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Inhibition of dengue virus RNA synthesis by an adenosine nucleoside.

ABSTRACT: We recently reported that (2R,3R,4R,5R)-2-(4-amino-pyrrolo[2,3-d]pyrimidin-7-yl)-3-ethynyl-5-hydroxy-methyl-tetrahydro-furan-3,4-diol is a potent inhibitor of dengue virus (DENV), with 50% effective concentration (EC(50)) and cytotoxic concentration (CC(50)) values of 0.7 microM and >100 microM, respectively. Here we describe the synthesis, structure-activity relationship, and antiviral characterization of the inhibitor. In an AG129 mouse model, a single-dose treatment of DENV-infected mice with the compound suppressed peak viremia and completely prevented death. Mode-of-action analysis using a DENV replicon indicated that the compound blocks viral RNA synthesis. Recombinant adenosine kinase could convert the compound to a monophosphate form. Suppression of host adenosine kinase, using a specific inhibitor (iodotubercidin) or small interfering RNA (siRNA), abolished or reduced the compound's antiviral activity in cell culture. Studies of rats showed that (14)C-labeled compound was converted to mono-, di-, and triphosphate metabolites in vivo. Collectively, the results suggest that this adenosine inhibitor is phosphorylated to an active (triphosphate) form which functions as a chain terminator for viral RNA synthesis.

SUBMITTER: Chen YL

PROVIDER: S-EPMC2897296 | biostudies-literature | 2010 Jul

REPOSITORIES: biostudies-literature

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Inhibition of dengue virus RNA synthesis by an adenosine nucleoside.

Chen Yen-Liang YL Yin Zheng Z Duraiswamy Jeyaraj J Schul Wouter W Lim Chin Chin CC Liu Boping B Xu Hao Ying HY Qing Min M Yip Andy A Wang Gang G Chan Wai Ling WL Tan Hui Pen HP Lo Melissa M Liung Sarah S Kondreddi Ravinder Reddy RR Rao Ranga R Gu Helen H He Handan H Keller Thomas H TH Shi Pei-Yong PY

Antimicrobial agents and chemotherapy 20100510 7

We recently reported that (2R,3R,4R,5R)-2-(4-amino-pyrrolo[2,3-d]pyrimidin-7-yl)-3-ethynyl-5-hydroxy-methyl-tetrahydro-furan-3,4-diol is a potent inhibitor of dengue virus (DENV), with 50% effective concentration (EC(50)) and cytotoxic concentration (CC(50)) values of 0.7 microM and >100 microM, respectively. Here we describe the synthesis, structure-activity relationship, and antiviral characterization of the inhibitor. In an AG129 mouse model, a single-dose treatment of DENV-infected mice with ...[more]

PMID: 20457821

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Project description:BackgroundDengue virus-host cell interaction initiates when the virus binds to the attachment receptors followed by endocytic internalization of the virus particle. Successful entry into the cell is necessary for infection initiation. Currently, there is no protective vaccine or antiviral treatment for dengue infection. Targeting the viral entry pathway has become an attractive therapeutic strategy to block infection. This study aimed to investigate the effect of silencing the GRP78 and clathrin-mediated endocytosis on dengue virus entry and multiplication into HepG2 cells.Methodology/principal findingsHepG2 cells were transfected using specific siRNAs to silence the cellular surface receptor (GRP78) and clathrin-mediated endocytosis pathway. Gene expression analysis showed a marked down-regulation of the targeted genes (87.2%, 90.3%, and 87.8% for GRP78, CLTC, and DNM2 respectively) in transfected HepG2 cells when measured by RT-qPCR. Intracellular and extracellular viral RNA loads were quantified by RT-qPCR to investigate the effect of silencing the attachment receptor and clathrin-mediated endocytosis on dengue virus entry. Silenced cells showed a significant reduction of intracellular (92.4%) and extracellular viral RNA load (71.4%) compared to non-silenced cells. Flow cytometry analysis showed a marked reduction of infected cells (89.7%) in silenced HepG2 cells compared to non-silenced cells. Furthermore, the ability to generate infectious virions using the plaque assay was reduced 1.07 log in silenced HepG2 cells.Conclusions/significanceSilencing the attachment receptor and clathrin-mediated endocytosis using siRNA could inhibit dengue virus entry and multiplication into HepG2 cells. This leads to reduction of infected cells as well as the viral load, which might function as a unique and promising therapeutic agent for attenuating dengue infection and prevent the development of dengue fever to the severe life-threatening DHF or DSS. Furthermore, a decrease of viremia in humans can result in the reduction of infected vectors and thus, halt of the transmission cycle.

Project description:Zika virus (ZIKV) remains a potentially significant public health concern because it can cause teratogenic effects, such as microcephaly in newborns and neurological disease, like Guillain-Barré syndrome. Together with efforts to develop a vaccine, the discovery of antiviral molecules is important to control ZIKV infections and to prevent its most severe symptoms. Here, we report the development of small nonnucleoside inhibitors (NNIs) of ZIKV RNA-dependent RNA polymerase (RdRp) activity. These NNIs target an allosteric pocket (N pocket) located next to a putative hinge region between the thumb and the palm subdomains that was originally described for dengue virus (DENV) RdRp. We first tested the activity of DENV RdRp N-pocket inhibitors against ZIKV RdRp, introduced chemical modifications into these molecules, and assessed their potency using both enzymatic and cell-based assays. The most potent compound had a 50% inhibitory concentration value of 7.3 μM and inhibited ZIKV replication in a cell-based assay with a 50% effective concentration value of 24.3 μM. Importantly, we report four high-resolution crystal structures detailing how these NNIs insert into the N pocket of ZIKV RdRp. Our observations point to subtle differences in the size, shape, chemical environment, and hydration of the N pocket from ZIKV RdRp from those of the N pocket from DENV RdRp that are crucial for the design of improved antiviral inhibitors with activity against ZIKV.IMPORTANCE Zika virus belongs to the Flavivirus genus, which comprises several important human pathogens. There is currently neither an approved vaccine nor antiviral drugs available to prevent infection by ZIKV. The nonstructural protein 5 (NS5) polymerase, which is responsible for replicating the viral RNA genome, represents one of the most promising targets for antiviral drug development. Starting from compounds recently developed against dengue virus NS5, we designed and synthesized inhibitors targeting Zika virus NS5. We show that these novel compounds inhibit viral replication by targeting the polymerase activity. High-resolution X-ray crystallographic structures of protein-inhibitor complexes demonstrated specific binding to an allosteric site within the polymerase, called the N pocket. This work paves the way for the future structure-based design of potent compounds specifically targeting ZIKV RNA polymerase activity.

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Inhibition of dengue virus RNA synthesis by an adenosine nucleoside.

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Inhibition of dengue virus RNA synthesis by an adenosine nucleoside.

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