Aurintricarboxylic Acid Modulates the Affinity of Hepatitis C Virus NS3 Helicase for Both Nucleic Acid and ATP

Shadrick, William R.; Mukherjee, Sourav; Hanson, Alicia M.; Sweeney, Noreena L.; Frick, David N.

doi:10.1021/bi4006495

Cited by 35 publications

(45 citation statements)

References 47 publications

(135 reference statements)

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“…This result confirms that the two enzymatic functions are closely intertwined. Although ATA results in the same effects on the MTase and GT, its role is more elusive since it can inhibit a large range of proteins, including hepatitis C virus (HCV) NS3 or influenza virus neuraminidase (65,66). Concerning ribavirin and its phosphorylated derivatives, it is commonly accepted that it is a broadspectrum antiviral molecule that probably inhibits the cellular enzyme IMP dehydrogenase (IMPDH) through 5=-monophosphate ribavirin, thus depleting the cellular GTP pool required for virus replication.…”

Section: Discussionmentioning

confidence: 99%

mRNA Capping by Venezuelan Equine Encephalitis Virus nsP1: Functional Characterization and Implications for Antiviral Research

Guillén

Rabah

et al. 2015

J Virol

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Alphaviruses are known to possess a unique viral mRNA capping mechanism involving the viral nonstructural protein nsP1. This enzyme harbors methyltransferase (MTase) and nsP1 guanylylation (GT) activities catalyzing the transfer of the methyl group from S-adenosylmethionine (AdoMet) to the N7 position of a GTP molecule followed by the formation of an m 7 GMPnsP1 adduct. Subsequent transfer of m 7 GMP onto the 5= end of the viral mRNA has not been demonstrated in vitro yet. Here we report the biochemical characterization of Venezuelan equine encephalitis virus (VEEV) nsP1. We have developed enzymatic assays uncoupling the different reactions steps catalyzed by nsP1. The MTase and GT reaction activities were followed using a nonhydrolyzable GTP (GIDP) substrate and an original Western blot assay using anti-m 3 G/m 7 G-cap monoclonal antibody, respectively. The GT reaction is stimulated by S-adenosyl-L-homocysteine (Ado-Hcy), the product of the preceding MTase reaction, and metallic ions. The covalent linking between nsP1 and m 7 GMP involves a phosphamide bond between the nucleotide and a histidine residue. Final guanylyltransfer onto RNA was observed for the first time with an alphavirus nsP1 using a 5=-diphosphate RNA oligonucleotide whose sequence corresponds to the 5= end of the viral genome. Alanine scanning mutagenesis of residues H37, H45, D63, E118, Y285, D354, R365, N369, and N375 revealed their respective roles in MT and GT reactions. Finally, the inhibitory effects of sinefungin, aurintricarboxylic acid (ATA), and ribavirin triphosphate on MTase and capping reactions were investigated, providing possible avenues for antiviral research. IMPORTANCEEmergence or reemergence of alphaviruses represents a serious health concern, and the elucidation of their replication mechanisms is a prerequisite for the development of specific inhibitors targeting viral enzymes. In particular, alphaviruses are able, through an original reaction sequence, to add to their mRNA a cap required for their protection against cellular nucleases and initiation of viral proteins translation. In this study, the capping of a 5= diphosphate synthetic RNA mimicking the 5= end of an alphavirus mRNA was observed in vitro for the first time. The different steps for this capping are performed by the nonstructural protein 1 (nsP1). Reference compounds known to target the viral capping inhibited nsP1 enzymatic functions, highlighting the value of this enzyme in antiviral development. E mergence or reemergence of alphaviruses represents a serious health concern, as exemplified by the worldwide epidemics of Chikungunya virus during the last 10 years (1). The Alphavirus genus comprises 31 species that together with the genus Rubivirus forms the Togaviridae family. Alphaviruses have been classified on the basis of their geographical distribution. Alphaviruses circulating in the Old World (OW) most commonly cause febrile illness and painful arthralgias or polyarthralgias, particularly in the small joints (2). In contrast, New World (NW) alph...

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Section: Discussionmentioning

confidence: 99%

mRNA Capping by Venezuelan Equine Encephalitis Virus nsP1: Functional Characterization and Implications for Antiviral Research

Guillén

Rabah

et al. 2015

J Virol

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“…A possible explanation is masking by ATA of the calcineurininteracting region in PMCA4 (amino acids 428 to 651 in human PMCA4b) [9]. Supporting this possibility, ATA inhibits hepatitis C helicase by a similar mechanism, binding to a defined region in the enzyme and thereby blocking interaction with nucleic acids [35]. However, a different picture emerges from analysis of the effect of ATA on PMCA4 binding to endothelial nitric oxide synthase (eNOS), which interacts with the same PMCA4 domain as calcineurin [19].…”

Section: Discussionmentioning

confidence: 99%

Selective inhibition of plasma membrane calcium ATPase 4 improves angiogenesis and vascular reperfusion

Kurusamy

López-Maderuelo

Little

et al. 2017

Journal of Molecular and Cellular Cardiology

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Aims: Ischaemic cardiovascular disease is a major cause of morbidity and mortality worldwide. Despite promising results from pre-clinical animal models, VEGF-based strategies for therapeutic angiogenesis have yet to achieve successful reperfusion of ischaemic tissues in patients. Failure to restore efficient VEGF activity in the ischaemic organ remains a major problem in current pro-angiogenic therapeutic approaches. Plasma membrane calcium ATPase 4 (PMCA4) negatively regulates VEGF-activated angiogenesis via inhibition of the calcineurin/NFAT signalling pathway. PMCA4 activity is inhibited by the small molecule aurintricarboxylic acid (ATA). We hypothesize that inhibition of PMCA4 with ATA might enhance VEGF-induced angiogenesis. Methods and results: We show that inhibition of PMCA4 with ATA in endothelial cells triggers a marked increase in VEGF-activated calcineurin/NFAT signalling that translates into a strong increase in endothelial cell motility and blood vessel formation. ATA enhances VEGF-induced calcineurin signalling by disrupting the interaction between PMCA4 and calcineurin at the endothelial-cell membrane. ATA concentrations at the nanomolar range, that efficiently inhibit PMCA4, had no deleterious effect on endothelial-cell viability or zebrafish embryonic development. However, high ATA concentrations at the micromolar level impaired endothelial cell viability and tubular morphogenesis, and were associated with toxicity in zebrafish embryos. In mice undergoing experimentally-induced hindlimb ischaemia, ATA treatment significantly increased the reperfusion of post-ischaemic limbs. Conclusions: Our study provides evidence for the therapeutic potential of targeting PMCA4 to improve VEGFbased pro-angiogenic interventions. This goal will require the development of refined, highly selective versions of ATA, or the identification of novel PMCA4 inhibitors.

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“…Somewhat surprisingly, most compounds tested did not effect observed rates of DNA strand separation, even when supplied at concentrations as high as 1 mM (Table 1). Primuline [18] and aurintricarboxylic acid [35] were included as positive controls.…”

Section: Hcv Ns3 Helicase Enzymatic Assaysmentioning

confidence: 99%

In silico identification, design and synthesis of novel piperazine-based antiviral agents targeting the hepatitis C virus helicase

Bassetto

Leyssen

Neyts

et al. 2017

European Journal of Medicinal Chemistry

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A structure-based virtual screening of commercial compounds was carried out on the HCV NS3 helicase structure, with the aim to identify novel inhibitors of HCV replication. Among a selection of 13 commercial structures, one compound was found to inhibit the subgenomic HCV replicon in the low micromolar range. Different series of new piperazine-based analogues were designed and synthesised, and among them, several novel structures exhibited antiviral activity in the HCV replicon assay. Some of the new compounds were also found to inhibit HCV NS3 helicase function in vitro, and one directly bound NS3 with a dissociation constant of 570 ± 270 nM.

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Aurintricarboxylic Acid Modulates the Affinity of Hepatitis C Virus NS3 Helicase for Both Nucleic Acid and ATP

Cited by 35 publications

References 47 publications

mRNA Capping by Venezuelan Equine Encephalitis Virus nsP1: Functional Characterization and Implications for Antiviral Research

mRNA Capping by Venezuelan Equine Encephalitis Virus nsP1: Functional Characterization and Implications for Antiviral Research

Selective inhibition of plasma membrane calcium ATPase 4 improves angiogenesis and vascular reperfusion

In silico identification, design and synthesis of novel piperazine-based antiviral agents targeting the hepatitis C virus helicase

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