The monoethyl ester of meconic acid is an active site inhibitor of HCV NS5B RNA-dependent RNA polymerase

Pace, Paola; Nizi, Emanuela; Pacini, Barbara; Pesci, Silvia; Matassa, Victor G.; Francesco, Raffaele De; Altamura, Sergio; Summa, Vincenzo

doi:10.1016/j.bmcl.2004.03.087

Cited by 50 publications

(31 citation statements)

References 18 publications

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“…As the anionic ATA molecule harbours a keto carboxylic acid functional group, a pharmacophoric feature of the DKA series, we speculated that its inhibitory potency might be dependent on the nature of the divalent cation used in the assay, similar to that reported for some NS5B pyrophosphate mimetic inhibitors [14][15][16][17]. As expected, the IC 50 …”

Section: Identification Of Ata As a Potent Inhibitor Of The Rdrp Actisupporting

confidence: 66%

“…Binding of ATA to NS5B was examined by competition displacement assay employing [ 14 C]-foscarnet as a probe and nitrocellulose membrane filter assembly [17]. Briefly, 9 µg of NS5BC∆21 and 0.2 µCi [ …”

Section: Competition Displacement Assaysmentioning

confidence: 99%

“…Pyrophosphate mimetics, sometimes categorized within the NNI class, represents a third group of NS5B inhibitors. Representative members of this group include derivatives of dihydroxypyrimidine carboxylic acid and diketo acid (DKA) [14][15][16][17]. These compounds function as product-like analogues and are presumed to inhibit NS5B through an interaction with the catalytic metal ion at the enzyme's active site.…”

mentioning

confidence: 99%

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Characterization of Aurintricarboxylic Acid as a Potent Hepatitis C Virus Replicase Inhibitor

Chen

Waffo

Basu

et al. 2009

Antivir Chem Chemother

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Background: Hepatitis C virus (HCV) NS5B is an essential component of the viral replication machinery and an important target for antiviral intervention. Aurintricarboxylic acid (ATA), a broad-spectrum antiviral agent, was evaluated and characterized for its anti-NS5B activity in vitro and in HCV replicon cells. Methods: Recombinant NS5B, HCV replicase and Huh-7 cells harbouring the subgenomic HCV replicon of genotype 1b were employed for biochemical and mechanistic investigations. Results: Analysis of ATA activity in vitro yielded equipotent inhibition of recombinant NS5B and HCV replicase in the submicromolar range (50% inhibition concentration [IC 50 ] approximately 150 nM). Biochemical and mechanistic studies revealed a bimodal mechanism of ATA inhibition with characteristics of pyrophosphate mimics and non-nucleoside inhibitors. Molecular modelling and competition displacement studies were consistent with these parameters, suggesting that ATA might bind to the benzothiadiazine allosteric pocket 3 of NS5B or at its catalytic centre. Kinetic studies revealed a mixed mode of ATA inhibition with respect to both RNA and UTP substrates. Under single-cycle assay conditions, ATA inhibited HCV NS5B initiation and elongation from pre-bound RNA, but with ≥fivefold decreased potency compared with continuous polymerization conditions. The IC 50 value of ATA for the native replicase complex was 145 nM. In HCV replicon cells, ATA treatment ablated HCV RNA replication (50% effective concentration =75 nM) with concomitant decrease in NS5B expression and no apparent cytotoxic effects. Conclusions: This study identified ATA as a potent anti-NS5B inhibitor and suggests that its unique mode of action might be exploited for structural refinement and development of novel anti-NS5B agents.Hepatitis C virus (HCV) is a significant human bloodborne pathogen affecting an estimated 3% of the world's population, of whom 80% progress to chronic infection [1,2]. Sequelae include fibrosis, cirrhosis and hepatocellular carcinoma, making HCV the leading cause of liver transplantation in the USA [2,3]. The currently approved anti-HCV therapy of pegylated interferon-α (PEG-IFN-α) in combination with ribavirin exhibit sustained virological response (SVR) rates of 40-50% for genotype 1 and ≤80% for genotypes 2 and 3, and are associated with severe side effects resulting in limited patient compliance [4]. Additional challenges are posed by the high cost of therapy and the emergence of HCV quasispecies during treatment [5]; therefore, it is paramount to develop new and improved anti-HCV therapeutic agents. A prime area of focus is the development of small molecule inhibitors targeting the essential viral enzymes.HCV is an enveloped, single-stranded RNA virus with approximately 9.6 kb genome of positive polarity that encodes three structural and seven non-structural (NS) proteins [6,7]. Among these, the NS5B RNAdependent RNA polymerase (RdRp), a crucial and unique component of the viral replication machinery with no functional equivalent in the ho...

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Section: Identification Of Ata As a Potent Inhibitor Of The Rdrp Actisupporting

confidence: 66%

Section: Competition Displacement Assaysmentioning

confidence: 99%

mentioning

confidence: 99%

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Characterization of Aurintricarboxylic Acid as a Potent Hepatitis C Virus Replicase Inhibitor

Chen

Waffo

Basu

et al. 2009

Antivir Chem Chemother

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“…This class of compounds has been shown to inhibit HCV replication in both subgenomic replicons and HCV-infected chimpanzees (5,19,41,49). The pyrophosphate mimics, including dihydroxypyrimidine carboxylic acids and diketoacid derivatives, are believed to inhibit RdRp activity through an interaction with the catalytic metal ions in the enzyme active site (42,50,51). For nonnucleoside analogs, several structurally distinct series have been identified, including benzothiadiazines, benzimidazoles/diamides, substituted pyranones, and disubstituted phenylalanine/thiophene amides (13,22,25,33,47,52,57).…”

mentioning

confidence: 99%

Mutations Conferring Resistance to a Hepatitis C Virus (HCV) RNA-Dependent RNA Polymerase Inhibitor Alone or in Combination with an HCV Serine Protease Inhibitor In Vitro

Pilot‐Matias

et al. 2005

Antimicrob Agents Chemother

121

112

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Compounds A-782759 (an N-1-aza-4-hydroxyquinolone benzothiadiazine) and BILN-2061 are specific antihepatitis C virus (HCV) agents that inhibit the RNA-dependent RNA polymerase and the NS3 serine protease, respectively. Both compounds display potent activity against HCV replicons in tissue culture. In order to characterize the development of resistance to these anti-HCV agents, HCV subgenomic 1b-N replicon cells were cultured with A-782759 alone or in combination with BILN-2061 at concentrations 10 times above their corresponding 50% inhibitory concentrations in the presence of neomycin. Single substitutions in the NS5B polymerase gene (H95Q, N411S, M414L, M414T, or Y448H) resulted in substantial decreases in susceptibility to A-782759. Similarly, replicons containing mutations in the NS5B polymerase gene (M414L or M414T), together with single mutations in the NS3 protease gene (A156V or D168V), conferred high levels of resistance to both A-782759 and BILN-2061. However, the A-782759-resistant mutants remained susceptible to nucleoside and two other classes of nonnucleoside NS5B polymerase inhibitors, as well as interferon. In addition, we found that the frequency of replicons resistant to both compounds was significantly lower than the frequency of resistance to the single compound. Furthermore, the dually resistant mutants displayed significantly reduced replication capacities compared to the wild-type replicon. These findings provide strategic guidance for the future treatment of HCV infection.Hepatitis C virus (HCV) is a leading cause of chronic liver disease, affecting over 4 million Americans and about 170 million people worldwide. The current standard of care for chronic HCV infection involves extended dosing with alpha interferon (IFN-␣) and ribavirin (10,14,18). However, these regimens have limited clinical benefit due to poor tolerability and limited efficacy (only approximately half of genotype 1 HCV-infected individuals have a sustained virological response, whereas the response rate improves significantly [ϳ80%] when genotypes 2 and 3 are treated) (9,11,35). Therefore, development of inhibitors against virally encoded targets is urgently needed.The HCV genome is a 9.6-kb single-stranded RNA of positive polarity encoding a large polyprotein, which is the precursor of at least 10 mature viral proteins: C, E1, E2, p7, NS2, NS3, NS4A, NS4B, NS5A, and NS5B (2). The HCV polymerase encoded by nonstructural protein 5B (NS5B) is responsible for HCV RNA-dependent RNA polymerase (RdRp) and terminal transferase activities (4,31,32). The N-terminal domain (approximately 180 amino acids) of NS3 and the small hydrophobic NS4A protein compose a heterodimeric enzyme catalyzing the posttranslational processing of the HCV NS proteins (1, 21). Both NS5B RdRp and NS3 serine protease are believed to be components of the HCV replication complex, responsible for viral RNA replication, and have been shown to be indispensable for HCV replication in chimpanzees (26).To date, a number of distinct classes of NS5B RdRp inhibitors...

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“…This compound was more potent than 1, but the chemical instability prevented further exploration of this scaffold. 20 In the search for chemically and biologically stable diketoacid replacements 2-aryl-5,6-dihydroxy-pyrimidine-4-carboxylic acid 3 was identified. 21 .…”

Section: Figurementioning

confidence: 99%

Inhibitors of the hepatitis C virus RNA-dependent RNA polymerase

Attenni¹,

Avolio²,

Colarusso³

et al. 2006

Arkivoc

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Infections caused by Hepatitis C Virus (HCV) are a significant world health problem for which novel therapies are in urgent demand. The polymerase of HCV is responsible for the replication of viral RNA. We recently disclosed diketoacids 1 and dihydroxypyrimidine carboxylates 3 as novel, reversible inhibitors of the HCV NS5B polymerase. We report here the further development of 3 into the more potent 5,6-dihydroxy-2-(2-thienyl)pyrimidine-4-carboxylic acids. The structure activity relationship of these inhibitors is discussed in the context of their physicochemical properties, supporting the proposed binding model, which involves pyrophosphate like chelation of the active site Mg-ions. We also report on the discovery and synthesis of two related scaffolds, the N1-substituted pyrimidones and pyridine-N-oxides.

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The monoethyl ester of meconic acid is an active site inhibitor of HCV NS5B RNA-dependent RNA polymerase

Cited by 50 publications

References 18 publications

Characterization of Aurintricarboxylic Acid as a Potent Hepatitis C Virus Replicase Inhibitor

Characterization of Aurintricarboxylic Acid as a Potent Hepatitis C Virus Replicase Inhibitor

Mutations Conferring Resistance to a Hepatitis C Virus (HCV) RNA-Dependent RNA Polymerase Inhibitor Alone or in Combination with an HCV Serine Protease Inhibitor In Vitro

Inhibitors of the hepatitis C virus RNA-dependent RNA polymerase

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