Mechanism and specificity of a symmetrical benzimidazolephenylcarboxamide helicase inhibitor

Craig A. Belon, Yoji D. High, Tse I. Lin, Frederik Pauwels, David N. Frick

Research output: Contribution to journalArticle

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Abstract

This study examines the effects of 1-N, 4-N-bis[4-(1H-benzimidazol-2-yl) phenyl]benzene-l, 4-dicarboxamide ((BIP)2B) on the NS3 helicase encoded by the hepatitis C virus (HCV). Molecular beaconbased helicase assays were used to show that (BIP)2B inhibits the ability of HCV helicase to separate a variety of RNA and DNA duplexes with half-maximal inhibitory concentrations ranging from 0.7 to 5 μM, depending on the nature of the substrate. In single turnover assays, (BIP)2B only inhibited unwinding reactions when it was preincubated with the helicase-nucleic acid complex. (BIP)2B quenched NS3 intrinsic protein fluorescence with an apparent dissociation constant of 5μM, and in the presence of (BIP)2B, HCV helicase did not appear to interact with a fluorescent DNA oligonucleotide. In assays monitoring HCV helicasecatalyzed ATP hydrolysis, (BIP)2B only inhibited helicase-catalyzed ATP hydrolysis in the presence of intermediate concentrations of RNA, suggesting RNA and (BIP)2B compete for the same binding site. HCV helicases isolated from various HCV genotypes were similarly sensitive to (BIP)2B, with half-maximal inhibitory concentrations ranging from 0.7 to 2.4 μM. (BIP)2B also inhibited ATP hydrolysis catalyzed by related helicases from Dengue virus, Japanese encephalitis virus, and humans. (BIP)2B appeared to bind the HCV and human proteins with similar affinity (Ki = 7 and 8μM, respectively), but it bound the flavivirus proteins up to 270 times more tightly. Results are discussed in light of a molecular model of a (BIP) 2B-HCV helicase complex, which is unable to bind nucleic acid, thus preventing the enzyme from separating doublestranded nucleic acid.

Original languageEnglish (US)
Pages (from-to)1822-1832
Number of pages11
JournalBiochemistry
Volume49
Issue number9
DOIs
StatePublished - Mar 9 2010
Externally publishedYes

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Viruses
Hepacivirus
Nucleic Acids
Hydrolysis
Adenosine Triphosphate
RNA
Assays
Japanese Encephalitis Virus
Flavivirus
Dengue Virus
Proteins
Molecular Models
DNA
Benzene
Oligonucleotides
Fluorescence
Binding Sites
Genotype
Enzymes
Monitoring

ASJC Scopus subject areas

  • Biochemistry

Cite this

Mechanism and specificity of a symmetrical benzimidazolephenylcarboxamide helicase inhibitor. / Belon, Craig A.; High, Yoji D.; Lin, Tse I.; Pauwels, Frederik; Frick, David N.

In: Biochemistry, Vol. 49, No. 9, 09.03.2010, p. 1822-1832.

Research output: Contribution to journalArticle

Belon, Craig A. ; High, Yoji D. ; Lin, Tse I. ; Pauwels, Frederik ; Frick, David N. / Mechanism and specificity of a symmetrical benzimidazolephenylcarboxamide helicase inhibitor. In: Biochemistry. 2010 ; Vol. 49, No. 9. pp. 1822-1832.
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