piperidines and Orthomyxoviridae-Infections

Infection with seasonal influenza A virus (IAV) leads to lung inflammation and respiratory failure, a main cause of death in influenza-infected patients. Previous experiments in our laboratory indicate that Bruton's tyrosine kinase (Btk) plays a substantial role in regulating inflammation in the respiratory region during acute lung injury in mice; therefore, we sought to determine if blocking Btk activity has a protective effect in the lung during influenza-induced inflammation. The Btk inhibitor ibrutinib (also known as PCI-32765) was administered intranasally to mice starting 72 h after lethal infection with IAV. Our data indicate that treatment with the Btk inhibitor not only reduced weight loss and led to survival, but also had a dramatic effect on morphological changes to the lungs, in IAV-infected mice. Attenuation of lung inflammation indicative of acute lung injury, such as alveolar hemorrhage, interstitial thickening, and the presence of alveolar exudate, together with reduced levels of the inflammatory mediators TNFα, IL-1β, IL-6, KC, and MCP-1, strongly suggests amelioration of the pathological immune response in the lungs to promote resolution of the infection. Finally, we observed that blocking Btk specifically in the alveolar compartment led to significant attenuation of neutrophil extracellular traps released into the lung in vivo and neutrophil extracellular trap formation in vitro. Our innovative findings suggest that Btk may be a new drug target for influenza-induced lung injury, and, in general, that immunomodulatory treatment may be key in treating lung dysfunction driven by excessive inflammation.

Topics: Acute Lung Injury; Adenine; Agammaglobulinaemia Tyrosine Kinase; Animals; Cytokines; Extracellular Traps; Influenza A Virus, H1N1 Subtype; Macrophages, Alveolar; Mice; Orthomyxoviridae Infections; Piperidines; Protein Kinase Inhibitors; Pyrazoles; Pyrimidines

Influenza viruses, the main cause of respiratory tract diseases, cause high morbidity and mortality in humans. Excessive inflammation in the lungs is proposed to be a hallmark for the severe influenza virus infection, especially influenza A virus infection. Strategies against inflammation induced by influenza A virus infection could be a potential anti-influenza therapy. Here, lethal dose of mouse-adapted H1N1 strain PR8A/PR/8/34 was inoculated C57BL/6 mice to detect the anti-influenza activity of andrographolide, the active component of traditional Chinese medicinal herb Andrographis paniculata, with or without influenza virus entry inhibitor CL-385319. Treatment was initiated on 4 days after infection. The survival rate, body weight, lung pathology, viral loads, cytokine expression were monitored in 14 days post inoculation. The combination group had the highest survival rate. Andrographolide treatment could increase the survival rate, diminish lung pathology, decrease the virus loads and the inflammatory cytokines expression induced by infection. Mechanism studies showed the NF-κB and JAK-STAT signaling pathway were involved in the activity of andrographolide. In conclusion, combination of virus entry inhibitor with immunomodulator might be a promising therapeutic approach for influenza.

Topics: A549 Cells; Andrographis; Animals; Benzamides; Cell Line; Cytokines; Disease Models, Animal; Diterpenes; Dogs; Drug Therapy, Combination; Female; Humans; Inflammation; Influenza A Virus, H1N1 Subtype; Janus Kinases; Madin Darby Canine Kidney Cells; Mice; Mice, Inbred C57BL; NF-kappa B; Orthomyxoviridae Infections; Piperidines; STAT1 Transcription Factor; STAT2 Transcription Factor; Virus Internalization

The influenza virus PA endonuclease, which cleaves capped host pre-mRNAs to initiate synthesis of viral mRNA, is a prime target for antiviral therapy. The diketo acid compound L-742,001 was previously identified as a potent inhibitor of the influenza virus endonuclease reaction, but information on its precise binding mode to PA or potential resistance profile is limited. Computer-assisted docking of L-742,001 into the crystal structure of inhibitor-free N-terminal PA (PA-Nter) indicated a binding orientation distinct from that seen in a recent crystallographic study with L-742,001-bound PA-Nter (R. M. DuBois et al., PLoS Pathog. 8:e1002830, 2012). A comprehensive mutational analysis was performed to determine which amino acid changes within the catalytic center of PA or its surrounding hydrophobic pockets alter the antiviral sensitivity to L-742,001 in cell culture. Marked (up to 20-fold) resistance to L-742,001 was observed for the H41A, I120T, and G81F/V/T mutant forms of PA. Two- to 3-fold resistance was seen for the T20A, L42T, and V122T mutants, and the R124Q and Y130A mutants were 3-fold more sensitive to L-742,001. Several mutations situated at noncatalytic sites in PA had no or only marginal impact on the enzymatic functionality of viral ribonucleoprotein complexes reconstituted in cell culture, consistent with the less conserved nature of these PA residues. Our data provide relevant insights into the binding mode of L-742,001 in the PA endonuclease active site. In addition, we predict some potential resistance sites that should be taken into account during optimization of PA endonuclease inhibitors toward tight binding in any of the hydrophobic pockets surrounding the catalytic center of the enzyme.

Topics: Animals; Antiviral Agents; Catalytic Domain; Cells, Cultured; Computational Biology; Dogs; Drug Resistance, Viral; Enzyme Inhibitors; Humans; Hydroxybutyrates; Molecular Structure; Mutation; Orthomyxoviridae; Orthomyxoviridae Infections; Piperidines; Protein Conformation; RNA-Dependent RNA Polymerase; Viral Proteins; Virus Internalization; Virus Replication

The principal aim of the study was to determine the influence of influenza A virus infection on capsaicin-induced relaxation responses in mouse isolated tracheal segments and clarify the underlying mechanisms. Anesthetized mice were intranasally inoculated with influenza A/PR-8/34 virus (VIRUS) or vehicle (SHAM), and 4 days later tracheal segments were harvested for isometric tension recording and biochemical and histologic analyses. Capsaicin induced dose-dependent relaxation responses in carbachol-contracted SHAM trachea (e.g., 10 μM capsaicin produced 66 ± 4% relaxation; n = 11), which were significantly inhibited by capsazepine [transient receptor potential vanilloid type 1 (TRPV1) antagonist], (2S,3S)-3-{[3,5-bis(trifluoromethyl)phenyl]methoxy}-2-phenylpiperidine hydrochloride (L-733,060) [neurokinin 1 (NK₁) receptor antagonist], indomethacin [cyclooxygenase (COX) inhibitor], and the combination of 6-isopropoxy-9-oxoxanthene-2-carboxylic acid (AH6809) and 7-[5α-([1S,1α(Z)-biphenyl]-4-ylmethoxy)-2β-(4-morpholinyl)-3-oxocyclopentyl]-4-heptenoic acid, calcium salt, hydrate (AH23848) [E-prostanoid (EP)₂ and EP₄ receptor antagonists, respectively], indicating that capsaicin-induced relaxation involved the TRPV1-mediated release of substance P (SP), activation of epithelial NK₁ receptors, and production of COX products capable of activating relaxant EP₂/EP₄ receptors. Consistent with this postulate, capsaicin-induced relaxation was associated with the significant release of SP and prostaglandin E₂ (PGE₂) from mouse tracheal segments. As expected, influenza A virus infection was associated with widespread disruption of the tracheal epithelium. Tracheal segments from VIRUS mice responded weakly to capsaicin (7 ± 3% relaxation) and were 25-fold less responsive to SP than tracheas from SHAM mice. In contrast, relaxation responses to exogenous PGE₂ and the β-adrenoceptor agonist isoprenaline were not inhibited in VIRUS trachea. Virus infection was associated with impaired capsaicin-induced release of PGE₂, but the release of SP was not affected. In summary, influenza A virus infection profoundly inhibits capsaicin- and SP-induced relaxation responses, most likely by inhibiting the production of PGE₂.

Topics: Animals; Biphenyl Compounds; Bronchoalveolar Lavage Fluid; Capsaicin; Carbachol; Cyclooxygenase Inhibitors; Dinoprostone; Dose-Response Relationship, Drug; Indomethacin; Inflammation; Influenza A virus; Isoproterenol; Leukocytes; Male; Mice; Mice, Inbred BALB C; Models, Biological; Muscle Contraction; Muscle Relaxation; Muscle, Smooth; Orthomyxoviridae Infections; Piperidines; Prostaglandin Antagonists; Respiratory Mucosa; Specific Pathogen-Free Organisms; Substance P; Trachea; TRPV Cation Channels; Xanthones

Infection with influenza virus induces severe pulmonary immune pathology that leads to substantial human mortality. Although antiviral therapy is effective in preventing infection, no current therapy can prevent or treat influenza-induced lung injury. Previously, we reported that influenza-induced pulmonary immune pathology is mediated by inflammatory monocytes trafficking to virus-infected lungs via CCR2 and that influenza-induced morbidity and mortality are reduced in CCR2-deficient mice. In this study, we evaluated the effect of pharmacologically blocking CCR2 with a small molecule inhibitor (PF-04178903) on the entry of monocytes into lungs and subsequent morbidity and mortality in influenza-infected mice. Subcutaneous injection of mice with PF-04178903 was initiated 1 d prior to infection with influenza strain H1N1A/Puerto Rico/8/34. Compared with vehicle controls, PF-04178903-treated mice demonstrated a marked reduction in mortality (75 versus 0%) and had significant reductions in weight loss and hypothermia during subsequent influenza infection. Drug-treated mice also displayed significant reductions in bronchoalveolar lavage fluid total protein, albumin, and lactose dehydrogenase activity. Administration of PF-04178903 did not alter viral titers, severity of secondary bacteria infections (Streptococcus pneumoniae), or levels of anti-influenza-neutralizing Abs. Drug-treated mice displayed an increase in influenza nucleoprotein-specific cytotoxic T cell activity. Our results suggest that CCR2 antagonists may represent an effective prophylaxis against influenza-induced pulmonary immune pathology.

Topics: Animals; Anti-Inflammatory Agents, Non-Steroidal; Antiviral Agents; Cell Migration Inhibition; Female; Humans; Influenza A Virus, H1N1 Subtype; Lung; Mice; Mice, Inbred C57BL; Orthomyxoviridae Infections; Piperazines; Piperidines; Pneumonia, Pneumococcal; Pyridines; Receptors, CCR2; Survival Analysis

The antiviral effect of 9-MS was examined in mice infected with mouse-adapted influenza A2 (H2N2) virus. Either a single or a continuous prophylactic administration of 9-MS protected mice from virus infection, and comparison of the minimal effective dose with the lethal dose gave a therapeutic index of approximately 60. When the treatment was started after infection, however, no antiviral effect was demonstrated. A potent virus-inhibitory factor was detected in the lung and serum after a single intraperitoneal administration of 9-MS in uninfected mice, and on the basis of chemical characteristics of this inhibitory factor, it was assumed to be an interferon. These results suggest that the protective activity of the antibiotic in mice is due to interferon induction.

Topics: Animals; Dose-Response Relationship, Drug; Drug Administration Schedule; Female; Influenza A virus; Interferon Inducers; Male; Mice; Orthomyxoviridae Infections; Piperidines; Piperidones

The antiviral effect of 9-methylstreptimidone (9-MS) was examined in mice infected with mouse-adapted influenza A(2) (H(2)N(2)) virus. Both a single and continuous prophylactic administration of 9-MS protected mice from virus infection, and comparison between the minimal effective and the 50% lethal dose gave a therapeutic index of 60. When the treatment was started after infection, however, no antiviral effect was demonstrated. After a single intraperitoneal administration of 9-MS, a highly potent virus-inhibitory factor was detected in the lungs (10 h later) and the sera (16 h later) of uninfected mice, which was assumed to be an interferon on the basis of the biological characteristics. These results suggest that the protective activity of the antibiotic is due to interferon induction in mice.

Topics: Animals; Anti-Bacterial Agents; Antiviral Agents; Female; Interferon Inducers; Lung; Male; Mice; Mice, Inbred Strains; Orthomyxoviridae Infections; Piperidines; Piperidones

Topics: Amantadine; Animals; Antiviral Agents; Azepines; Bridged-Ring Compounds; Infrared Rays; Magnetic Resonance Spectroscopy; Mice; Orthomyxoviridae Infections; Piperidines; Spectrum Analysis; Spiro Compounds