BPR3P0128, a non-nucleoside RNA-dependent RNA polymerase inhibitor, inhibits SARS-CoV-2 variants of concern and exerts synergistic antiviral activity in combination with remdesivir.

Viral RNA-dependent RNA polymerase (RdRp), a highly conserved molecule in RNA viruses, has recently emerged as a promising drug target for broad-acting inhibitors. Through a Vero E6-based anti-cytopathic effect assay, we found that BPR3P0128, which incorporates a quinoline core similar to hydroxychloroquine, outperformed the adenosine analog remdesivir in inhibiting RdRp activity (EC50 = 0.66 µM and 3 µM, respectively). BPR3P0128 demonstrated broad-spectrum activity against various severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants of concern. When introduced after viral adsorption, BPR3P0128 significantly decreased SARS-CoV-2 replication; however, it did not affect the early entry stage, as evidenced by a time-of-drug-addition assay. This suggests that BPR3P0128's primary action takes place during viral replication. We also found that BPR3P0128 effectively reduced the expression of proinflammatory cytokines in human lung epithelial Calu-3 cells infected with SARS-CoV-2. Molecular docking analysis showed that BPR3P0128 targets the RdRp channel, inhibiting substrate entry, which implies it operates differently-but complementary-with remdesivir. Utilizing an optimized cell-based minigenome RdRp reporter assay, we confirmed that BPR3P0128 exhibited potent inhibitory activity. However, an enzyme-based RdRp assay employing purified recombinant nsp12/nsp7/nsp8 failed to corroborate this inhibitory activity. This suggests that BPR3P0128 may inhibit activity by targeting host-related RdRp-associated factors. Moreover, we discovered that a combination of BPR3P0128 and remdesivir had a synergistic effect-a result likely due to both drugs interacting with separate domains of the RdRp. This novel synergy between the two drugs reinforces the potential clinical value of the BPR3P0128-remdesivir combination in combating various SARS-CoV-2 variants of concern.

Heme oxygenase-1 inhibits DENV-induced endothelial hyperpermeability and serves as a potential target against dengue hemorrhagic fever.

Dengue virus (DENV) is a cause of vascular endothelial dysfunction and vascular leakage, which are characterized as hallmarks of dengue hemorrhagic fever or dengue shock syndrome, which become a severe global health emergency with substantial morbidity and mortality. Currently, there are still no promising therapeutics to alleviate the dengue-associated vascular hemorrhage in a clinical setting. In the present study, we first observed that heme oxygenase-1 (HO-1) expression level was highly suppressed in severe DENV-infected patients. In contrast, the overexpression of HO-1 could attenuate DENV-induced pathogenesis, including plasma leakage and thrombocytopenia, in an AG129 mouse model. Our data indicate that overexpression of HO-1 or its metabolite biliverdin can maintain endothelial integrity upon DENV infection in vitro and in vivo. We further characterized the positive regulatory effect of HO-1 on the endothelial adhesion factor vascular endothelial-cadherin to decrease DENV-induced endothelial hyperpermeability. Subsequently, we confirmed that two medicinal plant-derived compounds, andrographolide, and celastrol, widely used as a nutritional or medicinal supplement are useful to attenuate DENV-induced plasma leakage through induction of the HO-1 expression in DENV-infected AG129 mice. In conclusion, our findings reveal that induction of the HO-1 signal pathway is a promising option for the treatment of DENV-induced vascular pathologies.

MicroRNA-155 inhibits dengue virus replication by inducing heme oxygenase-1-mediated antiviral interferon responses.

MicroRNAs (miRNAs) have been reported to directly alter the virus life cycle and virus-host interactions, and so are considered promising molecules for controlling virus infection. In the present study, we observed that miR-155 time-dependently downregulated upon dengue virus (DENV) infection. In contrast, exogenous overexpression of miR-155 appeared to limit viral replication in vitro, suggesting that the low levels of miR-155 would be beneficial for DENV replication. In vivo, overexpression of miR-155 protected ICR suckling mice from the life-threatening effects of DENV infection and reduced virus propagation. Further investigation revealed that the anti-DENV activity of miR-155 was due to target Bach1, resulting in the induction of the heme oxygenase-1 (HO-1)-mediated inhibition of DENV NS2B/NS3 protease activity, ultimately leading to induction of antiviral interferon responses, including interferon-induced protein kinase R (PKR), 2'-5'-oligoadenylate synthetase 1 (OAS1), OAS2, and OAS3 expression, against DENV replication. Collectively, our results provide a promising new strategy to manage DENV infection by modulation of miR-155 expression.

Discovery of 4-Anilinoquinolinylchalcone Derivatives as Potential NRF2 Activators.

Activation of nuclear factor erythroid-2-related factor 2 (NRF2) has been proven to be an effective means to prevent the development of cancer, and natural curcumin stands out as a potent NRF2 activator and cancer chemopreventive agent. In this study, we have synthesized a series of 4-anilinoquinolinylchalcone derivatives, and used a NRF2 promoter-driven firefly luciferase reporter stable cell line, the HaCaT/ARE cells, to screen a panel of these compounds. Among them, (E)-3-{4-[(4-acetylphenyl)amino]quinolin-2-yl}-1-(4-fluorophenyl)prop-2-en-1-one (13b) significantly increased NRF2 activity in the HaCaT cell with a half maximal effective concentration (EC50) value of 1.95 µM. Treatment of compound 13b upregulated HaCaT cell NRF2 expression at the protein level. Moreover, the mRNA level of NRF2 target genes, heme oxygenase-1 (HO-1), glutamate-cysteine ligase catalytic subunit (GCLC), and glucose-6-phosphate dehydrogenase (G6PD) were significantly increased in HaCaT cells upon the compound 13b treatment. The molecular docking results exhibited that the small molecule 13b is well accommodated by the bound region of Kelch-like ECH-associated protein 1 (Keap1)-Kelch and NRF2 through stable hydrogen bonds and hydrophobic interaction, which contributed to the enhancement of affinity and stability between the ligand and receptor. Compound 13b has been identified as the lead compound for further structural optimization.

Prostasin Impairs Epithelial Growth Factor Receptor Activation to Suppress Dengue Virus Propagation.

BACKGROUND: Dengue virus (DENV), a common and widely spread arbovirus, causes life-threatening diseases, such as dengue hemorrhagic fever or dengue shock syndrome. There is currently no effective therapeutic or preventive treatment for DENV infection. METHODS: Next-generation sequencing analysis revealed that prostasin expression was decreased upon DENV infection. Prostasin expression levels were confirmed by real-time quantitative polymerase chain reaction in patients with dengue fever and a DENV-infected mice model. Short hairpin RNA against EGFR and LY294002 were used to investigate the molecular mechanism. RESULTS: Based on clinical studies, we first found relatively low expression of prostasin, a glycosylphosphatidyl inositol-anchored membrane protease, in blood samples from patients with dengue fever compared with healthy individuals and a high correlation of prostasin expression and DENV-2 RNA copy number. DENV infection significantly decreased prostasin RNA levels of in vivo and in vitro models. By contrast, exogenous expression of prostasin could protect ICR suckling mice from life-threatening DENV-2 infection. Mechanistic studies showed that inhibition of DENV propagation by prostasin was due to reducing expression of epithelial growth factor receptor, leading to suppression of the Akt/NF-κB-mediated cyclooxygenase-2 signaling pathway. CONCLUSION: Our results demonstrate that prostasin expression is a noteworthy clinical feature and a potential therapeutic target against DENV infection.

MicroRNA-let-7c suppresses hepatitis C virus replication by targeting Bach1 for induction of haem oxygenase-1 expression.

MicroRNAs are small noncoding RNAs that are central factors between hepatitis C virus (HCV) and host cellular factors for viral replication and liver disease progression, including liver fibrosis, cirrhosis and hepatocellular carcinoma. In the present study, we found that overexpressing miR-let-7c markedly reduced HCV replication because it induced haem oxygenase-1 (HO-1) expression by targeting HO-1 transcriptional repressor Bach1, ultimately leading to stimulating an antiviral interferon response and blockade of HCV viral protease activity. In contrast, the antiviral actions of miR-let-7c were attenuated by miR-let-7c inhibitor treatment, exogenously expressing Bach1 or suppressing HO-1 activity and expression. A proposed model indicates a key role for miR-let-7c targeting Bach1 to transactivate HO-1-mediated antiviral actions against HCV. miR-let-7c may serve as an attractive target for antiviral development.

Liraglutide Inhibits Hepatitis C Virus Replication Through an AMP Activated Protein Kinase Dependent Mechanism.

Insulin resistance and diabetes are both associated with chronic hepatitis C virus (HCV) infection, and the glucagon-like peptide-1(GLP-1) receptor agonist, liraglutide, is a common therapy for diabetes. Our aim was to investigate whether liraglutide treatment can inhibit HCV replication. A cell culture-produced HCV infectious system was generated by transfection of in vitro-transcribed genomic JFH-1 ribonucleic acid (RNA) into Huh-7.5 cells. Total RNA samples were extracted to determine the efficiency of HCV replication. The Ava5 cells were treated with liraglutide and cell viability was calculated. A Western blot analysis of the protein expression was performed. The immunoreactive blot signals were also detected. Liraglutide activated GLP-1 receptors in the HCV infectious system, and inhibited subgenomic HCV RNA replication in the HuH-7.5 cells. The Western blot analysis revealed both HCV protein and replicon RNA were reduced after treatment with liraglutide in a dose-dependent manner. Liraglutide decreased the cell viability of HCV RNA at an optimum concentration of 120 µg/mL, activated the 5' adenosine monophosphate-activated protein kinase (AMPK) and the phosphorylated- transducer of regulated cyclic adenosine monophosphate (CAMP) response element-binding protein 2 (TORC2), thereby decreasing the cell viability of phosphoenolpyruvate carboxykinase (PEPCK) and G6pase RNA Therefore, we conclude that liraglutide can inhibit HCV replication via an AMPK/TORC2-dependent pathway.

Lipoprotein lipase liberates free fatty acids to inhibit HCV infection and prevent hepatic lipid accumulation.

Lipoprotein lipase (LPL) has been identified as an anti-hepatitis C virus (HCV) host factor, but the cellular mechanism remains elusive. Here, we investigated the cellular mechanism of LPL involving in anti-HCV. The functional activation of peroxisome proliferator-activated receptor (PPAR) α signal by LPL transducing into hepatocytes was investigated in HCV-infected cells, primary human hepatocytes, and in HCV-core transgenic mice. The result showed that the levels of transcriptional transactivity and nuclear translocation of PPARα in Huh7 cells and primary human hepatocytes were elevated by physiologically ranged LPL treatment of either very-low density lipoprotein or HCV particles. The LPL-induced hepatic PPARα activation was weakened by blocking the LPL enzymatic activity, and by preventing the cellular uptake of free unsaturated fatty acids with either albumin chelator or silencing of CD36 translocase. The knockdowns of PPARα and CD36 reversed the LPL-mediated suppression of HCV infection. Furthermore, treatment with LPL, like the direct activation of PPARα, not only reduced the levels of apolipoproteins B, E, and J, which are involved in assembly and release of HCV virions, but also alleviated hepatic lipid accumulation induced by core protein. HCV-core transgenic mice exhibited more hepatic miR-27b, which negatively regulates PPARα expression, than did the wild-type controls. The induction of LPL activity by fasting in the core transgenic mice activated PPARα downstream target genes that are involved in fatty acid ß-oxidation. Taken together, our study reveals dual beneficial outcomes of LPL in anti-HCV and anti-steatosis and shed light on the control of chronic hepatitis C in relation to LPL modulators.

Association between cryoglobulinemia and liver fibrosis in chronic hepatitis C patients.

BACKGROUND AND AIM: The prevalence of mixed cryoglobulinemia is 15-50% in chronic hepatitis C (CHC) patients, and these patients are in an increased risk of liver fibrosis and cirrhosis, but it is controversial. This study aimed to reveal the prevalence of mixed cryoglobulinemia in Asian population and to determine the relationship between presence of serum cryoglobulinemia and liver fibrosis in CHC patients with or without liver biopsy. METHODS: In total, 2255 treatment-naïve patients retrospectively enrolled in our study. Serum cryoglobulinemia precipitation, liver biopsy, and four indexes of fibrosis (FIB4) were assessed to detect the associated factors. RESULTS: Three hundred sixty-four (32%) out of 1135 liver biopsy patients and 341 (30.4%) out of 1120 non-biopsy patients were positive for serum cryoglobulinemia. Multivariate analysis revealed that male gender, hepatitis C virus RNA, platelet and advanced fibrosis (odds ratio [OR] 1.40, 95% confidence interval [CI] 1.05-1.87, P = 0.021) were significantly associated with the presence of cryoglobulinemia in the liver biopsy proven patients. The presence of serum cryoglobulinemia (OR 1.43, 95% CI 1.04-1.96, P = 0.026) was associated with advanced liver fibrosis (F3 and F4) by multivariate logistic regression analysis. In patients without liver biopsy, FIB4 (OR 1.72, 95% CI 1.30-2.27, P = 0.0001) was associated with the presence of serum cryoglobulinemia, and also cryoglobulinemia (OR 1.74, 95% CI 1.32-2.30, P = 0.0001) was associated with high FIB4 (≥ 3.25) patients. CONCLUSION: The prevalence of the presence of serum cryoglobulinemia is 30.4-32% in CHC patients and associated with advanced fibrosis in liver biopsy proven patients and high-FIB4 (≥ 3.25) patients without liver biopsy.

Bioactive Phenolic Components from the Twigs of Atalantia buxifolia.

Five new compounds named buxifoximes A-C (1-3), buxifobenzoate (4), and 7- O-(7'-peroxygeranyl) coumarin (5), together with 25 known compounds, were identified from the twigs of Atalantia buxifolia. Compounds 1-3 are unique secondary metabolites with the aldoxime functionality. The structures of the isolates were determined on the basis of spectroscopic data analyses, and the structure of 1 was confirmed by an X-ray single-crystallographic analysis. With respect to bioactivity, antidengue virus, anti-inflammatory, and cytotoxic activities of all purified compounds were tested and evaluated. Compound 1 showed a significant anti-inflammatory effect by inhibiting superoxide anion generation with an IC50 value of 4.8 ± 0.7 µM. Among the acridone alkaloids, 5-hydroxy- N-methylseverifoline (23) exhibited antidengue activity (IC50 = 5.3 ± 0.4 µM), and atalaphyllinine (20) demonstrated cytotoxicity (IC50 = 6.5 ± 0.0 µM) against the human liver cancer cell line, HepG2.

Discovery of 3-Amino-2-Hydroxypropoxyisoflavone Derivatives as Potential Anti-HCV Agents.

Synthesis and anti-hepatitis C virus (anti-HCV) effects of certain 3-amino-2-hydroxy-propoxy isoflavone derivatives, 6a⁻i, were described. The known 3-(3,4-dimethoxyphenyl)-7-(oxiran-2-ylmethoxy)-4H-chromen-4-one (5) was reacted with substituted amines to give the desired isoflavone derivatives, 6a⁻i. Among them, 7-{3-[(3,4-dimethoxy-phenethyl)amino]-2-hydroxypropoxy}-3-(3,4-dimethoxyphenyl)-4H-chromen-4-one (6b) was the most active, exhibiting approximately 2-fold higher anti-HCV effects than standard antiviral drug ribavirin (EC50 of 6.53 vs. 13.16 µM). In addition, compound 6b was less cytotoxic than ribavirin. The selectivity index (SI) of 6b is approximately 2.6-fold higher than ribavirin. The compounds 6e, 6h, and 6i were also found to possess higher anti-HCV effects than ribavirin. Compound 6b was found to inhibit the HCV RNA expression in Ava5 cells in a dose-dependent manner; furthermore, we found that the antiviral mechanism of compounds 6b, 6e, 6h, and 6i gave rise to induction of HO-1 expression. With the HO-1 promoter-based analysis, we found compounds 6b, 6e, 6h, and 6i induced HO-1 expression through increasing Nrf-2 binding activity. Taken together, compound 6b may serve as a potential lead compound for developing novel anti-HCV agents.

Butyrolactones and Diketopiperazines from Marine Microbes: Inhibition Effects on Dengue Virus Type 2 Replication.

Two new compounds, 4S,10R-dihydroxy-11-methyl-dodec-2-en-1,4-olide (1) (butyrolactone-type) and cyclo-(4-trans-6-dihydroxy-proline-D-leucine) (2) (diketopiperazine-type), as well as one known 4S,10-dihydroxy-10-methyl-dodec-2-en-1,4-olide (3) and three known diketopiperazines, cyclo-(L-proline-L-leucine) (4), cyclo-(4-trans-hydroxy-L-proline-L-leucine) (5), and cyclo-(4-trans-hydroxy-L-proline-L-phenylalanine) (6), were isolated from the ethyl acetate extracts of Streptomyces gougerotii GT and Microbulbifer variabilis C-03. Compounds 3, 4, 5, and 6 exhibited a significant reduction effect on dengue virus type 2 replication with EC50 values of 21.2, 16.5, 12.3, and 11.2 µM, respectively.

Inhibition of dengue virus replication by novel inhibitors of RNA-dependent RNA polymerase and protease activities.

Dengue virus (DENV) is the leading mosquito-transmitted viral infection in the world. With more than 390 million new infections annually, and up to 1 million clinical cases with severe disease manifestations, there continues to be a need to develop new antiviral agents against dengue infection. In addition, there is no approved anti-DENV agents for treating DENV-infected patients. In the present study, we identified new compounds with anti-DENV replication activity by targeting viral replication enzymes - NS5, RNA-dependent RNA polymerase (RdRp) and NS3 protease, using cell-based reporter assay. Subsequently, we performed an enzyme-based assay to clarify the action of these compounds against DENV RdRp or NS3 protease activity. Moreover, these compounds exhibited anti-DENV activity in vivo in the ICR-suckling DENV-infected mouse model. Combination drug treatment exhibited a synergistic inhibition of DENV replication. These results describe novel prototypical small anti-DENV molecules for further development through compound modification and provide potential antivirals for treating DENV infection and DENV-related diseases.

Ecdysones from Zoanthus spp. with inhibitory activity against dengue virus 2.

Bioassay-guided fractionation of an ethanolic extract of Zoanthus spp. collected in Taiwan has resulted in the isolation of one new ecdysone, zoanthone A (1), along with thirteen known compounds (2-14). The structures of these compounds were determined by spectroscopic methods, especially 2D NMR analyses. The in vitro antiviral activities of all isolated ecdysones (1-14) against dengue virus type 2 (DENV-2) were evaluated using DENV infectious system. New compound (1) exhibited potent antiviral activity (EC50=19.61 ± 2.46 µM) with a selectivity index (CC50/EC50) value of 36.7. The structure-activity relationships of isolated ecdysones against DENV-2 were concluded. Molecular docking information of 3 and NS5 polymerase was performed either.

Anti-Dengue Virus Constituents from Formosan Zoanthid Palythoa mutuki.

A new marine ecdysteroid with an α-hydroxy group attaching at C-4 instead of attaching at C-2 and C-3, named palythone A (1), together with eight known compounds (2-9) were obtained from the ethanolic extract of the Formosan zoanthid Palythoa mutuki. The structures of those compounds were mainly determined by NMR spectroscopic data analyses. The absolute configuration of 1 was further confirmed by comparing experimental and calculated circular dichroism (CD) spectra. Anti-dengue virus 2 activity and cytotoxicity of five isolated compounds were evaluated using virus infectious system and [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt (MTS) assays, respectively. As a result, peridinin (9) exhibited strong antiviral activity (IC50 = 4.50 ± 0.46 µg/mL), which is better than that of the positive control, 2'CMC. It is the first carotene-like substance possessing anti-dengue virus activity. In addition, the structural diversity and bioactivity of the isolates were compared by using a ChemGPS-NP computational analysis. The ChemGPS-NP data suggested natural products with anti-dengue virus activity locate closely in the chemical space.

Apolipoprotein J, a glucose-upregulated molecular chaperone, stabilizes core and NS5A to promote infectious hepatitis C virus virion production.

BACKGROUND & AIMS: Hepatitis C virus (HCV) infection leads to glucose abnormality. HCV depends on lipid droplets (LDs) and very-low density lipoproteins for assembly/releasing; however, the components and locations for this process remain unidentified. Apolipoprotein J (ApoJ), upregulated by glucose, functions as Golgi chaperone of secreted proteins and resides abundantly in very-low density lipoproteins. This study investigates the interplay between glucose, ApoJ and HCV virion production. METHODS: The effects of high glucose on ApoJ expression and HCV production were evaluated with cultivated HuH7.5, primary human hepatocytes, and in treatment naive chronic hepatitis C patients. How ApoJ affects HCV lifecycle was assessed using siRNA knockdown strategy in JFH1 infected and subgenomic replicon cells. The interactions and locations of ApoJ with viral and host components were examined by immunoprecipitation, immunofluorescence and subcellular fractionation experiments. RESULTS: HCV infection increased ApoJ expression, which in parallel with HCV infectivity was additionally elevated with high glucose treatment. Serum ApoJ correlated positively with fasting blood glucose concentration and HCV-RNA titre in patients. ApoJ silencing reduced intracellular and extracellular HCV infectivity and extracellular HCV-RNA, but accumulated intracellular HCV-RNA in HCV-infected cells. ApoJ interacted with HCV core and NS5A and stabilized the dual protein complex. HCV infection dispersed cytoplasmic ApoJ from the compact zones of the Golgi to encircle LDs, where co-localization of the core, NS5A, HCV-RNA, subcellular markers for LDs, endoplasmic reticulum (ER), Golgi, and membrane contact sites occurred. CONCLUSIONS: ApoJ facilitates infectious HCV particle production via stabilization of core/NS5A, which might surround LDs at the ER-Golgi membrane contact site.

Production of a neutralizing antibody against envelope protein of dengue virus type 2 using the linear array epitope technique.

Dengue virus (DENV; genus Flavivirus) contains a positive-stranded RNA genome. Binding of DENV to host cells is mediated through domain III of the viral envelope protein. Many therapeutic mAbs against domain III have been generated and characterized because of its high antigenicity. We have previously established a novel PCR method named the linear array epitope (LAE) technique for producing monoclone-like polyclonal antibodies. To prove this method could be utilized to produce antibody against epitopes with low antigenicity, a region of 10 aa (V365NIEAEPPFG374) from domain III of the envelope protein in DENV serotype 2 (DENV2) was selected to design the primers for the LAE technique. A DNA fragment encoding 10 directed repeats of these 10 aa for producing the tandem-repeated peptides was obtained and fused with glutathione S-transferase (GST)-containing vector. This fusion protein (GST-Den EIII10-His6) was purified from Escherichia coli and used as antigen for immunizing rabbits to obtain the polyclonal antibody. Furthermore, the EIII antibody could recognize envelope proteins either ectopically overexpressed or synthesized by DENV2 infection using Western blot and immunofluorescence assays. Most importantly, this antibody was also able to detect DENV2 virions by ELISA, and could block viral entry into BHK-21 cells as shown by immunofluorescence and quantitative real-time PCR assays. Taken together, the LAE technique could be applied successfully for the production of antibodies against antigens with low antigenicity, and shows high potential to produce antibodies with good quality for academic research, diagnosis and even therapeutic applications in the future.

The antiproliferative effect of C2-ceramide on lung cancer cells through apoptosis by inhibiting Akt and NFκB.

The anticancer effects of ceramide have been reported in many types of cancers but less in lung cancer. In this study, we used C2-ceramide to further investigate its possible anticancer effects and mechanisms on non-small cell lung cancer (NSCLC) H1299 cells. The result of cell proliferation in terms of trypan blue assay showed high dose of C2-ceramide inhibited cell survival after 24 h treatment. The flow cytometry-based assays indicated the effect of apoptosis, chromatin condensation, and G1 arrest in terms of Annexin V/propidium iodide (PI), DAPI, and PI stainings, respectively. Moreover, the decreased protein level of p-Akt, p-NFκB, survivin and cyclin A2 were detected by Western blot assay. Taken together, these results indicated the antiproliferative effect of C2-ceramide is majorly responsible for cell apoptosis in lung cancer H1299 cells.

Limonoids from the seeds of Swietenia macrophylla with inhibitory activity against dengue virus 2.

Fractionation of an ethanol-soluble extract of the seeds of Swietenia macrophylla yielded six new limonoids, swielimonoids A-F (1-6), along with 20 known compounds. Compounds 1 and 2, mexicanolide-type limonoids, were assigned with an α,ß-unsaturated δ-lactone moiety (ring D) and a C═C bond between C-8 and C-30. Compounds 3-6 could be categorized as highly oxygenated phragmalin-type limonoids. The structures of these new compounds were elucidated through the interpretation of spectroscopic data. The antidengue virus 2 activities of the isolated components from S. macrophylla were investigated, and of 12 compounds subjected to bioassay, compounds 2 and 7-10 were found to show inhibitory activity in the range 3.5 to 12.5 µM. Among these, the new limonoid 2 exhibited significant antiviral activity (EC50 = 7.2 ± 1.33 µM) with a selectivity index (CC50/EC50) value of >27.7.

Very low-density lipoprotein/lipo-viro particles reverse lipoprotein lipase-mediated inhibition of hepatitis C virus infection via apolipoprotein C-III.

OBJECTIVE: Circulating hepatitis C virus (HCV) virions are associated with triglyceride-rich lipoproteins, including very low-density lipoprotein (VLDL) and low-density lipoprotein (LDL), designated as lipo-viro-particles (LVPs). Previous studies showed that lipoprotein lipase (LPL), a key enzyme for hydrolysing the triglyceride in VLDL to finally become LDL, may suppress HCV infection. This investigation considers the regulation of LPL by lipoproteins and LVPs, and their roles in the LPL-mediated anti-HCV function. DESIGN: The lipoproteins were fractionated from normolipidemic blood samples using iodixanol gradients. Subsequent immunoglobulin-affinity purification from the canonical VLDL and LDL yielded the corresponding VLDL-LVP and LDL-LVP. Apolipoprotein (apo) Cs, LPL activity and HCV infection were quantified. RESULTS: A higher triglyceride/cholesterol ratio of LDL was found more in HCV-infected donors than in healthy volunteers, and the triglyceride/cholesterol ratio of LDL-LVP was much increased, suggesting that the LPL hydrolysis of triglyceride may be impaired. VLDL, VLDL-LVP, LDL-LVP, but not LDL, suppressed LPL lipolytic activity, which was restored by antibodies that recognised apoC-III/-IV and correlated with the steadily abundant apoC-III/-IV quantities in those particles. In a cell-based system, treatment with VLDL and LVPs reversed the LPL-mediated inhibition of HCV infection in apoC-III/-IV-dependent manners. A multivariate logistic regression revealed that plasma HCV viral loads correlated negatively with LPL lipolytic activity, but positively with the apoC-III content of VLDL. Additionally, apoC-III in VLDL was associated with a higher proportion of HCV-RNA than was IgG. CONCLUSION: This study reveals that LPL is an anti-HCV factor, and that apoC-III in VLDL and LVPs reduces the LPL-mediated inhibition of HCV infection.