Mayuko Fukuoka

Structure-based discovery of anti-influenza virus A compounds among medicines.

BACKGROUND Influenza A virus (IAV) infection is nowadays a major public health concern, in particular since the 2009 H1N1 flu pandemic. The outbreak of IAV strains resistant to currently available drugs, such as oseltamivir or zanamivir targeting the neuraminidase, is a real threat for humans as well as for animals. Thus the development of anti-IAV drugs with a novel action mechanism may be an urgent theme. METHODS We performed a docking simulation targeting the interface of PA interacting with PB1 using a drug database including ~4000 compounds. We then conducted cell viability assay and plaque assay using IAV/WSN/33. Finally we examined their anti-IAV mechanism by surface plasmon resonance and IAV replicon assay. RESULTS We found that benzbromarone, diclazuril, and trenbolone acetate had strong anti-IAV activities. We confirmed that benzbromarone and diclazuril bound with PA subunit, and decreased the transcriptional activity of the viral RNA polymerase. CONCLUSIONS Benzbromarone and diclazuril had strong anti-IAV activities with novel action mechanism, i.e. inhibition of viral RNA polymerase. GENERAL SIGNIFICANCE Since benzbromarone and diclazuril are already used in public as medicines, these could be the candidates for alternatives in case of an outbreak of IAV which is resistant to pre-existing anti-IAV drugs.

Caffeoylquinic acids are major constituents with potent anti-influenza effects in brazilian green propolis water extract.

Influenza A viral infections reached pandemic levels in 1918, 1957, 1968, and, most recently, in 2009 with the emergence of the swine-origin H1N1 influenza virus. The development of novel therapeutics or prophylactics for influenza virus infection is urgently needed. We examined the evaluation of the anti-influenza virus (A/WSN/33 (H1N1)) activity of Brazilian green propolis water extract (PWE) and its constituents by cell viability and real-time PCR assays. Our findings showed strong evidence that PWE has an anti-influenza effect and demonstrate that caffeoylquinic acids are the active anti-influenza components of PWE. Furthermore, we have found that the amount of viral RNA per cell remained unchanged even in the presence of PWE, suggesting that PWE has no direct impact on the influenza virus but may have a cytoprotective activity by affecting internal cellular process. These findings indicate that caffeoylquinic acids are the active anti-influenza components of PWE. Above findings might facilitate the prophylactic application of natural products and the realization of novel anti-influenza drugs based on caffeoylquinic acids, as well as further the understanding of cytoprotective intracellular mechanisms in influenza virus-infected cells.

3,4-Dicaffeoylquinic Acid, a Major Constituent of Brazilian Propolis, Increases TRAIL Expression and Extends the Lifetimes of Mice Infected with the Influenza A Virus

Brazilian green propolis water extract (PWE) and its chemical components, caffeoylquinic acids, such as 3,4-dicaffeoylquinic acid (3,4-diCQA), act against the influenza A virus (IAV) without influencing the viral components. Here, we evaluated the anti-IAV activities of these compounds in vivo. PWE or PEE (Brazilian green propolis ethanol extract) at a dose of 200 mg/kg was orally administered to Balb/c mice that had been inoculated with IAV strain A/WSN/33. The lifetimes of the PWE-treated mice were significantly extended compared to the untreated mice. Moreover, oral administration of 3,4-diCQA, a constituent of PWE, at a dose of 50 mg/kg had a stronger effect than PWE itself. We found that the amount of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) mRNA in the mice that were administered 3,4-diCQA was significantly increased compared to the control group, while H1N1 hemagglutinin (HA) mRNA was slightly decreased. These data indicate that PWE, PEE or 3,4-diCQA possesses a novel and unique mechanism of anti-influenza viral activity, that is, enhancing viral clearance by increasing TRAIL.

Effects of ligand binding on the stability of aldo-keto reductases: Implications for stabilizer or destabilizer chaperones.

Ligands such as enzyme inhibitors stabilize the native conformation of a protein upon binding to the native state, but some compounds destabilize the native conformation upon binding to the non‐native state. The former ligands are termed “stabilizer chaperones” and the latter ones “destabilizer chaperones.” Because the stabilization effects are essential for the medical chaperone (MC) hypothesis, here we have formulated a thermodynamic system consisting of a ligand and a protein in its native‐ and non‐native state. Using the differential scanning fluorimetry and the circular dichroism varying the urea concentration and temperature, we found that when the coenzyme NADP+ was absent, inhibitors such as isolithocholic acid stabilized the aldo–keto reductase AKR1A1 upon binding, which showed actually the three‐state folding, but destabilized AKR1B10. In contrast, in the presence of NADP+, they destabilized AKR1A1 and stabilized AKR1B10. To explain these phenomena, we decomposed the free energy of stabilization (ΔΔG) into its enthalpy (ΔΔH) and entropy (ΔΔS) components. Then we found that in a relatively unstable protein showing the three‐state folding, native conformation was stabilized by the negative ΔΔH in association with the negative ΔΔS, suggesting that the stabilizer chaperon decreases the conformational fluctuation of the target protein or increase its hydration. However, in other cases, ΔΔG was essentially determined by the delicate balance between ΔΔH and ΔΔS. The proposed thermodynamic formalism is applicable to the system including multiple ligands with allosteric interactions. These findings would promote the development of screening strategies for MCs to regulate the target conformations.

Comparison of the Efficacy of Alpha-Lactalbumin from Equine, Bovine, and Human Milk in the Growth of Intestinal IEC-6 Cells

Native alpha-lactalbumins (α-LA) from equine, bovine, and human milk were not cytotoxic. However, after treatment with trifluoroethanol (TFE), all three α-LAs exhibited cytotoxicity. Toxic potencies were distinctly different among them. Equine α-LA was the most robust, bovine α-LA was moderate, and human α-LA was weak. There were no significant structural changes as between the native and the TFE-treated α-LAs.

Effects of heat treatment on conformation and cell growth activity of alpha- lactalbumin and beta-lactoglobulin from market milk

Heat processes, low temperature for long time (LTLT) pasteurization and ultra-heat treatment (UHT) sterilization, are essential for commercial market milk to improve the shelf life of raw milk and ensure microbial safety. We evaluated the effects of heat experience on the molecular properties of α-lactalbumin (α-LA) and β-lactoglobulin (β-LG) isolated from four types of market milk such as LTLT-A (66°C for 30 min), LTLT-B (65°C for 30 min), UHT-I (130°C for 2 s, indirect heating) and UHT-D (135°C for 2 s, direct heating) samples. We examined molecular conformations using circular dichroism spectrum measurement and cell growth activity using the WST-1 method for the proteins. α-LA isolated from each of these four types of market milk displayed no significant structural difference as compared to raw milk α-LA, while α-LA of UHT-I only inhibited cell growth of an intestinal epithelial cell line more potently than raw milk α-LA. In the case of β-LG, only the UHT-I sample demonstrated a drastic change in structure, while it did not exhibit any cytotoxicity. We found that cell viability effects of α-LA and β-LG are attributable to the type of UHT; indirect and direct. These findings indicate that the effect of heat treatment on whey proteins should carefully be investigated further.

Evaluation of compound selectivity of aldo-keto reductases using differential scanning fluorimetry.

Inhibitors of AKR1B10 belonging to the aldo-keto reductase (AKR) superfamily are considered promising candidates for anti-cancer drugs. AKR1B1, a structurally similar isoform of AKR1B10, is involved in glucose metabolism. Thus, selective inhibition of AKR1B10 is required for the development of anti-cancer drugs. In this study, we first compared correlations between melting temperature and the 50% inhibition concentration obtained from differential scanning fluorimetry (DSF) and an enzyme inhibitory experiment, respectively, and a good correlation was found, except for compounds with low solubility. This result indicates that the DSF method is useful for drug screening for the AKR superfamily. We then evaluated their selectivity as inhibitors against all seven major human AKR1 family proteins and found that C18 is most specific for AKR1B10.

Logical design of an anti‐cancer agent targeting the plant homeodomain in Pygopus2

Pygopus2 (Pygo2) is a component of the Wnt signaling pathway, which is required for β‐catenin mediated transcription. Plant homeodomain (PHD) finger in Pygo2 intercalates the methylated histone 3 (H3K4me) tail and HD1 domain of BCL9 that binds to β‐catenin. Thus, PHD finger may be a potential target for the logical design of an anti‐cancer drug. Here, we found that Spiro[2H‐naphthol[1,2‐b]pyran‐2,4′‐piperidine]‐1′ethanol,3,4‐dihydro‐4‐hydroxy‐α‐(6‐methyl‐1H‐indol‐3‐yl)) termed JBC117 interacts with D339, A348, R356, V376 and A378 in PHD corresponding to the binding sites with H3K4me and/or HD1, and has strong anti‐cancer effects. For colon (HCT116) and lung (A549) cancer cell lines, IC50 values were 2.6 ± 0.16 and 3.3 ± 0.14 μM, respectively, while 33.80 ± 0.15 μM for the normal human fibroblast cells. JBC117 potently antagonized the cellular effects of β‐catenin‐dependent activity and also inhibited the migration and invasion of cancer cells. In vivo studies showed that the survival time of mice was significantly prolonged by the subcutaneous injection of JBC117 (10 mg/kg/day). In conclusion, JBC117 is a novel anti‐cancer lead compound targeting the PHD finger of Pygo2 and has a therapeutic effect against colon and lung cancer.