Searching for a new anti-HCV therapy: Synthesis and properties of tropolone derivatives

Boguszewska-Chachulska, Anna M.; Krawczyk, Mariusz; Najda, Andżelika; Kopanska, Katarzyna S.; Stankiewicz-Drogoń, Anna; Zagórski-Ostoja, Włodzimierz; Bretner, Maria

doi:10.1016/j.bbrc.2006.01.015

Cited by 34 publications

(21 citation statements)

References 33 publications

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“…The fluorometric assay was performed as described by Boguszewska-Chachulska et al (3), with some modifications. Helicase assays (in 60 l) were performed in 30 mM Tris-HCl (pH 7.5), 6 mM MnCl 2 , 0.075% Triton X-100, and 0.05% sodium azide, with 10 nM double-stranded DNA (dsDNA) substrate, 1.5 mM ATP, and 125 nM capture strand.…”

Section: Methodsmentioning

confidence: 99%

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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: Methodsmentioning

confidence: 99%

“…The ATPase assay was performed as described previously (3,4). The peptide was tested up to a concentration of 100 M. The reactions were carried out in 25 l of reaction buffer with 100 nM helicase for 60 min at 30°C.…”

Section: Methodsmentioning

confidence: 99%

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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“…Recently, a study with similar compounds based on our results was published (Boguszewska-Chachulska et al, 2006). This study also included DBMMTR, which displayed much less (~100-fold) inhibitory potential.…”

Section: Discussionmentioning

confidence: 92%

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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“…Tropolone and its derivatives have been reported as antimicrobial agents. It also possess antiviral, antitumor, antioxidant and enzyme inhibiting properties [3][4][5][6][7]. The title compound exhibit antiviral activities [7], while the isopropyl derivatives of tropolone (β-thujaplicin and γ-thujaplicin) were reported to have antifungal and antibacterial properties [6].…”

Section: Discussionmentioning

confidence: 99%

Crystal structure of 3,5,7-tris(morpholinomethyl)tropolone·0.67 hydrate, C₂₂H₃₃N₃O₅·0.67H₂O

Schutte‐Smith

Bungu

Steyl

et al. 2016

Zeitschrift Für Kristallographie - New Crystal Structures

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Searching for a new anti-HCV therapy: Synthesis and properties of tropolone derivatives

Cited by 34 publications

References 33 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

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

Crystal structure of 3,5,7-tris(morpholinomethyl)tropolone·0.67 hydrate, C₂₂H₃₃N₃O₅·0.67H₂O

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Searching for a new anti-HCV therapy: Synthesis and properties of tropolone derivatives

Cited by 34 publications

References 33 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

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

Crystal structure of 3,5,7-tris(morpholinomethyl)tropolone·0.67 hydrate, C22H33N3O5·0.67H2O

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Crystal structure of 3,5,7-tris(morpholinomethyl)tropolone·0.67 hydrate, C₂₂H₃₃N₃O₅·0.67H₂O