Triaryl Pyrazoline Compound Inhibits Flavivirus RNA Replication

Puig-Basagoiti, Francesc; Tilgner, Mark; Forshey, Brett M.; Philpott, Sean; Espina, Noel; Wentworth, David E.; Goebel, Scott J.; Masters, Paul S.; Falgout, Barry; Ren, Ping; Ferguson, David M.; Shi, Pei Yong

doi:10.1128/aac.50.4.1320-1329.2006

Cited by 109 publications

(114 citation statements)

References 54 publications

(54 reference statements)

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“…The viral inocula were washed and replaced with DMEM plus 10% FBS containing various concentrations of SIN. Culture supernatants were collected at 48 h post-infection, and viral titers were determined by plaque assay (42). For the cytotoxicity assay, baby hamster kidney cells were incubated with various concentrations of SIN and assayed for cell viability at 48 h post-treatment using an 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay kit (American Type Culture Collection).…”

Section: Methodsmentioning

confidence: 99%

“…Interestingly, DENV-2 was more sensitive to SIN inhibition than was YFV; at 250 M, SIN reduced DENV-2 and YFV titers by about 6-fold and 50%, respectively. A cell proliferation-based MTT assay (42) showed no cytotoxicity when uninfected cells were treated with SIN at concentrations up to 300 M, indicating that the observed antiviral activity was not due to compoundmediated cytotoxicity (Fig. 1C).…”

Section: Inhibition Of Flavivirus Replication By Sin-sin Differs Frommentioning

confidence: 99%

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Structural and Functional Analyses of a Conserved Hydrophobic Pocket of Flavivirus Methyltransferase

Dong

Liu

Zou

et al. 2010

Journal of Biological Chemistry

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The flavivirus methyltransferase (MTase) sequentially methylates the N7 and 2-O positions of the viral RNA cap (GpppA-RNA 3 m 7 GpppA-RNA 3 m 7 GpppAm-RNA), using S-adenosyl-L-methionine (AdoMet) as a methyl donor. We report here that sinefungin (SIN), an AdoMet analog, inhibits several flaviviruses through suppression of viral MTase. The crystal structure of West Nile virus MTase in complex with SIN inhibitor at 2.0-Å resolution revealed a flavivirus-conserved hydrophobic pocket located next to the AdoMet-binding site. The pocket is functionally critical in the viral replication and cap methylations. In addition, the N7 methylation efficiency was found to correlate with the viral replication ability. Thus, SIN analogs with modifications that interact with the hydrophobic pocket are potential specific inhibitors of flavivirus MTase.The genus Flavivirus in the family Flaviviridae is composed of more than 70 viruses (1). Many flaviviruses are arthropodborne and cause significant human disease. The four serotypes of Dengue virus (DENV), 5 yellow fever virus (YFV), West Nile virus (WNV), Japanese encephalitis virus, and tick-borne encephalitis virus, are categorized as global emerging pathogens and are also National Institutes of Health NIAID Priority Pathogens (2). The World Health Organization has estimated annual human cases of more than 50 million, 200,000, and 50,000 for DENV (3), YFV (4), and Japanese encephalitis virus (5), respectively. Approximately 2.5 billion people are at risk of DENV infection (3). Since 1999, WNV has spread rapidly throughout the Western Hemisphere (6). Vaccines for humans are currently available only for YFV, Japanese encephalitis virus, and tick-borne encephalitis virus (2). No clinically approved antiviral therapy is available for treatment of flavivirus infections. Therefore, the development of vaccines and antiviral agents for prevention and treatment of flavivirus infections is a clear public health priority (6).The flavivirus genomic RNA is single-stranded and of positive (i.e. mRNA) polarity. A type I cap (see details below) is present at the 5Ј end, followed by the conserved dinucleotide sequence 5Ј-AG-3Ј (m 7 GpppAmG) (7). The 3Ј end of the genome terminates with 5Ј-CU OH -3Ј rather than with a poly(A) tract (8). The viral genome is ϳ11 kb in length, consisting of a 5Ј UTR, a single long open reading frame (ORF), and a 3Ј UTR (9). The single ORF encodes a polyprotein that is co-and posttranslationally processed by viral and cellular proteases into three structural proteins (capsid (C), premembrane (prM), or membrane (M), and envelope (E)), and seven nonstructural proteins (NS1, NS2a, NS2b, NS3, NS4a, NS4b, and NS5) (10). The nonstructural proteins are assumed to be involved primarily in the replication of viral RNA, as components of a replicase complex. Among the NS proteins, NS3 is a multifunctional protein with activities of a serine protease, an RNA triphosphatase, a nucleoside triphosphatase, and a helicase (11-13); NS5 has the functions of an RNA-dependent RNA polymerase...

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Section: Methodsmentioning

confidence: 99%

Section: Inhibition Of Flavivirus Replication By Sin-sin Differs Frommentioning

confidence: 99%

Structural and Functional Analyses of a Conserved Hydrophobic Pocket of Flavivirus Methyltransferase

Dong

Liu

Zou

et al. 2010

Journal of Biological Chemistry

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“…The following viruses were used in the viral titer inhibition assay: DENV-2 (strain New Guinea C), WNV (strain 3356), YFV (17D vaccine strain), WEEV (strain Cova 746), and VSV (New Jersey serotype). Plaque assay was performed as reported previously (38 Expression of the fusion protein was induced with 1 mM isopropyl-␤-D-thiogalactoside at an optical density at 600 nm of 0.6. Cells were grown for another 3 h at 37°C in Terrific broth medium.…”

Section: Methodsmentioning

confidence: 99%

Inhibition of Dengue Virus through Suppression of Host Pyrimidine Biosynthesis

Wang

Bushell

Qing

et al. 2011

J Virol

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Viral replication relies on the host to supply nucleosides. Host enzymes involved in nucleoside biosynthesis are potential targets for antiviral development. Ribavirin (a known antiviral drug) is such an inhibitor that suppresses guanine biosynthesis; depletion of the intracellular GTP pool was shown to be the major mechanism to inhibit flavivirus. Along similar lines, inhibitors of the pyrimidine biosynthesis pathway could be targeted for potential antiviral development. Here we report on a novel antiviral compound (NITD-982) that inhibits host dihydroorotate dehydrogenase (DHODH), an enzyme required for pyrimidine biosynthesis. The inhibitor was identified through screening 1.8 million compounds using a dengue virus (DENV) infection assay. The compound contains an isoxazole-pyrazole core structure, and it inhibited DENV with a 50% effective concentration (EC 50 ) of 2.4 nM and a 50% cytotoxic concentration (CC 50 ) of >5 M. NITD-982 has a broad antiviral spectrum, inhibiting both flaviviruses and nonflaviviruses with nanomolar EC 90 s. We also show that (i) the compound inhibited the enzymatic activity of recombinant DHODH, (ii) an NITD-982 analogue directly bound to the DHODH protein, (iii) supplementing the culture medium with uridine reversed the compoundmediated antiviral activity, and (iv) DENV type 2 (DENV-2) variants resistant to brequinar (a known DHODH inhibitor) were cross resistant to NITD-982. Collectively, the results demonstrate that the compound inhibits DENV through depleting the intracellular pyrimidine pool. In contrast to the in vitro potency, the compound did not show any efficacy in the DENV-AG129 mouse model. The lack of in vivo efficacy is likely due to the exogenous uptake of pyrimidine from the diet or to a high plasma protein-binding activity of the current compound.

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“…The DNA-based replicons are more stable than RNA-based constructs and the replicons can be directly transfected (thus without in vitro transcription) (5,6). Reporter replicon systems have been used for the analysis of flavivirus replication mechanisms and for conducting high-throughput screening for antiviral compounds (8)(9)(10)(11)(12)(13)(14)(15). The incorporation of firefly and Renilla luciferase reporter genes into DENV replicon constructs has facilitated the study of this virus.…”

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confidence: 99%

Development of a Novel Dengue-1 Virus Replicon System Expressing Secretory <i>Gaussia</i> luciferase for Analysis of Viral Replication and Discovery of Antiviral Drugs

et al. 2014

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SUMMARY: Replicon systems have been used for high-throughput screening of anti-dengue virus (anti-DENV) inhibitors and for understanding mechanisms of viral replication. In the present study, we constructed novel DENV-1 replicons encoding Gaussia luciferase that was secreted into the culture medium. Two types of constructs were generated: RNA-based and DNA-based. Each type was translated in an internal ribosome entry site (IRES)-dependent or IRES-independent manner. Among these constructs, the DNA-based replicon employing IRES-dependent translation (DGL2) produced the highest titer. Luciferase levels in the culture medium revealed that the DGL2 replicon was inhibited by ribavirin (a well-known DENV inhibitor) at levels similar to those measured for drug inhibition of multi-round DENV-1 infection. These results indicate that the DNA-based IRES-driven DENV-1 replicon may facilitate studies on viral replication and antiviral compound discovery.Dengue virus (four serotypes [DENV-1-4]), which belongs to the genus Flavivirus of the family Flaviviridae, is transmitted to humans by Aedes mosquitoes and is the etiologic agent of dengue fever and dengue hemorrhagic fever (1). DENV annually infects 50-100 million humans in tropical and sub-tropical regions, posing a considerable public health threat in over 100 countries (2). However, at present, no specific antiviral drug or licensed vaccine is available for DENV infection (3). Therefore, the development of effective prophylactic and therapeutic measures is urgently required. The virus is an enveloped RNA virus with a single-stranded positive-sense genome of approximately 11 kb. A single long open reading frame (ORF) encodes a polyprotein that is processed by cellular and viral proteases into 3 structural (C, prM, and E) and 7 non-structural (NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5) proteins. The structural proteins form the virus particle, whereas the NS proteins are involved in the replication of the viral genome.A subgenomic replicon is a self-replicating segment of the viral genome that lacks the genes encoding structural proteins. In general, a subgenomic replicon is established by transfection of in vitro transcribed RNA. Recently, DNA-based replicon systems, in which transcription is controlled by a cytomegalovirus (CMV) promoter, have been developed from alphavirus, arterivirus, and flavivirus (4-7). The DNA-based replicons are more stable than RNA-based constructs and the replicons can be directly transfected (thus without in vitro transcription) (5,6). Reporter replicon systems have been used for the analysis of flavivirus replication mechanisms and for conducting high-throughput screening for antiviral compounds (8-15). The incorporation of firefly and Renilla luciferase reporter genes into DENV replicon constructs has facilitated the study of this virus. Recently, genes encoding secretory luciferases have been used in studies on other viruses such as retrovirus, togavirus, herpesvirus, and flavivirus (16)(17)(18)(19).To generate a more useful reporter replic...

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Triaryl Pyrazoline Compound Inhibits Flavivirus RNA Replication

Cited by 109 publications

References 54 publications

Structural and Functional Analyses of a Conserved Hydrophobic Pocket of Flavivirus Methyltransferase

Structural and Functional Analyses of a Conserved Hydrophobic Pocket of Flavivirus Methyltransferase

Inhibition of Dengue Virus through Suppression of Host Pyrimidine Biosynthesis

Development of a Novel Dengue-1 Virus Replicon System Expressing Secretory <i>Gaussia</i> luciferase for Analysis of Viral Replication and Discovery of Antiviral Drugs

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