A Novel p38 Mitogen Activated Protein Kinase (MAPK) Specific Inhibitor Suppresses Respiratory Syncytial Virus and Influenza A Virus Replication by Inhibiting Virus-Induced p38 MAPK Activation
Myung-Soo Choi, Jinyuk Heo, Chae-Min Yi, Junsu Ban, Noh-Jin Lee, Na-Rae Lee, Sang Won Kim, Nam-Jung Kim, Kyung-Soo Inn
Abstract
Respiratory syncytial virus (RSV) and influenza A virus are leading causes of acute lower respiratory infectious disease. Respiratory diseases caused by RSV and influenza A result in significant economic burden and can be life-threatening, especially in immunocompromised individuals. It has been revealed that p38 mitogen-activated protein kinase (MAPK) activity in host cells is crucial for infection and replication of RSV and influenza A virus. Therefore, inhibition of p38 MAPK activity has been suggested as a potential antiviral therapeutic strategy. However, earlier p38 MAPK inhibitors possess low selectivity and high toxicity, underscoring the need for improved inhibitors. In this study, we report the synthesis of a novel p38 MAPK inhibitor, NJK14047, with high kinase selectivity. We demonstrate that NJK14047 inhibits RSV- and influenza A-mediated p38 MAPK activation in epithelial cells. Treatment with NJK14047 results in decreased viral replication and viral mRNA synthesis. Additionally, NJK14047 greatly diminishes secretion of interleukin-6 from infected cells, suggesting it can ameliorate immunopathological responses to RSV and influenza A. Collectively, these results suggest NJK14047 has therapeutic potential to treat respiratory viral infections by suppressing p38 MAPK activation, an essential step in viral infection.
Introduction
Respiratory diseases caused by viral infection with respiratory syncytial virus (RSV) and influenza A virus are major global public health threats. RSV infection is a leading cause of acute lower respiratory tract infectious disease and pediatric hospitalization. In high-risk groups, such as infants and immunocompromised patients, RSV-induced bronchiolitis causes significant mortality and morbidity. Most RSV-infected infants under two years of age experience bronchiolitis and pneumonia. No effective RSV-specific vaccines or therapies are currently available despite the prevalence and severity of infections. Ribavirin is approved but limited by adverse effects. Since RSV triggers immunopathology including inflammation, it is critical to develop new agents that prevent RSV infection or excessive immune responses.
Influenza A virus, a single-stranded RNA virus, infects 5-10% of adults and 20-30% of children yearly, causing significant mortality worldwide. Antigenic variation through gene reassortment complicates vaccination efforts.
p38 mitogen-activated protein kinase (MAPK) plays central roles in inflammation and stress responses and has been considered a target for treatment of cancer and autoimmune diseases. Recent findings show that respiratory viruses including RSV and influenza A activate p38 MAPK via Toll-like receptor 4 (TLR4) during early infection stages; this activation is critical for viral entry and replication. Inhibition of p38 MAPK protected mice from mortality caused by excessive cytokine secretion during H5N1 infection. We previously demonstrated that berberine, a natural anti-inflammatory compound, suppresses RSV and influenza A replication through p38 MAPK inhibition. These data support p38 MAPK inhibition as a viable strategy to treat both inflammatory and infectious respiratory diseases.
Although various p38 MAPK inhibitors have been developed, none have reached clinical use due to adverse effects and limited efficacy. We previously designed and synthesized N-cyclopropylbenzamide-benzophenone hybrid compounds to inhibit p38 MAPK with high selectivity. NJK14047 displayed potent inhibition of p38α MAPK with high kinase specificity. We evaluated its antiviral activity against RSV and influenza A, hypothesizing that specific p38 MAPK inhibition could effectively treat respiratory viral infections.
Materials and Methods
Chemicals, Cells, and Viruses
NJK14047 was synthesized as described previously. SB203580 was purchased commercially. Human alveolar basal epithelial adenocarcinoma A549 cells, human larynx carcinoma HEp-2 cells, and murine macrophage Raw 264.7 cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) with antibiotics and fetal bovine serum. RSV A2 strain was propagated in HEp-2 cells; influenza A/PR/8/34 (H1N1) virus was propagated in embryonated eggs.
Cell Cytotoxicity Assay
Cytotoxicity of NJK14047 was assessed by MTT assay following standard protocols.
Immunoblotting
Phosphorylation status of p38 MAPK was analyzed by immunoblotting. A549 cells were infected with RSV or influenza A virus at specified multiplicities of infection (MOI) and treated with inhibitors. Lysates were analyzed using antibodies specific for total and phosphorylated p38 MAPK and influenza NS1 protein.
Plaque Assay
A549 cells were pretreated with vehicle, SB203580, or NJK14047 before RSV infection. After incubation, culture supernatants were serially diluted and viral titers were determined by plaque assay on HEp-2 cells.
Quantitative Reverse-Transcription PCR (qRT-PCR)
Viral mRNA synthesis and cytokine gene expression were measured by qRT-PCR from RNA isolated from infected cells treated with compounds.
Immunofluorescence Staining
RSV nucleocapsid (N) protein was detected by immunofluorescence to monitor viral uncoating.
Enzyme-Linked Immunosorbent Assay (ELISA)
Levels of interleukin-6 (IL-6) in culture supernatants were quantified by ELISA.
Statistical Analysis
Data comparisons were performed using Student’s t-test; p-values ≤0.05 were considered significant.
Results and Discussion
Inhibition of RSV- and Influenza A Infection-Mediated p38 MAPK Activation by NJK14047
NJK14047 was identified as a selective p38 MAPK inhibitor. NJK14047 suppressed lipopolysaccharide (LPS)-induced p38 MAPK phosphorylation in Raw cells, demonstrating TLR4-mediated inhibition. RSV and influenza A virus infection triggered robust p38 MAPK phosphorylation in A549 cells, which was dose-dependently suppressed by NJK14047. NJK14047 was non-cytotoxic up to 50 μM, confirming the antiviral effects were not due to toxicity.
Antiviral Effect of NJK14047
NJK14047 treatment reduced RSV progeny titers by up to 97% and suppressed influenza A virus replication as shown by decreased NS1 protein expression in a dose-dependent manner. NJK14047 was more effective than SB203580, likely due to its higher potency.
Suppression of Viral mRNA Synthesis by NJK14047
NJK14047 inhibited the synthesis of RSV fusion protein (F), RSV nonstructural protein 2 (NS2), and influenza nucleoprotein (NP) mRNAs in infected cells. Inhibition of viral uncoating by NJK14047 was confirmed by reduced fluorescence in assays detecting exposed RSV nucleocapsid protein.
Suppression of RSV- and Influenza A-Mediated IL-6 Production by NJK14047
NJK14047 dose-dependently decreased IL-6 mRNA and protein secretion in infected cells, suggesting suppression of virus-induced inflammatory responses, likely by direct inhibition of p38 MAPK and reduced viral load.
Conclusion
NJK14047 effectively inhibits virus-induced p38 MAPK activation and suppresses replication of RSV and influenza A viruses while attenuating associated inflammatory responses. This highly selective p38 MAPK inhibitor holds promise as a therapeutic agent against respiratory viral infections.