibility to influenza viruses through the inhibition of endosomal acidification, thus interfering with downstream virus replication, including that of known drug-resistant strains. In addition, combinatorial treatment of the host-targeting diphyllin with pathogen-targeting therapeutics (oseltamivir and amantadine) demonstrates enhanced antiviral effects and cell protection in vitro.

Guidance for clinical and public health laboratories testing for influenza virus antiviral drug susceptibility in Europe

Two classes of antiviral drugs are licensed in Europe for treatment and prophylaxis of influenza; the M2 ion-channel blockers amantadine and rimantadine acting against type A influenza viruses only and the neuraminidase enzyme inhibitors zanamivir and oseltamivir acting against type A and type B influenza viruses.This guidance document was developed for but not limited to the European Union (EU) and other European Economic Area (EEA) countries on how and when to test for influenza virus antiviral drug susceptibility. It is aimed at clinical and influenza surveillance laboratories carrying out antiviral drug susceptibility testing on influenza viruses from patients suspected of harbouring viruses with reduced susceptibility or for the monitoring of the emergence of such among circulating viruses, respectively. Therefore, the guidance should not be read as a directive or an algorithm for treatment.Monitoring for emergence of influenza viruses with reduced drug susceptibility in hospitalized cases is crucial for decision making on possible changes to antiviral treatment. Therefore, it is important to test for antiviral susceptibility in certain patient groups, such as patients treated with influenza antiviral drugs. It is also important to determine the frequency of viruses with natural (not related to drug use) reduced susceptibility among community and hospitalized cases, as this knowledge is essential for making empirical antiviral treatment decisions. Furthermore, testing of specimens from community influenza patients is needed to determine the frequency of viruses with reduced susceptibility and good viral fitness that are readily transmissible, as they may become dominant among circulating viruses.Phenotypic neuraminidase enzyme inhibition assays are recommended to determine the level of inhibition of the neuraminidase enzyme by antiviral drugs as a measure of drug susceptibility of the virus. Genotypic assays are recommended to identify amino acid substitutions in the neuraminidase and M2 ion-channel proteins that have been associated with reduced antiviral susceptibility previously. By 2012 all circulating seasonal influenza A(H1N1)pdm09 and A(H3N2) viruses were naturally resistant to the M2 ion-channel blockers, so priority should be given to testing for neuraminidase inhibitor susceptibility.

A cell-based screening system for influenza A viral RNA transcription/replication inhibitors

Although two classes of antivirals, NA inhibitors and M2 ion channel blockers, are licensed for influenza treatment, dual resistant mutants, including highly pathogenic H5N1 viruses, have appeared. Alternative treatment options are, therefore, needed. Influenza A viral RNA (vRNA) transcription/replication is a promising target for antiviral development, since it is essential for virus replication. Accordingly, an efficient and reliable method to identify vRNA transcription/replication inhibitors is desirable. Here, we developed a cell-based screening system by establishing a cell line that stably expresses influenza viral ribonucleoprotein complex (vRNP). Compound library screening using this cell line allowed us to identify a compound that inhibits vRNA transcription/replication by using reporter protein expression from virus-like RNA as a readout and virus replication in vitro. vRNP-expressing cells have potential as a simple and convenient high-throughput screening (HTS) system, and, thus, are promising to identify vRNA transcription/replication inhibitors for various RNA viruses, especially for primary screens.

Combination Chemotherapy for Influenza

The emergence of pandemic H1N1 influenza viruses in April 2009 and the continuous evolution of highly pathogenic H5N1 influenza viruses underscore the urgency of novel approaches to chemotherapy for human influenza infection. Anti-influenza drugs are currently limited to the neuraminidase inhibitors (oseltamivir and zanamivir) and to M2 ion channel blockers (amantadine and rimantadine), although resistance to the latter class develops rapidly. Potential targets for the development of new anti-influenza agents include the viral polymerase (and endonuclease), the hemagglutinin, and the non-structural protein NS1. The limitations of monotherapy and the emergence of drug-resistant variants make combination chemotherapy the logical therapeutic option. Here we review the experimental data on combination chemotherapy with currently available agents and the development of new agents and therapy targets.

5'PPP-RNA induced RIG-I activation inhibits drug-resistant avian H5N1 as well as 1918 and 2009 pandemic influenza virus replication

BackgroundEmergence of drug-resistant strains of influenza viruses, including avian H5N1 with pandemic potential, 1918 and 2009 A/H1N1 pandemic viruses to currently used antiviral agents, neuraminidase inhibitors and M2 Ion channel blockers, underscores the importance of developing novel antiviral strategies. Activation of innate immune pathogen sensor Retinoic Acid Inducible Gene-I (RIG-I) has recently been shown to induce antiviral state.ResultsIn the present investigation, using real time RT-PCR, immunofluorescence, immunoblot, and plaque assay we show that 5'PPP-containing single stranded RNA (5'PPP-RNA), a ligand for the intracytoplasmic RNA sensor, RIG-I can be used as a prophylactic agent against known drug-resistant avian H5N1 and pandemic influenza viruses. 5'PPP-RNA treatment of human lung epithelial cells inhibited replication of drug-resistant avian H5N1 as well as 1918 and 2009 pandemic influenza viruses in a RIG-I and type 1 interferon dependant manner. Additionally, 5'PPP-RNA treatment also inhibited 2009 H1N1 viral replication in vivo in mice.ConclusionsOur findings suggest that 5'PPP-RNA mediated activation of RIG-I can suppress replication of influenza viruses irrespective of their genetic make-up, pathogenicity, and drug-sensitivity status.

Current and future antiviral therapy of severe seasonal and avian influenza

The currently circulating H3N2 and H1N1 subtypes of influenza A virus cause a transient, febrile upper respiratory illness in most adults and children (“seasonal influenza”), but infants, the elderly, immunodeficient and chronically ill persons may develop life-threatening primary viral pneumonia or complications such as bacterial pneumonia. By contrast, avian influenza viruses such as the H5N1 virus that recently emerged in Southeast Asia can cause severe disease when transferred from domestic poultry to previously healthy people (“avian influenza”). Most H5N1 patients present with fever, cough and shortness of breath that progress rapidly to adult respiratory distress syndrome. In seasonal influenza, viral replication remains confined to the respiratory tract, but limited studies indicate that H5N1 infections are characterized by systemic viral dissemination, high cytokine levels and multiorgan failure. Gastrointestinal infection and encephalitis also occur. The licensed anti-influenza drugs (the M2 ion channel blockers, amantadine and rimantadine, and the neuraminidase inhibitors, oseltamivir and zanamivir) are beneficial for uncomplicated seasonal influenza, but appropriate dosing regimens for severe seasonal or H5N1 viral infections have not been defined. Treatment options may be limited by the rapid emergence of drug-resistant viruses. Ribavirin has also been used to a limited extent to treat influenza. This article reviews licensed drugs and treatments under development, including high-dose oseltamivir; parenterally administered neuraminidase inhibitors, peramivir and zanamivir; dimeric forms of zanamivir; the RNA polymerase inhibitor T-705; a ribavirin prodrug, viramidine; polyvalent and monoclonal antibodies; and combination therapies.

Strategies of Development of Antiviral Agents Directed Against Influenza Virus Replication

In this review, we will discuss drug design based on proven and potential anti-influenza drug targets including viral hemagglutinin (HA), neuraminidase (NA), M2 ion channel, 3P polymerase complex, and host factors such as kinases. We have summarized influenza inhibitors based on their mode of actions. For instance, included are descriptions of (1) inhibitors of HA cleavage, such as nafamostat, camostat, gabexate, epsilon-aminocapronic acid and aprotinin, (2) inhibitors of fusion and entry, such as benzoquinones and hydroquinones, CL 385319, BMY-27709, stachyflin, and their analogues, (3) inhibitors of viral RNPs/polymerase/endonuclease, such as T-705, L-735,822, flutimide and their analogues, (4) inhibitors of MEK, such as PD 0325901, CI-1040 and ARRY-142886, and (5) inhibitors of NA such as DANA, FANA, zanamivir, and oseltamivir, etc. Although amantadine and rimantadine are not recommended for treating influenza virus infections because of drug resistance problem, these viral M2 ion channel blockers established a proof-of-concept that the endocytosis of virion into host cells can be a valid drug target because M2 protein is involved in the endocytosis process. The influenza polymerase complex not only catalyzes RNA polymerization but also encodes the "cap snatching" activity. After being exported from the nucleus to the cytoplasm, the newly synthesized vRNPs are assembled into virions at the plasma membrane. The progeny virions will then leave the host cells through the action of NA. The strategies for discovery of small molecule inhibitors of influenza virus replication based on each particular mechanism will be discussed. Finally, the lessons learned from the design of NA inhibitors (NAI) are also included. Many exciting opportunities await the cadre of virologists, medicinal chemists, and pharmacologists to design novel influenza drugs with favorable pharmacological and pharmacokinetic properties to combat this threatening infectious disease.

Antiviral therapy for influenza : a clinical and economic comparative review.

Each year influenza epidemics cause a considerable burden of disease. Vaccination against influenza A and B viruses has been and remains the cornerstone of influenza prevention, but antiviral therapy can serve as an important adjunct to vaccination in controlling the impact of the disease. Two classes of drugs are currently licensed in a large number of countries for the treatment of influenza. The M2 ion channel blockers or amantadanes (amantadine and rimantadine) are specific inhibitors of influenza A virus replication, whereas the neuraminidase inhibitors (zanamivir and oseltamivir) are active against influenza A and B viruses. Readily transmissible drug-resistant viruses develop frequently during amantadane treatment but not during neuraminidase inhibitor treatment. In this review, efficacy and safety data from randomised controlled trials are evaluated to gain an understanding of what we can and cannot expect from antiviral treatment. All four drugs shorten the course of influenza disease by approximately 1 day and relieve symptoms to some extent, but there is still uncertainty as to whether antiviral therapy leads to a reduction of serious complications and hospitalisation. The results of cost-effectiveness analyses are very diverse, in part because of differences in methodology but also because there is no consensus on what probabilities to assign to the key risks and benefits that form the basis of these studies. Consensus statements by advisory bodies in England and Germany recommend neuraminidase inhibitors for the therapy of influenza in high-risk individuals such as people over 65 years or under 2 years, and individuals with chronic cardiovascular, pulmonary or renal disease, diabetes mellitus or immunosuppression. However, there is no agreement as to whether antiviral therapy can be generally recommended for otherwise healthy children and adults. The availability of safe and effective antiviral therapy options should be kept in mind by the practising clinician, while more specific recommendations and policy formulation will depend on additional efficacy data that include frequency of complications and hospitalisation as outcome measures.