Nucleotide triphosphatase/helicase of hepatitis C virus as a target for antiviral therapy

Borowski, Peter; Schalinski, Sarah; Schmitz, Herbert

doi:10.1016/s0166-3542(02)00096-7

Cited by 73 publications

(61 citation statements)

References 79 publications

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“…As presented above, the p14 peptide possesses a potent anti-helicase activity with an IC 50 of 725 nM in the fluorometric helicase activity assay, indicating that it is one of the most efficient NS3 helicase inhibitors cited in the literature (3,8,19), as well as when compared to other inhibitors tested in the same system, for which IC 50 values exceeded 17 M (3). Subsequent experiments with various concentrations of the enzyme and substrate enabled us to reveal more facts on the mechanism of action of the peptide.…”

Section: Discussionmentioning

confidence: 74%

NS3 Peptide, a Novel Potent Hepatitis C Virus NS3 Helicase Inhibitor: Its Mechanism of Action and Antiviral Activity in the Replicon System

Gozdek

Zhukov

Polkowska

et al. 2008

Antimicrob Agents Chemother

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Hepatitis C virus (HCV) chronic infections represent one of the major and still unresolved health problems because of low efficiency and high cost of current therapy. Therefore, our studies centered on a viral protein, the NS3 helicase, whose activity is indispensable for replication of the viral RNA, and on its peptide inhibitor that corresponds to a highly conserved arginine-rich sequence of domain 2 of the helicase. The NS3 peptide (p14) was expressed in bacteria. Its 50% inhibitory activity in a fluorometric helicase assay corresponded to 725 nM, while the ATPase activity of NS3 was not affected. Nuclear magnetic resonance (NMR) studies of peptide-protein interactions using the relaxation filtering technique revealed that p14 binds directly to the full-length helicase and its separately expressed domain 1 but not to domain 2. Changes in the NMR chemical shift of backbone amide nuclei ( 1 H and 15 N) of domain 1 or p14, measured during complex formation, were used to identify the principal amino acids of both domain 1 and the peptide engaged in their interaction. In the proposed interplay model, p14 contacts the clefts between domains 1 and 2, as well as between domains 1 and 3, preventing substrate binding. This interaction is strongly supported by cross-linking experiments, as well as by kinetic studies performed using a fluorometric assay. The antiviral activity of p14 was tested in a subgenomic HCV replicon assay that showed that the peptide at micromolar concentrations can reduce HCV RNA replication.Hepatitis C virus (HCV) is a positive-strand RNA virus of the Flaviviridae family (11). HCV infection, affecting 3% of the world population, leads to chronic hepatitis in up to 85% of the cases, in 10 to 20% of the cases it develops into cirrhosis that requires constant treatment and provokes permanent infirmity, while 1 to 5% of chronically infected patients are diagnosed with hepatocellular carcinoma (9). No efficient treatment exists; even the new dual therapy with pegylated interferon alpha 2a or 2b and ribavirin is effective only in up to 60% of the cases, depending on the genotype of the virus and the duration of the treatment (21). To date, no vaccine against HCV has been developed in spite of numerous attempts and advanced trials, principally because of the high variability of the RNA genome and association of HCV particles with host lipoproteins and immunoglobulins (1, 15). Thus, nonstructural proteins involved in viral replication are being examined as targets of antiviral therapy. One of them is NS3 (serine protease/RNA helicase), whose helicase activity is indispensable for replication of the viral RNA (25). The helicase part of NS3 folds into three domains of comparable size (domains 1, 2, and 3) that form a triangular molecule. Five structures of the NS3 helicase have been resolved by X-ray crystallography. The latest resolved structure shows two helicases bound to a single DNA molecule and reveals an apparent interface between two protein molecules (33). The existence of oligomeric structures o...

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Section: Discussionmentioning

confidence: 74%

NS3 Peptide, a Novel Potent Hepatitis C Virus NS3 Helicase Inhibitor: Its Mechanism of Action and Antiviral Activity in the Replicon System

Gozdek

Zhukov

Polkowska

et al. 2008

Antimicrob Agents Chemother

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“…Small molecule inhibitors of HCV helicase and related helicases were recently reviewed by Borowski and his colleagues in references [174] and [175]. One of the most promising of these early inhibitors is 1-(2'-O-methyl-beta-D-ribofuranosyl)imidazo [4, 5-d]pyridazine-4, 7(5H, 6H)-dione (HMC-HO4) (Fig.…”

Section: Sf2 Helicase Inhibitorsmentioning

confidence: 99%

Understanding Helicases as a Means of Virus Control

Frick¹,

Lam²

2006

CPD

101

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Helicases are promising antiviral drug targets because their enzymatic activities are essential for viral genome replication, transcription, and translation. Numerous potent inhibitors of helicases encoded by herpes simplex virus, severe acute respiratory syndrome coronavirus, hepatitis C virus, Japanese encephalitis virus, West Nile virus, and human papillomavirus have been recently reported in the scientific literature. Some inhibitors have also been shown to decrease viral replication in cell culture and animal models. This review discusses recent progress in understanding the structure and function of viral helicases to help clarify how these potential antiviral compounds function and to facilitate the design of better inhibitors. The above helicases and all related viral proteins are classified here based on their evolutionary and functional similarities, and the key mechanistic features of each group are noted. All helicases share a common motor function fueled by ATP hydrolysis, but differ in exactly how the motor moves the protein and its cargo on a nucleic acid chain. The helicase inhibitors discussed here influence rates of helicase-catalyzed DNA (or RNA) unwinding by preventing ATP hydrolysis, nucleic acid binding, nucleic acid release, or by disrupting the interaction of a helicase with a required cofactor.

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“…Promising NTPase/helicase inhibitors are currently being tested for the treatment of herpes simplex virus (13)(14)(15) and hepatitis C viral infections (16). As a foundation to the development of specific SARS-CoV NTPase/helicase inhibitors, we have cloned, expressed, purified, and characterized the biochemical properties of the SARS-CoV NTPase/helicase domain.…”

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confidence: 99%

The Severe Acute Respiratory Syndrome (SARS) Coronavirus NTPase/Helicase Belongs to a Distinct Class of 5′ to 3′ Viral Helicases

Tanner

Watt

Chai

et al. 2003

Journal of Biological Chemistry

200

229

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The putative NTPase/helicase protein from severe acute respiratory syndrome coronavirus (SARS-CoV) is postulated to play a number of crucial roles in the viral life cycle, making it an attractive target for anti-SARS therapy. We have cloned, expressed, and purified this protein as an N-terminal hexahistidine fusion in Escherichia coli and have characterized its helicase and NTPase activities. The enzyme unwinds doublestranded DNA, dependent on the presence of a 5 singlestranded overhang, indicating a 5 to 3 polarity of activity, a distinct characteristic of coronaviridae helicases. We provide the first quantitative analysis of the polynucleic acid binding and NTPase activities of a Nidovirus helicase, using a high throughput phosphate release assay that will be readily adaptable to the future testing of helicase inhibitors. All eight common NTPs and dNTPs were hydrolyzed by the SARS helicase in a magnesium-dependent reaction, stimulated by the presence of either single-stranded DNA or RNA. The enzyme exhibited a preference for ATP, dATP, and dCTP over the other NTP/dNTP substrates. Homopolynucleotides significantly stimulated the ATPase activity (15-25-fold) with the notable exception of poly(G) and poly(dG), which were non-stimulatory. We found a large variation in the apparent strength of binding of different homopolynucleotides, with dT 24 binding over 10 times more strongly than dA 24 as observed by the apparent K m .

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Nucleotide triphosphatase/helicase of hepatitis C virus as a target for antiviral therapy

Cited by 73 publications

References 79 publications

NS3 Peptide, a Novel Potent Hepatitis C Virus NS3 Helicase Inhibitor: Its Mechanism of Action and Antiviral Activity in the Replicon System

NS3 Peptide, a Novel Potent Hepatitis C Virus NS3 Helicase Inhibitor: Its Mechanism of Action and Antiviral Activity in the Replicon System

Understanding Helicases as a Means of Virus Control

The Severe Acute Respiratory Syndrome (SARS) Coronavirus NTPase/Helicase Belongs to a Distinct Class of 5′ to 3′ Viral Helicases

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