Expression and Purification of Dengue Virus NS5 Polymerase and Development of a High-Throughput Enzymatic Assay for Screening Inhibitors of Dengue Polymerase

Gong, Edwin Yunhao; Kenens, Hannah; Ivens, Tania; Dockx, Koen; Vermeiren, Katrien; Vandercruyssen, Geneviève; Devogelaere, Benoit; Lory, Pedro; Kraus, Guenter

doi:10.1007/978-1-62703-484-5_19

Cited by 14 publications

(16 citation statements)

References 7 publications

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“…Several methods for high-throughput drug screening against virus RdPs have been described and validated 61–66 . However, there are several practical limitations to these approaches, such as the requirement for radioactive substances, entailing additional biosafety measures 63 , or an arduous experimental setup 65,66 when compared with fluorescence-based methods 61,62,64 . Indeed, an advantage of fluorescence-based methods over traditional approaches is the absence of radioactive compounds, which facilitates their broad use in different laboratory settings without specific facilities or training requirements.…”

Section: Discussionmentioning

confidence: 99%

Development of a fluorescence-based method for the rapid determination of Zika virus polymerase activity and the screening of antiviral drugs

Sáez-Álvarez

Arias

Águila

et al. 2019

Sci Rep

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Zika virus (ZIKV) is an emerging pathogen that has been associated with large numbers of cases of severe neurologic disease, including Guillain-Barré syndrome and microcephaly. Despite its recent establishment as a serious global public health concern there are no licensed therapeutics to control this virus. Accordingly, there is an urgent need to develop methods for the high-throughput screening of antiviral agents. We describe here a fluorescence-based method to monitor the real-time polymerization activity of Zika virus RNA-dependent RNA polymerase (RdRp). By using homopolymeric RNA template molecules, de novo RNA synthesis can be detected with a fluorescent dye, which permits the specific quantification and kinetics of double-strand RNA formation. ZIKV RdRp activity detected using this fluorescence-based assay positively correlated with traditional assays measuring the incorporation of radiolabeled nucleotides. We also validated this method as a suitable assay for the identification of ZIKV inhibitors targeting the viral polymerase using known broad-spectrum inhibitors. The assay was also successfully adapted to detect RNA polymerization activity by different RdRps, illustrated here using purified RdRps from hepatitis C virus and foot-and-mouth disease virus. The potential of fluorescence-based approaches for the enzymatic characterization of viral polymerases, as well as for high-throughput screening of antiviral drugs, are discussed.

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Section: Discussionmentioning

confidence: 99%

Development of a fluorescence-based method for the rapid determination of Zika virus polymerase activity and the screening of antiviral drugs

Sáez-Álvarez

Arias

Águila

et al. 2019

Sci Rep

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“…Novagen BL21 (DE3) competent cells (EMD Millipore, Etobicoke, ON, Canada) were transformed with the pET21aDENV2NS5FL plasmid for expression and purification of the DENV NS5 with a C-terminal LEHHHHHH tag using minor modifications of published protocols 29, 33, 48 . Briefly, the bacterial cells were cultured in LB medium supplemented with carbenicillin (100 µg/ml) at 37 °C until the OD600 reached 0.6–0.8.…”

Section: Methodsmentioning

confidence: 99%

Evaluation of Sofosbuvir (β-D-2′-deoxy-2′-α-fluoro-2′-β-C-methyluridine) as an inhibitor of Dengue virus replication #

Colby-Germinario

Hassounah

et al. 2017

Sci Rep

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We evaluated Sofosbuvir (SOF), the anti-hepatitis C virus prodrug of β-d-2′-deoxy-2′-α-fluoro-2′-β-C-methyluridine-5′-monophosphate, for potential inhibitory activity against DENV replication. Both cell-based and biochemical assays, based on use of purified DENV full-length NS5 enzyme, were studied. Cytopathic effect protection and virus yield reduction assays confirmed that SOF possessed anti-DENV activity in cell culture with a 50% effective concentration (EC50) of 4.9 µM and 1.4 µM respectively. Real-time RT-PCR verified that SOF inhibits generation of viral RNA with an EC50 of 9.9 µM. Purified DENV NS5 incorporated the active triphosphate form (SOF-TP) into nascent RNA, causing chain-termination. Relative to the natural UTP, the incorporation efficiency of SOF-TP was low (discrimination value = 327.5). In a primer extension assay, SOF-TP was active against DENV NS5 wild-type polymerase activity with an IC50 of 14.7 ± 2.5 µM. The S600T substitution in the B Motif of DENV polymerase conferred 4.3-fold resistance to SOF-TP; this was due to decreased incorporation efficiency rather than enhanced excision of the incorporated SOF nucleotide. SOF has antiviral activity against DENV replication. The high discrimination value in favor of UTP in enzyme assays may not necessarily preclude antiviral activity in cells. SOF may be worthy of evaluation against severe DENV infections in humans.

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“…Storage was in small aliquots at Ϫ80°C. NS5 polymerase domain of the DENV2 NGC strain (38), was transformed into Novagen Rosetta 2(DE3) competent cells (EMD Millipore, Etobicoke, ON, Canada) and used for purification of the NS5 RNA-dependent RNA polymerase (RdRp) domain with an N-terminal His 6 tag of the DENV2 NGC strain, based on previously described protocols with minor modifications (38,39). For construction of an S600T mutant DENV2 NS5 polymerase, we performed site-directed mutagenesis using the Stratagene Quick Change II XL site-directed mutagenesis kit on pET21a[His-Strep-DENV2_NS5 DNA].…”

Section: Methodsmentioning

confidence: 99%

Identification of a Pyridoxine-Derived Small-Molecule Inhibitor Targeting Dengue Virus RNA-Dependent RNA Polymerase

Colby-Germinario

Hassounah

et al. 2016

Antimicrob Agents Chemother

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The viral RNA-dependent RNA polymerase (RdRp) activity of the dengue virus (DENV) NS5 protein is an attractive target for drug design. Here, we report the identification of a novel class of inhibitor (i.e., an active-site metal ion chelator) that acts against DENV RdRp activity. DENV RdRp utilizes a two-metal-ion mechanism of catalysis; therefore, we constructed a small library of compounds, through mechanism-based drug design, aimed at chelating divalent metal ions in the catalytic site of DENV RdRp. We now describe a pyridoxine-derived small-molecule inhibitor that targets DENV RdRp and show that 5-benzenesulfonylmethyl-3-hydroxy-4-hydroxymethyl-pyridine-2-carboxylic acid hydroxyamide (termed DMB220) inhibited the RdRp activity of DENV serotypes 1 to 4 at low micromolar 50% inhibitory concentrations (IC 50s of 5 to 6.7 M) in an enzymatic assay. The antiviral activity of DMB220 against DENV infection was also verified in a cell-based assay and showed a 50% effective concentration (EC 50 ) of <3 M. Enzyme assays proved that DMB220 was competitive with nucleotide incorporation. DMB220 did not inhibit the enzymatic activity of recombinant HIV-1 reverse transcriptase and showed only weak inhibition of HIV-1 integrase strand transfer activity, indicating high specificity for DENV RdRp. S600T substitution in the DENV RdRp, which was previously shown to confer resistance to nucleoside analogue inhibitors (NI), conferred 3-fold hypersusceptibility to DMB220, and enzymatic analyses showed that this hypersusceptibility may arise from the decreased binding/incorporation efficiency of the natural NTP substrate without significantly impacting inhibitor binding. Thus, metal ion chelation at the active site of DENV RdRp represents a viable anti-DENV strategy, and DMB220 is the first of a new class of DENV inhibitor. D engue virus (DENV) belongs to the family Flaviviridae, a group of enveloped positive-sense single-stranded RNA viruses that includes the genera Hepacivirus (prototype, hepatitis C virus [HCV]), Flavivirus (prototype, yellow fever virus), and Pestivirus (prototype, bovine viral diarrhea virus) (1). Distinct from the hepaciviruses and pestiviruses that are not arthropodborne, the flaviviruses are transmitted by mosquitos and ticks. Dengue, the most prevalent arthropod-borne viral disease of humans, is caused by four serotypes (DENV1 to -4) and has had a major impact on global public health (2-4).Infection with any of the DENV serotypes may result in a wide spectrum of clinical symptoms ranging from a mild flu-like syndrome (known as dengue fever [DF]) to the most severe forms of the disease, which are characterized by coagulopathy, increased vascular fragility, and permeability (dengue hemorrhagic fever [DHF]). The latter may progress to hypovolemic shock (dengue shock syndrome [DSS]) (3, 5). Among the four serotypes, DENV2 is the most prevalent on a global scale, followed by DENV3, DENV1, and DENV4 (6). Dengue is endemic in over 100 tropical and subtropical countries, and the global incidence of dengue has grown ...

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Expression and Purification of Dengue Virus NS5 Polymerase and Development of a High-Throughput Enzymatic Assay for Screening Inhibitors of Dengue Polymerase

Cited by 14 publications

References 7 publications

Development of a fluorescence-based method for the rapid determination of Zika virus polymerase activity and the screening of antiviral drugs

Development of a fluorescence-based method for the rapid determination of Zika virus polymerase activity and the screening of antiviral drugs

Evaluation of Sofosbuvir (β-D-2′-deoxy-2′-α-fluoro-2′-β-C-methyluridine) as an inhibitor of Dengue virus replication #

Identification of a Pyridoxine-Derived Small-Molecule Inhibitor Targeting Dengue Virus RNA-Dependent RNA Polymerase

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