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Targeted inhibition of the hepatitis C internal ribosomal entry site genomic RNA with oligonucleotide conjugates.

Guerniou V, Gillet R, Berrée F, Carboni B, Felden B - Nucleic Acids Res. (2007)

Bottom Line: All these molecules inhibit, in a dose-dependent manner, the 'IRES-dependent' translation in vitro.The 5'-coupled imidazole conjugate reduces viral protein synthesis by half at a 300 nM concentration (IC50), corresponding to a 4-fold increase of activity when compared to the naked oligonucleotide.These new conjugates are now being tested for activity on infected hepatic cell lines.

View Article: PubMed Central - PubMed

Affiliation: Biochimie Pharmaceutique, Inserm U835, Upres JE 2311, Université de Rennes 1, France.

ABSTRACT
Hepatitis C is a major public health concern, with an estimated 170 million people infected worldwide and an urgent need for new drug development. An attractive therapeutic approach is to prevent the 'cap-independent' translation initiation of the viral proteins by interfering with both the structure and function of the hepatitis C viral internal ribosomal entry site (HCV IRES). Towards this goal, we report the design, synthesis and purification of novel bi-functional molecules containing DNA or RNA antisenses attached to functional groups performing RNA hydrolysis. These 5' or 3'-coupled conjugates bind the HCV IRES with affinity and specificity and elicit targeted hydrolysis of the viral genomic RNA after short (1 h) incubation at low (500 nM) concentration at 37 degrees C in vitro. Additional secondary cleavage sites are induced and their mapping within the RNA structure indicates that functional domains IIIb-e are excised from the IRES that, based on cryo-EM studies, becomes incapable of binding the small ribosomal subunit and initiation factor 3 (eIF3). All these molecules inhibit, in a dose-dependent manner, the 'IRES-dependent' translation in vitro. The 5'-coupled imidazole conjugate reduces viral protein synthesis by half at a 300 nM concentration (IC50), corresponding to a 4-fold increase of activity when compared to the naked oligonucleotide. These new conjugates are now being tested for activity on infected hepatic cell lines.

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Dose-dependent inhibition of viral protein synthesis by the DNA conjugates in vitro. The translation assays were performed at 30°C for 45 min. The Renilla luciferase protein levels measured in the presence of each conjugates were all divided by the protein level obtained in the absence of translation inhibitors. (a) Concentration-dependent viral translation inhibition of the HCV-IRES RNA by the non-specific oligonucleotide NS, the naked DNA antisense AS, the ‘naked-free imidazole’ in a one-to-one ratio or the 3′- or the 5′-imidazole-coupled oligonucleotides 4 and 1. (b) translation inhibition of the ECMV-IRES RNA by the naked (AS), the 3′- or the 5′-imidazole-coupled oligonucleotides 4 and 1. (c) Concentration-dependent translation inhibition of the HCV-IRES RNA by the naked 2′-O-Me RNA antisense RNA_AS or the 5′-imidazole-coupled 2′-O-Me-RNA oligonucleotides 5.
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Figure 5: Dose-dependent inhibition of viral protein synthesis by the DNA conjugates in vitro. The translation assays were performed at 30°C for 45 min. The Renilla luciferase protein levels measured in the presence of each conjugates were all divided by the protein level obtained in the absence of translation inhibitors. (a) Concentration-dependent viral translation inhibition of the HCV-IRES RNA by the non-specific oligonucleotide NS, the naked DNA antisense AS, the ‘naked-free imidazole’ in a one-to-one ratio or the 3′- or the 5′-imidazole-coupled oligonucleotides 4 and 1. (b) translation inhibition of the ECMV-IRES RNA by the naked (AS), the 3′- or the 5′-imidazole-coupled oligonucleotides 4 and 1. (c) Concentration-dependent translation inhibition of the HCV-IRES RNA by the naked 2′-O-Me RNA antisense RNA_AS or the 5′-imidazole-coupled 2′-O-Me-RNA oligonucleotides 5.

Mentions: Translation assays were performed using a Renilla luciferase reporter gene under the control of the HCV IRES (Figures 5a and c) or the ECMV IRES (Figure 5b). The IRES-Renilla luciferase RNA (IRES-RLuc) transcripts were first incubated with increasing concentrations of the conjugates and then with the reticulocyte lysate containing the ribosomes (see Material and Methods section). The translation reactions were performed for 45 min, a time at which no RNA degradation could be observed in the absence of the antisense oligonucleotides (data not shown). The Renilla luciferase protein levels were measured in the presence of the conjugates and compared to those obtained in the absence of the putative inhibitors. Direct comparisons between the conjugates were inferred from the determination of the concentration of the drug required to inhibit reporter gene translation by half (IC50). As a control, the effect of a mismatched oligonucleotide (NS) was tested and it doesn't inhibit the translation of the HCV IRES RNA (Figure 5a).Figure 5.


Targeted inhibition of the hepatitis C internal ribosomal entry site genomic RNA with oligonucleotide conjugates.

Guerniou V, Gillet R, Berrée F, Carboni B, Felden B - Nucleic Acids Res. (2007)

Dose-dependent inhibition of viral protein synthesis by the DNA conjugates in vitro. The translation assays were performed at 30°C for 45 min. The Renilla luciferase protein levels measured in the presence of each conjugates were all divided by the protein level obtained in the absence of translation inhibitors. (a) Concentration-dependent viral translation inhibition of the HCV-IRES RNA by the non-specific oligonucleotide NS, the naked DNA antisense AS, the ‘naked-free imidazole’ in a one-to-one ratio or the 3′- or the 5′-imidazole-coupled oligonucleotides 4 and 1. (b) translation inhibition of the ECMV-IRES RNA by the naked (AS), the 3′- or the 5′-imidazole-coupled oligonucleotides 4 and 1. (c) Concentration-dependent translation inhibition of the HCV-IRES RNA by the naked 2′-O-Me RNA antisense RNA_AS or the 5′-imidazole-coupled 2′-O-Me-RNA oligonucleotides 5.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC2175329&req=5

Figure 5: Dose-dependent inhibition of viral protein synthesis by the DNA conjugates in vitro. The translation assays were performed at 30°C for 45 min. The Renilla luciferase protein levels measured in the presence of each conjugates were all divided by the protein level obtained in the absence of translation inhibitors. (a) Concentration-dependent viral translation inhibition of the HCV-IRES RNA by the non-specific oligonucleotide NS, the naked DNA antisense AS, the ‘naked-free imidazole’ in a one-to-one ratio or the 3′- or the 5′-imidazole-coupled oligonucleotides 4 and 1. (b) translation inhibition of the ECMV-IRES RNA by the naked (AS), the 3′- or the 5′-imidazole-coupled oligonucleotides 4 and 1. (c) Concentration-dependent translation inhibition of the HCV-IRES RNA by the naked 2′-O-Me RNA antisense RNA_AS or the 5′-imidazole-coupled 2′-O-Me-RNA oligonucleotides 5.
Mentions: Translation assays were performed using a Renilla luciferase reporter gene under the control of the HCV IRES (Figures 5a and c) or the ECMV IRES (Figure 5b). The IRES-Renilla luciferase RNA (IRES-RLuc) transcripts were first incubated with increasing concentrations of the conjugates and then with the reticulocyte lysate containing the ribosomes (see Material and Methods section). The translation reactions were performed for 45 min, a time at which no RNA degradation could be observed in the absence of the antisense oligonucleotides (data not shown). The Renilla luciferase protein levels were measured in the presence of the conjugates and compared to those obtained in the absence of the putative inhibitors. Direct comparisons between the conjugates were inferred from the determination of the concentration of the drug required to inhibit reporter gene translation by half (IC50). As a control, the effect of a mismatched oligonucleotide (NS) was tested and it doesn't inhibit the translation of the HCV IRES RNA (Figure 5a).Figure 5.

Bottom Line: All these molecules inhibit, in a dose-dependent manner, the 'IRES-dependent' translation in vitro.The 5'-coupled imidazole conjugate reduces viral protein synthesis by half at a 300 nM concentration (IC50), corresponding to a 4-fold increase of activity when compared to the naked oligonucleotide.These new conjugates are now being tested for activity on infected hepatic cell lines.

View Article: PubMed Central - PubMed

Affiliation: Biochimie Pharmaceutique, Inserm U835, Upres JE 2311, Université de Rennes 1, France.

ABSTRACT
Hepatitis C is a major public health concern, with an estimated 170 million people infected worldwide and an urgent need for new drug development. An attractive therapeutic approach is to prevent the 'cap-independent' translation initiation of the viral proteins by interfering with both the structure and function of the hepatitis C viral internal ribosomal entry site (HCV IRES). Towards this goal, we report the design, synthesis and purification of novel bi-functional molecules containing DNA or RNA antisenses attached to functional groups performing RNA hydrolysis. These 5' or 3'-coupled conjugates bind the HCV IRES with affinity and specificity and elicit targeted hydrolysis of the viral genomic RNA after short (1 h) incubation at low (500 nM) concentration at 37 degrees C in vitro. Additional secondary cleavage sites are induced and their mapping within the RNA structure indicates that functional domains IIIb-e are excised from the IRES that, based on cryo-EM studies, becomes incapable of binding the small ribosomal subunit and initiation factor 3 (eIF3). All these molecules inhibit, in a dose-dependent manner, the 'IRES-dependent' translation in vitro. The 5'-coupled imidazole conjugate reduces viral protein synthesis by half at a 300 nM concentration (IC50), corresponding to a 4-fold increase of activity when compared to the naked oligonucleotide. These new conjugates are now being tested for activity on infected hepatic cell lines.

Show MeSH
Related in: MedlinePlus