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Dengue is a major health threat and the number of symptomatic infections caused by the four dengue serotypes is estimated to be 96 million1with annually around 10,000 deaths2. However, no antiviral drugs are available for the treatment or prophylaxis of dengue. We recently described the interaction between non-structural proteins NS3 and NS4B as a promising target for the development of pan-serotype dengue virus (DENV) inhibitors3. Here we present JNJ-1802—a highly potent DENV inhibitor that blocks the NS3–NS4B interaction within the viral replication complex. JNJ-1802 exerts picomolar to low nanomolar in vitro antiviral activity, a high barrier to resistance and potent in vivo efficacy in mice against infection with any of the four DENV serotypes. Finally, we demonstrate that the small-molecule inhibitor JNJ-1802 is highly effective against viral infection with DENV-1 or DENV-2 in non-human primates. JNJ-1802 has successfully completed a phase I first-in-human clinical study in healthy volunteers and was found to be safe and well tolerated4. These findings support the further clinical development of JNJ-1802, a first-in-class antiviral agent against dengue, which is now progressing in clinical studies for the prevention and treatment of dengue.

The RV144 phase 3 vaccine trial in Thailand demonstrated that ALVAC-HIV (vCP1521) and AIDSVAX B/E administration over 6 months resulted in a 31% efficacy in preventing HIV acquisition. In this trial, we assessed the immunological effect of an additional vaccine boost to the RV144 regimen at varying intervals between the priming vaccine series and the boost.

Although there is no compelling evidence that vitamin C has antitumor activity in humans, clinical trials are testing the hypothesis that ascorbic acid (AA) will enhance the efficacy of arsenic trioxide (As2O3) in myeloma. In vitro, AA cytotoxicity depends on its interaction with free transition metal ions in culture media leading to the generation of H2O2 and other reactive oxygen species (ROSs). Therefore, to circumvent the extracellular in vitro pro-oxidant effects of AA, we loaded HL60, U266, and RPMI-8226 cells with vitamin C by incubation with dehydroascorbic acid (DHA). Loading cells in this manner resulted in prominent, dose-dependent protection of As2O3-treated cells as measured by viability, colony formation, and apoptosis assays. Glutathione depletion enhanced cell sensitivity to the cytotoxic effects of As2O3 and vitamin C loading provided protection. AA was found to generate cytotoxic concentrations of H2O2 in culture medium without cells and copper/iron chelators inhibited this reaction. However, AA did not generate H2O2 in simple buffer or human plasma. Direct incubation with AA resulted in increased intracellular ROSs, whereas DHA incubation decreased it. These results clarify an apparent paradox and indicate that vitamin C loading in HL60, U266, and RPMI-8226 cells ameliorates As2O3 cytotoxicity.

Although there is no compelling evidence that vitamin C has antitumor activity in humans, clinical trials are testing the hypothesis that ascorbic acid (AA) will enhance the efficacy of arsenic trioxide (As2O3) in myeloma. In vitro, AA cytotoxicity depends on its interaction with free transition metal ions in culture media leading to the generation of H2O2and other reactive oxygen species (ROSs). Therefore, to circumvent the extracellular in vitro pro-oxidant effects of AA, we loaded HL60, U266, and RPMI-8226 cells with vitamin C by incubation with dehydroascorbic acid (DHA). Loading cells in this manner resulted in prominent, dose-dependent protection of As2O3-treated cells as measured by viability, colony formation, and apoptosis assays. Glutathione depletion enhanced cell sensitivity to the cytotoxic effects of As2O3and vitamin C loading provided protection. AA was found to generate cytotoxic concentrations of H2O2in culture medium without cells and copper/iron chelators inhibited this reaction. However, AA did not generate H2O2in simple buffer or human plasma. Direct incubation with AA resulted in increased intracellular ROSs, whereas DHA incubation decreased it. These results clarify an apparent paradox and indicate that vitamin C loading in HL60, U266, and RPMI-8226 cells ameliorates As2O3cytotoxicity.

The granulocyte macrophage–colony-stimulating factor receptor (GM-CSF-R) is a heterodimer composed of 2 subunits, ? and ß, and ligand binding to the high-affinity receptor leads to signalling for the multiple actions of GM-CSF on target cells. In order to explore the role of the ? subunit in signalling, we used a yeast-2-hybrid system to identify proteins interacting with the intracellular domain of the GMR-?. A cDNA encoding a predicted protein of 198 amino acids, designated GRAP (GM-CSFreceptor ? subunit-associatedprotein), was isolated in experiments using the intracellular portion of GMR-? as bait. The interaction between GRAP and GMR-??was confirmed by coimmunoprecipitation in mammalian cells. GRAP mRNA is widely expressed in normal human and mouse tissues and in neoplastic human cell lines, but it is not restricted to cells or tissues that express GM-CSF receptors. Three discrete GRAP mRNA species were detected in human tissues and cells, with estimated sizes of 3.3, 3.1, and 1.3 kb. GRAP is highly conserved throughout evolution, and homologues are found in yeast. The GRAP locus in Saccharomyces cerevisiae was disrupted, and mutant yeast cells showed an inappropriate stress response under normal culture conditions, manifested by early accumulation of glycogen during the logarithmic growth phase. GRAP is, therefore, a highly conserved and widely expressed protein that binds to the intracellular domain of GMR-?, and it appears to play an important role in cellular metabolism.

Vitamin C is an antioxidant vitamin that has been hypothesized to antagonize the effects of antineoplastic drugs that generate reactive oxygen species. Leukemia (K562) and lymphoma (RL) cells pre-treated with dehydroascorbic acid, the commonly transported form of vitamin C, demonstrated a dose-dependent attenuation of cytotoxicity after treatment with the widely-used antineoplastic drugs, doxorubicin, cisplatin, vincristine, methotrexate and imatinib that ranged from 30–80% as measured by trypan blue exclusion and colony formation in methylcellulose. While treatment with vitamin C alone did not appreciably alter tumor growth compared with untreated mice with xenogeneic tumors (RL) (p=0.114), mice treated with vitamin C prior to doxorubicin administration had tumors that were, on average, four times larger than tumors in mice treated with doxorubicin alone (p=0.01) at day 32 of treatment. This result indicates that vitamin C treatment can attenuate antineoplastic therapy in vivo. Pretreatment of cells with vitamin C led to a dose-dependent decrease in apoptosis with all agents tested. At the highest concentrations of intracellular vitamin C, the decrease in apoptosis ranged from 37–82% as measured by TUNEL. There was no change in P-glycoprotein expression in vitamin C-treated cells and no difference in doxorubicin concentrations in the tumors of vitamin C-treated mice suggesting that the inhibitory effects of vitamin C are not due to generalized effects of antineoplastic drug uptake or efflux. When K562 and RL cells were treated with antineoplastic agents, we found that, as has been previously reported, doxorubicin and, to a lesser extent, cisplatin, increased the intracellular levels of ROS, while other agents had little discernible effect on ROS levels. In cells treated with chemotherapeutic drugs, as well as in untreated cells, pretreatment of cells with vitamin C caused a small (<15%) reduction in intracellular levels of ROS. This modest reduction in intracellular ROS levels appeared to be minor in comparison to the reduction in cytotoxicity. While treatment with N-acetylcysteine only attenuated the cytotoxic effects of cisplatin, vitamin C pretreatment attenuated the effects of all drugs tested, suggesting a distinct mechanism of action. We found that all of the antineoplastic agents tested caused rapid mitochondrial membrane depolarization. Treatment with vitamin C prior to exposure to antineoplastic agents prevented mitochondrial membrane depolarization. Mitochondrial membrane potentials in vitamin C-treated cells 6 hr after treatment with chemotherapeutic drugs were similar to untreated control cells (p<0.003). A mitochondrial site of action was further supported by the finding that vitamin C antagonizes the cytotoxic effects of actinonin, an inhibitor of peptide deformylase. These findings suggest that vitamin C supplementation during cancer treatment may detrimentally affect therapeutic response to a broad range of anti-cancer drugs and point to preserved mitochondrial membrane potential as a mechanism of action.

Vitamin C is an antioxidant vitamin that has been hypothesized to antagonize the effects of antineoplastic drugs that generate reactive oxygen species. Leukemia (K562) and lymphoma (RL) cells pre-treated with dehydroascorbic acid, the commonly transported form of vitamin C, demonstrated a dose-dependent attenuation of cytotoxicity after treatment with the widely-used antineoplastic drugs, doxorubicin, cisplatin, vincristine, methotrexate and imatinib that ranged from 30–80% as measured by trypan blue exclusion and colony formation in methylcellulose. While treatment with vitamin C alone did not appreciably alter tumor growth compared with untreated mice with xenogeneic tumors (RL) (p=0.114), mice treated with vitamin C prior to doxorubicin administration had tumors that were, on average, four times larger than tumors in mice treated with doxorubicin alone (p=0.01) at day 32 of treatment. This result indicates that vitamin C treatment can attenuate antineoplastic therapy in vivo. Pretreatment of cells with vitamin C led to a dose-dependent decrease in apoptosis with all agents tested. At the highest concentrations of intracellular vitamin C, the decrease in apoptosis ranged from 37–82% as measured by TUNEL. There was no change in P-glycoprotein expression in vitamin C-treated cells and no difference in doxorubicin concentrations in the tumors of vitamin C-treated mice suggesting that the inhibitory effects of vitamin C are not due to generalized effects of antineoplastic drug uptake or efflux. When K562 and RL cells were treated with antineoplastic agents, we found that, as has been previously reported, doxorubicin and, to a lesser extent, cisplatin, increased the intracellular levels of ROS, while other agents had little discernible effect on ROS levels. In cells treated with chemotherapeutic drugs, as well as in untreated cells, pretreatment of cells with vitamin C caused a small (<15%) reduction in intracellular levels of ROS. This modest reduction in intracellular ROS levels appeared to be minor in comparison to the reduction in cytotoxicity. While treatment with N-acetylcysteine only attenuated the cytotoxic effects of cisplatin, vitamin C pretreatment attenuated the effects of all drugs tested, suggesting a distinct mechanism of action. We found that all of the antineoplastic agents tested caused rapid mitochondrial membrane depolarization. Treatment with vitamin C prior to exposure to antineoplastic agents prevented mitochondrial membrane depolarization. Mitochondrial membrane potentials in vitamin C-treated cells 6 hr after treatment with chemotherapeutic drugs were similar to untreated control cells (p<0.003). A mitochondrial site of action was further supported by the finding that vitamin C antagonizes the cytotoxic effects of actinonin, an inhibitor of peptide deformylase. These findings suggest that vitamin C supplementation during cancer treatment may detrimentally affect therapeutic response to a broad range of anti-cancer drugs and point to preserved mitochondrial membrane potential as a mechanism of action.