Inhibition of the helicase activity of HCV NTPase/helicase by 1-beta-D-ribofuranosyl-1,2,4-triazole-3-carboxamide-5 '-triphosphate (ribavirin-TP).

Borowski, Peter; Lang, M; Niebuhr, Andreas; Haag, Annemarie; Schmitz, Herbert; Wiesch, Julian Schulze zur; Choe, Joonho; Siwecka, Maria A.; Kulikowski, Tadeusz

doi:10.18388/abp.2001_3908

Cited by 29 publications

(20 citation statements)

References 21 publications

(40 reference statements)

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“…4 ) at a capture strand to substrate molar ratio of 12.5–1. No substrate inhibition was observed, as opposed to results obtained with radioactive helicase assays where non‐saturating substrate concentrations were required [29,30]. Without capture strand the reaction was practically stopped (not shown), probably because of immediate reannealing of the separated strands.…”

Section: Resultsmentioning

confidence: 71%

Direct fluorometric measurement of hepatitis C virus helicase activity

Boguszewska-Chachulska

Krawczyk

Stankiewicz

et al. 2004

FEBS Letters

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The non-structural protein 3 (NS3) of hepatitis C virus (HCV) is a highly promising target for anti-HCV therapy because of its multiple enzymatic activities, such as RNAstimulated nucleoside triphosphatase, RNA helicase and serine protease. The helicase domain of NS3 as well as domain 2 of the helicase were expressed in a baculovirus system to obtain in high yield active proteins for prospective studies of complexes of the helicase with its inhibitors. A novel direct fluorometric test of helicase activity with a quenched DNA substrate, 3 0 labeled with a Cy3 dye and 5 0 labeled with a Black Hole Quencher, was developed and optimal reaction conditions established. This test based on fluorescence resonance energy transfer is simple and fast. It allows for direct measurements of enzyme activity, circumventing laborious and complicated radioactive techniques that are poorly reproducible. The results obtained encourage us to propose this new fluorescent assay as a method enabling high throughput screening of anti-helicase compounds.

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Section: Resultsmentioning

confidence: 71%

Direct fluorometric measurement of hepatitis C virus helicase activity

Boguszewska-Chachulska

Krawczyk

Stankiewicz

et al. 2004

FEBS Letters

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“…The DNA helicase activity of the HCV enzyme, monitored under optimal conditions [20,21] in the presence of 105 µ m ATP, corresponding to the K m for ATP in the ATPase reaction, was followed in the presence of varying concentrations of the benzimidazole and benzotriazole analogues (Fig. 2).…”

Section: Resultsmentioning

confidence: 99%

“…Ribavirin‐5′‐triphosphate, a potent classical competitive inhibitor of the NTPase activities of the West Nile virus (WNV) and HCV NTPase/helicases at low ATP concentrations (<< K m ), failed to inhibit the ATPase activities at high ATP concentrations (> K m ) and, indeed, even stimulated enzyme activity. By contrast, ribavirin‐5′‐triphosphate moderately inhibits the helicase activities of both enzymes by a mechanism that is independent of the ATP concentration [19–21], most likely due to occupation of the second nucleotide binding site.…”

mentioning

confidence: 99%

Halogenated benzimidazoles and benzotriazoles as inhibitors of the NTPase/helicase activities of hepatitis C and related viruses

Borowski

Deinert

Schalinski

et al. 2003

European Journal of Biochemistry

118

108

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A search has been initiated for lead inhibitors of the nonstructural protein 3 (NS3)-associated NTPase/helicase activities of hepatitis C virus, the related West Nile virus, Japanese encephalitis virus and the human mitochondrial Suv3 enzyme. Random screening of a broad range of unrelated low-molecular mass compounds, employing both RNA and DNA substrates, revealed that 4,5,6,7-tetrabromobenzotriazole (TBBT) hitherto known as a potent highly selective inhibitor of protein kinase 2, is a good inhibitor of the helicase, but not NTPase, activity of hepatitis C virus NTPase/helicase. The IC 50 is approximately 20 lM with a DNA substrate, but only 60 lM with an RNA substrate. Several related analogues of TBBT were enzymeand/or substrate-specific inhibitors. For example, 5,6-dichloro-1-(b-D-ribofuranosyl)benzotriazole (DRBT) was a good, and selective, inhibitor of the West Nile virus enzyme with an RNA substrate (IC 50 0.3 lM), but much weaker with a DNA substrate (IC 50 3 lM). Preincubation of the enzymes, but not substrates, with DRBT enhanced inhibitory potency, e.g. the IC 50 vs the hepatitis C virus helicase activity was reduced from 1.5 to 0.1 lM. No effect of preincubation was noted with TBBT, suggesting a different mode of interaction with the enzyme. The tetrachloro congener of TBBT, 4,5,6,7,-tetrachlorobenzotriazole (TCBT; a much weaker inhibitor of casein kinase 2) is also a much weaker inhibitor than TBBT of all four helicases. Kinetic studies, supplemented by comparison of ATP-binding sites, indicated that, unlike the case with casein kinase 2, the mode of action of the inhibitors vs the helicases is not by interaction with the catalytic ATP-binding site, but rather by occupation of an allosteric nucleoside/nucleotide binding site. The halogeno benzimidazoles and benzotriazoles included in this study are excellent lead compounds for the development of more potent inhibitors of hepatitis C virus and other viral NTPase/helicases.

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“…Nevertheless, the helicase activity could be experimentally documented only in a few members of the Flaviviridae family (Fig. 2) (Suzich et al, 1993;Gwack et al, 1999;Li et al, 1999;Gu et al, 2000;Utama et al, 2000;Borowski et al, 2001a;2001b). All the enzymes exhibit a 3¢to 5¢-directionality with respect to the template strand (Kadare & Haenni, 1997) and, in contrast to the majority of the NTPase/helicases described, they are capable of unwinding DNA/DNA and RNA/RNA homoduplexes and RNA/DNA heteroduplexes (Tai et al, 1996 and own observations).…”

Section: Structure and Function Of Ns3 Ntpase/helicasementioning

confidence: 96%

NTPase/helicase of Flaviviridae: inhibitors and inhibition of the enzyme.

Borowski¹,

Niebuhr²,

Schmitz³

et al. 2002

Acta Biochim Pol

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RNA nucleoside triphosphatases (NTPase)/helicases represent a large family of proteins that are ubiquitously distributed over a wide range of organisms. The enzymes play essential role in cell development and differentiation, and some of them are involved in transcription and replication of viral single-stranded RNA genomes. The enzymatic activities of a NTPase/helicase were also detected in the carboxyl-terminal non-structural protein 3 (NS3) of members of the Flaviviridae family. The crucial role of the enzyme for the virus life cycle was demonstrated in knock out experiments and by using NTPase/helicase specific inhibitors. This makes the enzyme an attractive target for development of Flaviviridae-specific antiviral therapies. This review will summarize our knowledge about the function and structure of the enzyme, update the spectrum of inhibitors of the enzymatic activities of the NTPase/helicase and describe the different mechanisms by which the compounds act. Some of the compounds reviewed herein could show potential utility as antiviral agents against Flaviviridae viruses.

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Inhibition of the helicase activity of HCV NTPase/helicase by 1-beta-D-ribofuranosyl-1,2,4-triazole-3-carboxamide-5 '-triphosphate (ribavirin-TP).

Cited by 29 publications

References 21 publications

Direct fluorometric measurement of hepatitis C virus helicase activity

Direct fluorometric measurement of hepatitis C virus helicase activity

Halogenated benzimidazoles and benzotriazoles as inhibitors of the NTPase/helicase activities of hepatitis C and related viruses

NTPase/helicase of Flaviviridae: inhibitors and inhibition of the enzyme.

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