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Conformational inhibition of the hepatitis C virus internal ribosome entry site RNA.

Parsons J, Castaldi MP, Dutta S, Dibrov SM, Wyles DL, Hermann T - Nat. Chem. Biol. (2009)

Bottom Line: The internal ribosome entry site (IRES), a highly conserved structured element of the hepatitis C virus (HCV) genomic RNA, is an attractive target for antiviral drugs.Here we show that benzimidazole inhibitors of the HCV replicon act by conformational induction of a widened interhelical angle in the IRES subdomain IIa, which facilitates the undocking of subdomain IIb from the ribosome and ultimately leads to inhibition of IRES-driven translation in HCV-infected cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry and Biochemistry, University of California, San Diego, La Jolla, California, USA.

ABSTRACT
The internal ribosome entry site (IRES), a highly conserved structured element of the hepatitis C virus (HCV) genomic RNA, is an attractive target for antiviral drugs. Here we show that benzimidazole inhibitors of the HCV replicon act by conformational induction of a widened interhelical angle in the IRES subdomain IIa, which facilitates the undocking of subdomain IIb from the ribosome and ultimately leads to inhibition of IRES-driven translation in HCV-infected cells.

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Normalized FRET signal for titrations of Cy3/Cy5-labeled IIa-2 RNA with benzimidazole ligands (a, compound 1; b, precursor 2) in the presence of 2mM Mg2+. Fitting of dose-response curves resulted in EC50 values for ligand binding of 600±80nM for compound 1 and 90±75μM for precursor 2. An interhelical angle of 112±5° was calculated from asymptotic FRET efficiencies for the ligand-bound state of the IIa RNA. Error bars represent ± s.d. calculated from three independent titrations.
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Figure 2: Normalized FRET signal for titrations of Cy3/Cy5-labeled IIa-2 RNA with benzimidazole ligands (a, compound 1; b, precursor 2) in the presence of 2mM Mg2+. Fitting of dose-response curves resulted in EC50 values for ligand binding of 600±80nM for compound 1 and 90±75μM for precursor 2. An interhelical angle of 112±5° was calculated from asymptotic FRET efficiencies for the ligand-bound state of the IIa RNA. Error bars represent ± s.d. calculated from three independent titrations.

Mentions: The FRET assay was then used to investigate ligand binding to the subdomain IIa. Therefore, IIa-2 RNA was titrated with 1 and the precursor 2 in the presence of 2mM Mg2+ (Fig. 2). Addition of the benzimidazoles resulted in dose-dependent quenching of FRET, suggesting that ligand binding at the internal loop induced a conformational change which led to a 23° widening of the interhelical angle of the IIa target. The EC50 value of the FRET quenching for 1 (600±80nM) corresponded well with the reported KD (720nM).8 The precursor 2 required higher concentrations for FRET quenching, suggesting weaker target binding in agreement with published data.8


Conformational inhibition of the hepatitis C virus internal ribosome entry site RNA.

Parsons J, Castaldi MP, Dutta S, Dibrov SM, Wyles DL, Hermann T - Nat. Chem. Biol. (2009)

Normalized FRET signal for titrations of Cy3/Cy5-labeled IIa-2 RNA with benzimidazole ligands (a, compound 1; b, precursor 2) in the presence of 2mM Mg2+. Fitting of dose-response curves resulted in EC50 values for ligand binding of 600±80nM for compound 1 and 90±75μM for precursor 2. An interhelical angle of 112±5° was calculated from asymptotic FRET efficiencies for the ligand-bound state of the IIa RNA. Error bars represent ± s.d. calculated from three independent titrations.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2770845&req=5

Figure 2: Normalized FRET signal for titrations of Cy3/Cy5-labeled IIa-2 RNA with benzimidazole ligands (a, compound 1; b, precursor 2) in the presence of 2mM Mg2+. Fitting of dose-response curves resulted in EC50 values for ligand binding of 600±80nM for compound 1 and 90±75μM for precursor 2. An interhelical angle of 112±5° was calculated from asymptotic FRET efficiencies for the ligand-bound state of the IIa RNA. Error bars represent ± s.d. calculated from three independent titrations.
Mentions: The FRET assay was then used to investigate ligand binding to the subdomain IIa. Therefore, IIa-2 RNA was titrated with 1 and the precursor 2 in the presence of 2mM Mg2+ (Fig. 2). Addition of the benzimidazoles resulted in dose-dependent quenching of FRET, suggesting that ligand binding at the internal loop induced a conformational change which led to a 23° widening of the interhelical angle of the IIa target. The EC50 value of the FRET quenching for 1 (600±80nM) corresponded well with the reported KD (720nM).8 The precursor 2 required higher concentrations for FRET quenching, suggesting weaker target binding in agreement with published data.8

Bottom Line: The internal ribosome entry site (IRES), a highly conserved structured element of the hepatitis C virus (HCV) genomic RNA, is an attractive target for antiviral drugs.Here we show that benzimidazole inhibitors of the HCV replicon act by conformational induction of a widened interhelical angle in the IRES subdomain IIa, which facilitates the undocking of subdomain IIb from the ribosome and ultimately leads to inhibition of IRES-driven translation in HCV-infected cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry and Biochemistry, University of California, San Diego, La Jolla, California, USA.

ABSTRACT
The internal ribosome entry site (IRES), a highly conserved structured element of the hepatitis C virus (HCV) genomic RNA, is an attractive target for antiviral drugs. Here we show that benzimidazole inhibitors of the HCV replicon act by conformational induction of a widened interhelical angle in the IRES subdomain IIa, which facilitates the undocking of subdomain IIb from the ribosome and ultimately leads to inhibition of IRES-driven translation in HCV-infected cells.

Show MeSH
Related in: MedlinePlus