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.

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.

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.

Lucidone suppresses hepatitis C virus replication by Nrf2-mediated heme oxygenase-1 induction.

Upon screening of plant-derived natural products against hepatitis C virus (HCV) in the replicon system, we demonstrate that lucidone, a phytocompound, isolated from the fruits of Lindera erythrocarpa Makino, significantly suppressed HCV RNA levels with 50% effective concentrations of 15 ± 0.5 µM and 20 ± 1.1 µM in HCV replicon and JFH-1 infectious assays, respectively. There was no significant cytotoxicity observed at high concentrations, with a 50% cytotoxic concentration of 620 ± 5 µM. In addition, lucidone significantly induced heme oxygenase-1 (HO-1) production and led to the increase of its product biliverdin for inducing antiviral interferon response and inhibiting HCV NS3/4A protease activity. Conversely, the anti-HCV activity of lucidone was abrogated by blocking HO-1 activity or silencing gene expression of HO-1 or NF-E2-related factor 2 (Nrf2) in the presence of lucidone, indicating that the anti-HCV action of lucidone was due to the stimulation of Nrf-2-mediated HO-1 expression. Moreover, the combination of lucidone and alpha interferon, the protease inhibitor telaprevir, the NS5A inhibitor BMS-790052, or the NS5B polymerase inhibitor PSI-7977, synergistically suppressed HCV RNA replication. These findings suggest that lucidone could be a potential lead or supplement for the development of new anti-HCV agent in the future.

Phellinus linteus mycelia extract in COVID-19 prevention and identification of its key metabolic compounds profiling using UPLC-QTOF-MS/MS spectrometry.

For centuries, food, herbal medicines, and natural products have been valuable resources for discovering novel antiviral drugs, uncovering new structure-activity relationships, and developing effective strategies to prevent/treat viral infections. One such resource is Phellinus linteus, a mushroom used in folk medicine in Taiwan, Japan, Korea, and China. In this rich historical context, the key metabolites of Phellinus linteus mycelia ethanolic extract (GKPL) impacting the entry of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) at multiple stages have yet to be explored. Thus, this study systematically identifies and assesses the inhibitory effect of GKPL on the SARS-CoV-2 virus. Initially, the concentrations and contact times of GKPL against SARS-CoV-2 pseudovirus were assessed in HepG2 cells. Subsequently, utilizing the Ultra Performance Liquid Chromatography-Quadrupole Time-of-Flight Mass Spectrometry method, potential biomarkers in the fungal extract were discerned. Metabolomic analysis identified 18 compounds in GKPL, with hispidin and hypholomine B present in the highest amounts. These compounds were isolated using chromatographic techniques and further identified through 1D NMR spectroscopic and mass spectrometry analysis. Hispidin and hypholomine B were found to inhibit the infection of SARS-CoV-2 pseudovirus by reducing angiotensin-converting enzyme 2 gene expression in HepG2, thereby decreasing viral entry. Moreover, hispidin and hypholomine B effectively block the spike receptor-binding domain, while hypholomine B, for the first time, showed significant inhibition of 3CL protease. This suggests that GKPL, enriched with hispidin and hypholomine B, has the potential to be used as an active ingredient against SARS-CoV-2.

Synthesis and anti-HCV activity evaluation of anilinoquinoline derivatives.

Hepatitis C virus (HCV) infection is a main cause of chronic liver disease, leading to liver cirrhosis and hepatocellular carcinoma (HCC). The objective of our research was to develop effective agents against viral replication. Here, we have synthesized a series of anilinoquinoline derivatives. Based on a cell-based HCV replicon system, we observed that 2-(3'-nitroanilino)quinoline (18) exhibited anti-HCV activity with a 50% effective concentration (EC(50)) value of 7µM and a selective index (SI) value of 10. In addition, compound 18 possessed the inhibitory effect on HCV NS3/4A protease activity. Therefore, we concluded that the compound 18 possessed a potent activity against HCV replication and could provide as a new lead compound as anti-HCV inhibitor.

Antigenic Cross-Reactivity Between SARS-CoV-2 S1-RBD and Its Receptor ACE2.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is an emerging virus responsible for the ongoing COVID-19 pandemic. SARS-CoV-2 binds to the human cell receptor angiotensin-converting enzyme 2 (ACE2) through its receptor-binding domain in the S1 subunit of the spike protein (S1-RBD). The serum levels of autoantibodies against ACE2 are significantly higher in patients with COVID-19 than in controls and are associated with disease severity. However, the mechanisms through which these anti-ACE2 antibodies are induced during SARS-CoV-2 infection are unclear. In this study, we confirmed the increase in antibodies against ACE2 in patients with COVID-19 and found a positive correlation between the amounts of antibodies against ACE2 and S1-RBD. Moreover, antibody binding to ACE2 was significantly decreased in the sera of some COVID-19 patients after preadsorption of the sera with S1-RBD, which indicated that antibodies against S1-RBD can cross-react with ACE2. To confirm this possibility, two monoclonal antibodies (mAbs 127 and 150) which could bind to both S1-RBD and ACE2 were isolated from S1-RBD-immunized mice. Measurement of the binding affinities by Biacore showed these two mAbs bind to ACE2 much weaker than binding to S1-RBD. Epitope mapping using synthetic overlapping peptides and hydrogen deuterium exchange mass spectrometry (HDX-MS) revealed that the amino acid residues P463, F464, E465, R466, D467 and E471 of S1-RBD are critical for the recognition by mAbs 127 and 150. In addition, Western blotting analysis showed that these mAbs could recognize ACE2 only in native but not denatured form, indicating the ACE2 epitopes recognized by these mAbs were conformation-dependent. The protein-protein interaction between ACE2 and the higher affinity mAb 127 was analyzed by HDX-MS and visualized by negative-stain transmission electron microscopy imaging combined with antigen-antibody docking. Together, our results suggest that ACE2-cross-reactive anti-S1-RBD antibodies can be induced during SARS-CoV-2 infection due to potential antigenic cross-reactivity between S1-RBD and its receptor ACE2.

An in vitro coupled transcription/translation reporter system for hepatitis C virus RNA-dependent RNA polymerase.

Hepatitis C virus (HCV) NS5B, an RNA-dependent RNA polymerase (RdRp), is an attractive target for antiviral agents. The in vitro RNA synthesis system based on radioisotopic readout is commonly used for polymerase inhibitor screening; however, this system generates large amounts of radioactive waste and is not amenable to high-throughput applications. To overcome this limitation, we generated pFLuc-(-)UTRΔC-RLuc, a bicistronic reporter vector, which allows effective and sensitive distinction of RdRp activity by using a cell-free coupled transcription/translation system. This reporter construct comprises the firefly luciferase (FLuc) and the Renilla luciferase (RLuc) genes in reverse orientation flanked by the two negative strands of the HCV 5'- and 3'-untranslated regions in which FLuc and RLuc reporter proteins are regulated by bacteriophage T7 polymerase and NS5B polymerase, respectively. The increase in RLuc activity was proportional to the amount of active RdRp. This cell-free dual reporter system was further validated using specific RdRp inhibitors. Hence, linear dose-response curves between RLuc activity and specific inhibitors were obtained, as was faster drug screening through real-time measurement of chemiluminescence. Moreover, this reporter system is suitable for robust in vitro screening because of a statistically acceptable Z' factor value of 0.79 under the antiviral screening condition in the 96-well format.

A cell-based reporter assay for inhibitor screening of hepatitis C virus RNA-dependent RNA polymerase.

The hepatitis C virus (HCV) NS5B, a RNA-dependent RNA polymerase (RdRp), is an attractive target for anti-HCV agents. The major disadvantages of the commonly used polymerase inhibitor screening involving the assessment of in vitro RNA synthesis are that it is incapable of demonstrating the cellular permeability and the cytotoxicity of compounds. To overcome these limitations, we created the BHK-NS5B-FRLuc reporter cell line that carries stably transfected NS5B and a bicistronic reporter gene, (+)FLuc-(-)UTR-RLuc, which can be used to simultaneously measure cellular toxicity and intracellular RdRp activity. The (+)FLuc-(-)UTR-RLuc construct comprises the firefly luciferase (FLuc) gene and the Renilla luciferase (RLuc) gene in reverse orientation flanked by both negative strands of the HCV 5'- and 3'-untranslated regions (UTRs), in which FLuc and RLuc reporter proteins are regulated by host polymerase and functional NS5B polymerase, respectively. The reporter system was validated with specific agents against NS5B polymerization. Additionally, this assay was placed in 96-well plates and had a Z'-factor value of approximately 0.75, which is amenable for facilitating high-throughput screening operations. Notably, in combination with the structured-based virtual screening, an imidazole derivative compound was evaluated as a candidate HCV RdRp inhibitor.

Avocado (Persea americana) fruit extract (2R,4R)-1,2,4-trihydroxyheptadec-16-yne inhibits dengue virus replication via upregulation of NF-κB-dependent induction of antiviral interferon responses.

Dengue virus (DENV) caused millions of infections around the world annually. Co-infection with different serotypes of DENV is associated with dengue hemorrhagic shock syndrome, leading to an estimate of 50% death rate. No approved therapies are currently available for the treatment of DENV infection. Hence, novel anti-DENV agents are urgently needed for medical therapy. Here we demonstrated that a natural product (2 R,4 R)-1,2,4-trihydroxyheptadec-16-yne (THHY), extracted from avocado (Persea americana) fruit, can inhibit DENV-2 replication in a concentration-dependent manner and efficiently suppresses replication of all DENV serotypes (1-4). We further reveal that the NF-κB-mediated interferon antiviral response contributes to the inhibitory effect of THHY on DENV replication. Using a DENV-infected ICR suckling mouse model, we found that THHY treatment caused an increased survival rate among mice infected with DENV. Collectively, these findings support THHY as a potential agent to control DENV infection.

ICR suckling mouse model of Zika virus infection for disease modeling and drug validation.

BACKGROUND: Zika virus (ZIKV) infection causes diseases ranging from acute self-limiting febrile illness to life-threatening Guillain-Barré Syndrome and other neurological disorders in adults. Cumulative evidence suggests an association between ZIKV infection and microcephaly in newborn infants. Given the host-range restrictions of the virus, a susceptible animal model infected by ZIKV must be developed for evaluation of vaccines and antivirals. In this study, we propose a convenient mouse model for analysis of neurological disorders caused by ZIKV. METHODOLOGY: Six-day-old immunocompetent ICR suckling mice were used in the experiment. Different inoculum virus concentrations, challenge routes, and challenge times were assessed. Viremic dissemination was determined in the liver, spleen, kidney, and brain through Western blot assay, plaque assay, absolute quantification real-time PCR, and histological observation. Azithromycin, a well-characterized anti-ZIKV compound, was used to evaluate the ICR suckling mouse model for antiviral testing. CONCLUSIONS: Signs of illness and neurological disease and high mortality rate were observed in mice injected with ZIKV intracerebrally (102 to 105) and intraperitoneally (103 to 105). Viremic dissemination was observed in the liver, spleen, kidney, and brain. ZIKV transmitted, rapid replicated, and induced monocyte infiltration into the brain approximately 5 to 6 days post inoculum. Azithromycin conferred protection against ZIKV-caused neurological and life-threatening diseases. The developed model of ZIKV infection and disease can be used for screening drugs against ZIKV and discovering the underlying mechanism of ZIKV pathogenesis.

Discovery of naphtho[1,2-d]oxazole derivatives as potential anti-HCV agents through inducing heme oxygenase-1 expression.

A number of naphtho[1,2-d]oxazole derivatives were synthesized and evaluated for their anti-HCV virus activity. Among them, compound 18 was the most active, exhibited approximately 21-folds more active anti-HCV activity (IC50 of 0.63 µM) than that of ribavirin (IC50 = 13.16 µM). Compound 18 was less cytotoxic than ribavirin, and the selective index (SI) of 18 is approximately 28-folds higher than that of ribavirin (229.10 v.s. 8.08). By using heme oxygenase-1 (HO-1) promoter-based assay and western blotting, compound 18 could induce HO-1 promoter activity, and protein expression. The antiviral effect of compound 18 was attenuated by HO-1 specific inhibitor SnPP treatment, which indicated that compound 18 suppressed HCV replication through inducing HO-1 expression. We further found that compound 18 reduced bach1 expression resulting in increasing the activity of Nrf-2 binding element. Moreover, the induction of HO-1 by compound 18 reduced HCV NS3/4A protease activity and induced the antiviral interferon responses. Therefore, compound 18 can be considered as a supplemental antiviral agent or a lead compound for further developing more effective agents against HCV replication.

Lucidone suppresses dengue viral replication through the induction of heme oxygenase-1.

Dengue virus (DENV) infection causes life-threatening diseases such as dengue hemorrhagic fever and dengue shock syndrome. Currently, there is no effective therapeutic agent or vaccine against DENV infection; hence, there is an urgent need to discover anti-DENV agents. The potential therapeutic efficacy of lucidone was first evaluated in vivo using a DENV-infected Institute of Cancer Research (ICR) suckling mouse model by monitoring body weight, clinical score, survival rate, and viral titer. We found that lucidone effectively protected mice from DENV infection by sustaining survival rate and reducing viral titers in DENV-infected ICR suckling mice. Then, the anti-DENV activity of lucidone was confirmed by western blotting and quantitative-reverse-transcription-polymerase chain reaction analysis, with an EC50 value of 25 ± 3 µM. Lucidone significantly induced heme oxygenase-1 (HO-1) production against DENV replication by inhibiting DENV NS2B/3 protease activity to induce the DENV-suppressed antiviral interferon response. The inhibitory effect of lucidone on DENV replication was attenuated by silencing of HO-1 gene expression or blocking HO-1 activity. In addition, lucidone-stimulated nuclear factor erythroid 2-related factor 2 (Nrf2), which is involved in transactivation of HO-1 expression for its anti-DENV activity. Taken together, the mechanistic investigations revealed that lucidone exhibits significant anti-DENV activity in in vivo and in vitro by inducing Nrf2-mediated HO-1 expression, leading to blockage of viral protease activity to induce the anti-viral interferon (IFN) response. These results suggest that lucidone is a promising candidate for drug development.

Lobohedleolide suppresses hepatitis C virus replication via JNK/c-Jun-C/EBP-mediated down-regulation of cyclooxygenase-2 expression.

Hepatitis C virus (HCV) chronically infects 2-3% people of the global population, which leads to liver cirrhosis and hepatocellular carcinoma. Drug resistance remains a serious problem that limits the effectiveness of US Food and Drug Administration (FDA)-approved direct-acting antiviral (DAA) drugs against HCV proteins. The objective of our study was to discover new antivirals from natural products to supplement current therapeutics. We demonstrated that lobohedleolide, isolated from the Formosan soft coral Lobophytum crassum, significantly reduced HCV replication in replicon cells and JFH-1 infection system, with EC50 values of 10 ± 0.56 and 22 ± 0.75 µM, respectively, at non-toxic concentrations. We further observed that the inhibitory effect of lobohedleolide on HCV replication is due to suppression of HCV-induced cyclooxygenase-2 (COX-2) expression. Based on deletion-mutant analysis of the COX-2 promoter, we identified CCAAT/enhancer-binding protein (C/EBP) as a key transcription factor for the down-regulation of COX-2 by lobohedleolide, through which lobohedleolide decreased the phosphorylation of c-Jun NH2-terminal protein kinase and c-Jun to suppress HCV-induced C/EBP expression. The combination treatment of lobohedleolide with clinically used HCV drugs synergistically reduced HCV RNA replication, indicating that lobohedleolide exhibited a high biomedical potential to be used as a supplementary therapeutic agent to control HCV infection.

Discovery of novel diarylpyrazolylquinoline derivatives as potent anti-dengue virus agents.

A number of diarylpyrazolylquinoline derivatives were synthesized and evaluated for their anti-dengue virus (DENV) activity. Among them, 6-fluoro-2-(1-(4-fluorophenyl)-3- (4-methoxyphenyl)-1H-pyrazol-5-yl)quinoline (11c), 2-[1,3-bis(4-methoxyphenyl)-1H-pyrazol- 5-yl]-6-fluoroquinoline (12c), and 4-[5-(6-fluoroquinolin-2-yl)-3-(4-methoxyphenyl)-1H-pyrazol- 1-yl]benzenesulfonamide (13c) exhibited approximately 10-folds more active anti-DENV-2 activity (IC50 of 1.36, 1.09 and 0.81 µM, respectively) than that of ribavirin (IC50 = 12.61 µM). Compound 13c was also potent inhibited other sero-types of DENV. It reduced DENV replication in both viral protein and mRNA levels, and no significant cell cytotoxicity was detected, with greater than 50% viability of Huh-7-DV-Fluc cells at a concentration of 200 µM. Furthermore, compound 13c can effectively protect mice from DENV infection by reducing disease symptoms and mortality of DENV-infected mice. It represents a potential antiviral agent to block DENV replication in vitro and in vivo. Structural optimization of the initial lead compound, 13c, and the detailed molecular mechanism of action are ongoing.

Self-Assembly DNA Polyplex Vaccine inside Dissolving Microneedles for High-Potency Intradermal Vaccination.

The strong immunogenicity induction is the powerful weapon to prevent the virus infections. This study demonstrated that one-step synthesis of DNA polyplex vaccine in microneedle (MN) patches can induce high immunogenicity through intradermal vaccination and increase the vaccine stability for storage outside the cold chain. More negative charged DNA vaccine was entrapped into the needle region of MNs followed by DNA polyplex formation with branched polyethylenimine (bPEI) pre-coated in the cavities of polydimethylsiloxane (PDMS) molds that can deliver more DNA vaccine to immune-cell rich epidermis with high transfection efficiency. Our data in this study support the safety and immunogenicity of the MN-based vaccine; the MN patch delivery system induced an immune response 3.5-fold as strong as seen with conventional intramuscular administration; the DNA polyplex formulation provided excellent vaccine stability at high temperature (could be stored at 45ºC for at least 4 months); the DNA vaccine is expected to be manufactured at low cost and not generate sharps waste. We think this study is significant to public health because there is a pressing need for an effective vaccination in developing countries.

Celastrol inhibits hepatitis C virus replication by upregulating heme oxygenase-1 via the JNK MAPK/Nrf2 pathway in human hepatoma cells.

BACKGROUND AND PURPOSE: Celastrol, a quinone methide triterpene isolated from the root extracts of Tripterygium wilfordii, can greatly induce the gene expression activity of heme oxygenase-1 (HO-1) to achieve disease prevention and control. HO-1 induction was recently shown to result in anti-HCV activity by inducing type I interferon and inhibiting hepatitis C virus (HCV) NS3/4A protease activity. The aim of the present study is to evaluate the anti-HCV activity of celastrol and characterize its mechanism of inhibition. METHODS: The anti-HCV activity of celastrol was evaluated using the HCV subgenomic replicon and HCVcc infection systems. The anti-HCV mechanism of celastrol targeting HO-1 expression was clarified using specific inhibitors against several signaling pathways. The transcriptional regulation of celastrol on target gene expression was determined using promoter-based reporter activity assay. The synergistic effect of celastrol and a numbers of clinically used anti-HCV drugs was determined via a drug combination assay. RESULTS: Celastrol inhibited HCV replication in both the HCV subgenomic and HCVcc infection systems with EC50 values of 0.37 ± 0.022 and 0.43 ± 0.019 µM, respectively. Celastrol-induced heme oxygenase 1 (HO-1) expression promoted antiviral interferon responses and inhibition of NS3/4A protease activity, thereby blocking HCV replication. These antiviral effects were abrogated by treatment with the HO-1-specific inhibitor SnMP or silencing of HO-1 expression by transfection of shRNA, which indicates that HO-1 induction contributes to the anti-HCV activity of celastrol. JNK mitogen-activated protein kinase and nuclear factor erythroid 2-related factor 2 (Nrf2) were confirmed to be involved in the inductive effect of celastrol on HO-1 expression. Celastrol exhibited synergistic effects in combination with interferon-alpha, the NS5A inhibitor daclatasvir, and the NS5B inhibitor sofosbuvir. CONCLUSION: Celastrol can serve as a potential supplement for blocking HCV replication. Targeting the JNK/Nrf2/HO-1 axis presents a promising strategy against HCV infection.

Celastrol inhibits dengue virus replication via up-regulating type I interferon and downstream interferon-stimulated responses.

ETHNOPHARMACOLOGICAL RELEVANCE AND AIM OF THE STUDY: Tripterygium wilfordii (lei gong teng; Thunder of God Vine), a member of the Celastraceae family, is a medicinal plant used to treat a range of illnesses. Celastrol is a quinone methide triterpene and the most abundant bioactive constituent isolated from the root extracts of T. wilfordii. Previous studies have shown that celastrol exhibits antiviral activity against HIV and SARS-CoV. To date, no investigations of the anti-DENV activity of celastrol have been reported. This work aimed to investigate the anti-DENV effect and possible mechanism of celastrol in vitro and in vivo. METHODS: A four-serotype DENV infection system was performed to determine the anti-DENV effect of celastrol by detecting DENV RNA replication and protein synthesis. The precise anti-DENV replication mechanism of celastrol was clarified using specific RNA silencing and specific inhibitor. In addition, the therapeutic efficacy of celastrol was evaluated by monitoring survival rates and clinical scores in a DENV-infected Institute of Cancer Research (ICR) suckling mouse model. RESULTS: Celastrol inhibited DENV-1, -2, -3, and -4 RNA replication with EC50 values of 0.19 ± 0.09, 0.12 ± 0.11, 0.16 ± 0.14, and 0.17 ± 0.08 µM, respectively. This antiviral effect of celastrol was associated with celastrol-induced interferon-α (IFN-α) expression and was attenuated by a specific inhibitor of the JAK-STAT signaling pathway downstream of IFN-α or specific shRNA. Furthermore, celastrol protected ICR suckling mice against life-threatening DENV infection. CONCLUSION: Celastrol represents a potential anti-DENV agent that induces IFN-α expression and stimulates a downstream antiviral response, making the therapy a promising drug or dietary supplement for the treatment of DENV-infected patients.

Cyclooxygenase-2 facilitates dengue virus replication and serves as a potential target for developing antiviral agents.

Cyclooxygenase-2 (COX-2) is one of the important mediators of inflammation in response to viral infection, and it contributes to viral replication, for example, cytomegalovirus or hepatitis C virus replication. The role of COX-2 in dengue virus (DENV) replication remains unclear. In the present study, we observed an increased level of COX-2 in patients with dengue fever compared with healthy donors. Consistent with the clinical data, an elevated level of COX-2 expression was also observed in DENV-infected ICR suckling mice. Using cell-based experiments, we revealed that DENV-2 infection significantly induced COX-2 expression and prostaglandin E2 (PGE2) production in human hepatoma Huh-7 cells. The exogenous expression of COX-2 or PGE2 treatment dose-dependently enhanced DENV-2 replication. In contrast, COX-2 gene silencing and catalytic inhibition sufficiently suppressed DENV-2 replication. In an ICR suckling mouse model, we identified that the COX-2 inhibitor NS398 protected mice from succumbing to life-threatening DENV-2 infection. By using COX-2 promoter-based analysis and specific inhibitors against signaling molecules, we identified that NF-κB and MAPK/JNK are critical factors for DENV-2-induced COX-2 expression and viral replication. Altogether, our results reveal that COX-2 is an important factor for DENV replication and can serve as a potential target for developing therapeutic agents against DENV infection.