Brivanib alaninate inhibited dengue virus proliferation through VEGFR2/AMPK pathway.

Dengue virus (DENV) is the most prevalent arthropod-borne viral disease of humans and has a major impact on global public health. There is no clinically approved drugs for DENV infection. Since intracellular VEGFR2 is increased in DENV infected patients, we thus hypothesized that VEGFR2 participated DENV proliferation and its inhibitors could be served as antivirals against DENV. Actually our results showed that VEGFR2 was induced by DENV infection. Also the agonist of VEGFR2, VEGF-A, promoted DENV proliferation. Therefore, we screened the inhibitors of VEGFR2 and found that brivanib alaninate (brivanib) showed the best anti-DENV ability with the lowest cellular cytotoxicity. Mechanically, our results indicated VEGFR2 directly interacted with PTP1B to dephosphorylate AMPK to provide lipid environment for viral replication. However, this effect could be inhibited by brivanib, which significantly reversed the reduction of AMPK phosphorylation caused by DENV infection, thus improving the cellular lipid environment. Moreover, the antiviral effect of brivanib could be reversed by AMPK inhibitor, Compound C. In addition, oral administration of brivianib (20-50 mg/kg/day) clearly improved the survival rate of DENV2 infection, and this effect was abolished in accompanied with Compound C (10mg/kg/day). Collectively, our study disclosed the mechanism of VEGFR2 in DENV2 and evaluated the antiviral ability of brivanib, which deserved more attention for clinical usage in DENV infection.

Neutrophil Extracellular Traps in Dengue Are Mainly Generated NOX-Independently.

Neutrophil extracellular traps (NETs) are increasingly recognized to play a role in the pathogenesis of viral infections, including dengue. NETs can be formed NADPH oxidase (NOX)-dependently or NOX-independently. NOX-independent NETs can be induced by activated platelets and are very potent in activating the endothelium. Platelet activation with thrombocytopenia and endothelial dysfunction are prominent features of dengue virus infection. We postulated that dengue infection is associated with NOX-independent NET formation, which is related to platelet activation, endothelial perturbation and increased vascular permeability. Using our specific NET assays, we investigated the time course of NET formation in a cohort of Indonesian dengue patients. We found that plasma levels of NETs were profoundly elevated and that these NETs were predominantly NOX-independent NETs. During early recovery phase (7-13 days from fever onset), total NETs correlated negatively with platelet number and positively with platelet P-selectin expression, the binding of von Willebrand factor to platelets and levels of Syndecan-1. Patients with gall bladder wall thickening, an early marker of plasma leakage, had a higher median level of total NETs. Ex vivo, platelets induced NOX-independent NET formation in a dengue virus non-structural protein 1 (NS1)-dependent manner. We conclude that NOX-independent NET formation is enhanced in dengue, which is most likely mediated by NS1 and activated platelets.

Identification of a Direct-Acting Antiviral Agent Targeting RNA Helicase via a Graphene Oxide Nanobiosensor.

Dengue virus (DENV), an arbovirus transmitted by mosquitoes, causes infectious diseases such as dengue fever, dengue hemorrhagic fever, and dengue shock syndrome. Despite the dangers posed by DENV, there are no approved antiviral drugs for treatment of DENV infection. Considering the potential for a global dengue outbreak, rapid development of antiviral agents against DENV infections is crucial as a preemptive measure; thus, the selection of apparent drug targets, such as the viral enzymes involved in the viral life cycle, is recommended. Helicase, a potential drug target in DENV, is a crucial viral enzyme that unwinds double-stranded viral RNA, releasing single-stranded RNA genomes during viral replication. Therefore, an inhibitor of helicase activity could serve as a direct-acting antiviral agent. Here, we introduce an RNA helicase assay based on graphene oxide, which enables fluorescence-based analysis of RNA substrate-specific helicase enzyme activity. This assay demonstrated high reliability and ability for high-throughput screening, identifying a new helicase inhibitor candidate, micafungin (MCFG), from an FDA-approved drug library. As a direct-acting antiviral agent targeting RNA helicase, MCFG inhibits DENV proliferation in cells and an animal model. Notably, in vivo, MCFG treatment reduced viremia, inflammatory cytokine levels, and viral loads in several tissues and improved survival rates by up to 40% in a lethal mouse model. Therefore, we suggest MCFG as a potential direct-acting antiviral drug candidate.

Matrix metalloproteinases in the pathogenesis of dengue viral disease: Involvement of immune system and newer therapeutic strategies.

Globally, the burden due to dengue infection is increasing with a recent estimate of 96 million progressing to the disease every year. Dengue pathogenesis and the factors influencing it are not completely known. It is now widely speculated that there is an important role of matrix metalloproteinases (MMPs) in the initiation and progression of dengue pathogenesis; however, their exact roles are not fully understood. Overactivation of matrix metalloproteinases may contribute to the severity of dengue pathogenesis. Cytokines and various other mediators of inflammation interact with the vascular endothelium and matrix metalloproteinases may be one of the components among them. Extensive plasma leakage into tissue spaces may result in a shock. It is evident in the literature that MMP2 and MMP9 increase in dengue patients is correlated with the severity of the disease; however, the underlying mechanism is still unknown. Activation of innate cells and adaptive immune cells which include, B and T cells, macrophages or monocytes and dendritic cells also contribute to the dengue pathology. Newer therapeutic strategies include microRNAs, such as miR-134 (targets MMP3 and MMP1) and MicroRNA-320d, (targets MMP/TIMP proteolytic system). The use of antibodies-based therapeutics like (Andecaliximab; anti-matrix metalloproteinase-9 antibody) is also suggested against MMPs in dengue. In this review, we summarize some recent developments associated with the involvement of immune cells and their mediators associated with the matrix metalloproteinases mediated dengue pathogenesis. We highlight that, there is still very little knowledge about the MMPs in dengue pathogenesis which needs attention and extensive investigations.

Phosphoproteomic analysis of dengue virus infected U937 cells and identification of pyruvate kinase M2 as a differentially phosphorylated phosphoprotein.

Dengue virus (DENV) is an arthropod-borne Flavivirus that can cause a range of symptomatic disease in humans. There are four dengue viruses (DENV 1 to 4) and infection with one DENV only provides transient protection against a heterotypic virus. Second infections are often more severe as the disease is potentiated by antibodies from the first infection through a process known as antibody dependent enhancement (ADE) of infection. Phosphorylation is a major post-translational modification that can have marked effects on a number of processes. To date there has been little information on the phosphorylation changes induced by DENV infection. This study aimed to determine global phosphoproteome changes induced by DENV 2 in U937 cells infected under an ADE protocol. A 2-dimensional electrophoretic approach coupled with a phosphoprotein-specific dye and mass spectroscopic analysis identified 15 statistically significant differentially phosphorylated proteins upon DENV 2 infection. One protein identified as significantly differentially phosphorylated, pyruvate kinase M2 (PKM2) was validated. Treatment with a PKM2 inhibitor modestly reduced levels of infection and viral output, but no change was seen in cellular viral protein levels, suggesting that PKM2 acts on exocytic virus release. While the effect of inhibition of PKM2 was relatively modest, the results highlight the need for a greater understanding of the role of phosphoproteins in DENV infection.

DNA-induced 2'3'-cGAMP enhances haplotype-specific human STING cleavage by dengue protease.

The cytosolic DNA sensor cGMP-AMP synthase (cGAS) synthesizes the noncanonical cyclic dinucleotide 2'3'-cGAMP to activate the adaptor protein stimulator of IFN genes (STING), thus awakening host immunity in response to DNA pathogen infection. However, dengue virus (DENV), an RNA virus without a DNA stage in its life cycle, also manipulates cGAS-STING-mediated innate immunity by proteolytic degradation of STING. Here, we found that the sensitivity of STING to DENV protease varied with different human STING haplotypes. Exogenous DNA further enhanced DENV protease's ability to interact and cleave protease-sensitive STING. DNA-enhanced STING cleavage was reduced in cGAS-knockdown cells and triggered by the cGAS product 2'3'-cGAMP. The source of DNA may not be endogenous mitochondrial DNA but rather exogenous reactivated viral DNA. Cells producing 2'3'-cGAMP by overexpressing cGAS or with DNA virus reactivation enhanced STING cleavage in neighboring cells harboring DENV protease. DENV infection reduced host innate immunity in cells with the protease-sensitive STING haplotype, whose homozygote genotype frequency was found significantly reduced in Taiwanese people with dengue fever. Therefore, the human STING genetic background and DNA pathogen coinfection may be the missing links contributing to DENV pathogenesis.

Targeting Endoplasmic Reticulum α-Glucosidase I with a Single-Dose Iminosugar Treatment Protects against Lethal Influenza and Dengue Virus Infections.

Influenza and dengue viruses present a growing global threat to public health. Both viruses depend on the host endoplasmic reticulum (ER) glycoprotein folding pathway. In 2014, Sadat et al. reported two siblings with a rare genetic defect in ER α-glucosidase I (ER Glu I) who showed resistance to viral infections, identifying ER Glu I as a key antiviral target. Here, we show that a single dose of UV-4B (the hydrochloride salt form of N-(9'-methoxynonyl)-1-deoxynojirimycin; MON-DNJ) capable of inhibiting Glu I in vivo is sufficient to prevent death in mice infected with lethal viral doses, even when treatment is started as late as 48 h post infection. The first crystal structure of mammalian ER Glu I will constitute the basis for the development of potent and selective inhibitors. Targeting ER Glu I with UV-4B-derived compounds may alter treatment paradigms for acute viral disease through development of a single-dose therapeutic regime.

Anti-inflammatory Compound Shows Therapeutic Safety and Efficacy against Flavivirus Infection.

Flaviviruses comprise several medically important viruses, including Japanese encephalitis virus, West Nile virus, dengue virus (DENV), yellow fever virus, and Zika virus (ZIKV). A large outbreak of DENV and ZIKV occurred recently, leading to many cases of illness and death. However, despite decades of effort, we have no clinically specific therapeutic drugs against DENV and ZIKV. Previous studies showed that inflammatory responses play a critical role in dengue and Zika virus pathogenesis. Thus, in this study, we examined a series of novel anti-inflammatory compounds and found that treatment with compound 2d could dose dependently reduce viral protein expression and viral progeny production in HEK-293 and Raw264.7 cells infected with four serotypes of DENV and ZIKV. In addition, considering medication safety, compound 2d could not suppress cyclooxygenase-1 (COX-1) enzymatic activities and thus could prevent the side effect of bleeding. Moreover, compound 2d significantly inhibited COX-2 enzymatic activities and prostaglandin E2 levels, associated with viral replication, compared to results with a selective COX-2 inhibitor, celecoxib. Furthermore, administering 5 mg/kg compound 2d to DENV-2-infected AG129 mice prolonged survival and reduced viremia and serum cytokine levels. Overall, compound 2d showed therapeutic safety and efficacy in vitro and in vivo and could be further developed as a potential therapeutic agent for flavivirus infection.

Dengue virus-elicited tryptase induces endothelial permeability and shock.

Dengue virus (DENV) infection causes a characteristic pathology in humans involving dysregulation of the vascular system. In some patients with dengue hemorrhagic fever (DHF), vascular pathology can become severe, resulting in extensive microvascular permeability and plasma leakage into tissues and organs. Mast cells (MCs), which line blood vessels and regulate vascular function, are able to detect DENV in vivo and promote vascular leakage. Here, we identified that a MC-derived protease, tryptase, is consequential for promoting vascular permeability during DENV infection, through inducing breakdown of endothelial cell tight junctions. Injected tryptase alone was sufficient to induce plasma loss from the circulation and hypovolemic shock in animals. A potent tryptase inhibitor, nafamostat mesylate, blocked DENV-induced vascular leakage in vivo. Importantly, in two independent human dengue cohorts, tryptase levels correlated with the grade of DHF severity. This study defines an immune mechanism by which DENV can induce vascular pathology and shock.

Targeting host metabolism by inhibition of acetyl-Coenzyme A carboxylase reduces flavivirus infection in mouse models.

Flaviviruses are (re)-emerging RNA viruses strictly dependent on lipid metabolism for infection. In the search for host targeting antivirals, we explored the effect of pharmacological modulation of fatty acid metabolism during flavivirus infection. Considering the central role of acetyl-Coenzyme A carboxylase (ACC) on fatty acid metabolism, we analyzed the effect of three small-molecule ACC inhibitors (PF-05175157, PF-05206574, and PF-06256254) on the infection of medically relevant flaviviruses, namely West Nile virus (WNV), dengue virus, and Zika virus. Treatment with these compounds inhibited the multiplication of the three viruses in cultured cells. PF-05175157 induced a reduction of the viral load in serum and kidney in WNV-infected mice, unveiling its therapeutic potential for the treatment of chronic kidney disease associated with persistent WNV infection. This study constitutes a proof of concept of the reliability of ACC inhibitors to become viable antiviral candidates. These results support the repositioning of metabolic inhibitors as broad-spectrum antivirals.

Direct evidence of a low barrier hydrogen bond in the catalytic triad of a Serine protease.

Serine proteases are one of the largest groups of enzymes, found in both eukaryotes and prokaryotes, and are responsible for many different functions. The detailed information about the hydrogen-bonds in the catalytic triad (Asp…His…Ser) of these enzymes is of importance in order to fully understand the mechanism of action. The aspartate of the triad is hydrogen bonded to the histidine but the exact nature of this bond has been under discussion for some time. It is either a common short ionic hydrogen bond (SIHB) or a delocalized low barrier hydrogen bond (LBHB) were the hydrogen bond is shorter. So far, the evidence for LBHB in proteins have not been conclusive. Here we show clear NMR evidence that LBHB does exist in NS3, a serine protease from Dengue. The one bond coupling constant between the hydrogen and nitrogen was shown to be only 52 Hz instead of the usual 90 Hz. This together with a 1H chemical shift of 19.93 ppm is evidence that the hydrogen bond distance between His and Asp is shorter than for SIHB. Our result clearly shows the existence of LBHB and will help in understanding the mechanism of the catalytic triad in the important group of serine proteases.

Mechanisms of Antiviral Activity of Iminosugars Against Dengue Virus.

The antiviral mechanism of action of iminosugars against many enveloped viruses, including dengue virus (DENV), HIV, influenza and hepatitis C virus, is believed to be mediated by inducing misfolding of viral N-linked glycoproteins through inhibition of host endoplasmic reticulum-resident α-glucosidase enzymes. This leads to reduced secretion and/or infectivity of virions and hence lower viral titres, both in vitro and in vivo. Free oligosaccharide analysis from iminosugar-treated cells shows that antiviral activity correlates with production of mono- and tri-glucosylated sugars, indicative of inhibition of ER α-glucosidases. We demonstrate that glucose-mimicking iminosugars inhibit isolated glycoprotein and glycolipid processing enzymes and that this inhibition also occurs in primary cells treated with these drugs. Galactose-mimicking iminosugars that have been tested do not inhibit glycoprotein processing but do inhibit glycolipid processing, and are not antiviral against DENV. By comparison, the antiviral activity of glucose-mimetic iminosugars that inhibit endoplasmic reticulum-resident α-glucosidases, but not glycolipid processing, demonstrates that inhibition of α-glucosidases is responsible for iminosugar antiviral activity against DENV. This monograph will review the investigations of many researchers into the mechanisms of action of iminosugars and the contribution of our current understanding of these mechanisms for optimising clinical delivery of iminosugars. The effects of iminosugars on enzymes other than glucosidases, the induction of ER stress and viral receptors will be also put into context. Data suggest that inhibition of α-glucosidases results in inhibited release of virus and is the primary antiviral mechanism of action of iminosugars against DENV.

Glutathionylation of dengue and Zika NS5 proteins affects guanylyltransferase and RNA dependent RNA polymerase activities.

It has been estimated for dengue infection that the global population at risk is 3.5 billion people, which makes dengue an important public health problem. The causative agents of dengue are dengue viruses. For dengue virus replication, the dengue virus NS5 protein is of special importance as it has several enzyme activities important for viral replication. Previous reports of phosphorylation and SUMOylation of dengue NS5 have shown these protein modifications have important consequences for NS5 functions. In this report we identify glutathionylation, another reversible post translation modification that impacts on NS5 enzyme activity. Using dengue virus infected cells we employed specific antibodies and mass spectrometry to identify 3 cysteine residues of NS5 protein as being glutathionylated. Glutathionylation is a post translational protein modification where glutathione is covalently attached to a cysteine residue. We showed glutathionylation occurs on 3 conserved cysteine residues of dengue NS5. Then we generated two flavivirus recombinant full length proteins, dengue NS5 and Zika NS5, to characterize two of the NS5 enzyme activities, namely, guanylyltransferase and RNA-dependent RNA polymerase activities. We show glutathionylation of dengue and Zika NS5 affects enzyme activities of the two flavivirus proteins. The data suggests that glutathionylation is a general feature of the flavivirus NS5 protein and the modification has the potential to modulate several of the NS5 enzyme functions.

Detection and quantification of dengue virus using a novel biosensor system based on dengue NS3 protease activity.

BACKGROUND: The traditional methods, plaque assays and immuno-focus assays, used to titrate infectious dengue virus (DENV) particles are time consuming and labor intensive. Here, we developed a DENV protease activity detection system (DENPADS) to visualize DENV infection in cells based on dengue protease activity. METHODOLOGY/PRINCIPAL FINDINGS: Dengue NS3 protease cleaves NS4B-NS5. BHK-21 cells stably expressing the sensor module comprising DENV-2 NS4 and the 10 amino-terminal amino acids of NS5 (N10NS5) fused with the SV40 nuclear localization signal (NLS) and Cre recombinase (Cre), were generated. Cre is constrained outside the nucleus in the absence of NS3 activity but translocates into the nucleus through NS4B-NS5 cleavage when cells are infected with DENV. Nuclear translocation of Cre can trigger the reporter system, which contains a cis-loxP-flanked mCherry with three continuous stop codons following an SV40 polyA tail cDNA upstream of EGFP or mHRP cDNA. Our results show that DENPADS is an efficient and accurate method to titrate 4 DENV serotypes in 24 hours. Compared with current virus titration methods, the entire process is easy to perform, and the data are easily acquired. CONCLUSIONS/SIGNIFICANCE: In this study, we demonstrate that DENPADS can be used to detect dengue viral infection through a fluorescence switch or HRP activity in the infected cells. This approach is sensitive with less incubation time and labor input. In addition, DENPADS can simultaneously evaluate the efficacy and cytotoxicity of potential anti-DENV candidates. Overall, DENPADS is a useful tool for dengue research.

Evaluation of creatine kinase and liver enzymes in identification of severe dengue.

BACKGROUND: Existing biomarkers such as AST, ALT and hematocrit have been associated with severe dengue but evidence are mixed. Recently, interests in creatine kinase as a dengue biomarker have risen. These biomarkers represent several underlying pathophysiological processes in dengue. Hence, we aimed to assess AST, ALT, CK and hematocrit in identification of severe dengue and to assess the correlational relationship amongst common biomarkers of dengue. METHODS: This was a retrospective cohort study of confirmed dengue patients who were warded in Kuala Lumpur Hospital between December 2014 and January 2015. CK, AST, ALT, hematocrit, platelet count, WBC and serum albumin were taken upon ward admission and repeated at timed intervals. Composite indices based on admission AST and ALT were analyzed. Correlation coefficients and coefficients of determination were computed. RESULTS: Among the 365 cases reviewed, twenty-two (6%) patients had severe dengue. AST and ALT were found to be good at identification of severe dengue. The AST2/ALT composite index was the most accurate (AUC 0.83; 95% CI 0.73 - 0.93). Optimal cutoff was 402 with a sensitivity of 59.1% (95% CI: 36.4 - 79.3%) and specificity of 92.4% (95% CI: 89.1 - 95.0%). Modified cutoff of 653 had a sensitivity of 40.9% (95% CI: 20.7 - 63.7%) and specificity of 97.4% (95% CI: 95.1 - 98.8%). Our analyses also suggested that several underlying biological processes represented by biomarkers tested were unrelated despite occurring in the same disease entity. Also, markers of plasma leakage were discordant and AST was likely hepatic in origin. CONCLUSIONS: The composite index AST2/ALT may be used as a marker for identification of severe dengue based on admission AST and ALT, with two choices of cutoff values, 402 and 653. AST is most likely of liver origin and CK does not provide additional value.

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.

Human heme oxygenase 1 is a potential host cell factor against dengue virus replication.

Dengue virus (DENV) infection and replication induces oxidative stress, which further contributes to the progression and pathogenesis of the DENV infection. Modulation of host antioxidant molecules may be a useful strategy for interfering with DENV replication. In this study, we showed that induction or exogenous overexpression of heme oxygenase-1 (HO-1), an antioxidant enzyme, effectively inhibited DENV replication in DENV-infected Huh-7 cells. This antiviral effect of HO-1 was attenuated by its inhibitor tin protoporphyrin (SnPP), suggesting that HO-1 was an important cellular factor against DENV replication. Biliverdin but not carbon monoxide and ferrous ions, which are products of the HO-1 on heme, mediated the HO-1-induced anti-DENV effect by non-competitively inhibiting DENV protease, with an inhibition constant (Ki) of 8.55 ± 0.38 µM. Moreover, HO-1 induction or its exogenous overexpression, rescued DENV-suppressed antiviral interferon response. Moreover, we showed that HO-1 induction by cobalt protoporphyrin (CoPP) and andrographolide, a natural product, as evidenced by a significant delay in the onset of disease and mortality, and virus load in the infected mice's brains. These findings clearly revealed that a drug or therapy that induced the HO-1 signal pathway was a promising strategy for treating DENV infection.

Identification and characterization of the role of c-terminal Src kinase in dengue virus replication.

We screened a siRNA library targeting human tyrosine kinases in Huh-7 cells and identified c-terminal Src kinase (Csk) as one of the kinases involved in dengue virus replication. Knock-down of Csk expression by siRNAs or inhibition of Csk by an inhibitor reduced dengue virus RNA levels but did not affect viral entry. Csk partially colocalized with viral replication compartments. Dengue infection was drastically reduced in cells lacking the three ubiquitous src family kinases, Src, Fyn and Yes. Csk knock-down in these cells failed to block dengue virus replication suggesting that the effect of Csk is via regulation of Src family kinases. Csk was found to be hyper-phosphorylated during dengue infection and inhibition of protein kinase A led to a block in Csk phosphorylation and dengue virus replication. Overexpression studies suggest an important role for the kinase and SH3 domains in this process. Our results identified a novel role for Csk as a host tyrosine kinase involved in dengue virus replication and provide further insights into the role of host factors in dengue replication.

Selection of suitable detergents for obtaining an active dengue protease in its natural form from E. coli.

Dengue protease is a two-component enzyme and is an important drug target against dengue virus. The protease activity and protein stability of dengue nonstructural protein 3 (NS3) require a co-factor region from a four-span membrane protein NS2B. A natural form of dengue protease containing full-length NS2B and NS3 protease domain NS2BFL-NS3pro will be useful for dengue drug discovery. In current study, detergents that can be used for protease purification were tested. Using a water soluble protease construct, 39 detergents were selected for both NS2B and NS2BFL-NS3pro purification. The results showed that 18 detergents were able to sustain the activity of the natural dengue protease and 11 detergents could be used for NS2B purification. The results obtained in this study will be useful for biochemical and biophysical studies on dengue protease.