Anti-influenza Drug Discovery Research Articles

Sangbaipi decoction exerted in vitro and in vivo anti-influenza effect through inhibiting viral proteins

Headings ethnopharmacological relevanceSangbaipi Decoction (SBPD) is an effective treatment for lung diseases caused by phlegm-heat obstruction according to Jingyue Quanshu, and soothes panting by purging the lung meridian. It is composed of anti-pyretic herbs (e.g., Scutellaria baicalensis Georgi and Coptis chinensis Franch.) and antitussive herbs (e.g., Cortex Mori and Armeniacae Semen Amarum). Therefore, we hypothesized that SBPD has therapeutic effects on lung injury caused by influenza virus. Aim of the studyThis study aimed to explore anti-influenza activity, active components, and mechanisms of SBPD. Materials and methodsThe anti-influenza activities of SBPD were determined in 48 h drug-treated MDCK cell model using CPE and plaque reduction assays, and 24 h drug-treated A549 cells using qRT-PCR. The in vivo efficacy of SBPD (1.0 g/kg/day and 0.5 g/kg/day) was evaluated in PR8 infected BALB/c mice. The chemical component was assessed through HPLC-Q-TOF MS/MS analysis. Network pharmacology was built via TCMSP, GeneCards, DisgeNet, OMIM, DrugBank databases, and Cytoscape software. Additionally, TOA, HI and NAI assays were employed to investigate impact on the virus replication cycle with different concentrations of SBPD (2.5 mg/mL, 1.25 mg/mL, or 0.625 mg/mL). ResultsIn MDCK infected with viruses A/PR/8/34, A/Hong Kong/1/68, or A/California/4/2009, the IC50 values of SBPD were 0.80 mg/mL, 1.20 mg/mL, and 1.25 mg/mL. In A549 cells, SBPD treatment reduced cytokine expression (e.g., TNF-α, IL-6, IL-1β) (p < 0.05). In PR8 infected BALB/c mice, SBPD improved the survival rate of infected mice, reduced lung index (p < 0.05), protected lung tissue from pathological damage, and regulated cytokine overexpression (p < 0.05). 29 components of SBPD were identified in SBPD treated mouse serum including some phytochemicals targeting influenza proteins. HI and NAI assays suggested the potential antiviral mechanism of SBPD through inhibition of HA and NA. ConclusionThis study is the first to demonstrate the anti-influenza and the anti-inflammatory effects of SBPD in vitro and in vivo. Its major anti-influenza phytochemicals were explored and its inhibitory effects on HA and NA protein were proved. It provides more options for anti-influenza drug discovery.

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.

Structure-based drug design, molecular dynamics simulation, ADMET, and quantum chemical studies of some thiazolinones targeting influenza neuraminidase

The genetic mutability of the influenza virus leads to the existence of drug-resistant strains which is dangerous, particularly with the lingering coronavirus disease (COVID-19). This necessitated the need for the search and discovery of more potential anti-influenza agents to avert future outbreaks. In furtherance of our previous in-silico studies on 5-benzyl-4-thiazolinones as anti-influenza neuraminidase (NA) inhibitors, molecule 11 was selected as the template scaffold for the structure-based drug design due to its good binding, pharmacokinetic profiling, and better NA inhibitory activity. As such, eighteen (18) new molecules (11a-r) were designed with better MolDock scores as compared with the template scaffold and the zanamivir reference drug. However, the dynamic stability of molecule 11a in the binding cavity of the NA target (3TI5) showed water-mediated hydrogen and hydrophobic bondings with the active residues such as Arg118, Ile149, Arg152, Ile222, Trp403, and Ile427 after the MD simulation for 100 ns. The drug-likeness and ADMET assessment of all designed molecules predicted non-violation of the stipulated thresholds of Lipinski’s rule and good pharmacokinetic properties respectively. In addition, the quantum chemical calculations also suggested the significant chemical reactivity of molecules with their smaller band energy gap, high electrophilicity, high softness, and low hardness. The results obtained in this study proposed a reliable in-silico viewpoint for anti-influenza drug discovery and development.Communicated by Ramaswamy H. Sarma

Recent Advances in Application of Computer-Aided Drug Design in Anti-Influenza A Virus Drug Discovery.

Influenza A is an acute respiratory infectious disease caused by the influenza A virus, which seriously threatens global human health and causes substantial economic losses every year. With the emergence of new viral strains, anti-influenza drugs remain the most effective treatment for influenza A. Research on traditional, innovative small-molecule drugs faces many challenges, while computer-aided drug design (CADD) offers opportunities for the rapid and effective development of innovative drugs. This literature review describes the general process of CADD, the viral proteins that play an essential role in the life cycle of the influenza A virus and can be used as therapeutic targets for anti-influenza drugs, and examples of drug screening of viral target proteins by applying the CADD approach. Finally, the main limitations of current CADD strategies in anti-influenza drug discovery and the field’s future directions are discussed.

Anti-Influenza Drug Discovery and Development: Targeting the Virus and Its Host by All Possible Means.

Infections by influenza virus constitute a major and recurrent threat for human health. Together with vaccines, antiviral drugs play a key role in the prevention and treatment of influenza virus infection and disease. Today, the number of antiviral molecules approved for the treatment of influenza is relatively limited, and their use is threatened by the emergence of viral strains with resistance mutations. There is therefore a real need to expand the prophylactic and therapeutic arsenal. This chapter summarizes the state of the art in drug discovery and development for the treatment of influenza virus infections, with a focus on both virus-targeting and host cell-targeting strategies. Novel antiviral strategies targeting other viral proteins or targeting the host cell, some of which are based on drug repurposing, may be used in combination to strengthen our therapeutic arsenal against this major pathogen.

Dihydromyricetin is a new inhibitor of influenza polymerase PB2 subunit and influenza-induced inflammation

Development of new and effective anti-influenza drugs is critical for the treatment of influenza virus infection. The polymerase basic 2 (PB2) subunit as a core subunit of influenza A virus RNA polymerase complex is considered to be an attractive drug target for anti-influenza drug discovery. Dihydromyricetin, as a natural flavonoid, has a wide range of biological activities, but its anti-influenza A virus activity is ambiguous. Here, we found dihydromyricetin could inhibit the replication of a variety of influenza A virus strains. Mechanism studies demonstrated that dihydromyricetin reduced viral polymerase activity via selective inhibition of viral PB2 subunit, and decreased relative amounts of viral mRNA and genomic RNA during influenza A virus infection. The binding affinity and molecular docking analyses revealed that dihydromyricetin interacted with the PB2 cap-binding pocket, functioned as a cap-binding competitor. Interestingly, dihydromyricetin also reduced cellular immune injury by inhibiting TLR3 signaling pathway. Additionally, combination treatment of dihydromyricetin with zanamivir exerted a synergistic anti-influenza effect. Altogether, our experiments reveal the antiviral and anti-inflammatory activities of dihydromyricetin in vitro against influenza virus infection, which provides a new insight into the development of novel anti-influenza drugs.

Discovery of 5-(5-fluoro-1H-pyrrolo[2,3-b]pyridin-3-yl)pyrazin-2(1H)-one derivatives as new potent PB2 inhibitors.

PB2 is an important subunit of influenza RNA-dependent RNA polymerase (RdRP) and has been recognized as a promising target for the treatment of influenza. We herein report the discovery of a new series of PB2 inhibitors containing the skeleton 5-(5-fluoro-1H-pyrrolo[2,3-b]pyridin-3-yl)pyrazin-2(1H)-one. Compound 12b is the most potent one, which showed KD values of 0.11 μM and 0.19 μM in surface plasmon resonance (SPR) and isothermal titration calorimetry (ITC) assays, respectively. In antiviral activity and cellular cytotoxicity assays, compound 12b showed an EC50 value of 1.025 μM and a CC50 value greater than 100 μM. Molecular docking was also used to predict the binding mode of 12b with PB2. Collectively, this study provides a promising lead compound for subsequent anti-influenza drug discovery targeting PB2.

Identification and characterization of influenza variants resistant to a viral endonuclease inhibitor

The influenza endonuclease is an essential subdomain of the viral RNA polymerase. It processes host pre-mRNAs to serve as primers for viral mRNA and is an attractive target for antiinfluenza drug discovery. Compound L-742,001 is a prototypical endonuclease inhibitor, and we found that repeated passaging of influenza virus in the presence of this drug did not lead to the development of resistant mutant strains. Reduced sensitivity to L-742,001 could only be induced by creating point mutations via a random mutagenesis strategy. These mutations mapped to the endonuclease active site where they can directly impact inhibitor binding. Engineered viruses containing the mutations showed resistance to L-742,001 both in vitro and in vivo, with only a modest reduction in fitness. Introduction of the mutations into a second virus also increased its resistance to the inhibitor. Using the isolated wild-type and mutant endonuclease domains, we used kinetics, inhibitor binding and crystallography to characterize how the two most significant mutations elicit resistance to L-742,001. These studies lay the foundation for the development of a new class of influenza therapeutics with reduced potential for the development of clinical endonuclease inhibitor-resistant influenza strains.

Recent advances in computer-aided drug design as applied to anti-influenza drug discovery.

Influenza is a seasonal and serious health threat, and the recent outbreak of H7N9 following the pandemic spread of H1N1 in 2009 has served to emphasize the importance of anti-influenza drug discovery. Zanamivir (Relenza&#8482;) and oseltamivir (Tamiflu(®)) are two antiviral drugs currently recommended by the CDC for treating influenza. Both are examples of the successful application of structure-based drug design strategies. These strategies have combined computer- based approaches, such as docking- and pharmacophore-based virtual screening with X-ray crystallographic structural analyses. Docking is a routinely used computational method to identify potential hits from large compound libraries. This method has evolved from simple rigid docking approaches to flexible docking methods to handle receptor flexibility and to enhance hit rates in virtual screening. Virtual screening approaches can employ both ligand-based and structurebased pharmacophore models depending on the available information. The exponential growth in computing power has increasingly facilitated the application of computer-aided methods in drug discovery, and they now play significant roles in the search for novel therapeutics. An overview of these computational tools is presented in this review, and recent advances and challenges will be discussed. The focus of the review will be anti-influenza drug discovery and how advances in our understanding of viral biology have led to the discovery of novel influenza protein targets. Also discussed will be strategies to circumvent the problem of resistance emerging from rapid mutations that has seriously compromised the efficacy of current anti-influenza therapies.

A GFP-tagged nucleoprotein-based aggregation assay for anti-influenza drug discovery and antibody development

Influenza is a viral pandemic that affects millions of people worldwide. Seasonal variations due to genetic shuffling and antigenic drifts in the influenza viruses have necessitated continual updating of therapeutics. The growing resistance to current influenza drugs has increased demand for new antivirals. The highly conserved nature of NP, a multi-functional viral protein that is serotypically distinct and abundantly expressed during infection, has led to its use in developing universal biotherapeutics and vaccines that could be effective against the virus, irrespective of its strain variations. Compounds causing aggregation of NP have recently been shown to be potent antivirals but require the development of new high-throughput assays capable of screening compounds with similar modes of action. Here, we describe the development of a new bioassay for the Influenza A nucleoprotein (NP). The assay was developed to quantify ligand-induced aggregation of a GFP-tagged NP and was validated with aggregation-inducing compounds such as nucleozin and a NP-specific antibody. The new NP-GFP aggregation assay can be performed with partially purified or mixtures of proteins and is amenable to a high-throughput format. Using this assay, we demonstrate the potential of a new anti-NP polyclonal antibody that we have obtained from chicken. This cost-effective high-yield source of anti-NP IgY has potential for large-scale production and development of therapeutic antibodies. The simplicity, speed and flexibility of this assay make it an invaluable tool for timely development of effective antivirals that can help to control future epidemics.

Exploring the molecular basis of dsRNA recognition by NS1 protein of influenza A virus using molecular dynamics simulation and free energy calculation

The frequent outbreak of influenza pandemic and the limited available anti-influenza drugs highlight the urgent need for the development of new antiviral drugs. The dsRNA-binding surface of nonstructural protein 1 of influenza A virus (NS1A) is a promising target. The detailed understanding of NS1A–dsRNA interaction will be valuable for structure-based anti-influenza drug discovery. To characterize and explore the key interaction features between dsRNA and NS1A, molecular dynamics simulation combined with MM-GBSA calculations were performed. Based on the MM-GBSA calculations, we find that the intermolecular van der Waals interaction and the nonpolar solvation term provide the main driving force for the binding process. Meanwhile, 17 key residues from NS1A were identified to be responsible for the dsRNA binding. Compared with the wild type NS1A, all the studied mutants S42A, T49A, R38A, R35AR46A have obvious reduced binding free energies with dsRNA reflecting in the reduction of the polar and/or nonpolar interactions. In addition, the structural and energy analysis indicate the mutations have a small effect to the backbone structures but the loss of side chain interactions is responsible for the decrease of the binding affinity. The uncovering of NS1A–dsRNA recognition mechanism will provide some useful insights and new chances for the development of anti-influenza drugs.

Anti-Influenza Drug Discovery: Structure−Activity Relationship and Mechanistic Insight into Novel Angelicin Derivatives

By using a cell-based high throughput screening campaign, a novel angelicin derivative 6a was identified to inhibit influenza A (H1N1) virus induced cytopathic effect in Madin-Darby canine kidney cell culture in low micromolar range. Detailed structure-activity relationship studies of 6a revealed that the angelicin scaffold is essential for activity in pharmacophore B, while meta-substituted phenyl/2-thiophene rings are optimal in pharmacophore A and C. The optimized lead 4-methyl-9-phenyl-8-(thiophene-2-carbonyl)-furo[2,3-h]chromen-2-one (8g, IC(50) = 70 nM) showed 64-fold enhanced activity compared to the high throughput screening (HTS) hit 6a. Also, 8g was found effective in case of influenza A (H3N2) and influenza B virus strains similar to approved anti-influenza drug zanamivir (4). Preliminary mechanistic studies suggest that these compounds act as anti-influenza agents by inhibiting ribonucleoprotein (RNP) complex associated activity and have the potential to be developed further, which could form the basis for developing additional defense against influenza pandemics.

Nucleoside monophosphate complex structures of the endonuclease domain from the influenza virus polymerase PA subunit reveal the substrate binding site inside the catalytic center.

Highly pathogenic influenza virus strains currently in circulation pose a significant risk of a global pandemic. Following the reported crystal structure of the endonuclease domain from the avian influenza virus polymerase PA subunit, here we report the results of a systematic X-ray crystallographic analysis of its complex with adenosine, uridine, and thymidine nucleoside monophosphates (NMPs). Electron density corresponding to the monophosphate moiety of each nucleotide was apparent in each NMP complex and bound to the catalytic metal. A hydrophobic site was found to contribute to nucleoside binding. The NMP complex structures should represent the conformation of the bound product after nuclease cleavage. Moreover, one solvent molecule was found to occupy an equivalent position to the second reported Mn(2+) ion, where it mediates the interaction between bound NMPs and the N-terminal PA domain in the presence of the Mg(2+) ion. The results presented here indicate a possible cleavage mechanism and identify a distinct nucleotide binding pocket. The identification of this binding pocket opens a new avenue for anti-influenza drug discovery, targeting the cap-dependent endonuclease, in response to the worldwide threat of influenza.