T-705 (favipiravir) activity against lethal H5N1 influenza A viruses

The neuraminidase inhibitors oseltamivir and zanamivi are used to treat H5N1 influenza. However, oseltamivir-resistant H5N1 viruses have been isolated from oseltamivir-treated patients. Moreover, reassortment between H5N1 viruses and oseltamvir-resistant human H1N1 viruses currently circulating could create oseltamivir-resistant H5N1 viruses, rendering the oseltamivir stockpile obsolete. Therefore, there is a need for unique and effective antivirals to combat H5N1 influenza viruses. The investigational drug T-705 (favipiravir; 6-fluoro-3-hydroxy-2-pyrazinecarboxamide) has antiviral activity against seasonal influenza viruses and a mouse-adapted H5N1 influenza virus derived from a benign duck virus. However, its efficacy against highly pathogenic H5N1 viruses, which are substantially more virulent, remains unclear. Here, we demonstrate that T-705 effectively protects mice from lethal infection with oseltamivir-sensitive or -resistant highly pathogenic H5N1 viruses. Furthermore, our biochemical analysis suggests that T-705 ribofuranosyl triphosphate, an active form of T-705, acts like purines or purine nucleosides in human cells and does not inhibit human DNA synthesis. We conclude that T-705 shows promise as a therapeutic agent for the treatment of highly pathogenic H5N1 influenza patients.     

Mechanism by which ma-xing-shi-gan-tang inhibits the entry of influenza virus

Ethnopharmacological relevance

Ma-xing-shi-gan-tang (MXSGT, aka maxing shigan powder), a Chinese herbal decoction, has been used for the treatment of the common cold, fever, and influenza virus infections. However, the underlying mechanisms of its activity against the influenza virus are not fully understood. In this study, we examined the antiviral effects of MXSGT in influenza-virus-infected MDCK cells and their underlying mechanisms, including the damage of the viral surface ultrastructure and the consequent inhibition of viral entry.

Materials and methods

The antiviral activity of nontoxic concentrations of MXSGT against influenza virus A/WSN/33 was examined by assaying (neutralization assay) its inhibition of the virus-induced cytopathic effects. The mode of MXSGT action was first examined with a time-of-addition assay of synchronized infections, followed by viral attachment and penetration assays. Viral endocytosis was evaluated with attachment and penetration assays. We also performed assays related to the inhibition of viral entry, such as neuraminidase activity, hemagglutinin activity, and phosphoinositide-3-kinase (PI3K)/AKT phosphorylation assays. The inhibition of viral replication was demonstrated by quantitative real-time PCR, immunoblotting, and immunofluorescence microscopy. The surface ultrastructure of the MXSGT-treated virus was revealed by atomic force microscopy.

Results

MXSGT exhibited an EC₅₀ of 0.83±0.41 mg/ml against influenza virus A/WSN/33 (H1N1), with broad-spectrum inhibitory activity against different strains of human influenza A viruses, including clinical oseltamivir-resistant isolates and an H1N1pdm strain. The synthesis of both viral RNA and protein was profoundly inhibited when the cells were treated with MXSGT. The time-of-addition assay demonstrated that MXSGT blocks the virus entry phase. This was confirmed with attachment and penetration assays, in which MXSGT showed similar inhibitory potencies (IC₅₀ of 0.58±0.07 and 0.47±0.08 mg/ml). High-resolution images and quantitative measurements made with atomic force microscopy confirmed that the viral surface structure was disrupted by MXSGT. We also established that viral entry, regulated by the PI3K/AKT signaling pathway, was abolished by MXSGT.

Conclusions

Our results give scientific support to the use of MXSGT in the treatment of influenza virus infections. MXSGT has potential utility in the management of seasonal pandemics of influenza virus infections, like other clinically available drugs.     

流感病毒神经氨酸酶抑制剂的设计策略及研究进展

流感对人类的健康构成很大威胁,尤其在流感暴发期,能够造成大量人员死亡。神经氨酸酶（NA）为流感病毒表面蛋白,在病毒的生命过程中起着重要作用,是抗流感药物设计的重要靶点。自从1983年NA结构被解析出来后,基于结构的药物设计以及计算化学的运用极大地促进了NA抑制剂（NAI）的发展,到目前为止,已有两种抗流感药物上市——扎那米韦和奥司他韦。本文将以这两种药物的开发为例,简要介绍NAI的设计策略及最近几年的研究进展。     

Antiviral resistance in influenza viruses circulating in Central and South America based on the detection of established genetic markers

Background Recent influenza antiviral resistance studies reveal an alarming increase in both adamantanes and neuraminidase inhibitors (NAIs) resistant viral strains worldwide, particularly in Asia, Europe and the United States. Objectives In this study, we have evaluated influenza virus resistance in Central and South America. Methods Influenza viruses, isolated from symptomatic patients throughout Central and South America in 2005–2008 were analyzed for inhibitor resistance. The M2 and NA genes of influenza viruses were sequenced and resistance was inferred by comparison with published sequences and known resistant mutations. Results Our results indicate that: (i) resistance to adamantanes was seen in the majority (95·5%) of the influenza A/H3N2 isolates but only in one isolate of the influenza A/H1N1 viruses; (ii) resistance to NAIs began to be detected in A/H1N1 isolates from Central America in 2008; and (iii) none of the influenza B viruses analyzed were resistant to NAIs. Conclusions These findings suggest a limited effectiveness of influenza inhibitors due to the detection of resistance among A/H1 and A/H3 viruses.     

计算机辅助药物设计在病毒酶抑制剂开发中的应用

计算机辅助药物设计是创新药物研究的新方法和新技术,近年来引起了研究机构和制药公司的高度重视。本文概述了CADD的设计方法,并例举yCADD在HIV-1整合酶抑制剂和感冒病毒神经氨酸酶抑制剂研发中的应用,阐述了CADD在酶抑制剂研究中应用前景。     

Zanamivir: an influenza virus neuraminidase inhibitor

Zanamivir is the first of two registered neuraminidase inhibitors for the treatment and prophylaxis of influenza. Relenza^®, an orally inhaled powder form of zanamivir, is currently approved in 19 countries for treatment, and in two for prophylaxis. Relenza reduces the time to alleviation of symptoms by 1 to 2 days in the influenza-positive population, if taken within 48 h of symptom onset, and in prophylaxis in family settings, it confers an 80% reduction in the odds of contracting influenza. The resistance profile of zanamivir is encouraging in the sense that there are still no reports of patients on acute therapy shedding drug-resistant virus. However, patient uptake of the inhaled drug has been insufficient to conclude that drug resistance will not be an issue in the future. All zanamivir-resistant variants selected in the laboratory so far have diminished viability.     

Molecular Docking and QSAR Studies on Substituted Acyl(thio)urea and Thiadiazolo [2,3-α] Pyrimidine Derivatives as Potent Inhibitors of Influenza Virus Neuraminidase

Surflex–Dock was employed to dock 36 thiourea and thiadiazolo [2,3-α] pyrimidine derivatives into neuraminidase 1a4g. Molecular docking results showed that hydrogen bonding, electrostatic, and hydrophobic features were important factors affecting inhibitory activities of these neuraminidase inhibitors. Moreover, there was a significant correlation between the predicted binding affinity (total scores) and experimental pIC₅₀ values with correlation coefficient r = 0.846 and p < 0.0001. Hologram quantitative structure–activity relationship, comparative molecular field analysis, and comparative molecular similarity indices analysis were used to develop quantitative structure–activity relationship models. Squared multiple correlation coefficients (r ²) of hologram quantitative structure–activity relationship, comparative molecular field analysis, and comparative molecular similarity indices analysis models were 0.899, 0.878, and 0.865, respectively. Squared cross-validated correlation coefficient (q ²) of hologram quantitative structure–activity relationship, comparative molecular field analysis, and comparative molecular similarity indices analysis models was in turn 0.628, 0.656, and 0.509. In addition, squared multiple correlation coefficients for test set (r ²_test) of hologram quantitative structure–activity relationship, comparative molecular field analysis, and comparative molecular similarity indices analysis models were 0.558, 0.667, and 0.566, respectively. The most active sample ID 2 was taken as a template molecule to design new molecules. Based on the comparative molecular field analysis model, new compounds were designed by LeapFrog. Seven new compounds with improved binding energy and predicted activities were finally obtained.     

The Trypanosoma cruzi trans-sialidase is a T cell-independent B cell mitogen and an inducer of non-specific Ig secretion

Polyclonal lymphocyte activation and hypergammaglobulinemia characterize the acute phase of many parasitic diseases, including Chagas' disease, a debilitating condition caused by Trypanosoma cruzi. Polyclonal lymphocyte activation correlates with disease susceptibility inT. cruzi infection. Thus, identifying factors that drive such reactivities should provide insight into mechanisms of parasite evasion from host immunity and of disease pathogenesis. Sensitization of mice with small doses of T. cruzi trans-sialidase (TS) turns the mice into highly susceptible hosts to T. cruzi. In addition, TS heterologously expressed in Leishmania major greatly enhances virulence of the parasite to mice. In attempt to study the mechanism of TS-induced virulence, we found that TS and its C-terminal long tandem repeat (LTR) are T-independent polyclonal activators for mouse B cells. While B cells deficient/defective in L-6, CD40 or Toll-like receptor-4 are similarly activated by TS as compared to wild-type cells, B cells from Bruton's tyrosine kinase-defective X-linked immunodeficient mice are remarkably insensitive to TS activation. TS-induced B cell activation in vitro is accompanied by Ig secretion independent of T cells. Furthermore, administration of TS into normal mice leads to non-specific Ig secretion that peaks 4-6 days after injection. Thus TS, through its LTR, induces abnormal polyclonal B cell activation and Ig secretion, which could explain in part its virulence-enhancing activity.     

Influence of Neuraminidase and N-Acetylneuraminic Acid on Basophil Histamine Release in Vitro

Since N-acetylneuraminic acid (NANA) in cell membrane glucocalyx mediates or modulates a variety of actions, such as mediator release, we examined a possible modulating role of this amino sugar in histamine release from human basophil leukocytes. Removal of NANA from the cell membrane by the enzyme neuraminidase caused a dose-dependent histamine release. Removal of smaller amounts of NANA enhanced histamine release induced by anti-IgE, Concanavalin A and the calcium ionophore A23187, and reduced the interval between addition of antigen and initiation of histamine release. Pretreatment with free NANA had the opposite effects, i.e. a diminished and delayed maximal histamine release. The hypothesis that NANA in the cell membrane modulates the cellular response to stimulation was further substantiated by demonstrating that the altered response was reflected by a change in the sensitivity of the cell to extracellular calcium. NANA in the cell membrane glucocalyx thus seems to modulate the basophil response to stimulation by modulating transmembraneous calcium transport.     

Isolation and characterization of influenza C virus inhibitor in rat serum

Two hemagglutination inhibitors for influenza C virus were isolated from pooled sera of normal rats by sequential chromatography on Blue Sepharose CL 6B, Ultrogel AcA 22, and DEAE-cellulose. The two inhibitors were identified as alpha 1-macroglobulin and murinoglobulin by comparison with the authentic samples. These inhibitors abolished the hemagglutination by influenza C virus strains but did not affect the hemagglutination by influenza A and B virus strains. Hemagglutination inhibition activity of both inhibitors was completely destroyed by incubation with influenza C virus at 37 degrees C but not with the other types of influenza virus, indicating that the inhibitors are specific for influenza C virus. The inhibitory activity was also destroyed by incubation with neuraminidase from Arthrobacter ureafaciens. By contrast, no activity was lost after treatment with neuraminidase from Vibrio cholerae. These results suggest that the sialic acid residue(s) which is cleavable by the former neuraminidase but not by the latter is essential for the hemagglutination inhibition. The two inhibitors were inactivated by treating with sodium hydroxide and methylamine but not with sodium metaperiodate.