Characterization of Imidazo[4,5-
            <i>d</i>
            ]Pyridazine Nucleosides as Modulators of Unwinding Reaction Mediated by West Nile Virus Nucleoside Triphosphatase/Helicase: Evidence for Activity on the Level of Substrate and/or Enzyme

Borowski, Peter; Lang, M; Haag, Annemarie; Schmitz, Herbert; Choe, Joonho; Chen, Huanming; Hosmane, Ramachandra S.

doi:10.1128/aac.46.5.1231-1239.2002

Cited by 59 publications

(54 citation statements)

References 32 publications

(28 reference statements)

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“…Differentiation of compound-mediated inhibition between viral translation and RNA synthesis could further be validated by a transient reporting replicon system (see below) (43). Enzymatic assays of WNV NS3 and NS5 (3,31,45) could also be used for more specific analysis. Alternatively, the mode of action of the compound could be identified by selection of resistant virus, followed by mapping the mutated gene and back-engineering of specific mutations into an infectious clone (38,46) for phenotypic verification.…”

Section: Discussionmentioning

confidence: 99%

High-Throughput Assays Using a Luciferase-Expressing Replicon, Virus-Like Particles, and Full-Length Virus for West Nile Virus Drug Discovery

Puig-Basagoiti

Deas

Ren

et al. 2005

Antimicrob Agents Chemother

110

112

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Many flaviviruses cause significant human disease worldwide. The development of flavivirus chemotherapy requires reliable high-throughput screening (HTS) assays. Although genetic systems have been developed for many flaviviruses, their usage in antiviral HTS assays has not been well explored. Here we compare three cell-based HTS assays for West Nile virus (WNV) drug discovery: (i) an assay that uses a cell line harboring a persistently replicating subgenomic replicon (containing a deletion of viral structural genes), (ii) an assay that uses packaged virus-like particles containing replicon RNA, and (iii) an assay that uses a full-length reporting virus. A Renilla luciferase gene was engineered into the replicon or into the full-length viral genome to monitor viral replication. Potential inhibitors could be identified through suppression of luciferase signals upon compound incubation. The antiviral assays were optimized in a 96-well format, validated with known WNV inhibitors, and proved useful in identifying a new inhibitor(s) through HTS of a compound library. In addition, because each assay encompasses multiple but discrete steps of the viral life cycle, the three systems could potentially be used to discriminate the mode of action of any inhibitor among viral entry (detected by assays ii and iii but not by assay i), replication (including viral translation and RNA synthesis; detected by assays i to iii), and virion assembly (detected by assay iii but not by assays i and ii). The approaches described in this study should be applicable to the development of cell-based assays for other flaviviruses.

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Section: Discussionmentioning

confidence: 99%

High-Throughput Assays Using a Luciferase-Expressing Replicon, Virus-Like Particles, and Full-Length Virus for West Nile Virus Drug Discovery

Puig-Basagoiti

Deas

Ren

et al. 2005

Antimicrob Agents Chemother

110

112

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“…The ATPase activity of the NTPase/helicase was determined as described previously (Borowski et al, 1999;Borowski et al, 2002a). Briefly, assays were performed with 0.5 pmol of HCV NTPase/helicase.…”

Section: Methodsmentioning

confidence: 99%

Tropolone and its Derivatives as Inhibitors of the Helicase Activity of Hepatitis C Virus Nucleotide Triphosphatase/helicase

Borowski

Lang

Haag

et al. 2007

Antivir Chem Chemother

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In this report, we demonstrate the interaction of the non-structural protein 3 (NS3) of hepatitis C virus (HCV) with alkaloide tropolone (2-hydroxy-2,4,6-heptatriene-1-one) and its derivatives. The compounds were biochemically screened separately against the ATPase and helicase activities of HCV NS3. In the investigations presented, alkaloide tropolone and its derivatives significantly inhibited the helicase activity of the viral protein when using a DNA substrate, with 50% inhibitory concentration values within a low micromolar range. The results using the RNA substrate were unexpected -none of the tropolone derivatives excerted any modulating influence towards the unwinding activity. Surprisingly, no influence of the nucleoside triphosphatase (NTPase) turnover was observed. Evidence is presented confirming that these compounds do not act by blocking the NTP-binding site, but by occupying an additional allosteric regulatory site. Further mechanisms of action, particularly of some of the derivatives, are discussed.

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“…Among the NS proteins, NS3 and NS5 -the major components of the so called 'replication complex' -appear to be the most promising targets for antiviral therapy because of their well-characterized enzymatic activities (Tan et A range of knock out experiments in which the helicase or polymerase activities were switched off demonstrated the crucial role of these enzymes in Flaviviridae replication (Matusan et al, 2001). Hence compounds that block or at least reduce the activities of NTPase/helicase or RdRp could act as inhibitors of viral replication (Borowski et al, 2002).…”

Section: Introductionmentioning

confidence: 99%

Synthesis and Evaluation of ATP-Binding Site Directed Potential Inhibitors of Nucleoside Triphosphatases/ Helicases and Polymerases of Hepatitis C and other Selected Flaviviridae Viruses

Bretner

Schalinski

Haag

et al. 2004

Antivir Chem Chemother

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Hepatitis C virus (HCV), West Nile virus (WNV), Japanese encephalitis virus ( JEV), and dengue virus (DENV) are members of Flaviviridae. They are deadly human pathogens and new and better therapeutic strategies are desperately needed. However, there are considerable barriers to the development of anti-Flaviviridae therapeutics, including the persistence of the virus, its genetic diversity during replication in the host and, for example, in the case of HCV, the lack of reproducible infectious culture systems. Similarities in the key Flaviviridae enzymes (with respect to conserved subdomain organization and arrangement of structural motifs) may indicate the potential usefulness of ribonucleoside analogues in the treatment of these viral infections. Over the last few years our understanding of the Flaviviridae replication cycle and the key enzymes involved in it has increased, giving hope for development of new antiviral agents.The Flaviviridae genome is a positive-stranded RNA, with a single open reading frame that translates into a single polyprotein. This polyprotein is co-and post-translationally processed by cellular and viral proteases into structural and non-structural (NS) proteins (Rosenberg, 2001). Among the NS proteins, NS3 and NS5 -the major components of the so called 'replication complex' -appear to be the most promising targets for antiviral therapy because of their well-characterized enzymatic activities (Tan et al.,

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Characterization of Imidazo[4,5- d ]Pyridazine Nucleosides as Modulators of Unwinding Reaction Mediated by West Nile Virus Nucleoside Triphosphatase/Helicase: Evidence for Activity on the Level of Substrate and/or Enzyme

Cited by 59 publications

References 32 publications

High-Throughput Assays Using a Luciferase-Expressing Replicon, Virus-Like Particles, and Full-Length Virus for West Nile Virus Drug Discovery

High-Throughput Assays Using a Luciferase-Expressing Replicon, Virus-Like Particles, and Full-Length Virus for West Nile Virus Drug Discovery

Tropolone and its Derivatives as Inhibitors of the Helicase Activity of Hepatitis C Virus Nucleotide Triphosphatase/helicase

Synthesis and Evaluation of ATP-Binding Site Directed Potential Inhibitors of Nucleoside Triphosphatases/ Helicases and Polymerases of Hepatitis C and other Selected Flaviviridae Viruses

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