Rosmarinic acid interferes with influenza virus A entry and replication by decreasing GSK3ß and phosphorylated AKT expression levels.

BACKGROUND: The purpose of this study was to examine the in vivo activity of rosmarinic acid (RA) - a phytochemical with antioxidant, anti-inflammatory, and antiviral properties - against influenza virus (IAV). An antibody-based kinase array and different in vitro functional assays were also applied to identify the mechanistic underpinnings by which RA may exert its anti-IAV activity. METHODS: We initially examined the potential efficacy of RA using an in vivo mouse model. A time-of-addition assay and an antibody-based kinase array were subsequently applied to investigate mechanism-of-action targets for RA. The hemagglutination inhibition assay, neuraminidase inhibition assay, and cellular entry assay were also performed. RESULTS: RA increased survival and prevented body weight loss in IAV-infected mice. In vitro experiments revealed that RA inhibited different IAV viruses - including oseltamivir-resistant strains. From a mechanistic point of view, RA downregulated the GSK3ß and Akt signaling pathways - which are known to facilitate IAV entry and replication into host cells. CONCLUSIONS: RA has promising preclinical efficacy against IAV, primarily by interfering with the GSK3ß and Akt signaling pathways.

Vitamin D in gynecological diseases.

BACKGROUND: Most reproductive system studies suggest the protective effects of vitamin D, but vitamin D deficiency and insufficiency are growing global health issues. The present study investigates the association between vitamin D deficiency/insufficiency and gynecologic diseases to identify illness risks at different serum vitamin D levels in Taiwan. METHODS: A total of 7699 female adults aged ≥20 years with results for both serum vitamin D and gynecologic-associated diseases were drawn from the Taiwan MJ cohort. We analyzed the correlation between serum vitamin D levels and results from reproductive system evaluations, including history of dysmenorrhea, results of Pap smear, high-risk human papillomavirus (HPV) infection of the cervix, mammography, and ultrasound of breast and pelvis. RESULTS: Over 80% of participants showed vitamin D deficiency/insufficiency. Participants with abnormal Pap smear results, high-risk HPV infection, and history of dysmenorrhea showed significantly lower levels of serum vitamin D (p < 0.001-0.05). Serum vitamin D deficiency was significantly associated with positive high-risk HPV infection of the cervix (p < 0.05) and dysmenorrhea (p < 0.001). After controlling for age as a confounding variable for each gynecologic disease, level of serum vitamin D was significantly associated with abnormal breast ultrasound (odds ratio = 0.724) and uterus ultrasound (odds ratio = 0.673 - 0.8), and dysmenorrhea (odds ratio = 0.829). CONCLUSION: Associations were found between vitamin D deficiency and endometriosis, uterine myoma, dysmenorrhea, abnormal Pap smear results, and high-risk HPV infection of the cervix. Therefore, vitamin D supplements may present a cost-effective benefit for the prevention and treatment of gynecologic diseases, and thus reduction of healthcare expenditures.

Design, Synthesis, and Biological Evaluation of Itaconic Acid Derivatives as Potential Anti-Influenza Agents.

Influenza A viruses (IAVs) have caused worldwide epidemics and pandemics by reassortment and generation of drug-resistant mutants, which render antivirals and current vaccinations no longer usable. In this study, an itaconic acid derivative 1 was identified from a chemical library of 20 000 compounds, by performing a cell-based screening assay, as a lead agent exhibiting anti-influenza A activity. Accordingly, a series of itaconic acid derivatives were designed and synthesized by adopting a rational design strategy to obtain more potent anti-influenza agents. The results of an in vitro pharmacological study showed that compounds 4 and 8 exhibited the most potent anti-IAV effect with half-maximal effective concentration values of 0.14 and 0.11 µM, respectively, in Madin-Darby canine kidney cells. The mechanism of action studies showed that lead agents 1 and 4 reduced virus replication by directly targeting IAV nucleoproteins and disrupting virus ribonucleoprotein export from the nucleus to the cytosol. On the basis of its high potential as an anti-IAV agent and its selectivity index >785, compound 4 was found to be a promising candidate for further development against IAVs.

Inhibition of endosomal fusion activity of influenza virus by Rheum tanguticum (da-huang).

Rhubarb (Rheum tanguticum; da-huang in Chinese medicine) is a herbal medicine that has been used widely for managing fever and removing toxicity. In this study, we investigated how rhubarb inhibits influenza virus during the early stage of the infectious cycle using different functional assays. A non-toxic ethanolic extract of rhubarb (Rex) inhibited several H1N1 subtypes of influenza A viruses in Madin-Darby canine kidney cells, including strains that are clinically resistant to oseltamivir. Time course analysis of Rex addition showed that viral entry was one of the steps that was inhibited by Rex. We also confirmed that Rex effectively inhibited viral attachment and penetration into the host cells. The inhibition of red blood cell haemolysis and cell-cell fusion by Rex suggests that Rex may block haemagglutinin-mediated fusion (virus-endosome fusion) during the fusion/uncoating step. Rex has the capacity to inhibit influenza viruses by blocking viral endocytosis. Thus, rhubarb might provide an alternative therapeutic approach when resistant viruses become more prevalent.

Anti-influenza drug discovery: identification of an orally bioavailable quinoline derivative through activity- and property-guided lead optimization.

From a high-throughput screening (HTS) hit with inhibitory activity against virus-induced cytophathic in the low micromolar range, we have developed a potent anti-influenza lead through careful optimization without compromising the drug-like properties of the compound. An orally bioavailable compound was identified as a lead agent with nanomolar activity against influenza, representing a 140-fold improvement over the initial hit.

Identification of BPR3P0128 as an inhibitor of cap-snatching activities of influenza virus.

The aim of this study was to identify the antiviral mechanism of a novel compound, BPR3P0128. From a large-scale screening of a library of small compounds, BPR3P compounds were found to be potent inhibitors of influenza viral replication in Madin-Darby canine kidney (MDCK) cells. BPR3P0128 exhibited inhibitory activity against both influenza A and B viruses. The 50% inhibitory concentrations were in the range of 51 to 190 nM in MDCK cells, as measured by inhibition-of-cytopathic-effect assays. BPR3P0128 appeared to target the viral replication cycle but had no effect on viral adsorption. The inhibition of cap-dependent mRNA transcription by BPR3P0128 was more prominent with a concurrent increase in cap-independent cRNA replication in a primer extension assay, suggesting a role of BPR3P0128 in switching transcription to replication. This reduction in mRNA expression resulted from the BPR3P-mediated inhibition of the cap-dependent endoribonuclease (cap-snatching) activities of nuclear extracts containing the influenza virus polymerase complex. No inhibition of binding of 5' viral RNA to the viral polymerase complex by this compound was detected. BPR3P0128 also effectively inhibited other RNA viruses, such as enterovirus 71 and human rhinovirus, but not DNA viruses, suggesting that BPR3P0128 targets a cellular factor(s) associated with viral PB2 cap-snatching activity. The identification of this factor(s) could help redefine the regulation of viral transcription and replication and thereby provide a potential target for antiviral chemotherapeutics.