Retracted and republished from: "The current state of research on influenza antiviral drug development: drugs in clinical trial and licensed drugs".

Influenza viruses (IVs) threaten global human health due to the high morbidity, infection, and mortality rates. Currently, the influenza drugs recommended by the Food and Drug Administration are oseltamivir, zanamivir, peramivir, and baloxavir marboxil. These recommended antivirals are currently effective for major subtypes of IVs as the compounds target conserved domains in neuraminidase or polymerase acidic (PA) protein. However, this trend may gradually change due to the selection of antiviral drugs and the natural evolution of IVs. Therefore, there is an urgent need to develop drugs related to the treatment of influenza to deal with the next pandemic. Here, we summarized the cutting-edge research in mechanism of action, inhibitory activity, and clinical efficacy of drugs that have been approved and drugs that are still in clinical trials for influenza treatment. We hope this review will provide up-to-date and comprehensive information on influenza antivirals and generate hypotheses for screens and development of new broad-spectrum influenza drugs in the near future.

Redox regulation of age-associated defects in generation and maintenance of T cell self-tolerance and immunity to foreign antigens.

Thymic atrophy reduces naive T cell production and contributes to increased susceptibility to viral infection with age. Expression of tissue-restricted antigen (TRA) genes also declines with age and has been thought to increase autoimmune disease susceptibility. We find that diminished expression of a model TRA gene in aged thymic stromal cells correlates with impaired clonal deletion of cognate T cells recognizing an autoantigen involved in atherosclerosis. Clonal deletion in the polyclonal thymocyte population is also perturbed. Distinct age-associated defects in the generation of antigen-specific T cells include a conspicuous decline in generation of T cells recognizing an immunodominant influenza epitope. Increased catalase activity delays thymic atrophy, and here, we show that it mitigates declining production of influenza-specific T cells and their frequency in lung after infection, but does not reverse declines in TRA expression or efficient negative selection. These results reveal important considerations for strategies to restore thymic function.

Design, synthesis and biological evaluation of novel 1, 3, 4-oxadiazole derivatives as potent neuraminidase inhibitors.

Neuraminidase (NA) is an important target in the development of anti-influenza virus drugs. Compounds containing 1,3, 4-oxadiazole heterocycles have good biological activity and have been proved to have wide applications in antibacterial and antiviral drugs. In this paper, a series of novel 1, 3, 4-oxadiazole neuraminidase inhibitors (6a-6l) were designed and synthesized and their inhibitory activities of NA was tested in vitro. The results displayed that compound 6d exerts the best inhibitory activity (IC50 = 0.027 µM), which was obviously lower than that of oseltamivir carboxylate (OSC) (IC50 = 0.082 µM). Molecular docking analysis showed that the 1, 3, 4-oxadiazole heterocycle plays crucial part in compound 6d, and it can interact with the key arginine triad (Arg118, Arg292 and Arg 371) at the NA S1 site. The good efficacy of 6d may also be attributed to the extension of the substituted aniline ring to the 150-cavitiy. The theoretical and experimental results may provide reference for development of new anti-influenza drugs.

Synthesis and antiviral activity of formycin derivatives with anti-influenza virus activity.

In a screening using our unique natural product library, the C-nucleoside antibiotic formycin A, which exerts strong anti-influenza virus activity, was rediscovered. Aiming to develop a new type of anti-influenza virus drug, we synthesized new derivatives of formycin and evaluated its anti-influenza virus activity. Structural modifications were focused on the base moiety and sugar portion, respectively, and >40 novel formycin derivatives were synthesized. Modification of the C-7 position of the pyrazolopyrimidine ring strongly contributed to improve the activity. In particular, excellent anti-influenza virus activity was observed in the NHMe (10), SMe (12), and SeMe (15) derivatives, in which heteroatoms were introduced. In addition, in the modification of the sugar moiety, the presence of a hydroxyl group and its stereochemistry greatly affected both the expression and intensity of the activity. Furthermore, the evaluation results of the 7-SEt derivative (29) and the 2'-modified derivative (59) suggested that structural modifications may reduce cytotoxicity.

Expression and characterization of a recombinant broadly-reactive monoclonal antibody against group 1 and 2 influenza viruses.

Production of broadly-reactive antibodies is critical for universal immunodiagnosis of rapidly-evolving influenza viruses. Most monoclonal antibodies (mAbs) are generated in mice using the hybridoma technology which involves labor- and time-consuming screening and low yield issues. In this study, a recombinant antibody based on a broadly-reactive mAb against the hemagglutinin (HA) stalk of H7N9 avian influenza virus was expressed in CHO cells and its biological characteristics, cross-reactivity and epitope recognition were identified. The variable genes of the parental antibody were amplified and cloned into the antibody-expressing plasmids containing the constant genes of murine IgG1. The recombinant antibody was expressed in high yield and purity in CHO cells and showed similar features to the parental antibody, including negative hemagglutination inhibition activity against H7N9 virus and high binding activity with the H7N9 HA protein. Notably, the recombinant antibody exhibited a broad reactivity with different influenza subtypes belonging to group 1 and group 2, which was associated with its recognition of a highly-conserved epitope in the stalk, as observed for the parental antibody. Our results suggest that cell-based antibody expression system can be utilized as an important alternative to the hybridoma technology for antibody production for influenza virus diagnostics.

Validation of crystal structure of 2-acetamidophenyl acetate: an experimental and theoretical study.

In this present study, we have determined the crystal structure of 2-acetamidophenyl acetate (2-AAPA) commonly used as influenza neuraminidase inhibitor, to analyze the polymorphism. Molecular docking and molecular dynamics have been performed for the 2-AAPA-neuraminidase complex as the ester-derived benzoic group shows several biological properties. The X-ray diffraction studies confirmed that the 2-AAPA crystals are stabilized by N-H···O type of intermolecular interactions. Possible conformers of 2-AAPA crystal structures were computationally predicted by ab initio methods and the stable crystal structure was identified. Hirshfeld surface analysis of both experimental and predicted crystal structure exhibits the intermolecular interactions associated with 2D fingerprint plots. The lowest docking score and intermolecular interactions of 2-AAPA molecule against influenza neuraminidase confirm the binding affinity of the 2-AAPA crystals. The quantum theory of atoms in molecules analysis of these intermolecular interactions was implemented to understand the charge density redistribution of the molecule in the active site of influenza neuraminidase to validate the strength of the interactions.Communicated by Ramaswamy H. Sarma.

Lead Optimization of Influenza Virus RNA Polymerase Inhibitors Targeting PA-PB1 Interaction.

Influenza viruses are responsible for contagious respiratory illnesses in humans and cause seasonal epidemics and occasional pandemics worldwide. Previously, we identified a quinolinone derivative PA-49, which inhibited the influenza virus RNA-dependent RNA polymerase (RdRp) by targeting PA-PB1 interaction. This paper reports the structure optimization of PA-49, which resulted in the identification of 3-((dibenzylamino)methyl)quinolinone derivatives with more potent anti-influenza virus activity. During the optimization, the hit compound 89, which was more active than PA-49, was identified. Further optimization and scaffold hopping of 89 led to the most potent compounds 100 and a 1,8-naphthyridinone derivative 118, respectively. We conclusively determined that compounds 100 and 118 suppressed the replication of influenza virus and exhibited anti-influenza virus activity against both influenza virus types A and B in the range of 50% effective concentration (EC50) = 0.061-0.226 µM with low toxicity (50% cytotoxic concentration (CC50) >10 µM).

Bifunctional Inhibitors of Influenza Virus Neuraminidase: Molecular Design of a Sulfonamide Linker.

The growing resistance of the influenza virus to widely used competitive neuraminidase inhibitors occupying the active site of the enzyme requires the development of bifunctional compounds that can simultaneously interact with other regulatory sites on the protein surface. When developing such an inhibitor and combining structural fragments that could be located in the sialic acid cavity of the active site and the adjacent 430-cavity, it is necessary to select a suitable linker not only for connecting the fragments, but also to ensure effective interactions with the unique arginine triad Arg118-Arg292-Arg371 of neuraminidase. Using molecular modeling, we have demonstrated the usefulness of the sulfonamide group in the linker design and the potential advantage of this functional group over other isosteric analogues.

Exopolysaccharide Produced by Plant-Derived Lactobacillus plantarum SN35N Exhibits Antiviral Activity.

A lactic acid bacterial strain, Lactobacillus plantarum SN35N, which has been isolated from the pear, secretes negatively charged acidic exopolysaccharide (EPS) to outside cells. We have previously found that the SN35N-derived acidic EPS inhibits the catalytic activity of hyaluronidase (EC 3.2.1.35) promoting inflammation. The aim of this study is to find other health benefits of EPS. EPS has been found to exhibit an inhibitory effect against the influenza virus (Alphainfluenzavirus Influenza A virus) and feline calicivirus (Vesivirus Feline calicivirus), which is recognized as a model of norovirus. Although more studies on the structure-function relationship of EPSs are needed, SN35N-derived EPS is a promising lead for developing not only anti-inflammatory agents, but also antiviral substances.

Potential of an Eco-Sustainable Probiotic-Cleaning Formulation in Reducing Infectivity of Enveloped Viruses.

The COVID-19 pandemic has deeply influenced sanitization procedures, and high-level disinfection has been massively used to prevent SARS-CoV-2 spread, with potential negative impact on the environment and on the threat of antimicrobial resistance (AMR). Aiming to overcome these concerns, yet preserving the effectiveness of sanitization against enveloped viruses, we assessed the antiviral properties of the Probiotic Cleaning Hygiene System (PCHS), an eco-sustainable probiotic-based detergent previously proven to stably abate pathogen contamination and AMR. PCHS (diluted 1:10, 1:50 and 1:100) was tested in comparison to common disinfectants (70% ethanol and 0.5% sodium hypochlorite), in suspension and carrier tests, according with the European UNI EN 14476:2019 and UNI EN 16777:2019 standards. Human alpha- and beta-coronaviruses hCoV-229E and SARS-CoV-2, human herpesvirus type 1, human and animal influenza viruses, and vaccinia virus were included in the study. The results showed that PCHS was able to inactivate 99.99% of all tested viruses within 1-2 h of contact, both in suspension and on surface. Notably, while control disinfectants became inactive within 2 h after application, the PCHS antiviral action persisted up to 24 h post-application, suggesting that its use may effectively allow a continuous prevention of virus spread via contaminated environment, without worsening environmental pollution and AMR concern.

Molecular Docking and Virtual Screening of an Influenza Virus Inhibitor That Disrupts Protein-Protein Interactions.

Influenza is an acute respiratory infection caused by the influenza virus, but few drugs are available for its treatment. Consequently, researchers have been engaged in efforts to discover new antiviral mechanisms that can lay the foundation for novel anti-influenza drugs. The viral RNA-dependent RNA polymerase (RdRp) is an enzyme that plays an indispensable role in the viral infection process, which is directly linked to the survival of the virus. Methods of inhibiting PB1-PB2 (basic polymerase 1-basic polymerase 2) interactions, which are a key part of RdRp enzyme activity, are integral in the design of novel antiviral drugs, a specific PB1-PB2 interactions inhibitor has not been reported. We have screened Enamine's database and conducted a parallel screening of multiple docking schemes, followed by simulations of molecular dynamics to determine the structure of a stable ligand-PB1 complex. We also calculated the free energy of binding between the screened compounds and PB1 protein. Ultimately, we screened and identified a potential PB1-PB2 inhibitor using the ADMET prediction model.

Exploration of the 2,3-dihydroisoindole pharmacophore for inhibition of the influenza virus PA endonuclease.

Seasonal influenza A and B viruses represent a global concern. Antiviral drugs are crucial to treat severe influenza in high-risk patients and prevent virus spread in case of a pandemic. The emergence of viruses showing drug resistance, in particular for the recently licensed polymerase inhibitor baloxavir marboxil, drives the need for developing alternative antivirals. The endonuclease activity residing in the N-terminal domain of the polymerase acidic protein (PAN) is crucial for viral RNA synthesis and a validated target for drug design. Its function can be impaired by molecules bearing a metal-binding pharmacophore (MBP) able to coordinate the two divalent metal ions in the active site. In the present work, the 2,3-dihydro-6,7-dihydroxy-1H-isoindol-1-one scaffold is explored for the inhibition of influenza virus PA endonuclease. The structure-activity relationship was analysed by modifying the substituents on the lipophilic moiety linked to the MBP. The new compounds exhibited nanomolar inhibitory activity in a FRET-based enzymatic assay, and a few compounds (15-17, 21) offered inhibition in the micromolar range, in a cell-based influenza virus polymerase assay. When investigated against a panel of PA-mutant forms, compound 17 was shown to retain full activity against the baloxavir-resistant I38T mutant. This was corroborated by docking studies providing insight into the binding mode of this novel class of PA inhibitors.

Plant-Based Natural Products and Extracts: Potential Source to Develop New Antiviral Drug Candidates.

Viral infections are among the most complex medical problems and have been a major threat to the economy and global health. Several epidemics and pandemics have occurred due to viruses, which has led to a significant increase in mortality and morbidity rates. Natural products have always been an inspiration and source for new drug development because of their various uses. Among all-natural sources, plant sources are the most dominant for the discovery of new therapeutic agents due to their chemical and structural diversity. Despite the traditional use and potential source for drug development, natural products have gained little attention from large pharmaceutical industries. Several plant extracts and isolated compounds have been extensively studied and explored for antiviral properties against different strains of viruses. In this review, we have compiled antiviral plant extracts and natural products isolated from plants reported since 2015.

Research progress of epigallocatechin-3-gallate (EGCG) on anti-pathogenic microbes and immune regulation activities.

At the end of 2019, the COVID-19 virus spread worldwide, infecting millions of people. Infectious diseases induced by pathogenic microorganisms such as the influenza virus, hepatitis virus, and Mycobacterium tuberculosis are also a major threat to public health. The high mortality caused by infectious pathogenic microorganisms is due to their strong virulence, which leads to the excessive counterattack by the host immune system and severe inflammatory damage of the immune system. This paper reviews the efficacy, mechanism and related immune regulation of epigallocatechin-3-gallate (EGCG) as an anti-pathogenic microorganism drug. EGCG mainly shows both direct and indirect anti-infection effects. EGCG directly inhibits early infection by interfering with the adsorption on host cells, inhibiting virus replication and reducing bacterial biofilm formation and toxin release; EGCG indirectly inhibits infection by regulating immune inflammation and antioxidation. At the same time, we reviewed the bioavailability and safety of EGCG in vivo. At present, the bioavailability of EGCG can be improved to some extent using nanostructured drug delivery systems and molecular modification technology in combination with other drugs. This study provides a theoretical basis for the development of EGCG as an adjuvant drug for anti-pathogenic microorganisms.

System-oriented optimization of multi-target 2,6-diaminopurine derivatives: Easily accessible broad-spectrum antivirals active against flaviviruses, influenza virus and SARS-CoV-2.

The worldwide circulation of different viruses coupled with the increased frequency and diversity of new outbreaks, strongly highlight the need for new antiviral drugs to quickly react against potential pandemic pathogens. Broad-spectrum antiviral agents (BSAAs) represent the ideal option for a prompt response against multiple viruses, new and re-emerging. Starting from previously identified anti-flavivirus hits, we report herein the identification of promising BSAAs by submitting the multi-target 2,6-diaminopurine chemotype to a system-oriented optimization based on phenotypic screening on cell cultures infected with different viruses. Among the synthesized compounds, 6i showed low micromolar potency against Dengue, Zika, West Nile and Influenza A viruses (IC50 = 0.5-5.3 µM) with high selectivity index. Interestingly, 6i also inhibited SARS-CoV-2 replication in different cell lines, with higher potency on Calu-3 cells that better mimic the SARS-CoV-2 infection in vivo (IC50 = 0.5 µM, SI = 240). The multi-target effect of 6i on flavivirus replication was also analyzed in whole cell studies (in vitro selection and immunofluorescence) and against isolated host/viral targets.

Rosin Derivatives as a Platform for the Antiviral Drug Design.

The increased complexity due to the emergence and rapid spread of new viral infections prompts researchers to search for potential antiviral and protective agents for mucous membranes among various natural objects, for example, plant raw materials, their individual components, as well as the products of their chemical modification. Due to their structure, resin acids are valuable raw materials of natural origin to synthesize various bioactive substances. Therefore, the purpose of this study was to confirm the possibility of using resin acid derivatives for the drug design. As a result, we studied the cytotoxicity and biological activity of resin acid derivatives. It was shown that a slight decrease in the viral load in the supernatants was observed upon stimulation of cells (II) compared with the control. When using PASS-online modeling (Prediction of Activity Spectra for Substances), the prediction of the biological activity spectrum showed that compound (I) is capable of exhibiting antiviral activity against the influenza virus. The use of the SWISS-ADME webserver to reveal the drug-like properties of compounds did not directly indicate the presence of antiviral activity. These results indicate the potential of resin acid derivatives as a starting point for extensive research in the study of biological activity.

Development of End-Spliced Dimeric Nanodiscs for the Improved Virucidal Activity of a Nanoperforator.

Lipid-bilayer nanodiscs (NDs) wrapped in membrane scaffold proteins (MSPs) have primarily been used to study membrane proteins of interest in a physiological environment. Recently, NDs have been employed in broader applications including drug delivery, cancer immunotherapy, bio-imaging, and therapeutic virucides. Here, we developed a method to synthesize a dimeric nanodisc, whose MSPs are circularly end-spliced, with long-term thermal stability and resistance to aggregation. The end-spliced nanodiscs (esNDs) were assembled using MSPs that were self-circularized inside the cytoplasm ofEscherichia colivia highly efficient protein trans-splicing. The esNDs demonstrated a consistent size and 4-5-fold higher stability against heat and aggregation than conventional NDs. Moreover, cysteine residues on trans-spliced circularized MSPs allowed us to modulate the formation of either monomeric nanodiscs (essNDs) or dimeric nanodiscs (esdNDs) by controlling the oxidation/reduction conditions and lipid-to-protein ratios. When the esdNDs were used to prepare an antiviral nanoperforator that induced the disruption of the viral membrane upon contact, antiviral activity was dramatically increased, suggesting that the dimerization of nanodiscs led to cooperativity between linked nanodiscs. We expect that controllable structures, long-term stability, and aggregation resistance of esNDs will aid the development of novel versatile membrane-mimetic nanomaterials with flexible designs and improved therapeutic efficacy.

Synthesis and In Vitro Evaluation of C-7 and C-8 Luteolin Derivatives as Influenza Endonuclease Inhibitors.

The part of the influenza polymerase PA subunit featuring endonuclease activity is a target for anti-influenza therapies, including the FDA-approved drug Xofluza. A general feature of endonuclease inhibitors is their ability to chelate Mg2+ or Mn2+ ions located in the enzyme's catalytic site. Previously, we screened a panel of flavonoids for PA inhibition and found luteolin and its C-glucoside orientin to be potent inhibitors. Through structural analysis, we identified the presence of a 3',4'-dihydroxyphenyl moiety as a crucial feature for sub-micromolar inhibitory activity. Here, we report results from a subsequent investigation exploring structural changes at the C-7 and C-8 positions of luteolin. Experimental IC50 values were determined by AlphaScreen technology. The most potent inhibitors were C-8 derivatives with inhibitory potencies comparable to that of luteolin. Bio-isosteric replacement of the C-7 hydroxyl moiety of luteolin led to a series of compounds with one-order-of-magnitude-lower inhibitory potencies. Using X-ray crystallography, we solved structures of the wild-type PA-N-terminal domain and its I38T mutant in complex with orientin at 1.9 Å and 2.2 Å resolution, respectively.

Differential role of sphingomyelin in influenza virus, rhinovirus and SARS-CoV-2 infection of Calu-3 cells.

Host cell lipids play a pivotal role in the pathogenesis of respiratory virus infection. However, a direct comparison of the lipidomic profile of influenza virus and rhinovirus infections is lacking. In this study, we first compared the lipid profile of influenza virus and rhinovirus infection in a bronchial epithelial cell line. Most lipid features were downregulated for both influenza virus and rhinovirus, especially for the sphingomyelin features. Pathway analysis showed that sphingolipid metabolism was the most perturbed pathway. Functional study showed that bacterial sphingomyelinase suppressed influenza virus and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) replication, but promoted rhinovirus replication. These findings suggest that sphingomyelin pathway can be a potential target for antiviral therapy, but should be carefully evaluated as it has opposite effects on different respiratory viruses. Furthermore, the differential effect of sphingomyelinase on rhinovirus and influenza virus may explain the interference between rhinovirus and influenza virus infection.