Targeted cell ablation in zebrafish using optogenetic transcriptional control.

Cell ablation is a powerful method for elucidating the contributions of individual cell populations to embryonic development and tissue regeneration. Targeted cell loss in whole organisms has been typically achieved through expression of a cytotoxic or prodrug-activating gene product in the cell type of interest. This approach depends on the availability of tissue-specific promoters, and it does not allow further spatial selectivity within the promoter-defined region(s). To address this limitation, we have used the light-inducible GAVPO transactivator in combination with two genetically encoded cell-ablation technologies: the nitroreductase/nitrofuran system and a cytotoxic variant of the M2 ion channel. Our studies establish ablative methods that provide the tissue specificity afforded by cis-regulatory elements and the conditionality of optogenetics. Our studies also demonstrate differences between the nitroreductase and M2 systems that influence their efficacies for specific applications. Using this integrative approach, we have ablated cells in zebrafish embryos with both spatial and temporal control.

A structure and knowledge-based combinatorial approach to engineering universal scFv antibodies against influenza M2 protein.

BACKGROUND: The influenza virus enters the host via hemagglutinin protein binding to cell surface sialic acid. Receptor-mediated endocytosis is followed by viral nucleocapsid uncoating for replication aided by the transmembrane viral M2 proton ion channel. M2 ectodomain (M2e) is a potential universal candidate for monoclonal antibody therapy owing to its conserved nature across influenza virus subtypes and its importance in viral propagation. METHODS: The phage-displayed naive human antibody libraries were screened against the short stretch of the N-terminal 10-mer peptide (SLLTEVETPI) of the M2e. ELISA, BLI, and flow cytometry assays were used to examine scFv binding to M2e epitopes. The scFv crystal structures were determined to examine the nature of the interactions. The potencies of the scFvs against the influenza virus were demonstrated by real-time PCR and confocal microscopy imaging. RESULTS: The four unique scFv clones were obtained from the scFv phage-display antibody libraries and shown to exhibit binding with the 10-mer conserved part of the M2e and with full-length M2 protein expressed on the HEK293T cells. The crystal structure of scFv AU1 with M2e peptide showed the peptide as a dimer in the parallel beta-sheet conformation bound at the interface of two scFv CDRs. The scFv AU1 significantly restricted the release of H1N1 virus progeny from the infected A549 cells. CONCLUSION: This structural and biochemical study showcased the binding of antibody scFv molecules with M2e peptide dimer, providing the structural insights for the function effect in terms of recognizing and restricting the release of new viral particles from an infected host cell.

8-O-(E-p-methoxycinnamoyl)harpagide Inhibits Influenza A Virus Infection by Suppressing Intracellular Calcium.

Calcium (Ca2+) dependent signaling circuit plays a critical role in influenza A virus (IAV) infection. The 8-O-(E-p-methoxycinnamoyl)harpagide (MCH) exhibits pharmacological activities that exert neuroprotective, hepatoprotective, anti-inflammatory and other biological effects. However, not have reports of antiviral effects. To investigate the antiviral activity of MCH on IAV-infected human lung cells mediated by calcium regulation. We examined the inhibitory effect of MCH on IAV infections and measured the level of viral proteins upon MCH treatment using Western blotting. We also performed molecular docking simulation with MCH and IAV M2 protein. Finally, we analyzed MCH's suppression of intracellular calcium and ROS (reactive oxygen species) in IAV-infected human lung cells using a flow cytometer. The results shown that MCH inhibited the infection of IAV and increased the survival of the infected human lung cells. The levels of IAV protein M1, M2, NS1 and PA were inhibited in MCH-treated human lung cells compared to that in infected and untreated cells. Also, docking simulation suggest that MCH interacted with M2 on its hydrophobic wall (L40 and I42) and polar amino acids (D44 and R45), which formed intermolecular contacts and were a crucial part of the channel gate along with W41. Lastly, MCH inhibited IAV infection by reducing intracellular calcium and mitochondrial Ca2+/ROS levels in infected human lung cells. Taken together, these data suggest that MCH inhibits IAV infection and increases the survival of infected human lung cells by suppressing calcium levels. These results indicate that MCH is useful for developing IAV treatments.

Salinomycin Inhibits Influenza Virus Infection by Disrupting Endosomal Acidification and Viral Matrix Protein 2 Function.

Screening of chemical libraries with 2,000 synthetic compounds identified salinomycin as a hit against influenza A and B viruses, with 50% effective concentrations ranging from 0.4 to 4.3 µM in cells. This compound is a carboxylic polyether ionophore that exchanges monovalent ions for protons across lipid bilayer membranes. Monitoring the time course of viral infection showed that salinomycin blocked nuclear migration of viral nuclear protein (NP), the most abundant component of the viral ribonucleoprotein (vRNP) complex. It caused cytoplasmic accumulation of NP, particularly within perinuclear endosomes, during virus entry. This was primarily associated with failure to acidify the endosomal-lysosomal compartments. Similar to the case with amantadine (AMT), proton channel activity of viral matrix protein 2 (M2) was blocked by salinomycin. Using purified retroviral Gag-based virus-like particles (VLPs) with M2, it was proved that salinomycin directly affects the kinetics of a proton influx into the particles but in a manner different from that of AMT. Notably, oral administration of salinomycin together with the neuraminidase inhibitor oseltamivir phosphate (OSV-P) led to enhanced antiviral effect over that with either compound used alone in influenza A virus-infected mouse models. These results provide a new paradigm for developing antivirals and their combination therapy that control both host and viral factors.IMPORTANCE Influenza virus is a main cause of viral respiratory infection in humans as well as animals, occasionally with high mortality. Circulation of influenza viruses resistant to the matrix protein 2 (M2) inhibitor, amantadine, is highly prevalent. Moreover, the frequency of detection of viruses resistant to the neuraminidase inhibitors, including oseltamivir phosphate (OSV-P) or zanamivir, is also increasing. These issues highlight the need for discovery of new antiviral agents with different mechanisms. Salinomycin as the monovalent cation-proton antiporter exhibited consistent inhibitory effects against influenza A and B viruses. It plays multifunctional roles by blocking endosomal acidification and by inactivating the proton transport function of M2, the key steps for influenza virus uncoating. Notably, salinomycin resulted in marked therapeutic effects in influenza virus-infected mice when combined with OSV-P, suggesting that its chemical derivatives could be developed as an adjuvant antiviral therapy to treat influenza infections resistant or less sensitive to existing drugs.

Tackling Influenza A virus by M2 ion channel blockers: Latest progress and limitations.

Influenza outbreaks cause pandemics in millions of people. The treatment of influenza remains a challenge due to significant genetic polymorphism in the influenza virus. Also, developing vaccines to protect against seasonal and pandemic influenza infections is constantly impeded. Thus, antibiotics are the only first line of defense against antigenically distinct strains or new subtypes of influenza viruses. Among several anti-influenza targets, the M2 protein of the influenza virus performs several activities. M2 protein is an ion channel that permits proton conductance through the virion envelope and the deacidification of the Golgi apparatus. Both these functions are critical for viral replication. Thus, targeting the M2 protein of the influenza virus is an essential target. Rimantadine and amantadine are two well-known drugs that act on the M2 protein. However, these drugs acquired resistance to influenza and thus are not recommended to treat influenza infections. This review discusses an overview of anti-influenza therapy, M2 ion channel functions, and its working principle. It also discusses the M2 structure and its role, and the change in the structure leads to mutant variants of influenza A virus. We also shed light on the recently identified compounds acting against wild-type and mutated M2 proteins of influenza virus A. These scaffolds could be an alternative to M2 inhibitors and be developed as antibiotics for treating influenza infections.

Influenza A Virus Infection Activates NLRP3 Inflammasome through Trans-Golgi Network Dispersion.

The NLRP3 inflammasome consists of NLRP3, ASC, and pro-caspase-1 and is an important arm of the innate immune response against influenza A virus (IAV) infection. Upon infection, the inflammasome is activated, resulting in the production of IL-1ß and IL-18, which recruits other immune cells to the site of infection. It has been suggested that in the presence of stress molecules such as nigericin, the trans-Golgi network (TGN) disperses into small puncta-like structures where NLRP3 is recruited and activated. Here, we investigated whether IAV infection could lead to TGN dispersion, whether dispersed TGN (dTGN) is responsible for NLRP3 inflammasome activation, and which viral protein is involved in this process. We showed that the IAV causes dTGN formation, which serves as one of the mechanisms of NLRP3 inflammasome activation in response to IAV infection. Furthermore, we generated a series of mutant IAVs that carry mutations in the M2 protein. We demonstrated the M2 proton channel activity, specifically His37 and Trp41 are pivotal for the dispersion of TGN, NLRP3 conformational change, and IL-1ß induction. The results revealed a novel mechanism behind the activation and regulation of the NLRP3 inflammasome in IAV infection.

Influenza-existing drugs and treatment prospects.

Influenza is a century-old disease that continues to baffle humans by its frequently changing nature, seasonal epidemics, and occasional pandemics. Approximately 9% of the world's population is infected by the influenza virus annually. The emergence of novel strains because of rapid mutations as well as interspecies disease contamination, limits the efficiency of strain-specific vaccines. Anti-influenza drugs such as neuraminidase inhibitors, M2 ion channel inhibitors, etc. have become the first line of defense in prophylaxis and early containment of the disease. But the growing drug resistance due to drug-induced selective pressure has also limited the efficacy of those drugs. Because we can't predict the next strain types, their virulence, or the severity of the next epidemic/pandemic caused by influenza virus, we ought to gear up for the development of novel anti-influenza drugs with a broad spectrum of reactivity against all strains and subtypes, better bioavailability, easier administrative pathways, and lesser adverse effects. Various new compounds with each having significantly different target molecules and pharmacologic activity have shown potential against influenza virus strains in laboratory situations as well as clinical trials. We should also consider combination therapy to boost the efficacy of existing drugs. This review is aiming to succinctly document the recent signs of progress regarding anti-influenza drugs both in the market and under investigation.

An effective molecular blocker of ion channel of M2 protein as anti-influenza a drug.

Design of a drug compound that can effectively bind to the M2 ion channel and block the diffusion of hydrogen ions (H+) through and inhibit influenza A virus replication is an important task. Known anti-influenza drugs amantadine and rimantadine have a weak effect on influenza A virus. A new class of positively charged, +2 e.u., molecules is proposed here to block diffusion of H+ ion through the M2 channel. Several drug candidates, derivatives of a lead compound (diazabicyclooctane), were proposed and investigated. Molecular dynamics of thermal fluctuations of M2 protein structure and ionization-conformation coupling of all the ionizable residues were simulated at physiological pH. The influence of the most probable mutations of key drug-binding amino acid residues in the M2 ion channel were investigated too. It is shown that the suggested new blocker has high binding affinity for the M2 channel. There are two in-channel binding sites of high affinity, the first one has H-bonds with two of four serine residues Ser-31A (B) or Ser-31C(D), and the second one has H-bonds with two of four histidine residues His-37A (B), or His-37C(D). The main advantage of the new drug molecule is the positive charge, +2 e.u., which creates a positive electrostatic potential barrier (in addition to a steric one) for a transfer of H+ ion through M2 channel and may serve as an effective anti-influenza A virus drug.Communicated by Ramaswamy H. Sarma.

Influenza Hemagglutinin and M2 ion channel priming by trypsin: Killing two birds with one stone.

Influenza A virus membrane fusion and disassembly, prerequisite processes for viral infectivity, depend on acidic pH. In a recent study, Zhirnov et al. reported an important finding-that influenza virions are not permeable to protons unless the hemagglutinin (HA) fusion protein is primed by trypsin cleavage. This raises the question of whether in the viral context the M2 ion channel requires priming prior to its activation by low pH. Here, it is hypothesized that both HA and M2 ion channel direct priming by trypsin is required for their sensitization by low pH.

Drug resistance in influenza A virus: the epidemiology and management.

Influenza A virus (IAV) is the sole cause of the unpredictable influenza pandemics and deadly zoonotic outbreaks and constitutes at least half of the cause of regular annual influenza epidemics in humans. Two classes of anti-IAV drugs, adamantanes and neuraminidase (NA) inhibitors (NAIs) targeting the viral components M2 ion channel and NA, respectively, have been approved to treat IAV infections. However, IAV rapidly acquired resistance against both classes of drugs by mutating these viral components. The adamantane-resistant IAV has established itself in nature, and a majority of the IAV subtypes, especially the most common H1N1 and H3N2, circulating globally are resistant to adamantanes. Consequently, adamantanes have become practically obsolete as anti-IAV drugs. Similarly, up to 100% of the globally circulating IAV H1N1 subtypes were resistant to oseltamivir, the most commonly used NAI, until 2009. However, the 2009 pandemic IAV H1N1 subtype, which was sensitive to NAIs and has now become one of the dominant seasonal influenza virus strains, has replaced the pre-2009 oseltamivir-resistant H1N1 variants. This review traces the epidemiology of both adamantane- and NAI-resistant IAV subtypes since the approval of these drugs and highlights the susceptibility status of currently circulating IAV subtypes to NAIs. Further, it provides an overview of currently and soon to be available control measures to manage current and emerging drug-resistant IAV. Finally, this review outlines the research directions that should be undertaken to manage the circulation of IAV in intermediate hosts and develop effective and alternative anti-IAV therapies.

Progress of small molecular inhibitors in the development of anti-influenza virus agents.

The influenza pandemic is a major threat to human health, and highly aggressive strains such as H1N1, H5N1 and H7N9 have emphasized the need for therapeutic strategies to combat these pathogens. Influenza anti-viral agents, especially active small molecular inhibitors play important roles in controlling pandemics while vaccines are developed. Currently, only a few drugs, which function as influenza neuraminidase (NA) inhibitors and M2 ion channel protein inhibitors, are approved in clinical. However, the acquired resistance against current anti-influenza drugs and the emerging mutations of influenza virus itself remain the major challenging unmet medical needs for influenza treatment. It is highly desirable to identify novel anti-influenza agents. This paper reviews the progress of small molecular inhibitors act as antiviral agents, which include hemagglutinin (HA) inhibitors, RNA-dependent RNA polymerase (RdRp) inhibitors, NA inhibitors and M2 ion channel protein inhibitors etc. Moreover, we also summarize new, recently reported potential targets and discuss strategies for the development of new anti-influenza virus drugs.

Imidazole-based pinanamine derivatives: Discovery of dual inhibitors of the wild-type and drug-resistant mutant of the influenza A virus.

We previously reported potent hit compound 4 inhibiting the wild-type influenza A virus A/HK/68 (H3N2) and A/M2-S31N mutant viruses A/WS/33 (H1N1), with its latter activity quite weak. To further increase its potency, a structure-activity relationship study of a series of imidazole-linked pinanamine derivatives was conducted by modifying the imidazole ring of this compound. Several compounds of this series inhibited the amantadine-sensitive virus at low micromolar concentrations. Among them, 33 was the most potent compound, which was identified as being active on an amantadine-sensitive virus through blocking of the viral M2 ion channel. Furthermore, 33 markedly inhibited the amantadine-resistant virus (IC50 = 3.4 µM) and its activity increased by almost 24-fold compared to initial compound, with its action mechanism being not M2 channel mediated.

New small-molecule drug design strategies for fighting resistant influenza A.

Influenza A virus is the major cause of seasonal or pandemic flu worldwide. Two main treatment strategies-vaccination and small molecule anti-influenza drugs are currently available. As an effective vaccine usually takes at least 6 months to develop, anti-influenza small molecule drugs are more effective for the first line of protection against the virus during an epidemic outbreak, especially in the early stage. Two major classes of anti-influenza drugs currently available are admantane-based M2 protein blockers (amantadine and rimantadine) and neuraminidase (NA) inhibitors (oseltamivir, zanamivir, and peramivir). However, the continuous evolvement of influenza A virus and the rapid emergence of resistance to current drugs, particularly to amantadine, rimantadine, and oseltamivir, have raised an urgent need for developing new anti-influenza drugs against resistant forms of influenza A virus. In this review, we first give a brief introduction of the molecular mechanisms behind resistance, and then discuss new strategies in small-molecule drug development to overcome influenza A virus resistance targeting mutant M2 proteins and neuraminidases, and other viral proteins not associated with current drugs.

Antivirals for influenza: a summary of a systematic review and meta-analysis of observational studies.

Despite the use of antivirals to treat patients with severe influenza, questions remain with respect to effects and safety. Although a recent systematic review has provided some indication of benefit, the analysis is limited by the quality of the available evidence from randomized controlled trials. To supplement the existing information, the authors conducted a systematic review of observational studies of antiviral treatment for influenza. This report summarises the findings of that review. Similar to the randomised trials, the confidence in the estimates of the effects for decision-making is low to very low primarily due to the risk of selection and publication bias in the observational studies. From these observational studies, the summary estimates suggest that oseltamivir may reduce mortality, hospitalisation and duration of symptoms compared with no treatment. Inhaled zanamivir may also reduce symptom duration and hospitalisations, but patients may experience more complications compared with no treatment. Earlier treatment with antivirals is generally associated with better outcomes than later treatment. Further high-quality evidence is needed to inform treatment guidelines because of the overall low to very low quality of evidence.