Tyrosine kinase/phosphatase inhibitors decrease dengue virus production in HepG2 cells.

Dengue virus is the causative agent of dengue fever, dengue hemorrhagic fever, and dengue shock syndrome. High rates of dengue virus replication and virion production are related to disease severity. To identify anti-DENV compounds, we performed cell-based ELISA testing to detect the level of DENV E protein expression. Among a total of 83 inhibitors, eight were identified as inhibitors with antiviral activity. Epidermal growth factor receptor inhibitor II (EGFR/ErbB-2/ErbB-4 inhibitor II) and protein tyrosine phosphatase inhibitor IV (PTP inhibitor IV) significantly inhibited dengue virus production and demonstrated low toxicity in hepatocyte cell lines. Our results suggest the efficacy of tyrosine kinase/phosphatase inhibitors in decreasing dengue virus production in HepG2 cells.

Melatonin Inhibits Dengue Virus Infection via the Sirtuin 1-Mediated Interferon Pathway.

Dengue virus (DENV) is the causative pathogen in the life-threatening dengue hemorrhagic fever and dengue shock syndrome. DENV is transmitted to humans via the bite of an infected Aedes mosquito. Approximately 100 million people are infected annually worldwide, and most of those live in tropical and subtropical areas. There is still no effective drug or vaccine for treatment of DENV infection. In this study, we set forth to investigate the effect of melatonin, which is a natural hormone with multiple pharmacological functions, against DENV infection. Treatment with subtoxic doses of melatonin dose-dependently inhibited DENV production. Cross-protection across serotypes and various cell types was also observed. Time-of-addition assay suggested that melatonin exerts its influence during the post-entry step of viral infection. The antiviral activity of melatonin partly originates from activation of the sirtuin pathway since co-treatment with melatonin and the sirtuin 1 (SIRT1) inhibitor reversed the effect of melatonin treatment alone. Moreover, melatonin could modulate the transcription of antiviral genes that aid in suppression of DENV production. This antiviral mechanism of melatonin suggests a possible new strategy for treating DENV infection.

Serine protease inhibitor AEBSF reduces dengue virus infection via decreased cholesterol synthesis.

Dengue virus (DENV) infection has evolved into a major global health menace and economic burden due to its intensity and geographic distribution. DENV infection in humans can cause a wide range of symptoms including dengue fever (DF), dengue hemorrhagic fever (DHF) and dengue shock syndrome (DSS). An antiviral agent that is effective against all four serotypes of DENV is urgently needed to prevent and to manage this condition. Reducing the viral load during the early phase of infection may minimize the chance of patients progressing to more severe DHF or DSS. In this study, we set forth to investigate the anti-viral effect of five commercially available protease inhibitors on DENV infection since both viral and host proteases can contribute to effective viral replication. Previously, the serine protease inhibitor AEBSF [4-(2-aminoethyl) benzene sulfonyl fluoride] has been shown to inhibit DENV NS3 protease activity. The results of the present study revealed that DENV genome replication and protein synthesis were significantly inhibited by AEBSF in a dose-dependent manner. AEBSF inhibited the expression of genes such as 3-hydroxy 3-methyl-glutaryl-CoA synthase (HMGCS), 3-hydroxy-3-methyl-glutaryl-CoA reductase (HMGCR), and low-density lipoprotein receptor (LDLR). Moreover, AEBSF significantly inhibited HMGCR activity and intracellular cholesterol synthesis after DENV infection. The anti-DENV effect of AEBSF was confirmed in all four DENV serotypes and in three different cell lines. These results indicate that AEBSF reduces DENV infection via both viral and host protease activities.

Drug repurposing of quinine as antiviral against dengue virus infection.

Dengue virus (DENV) disease outbreaks continue to develop across the globe with significant associated mortality and economic burden, yet no treatment has been approved to combat this virus. In an attempt to identify novel drug candidates as therapeutics for DENV infection, we evaluated four US Food and Drug Administration (FDA) approved drugs including aminolevullic acid, azelaic acid, mitoxantrone hydrochloride, and quinine sulfate, and tested their ability to inhibit DENV replication using focus-forming unit assay to quantify virus production. Of the four investigated compounds, quinine was found to have the most pronounced anti-DENV activity. Quinine inhibited DENV production of DENV by about 80% compared to untreated controls, while the other three drugs decreased virus production by only about 50%. Moreover, quinine inhibited DENV production of all four serotypes of DENV. Reduction in virus production was documented in three different cell lines of human origin. Quinine significantly inhibited DENV replication by reducing DENV RNA and viral protein synthesis in a dose-dependent manner. In addition, quinine ameliorated expression of genes related to innate immune response. These findings suggest the efficacy of quinine for stimulating antiviral genes to reduce DENV replication. The antiviral activity of quinine observed in this study may have applicability in the development of new drug therapies against DENV.

Coat protein complex I facilitates dengue virus production.

Dengue hemorrhagic fever is a life-threatening disease caused by the dengue virus (DENV). After DENV enters into host cells, it replicates to generate viral particles to infect other cells. DENV exploits components of the cellular trafficking pathway to achieve effective virion production. Understanding of the proteins required for this trafficking process is essential for revealing the pathogenesis of DENV infection. Coat protein complex and soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs), two host protein families in the cellular trafficking pathway, were investigated to elucidate their respective roles during DENV infection. Coat proteins (COPI and COPII) and SNAREs (STX 5 and NSF) were knocked down in a DENV-infected Huh7 cells by RNA interference. Depletion of COPI and COPII, but not of STX5 and NSF, decreased DENV production in DENV-infected Huh7 cells. DENV proteins, including DENV C, prM, E, and NS1, were significantly reduced in COPI-silenced DENV-infected Huh7 cells, when compared to those of control cells. COPI also facilitated DENV production in an endothelial cell line and in all DENV serotypes, indicating the importance of coat protein complex in facilitating DENV infection.

Role of ERK1/2 signaling in dengue virus-induced liver injury.

The liver is considered to be an important organ of dengue virus (DENV) replication and pathogenesis. However, molecular mechanisms of hepatic injury are still poorly understood. Modulation of Mitogen Activated Protein Kinases (MAPKs) was previously shown to affect DENV-induced apoptosis of hepatocytes in vitro. However, the in vivo role of ERK1/2, a member of the MAPK family, and the question whether its activation can facilitate cell survival or cell death, has not been thoroughly investigated. Therefore, the role of ERK1/2 in a mouse model of DENV infection was examined. Our results show that DENV induces phosphorylation of ERK1/2 and increases apoptosis. Inhibition of phosphorylated ERK1/2 by the selective ERK1/2 inhibitor, FR180204, limits hepatocyte apoptosis and reduces DENV-induced liver injury. Clinical parameters, including leucopenia, thrombocytopenia, transaminases and histology, show improvements after FR180204 treatment. The expression of cell death genes was further identified using real-time PCR array and Western blot analysis. Caspase-3 was significantly decreased in FR180204 treated DENV-infected mice compared to the levels of untreated DENV-infected mice suggesting the role of ERK1/2 signaling in immune-mediated liver injury during DENV infection.