Limits...
Identification and characterization of coumestans as novel HCV NS5B polymerase inhibitors.

Kaushik-Basu N, Bopda-Waffo A, Talele TT, Basu A, Costa PR, da Silva AJ, Sarafianos SG, Noël F - Nucleic Acids Res. (2008)

Bottom Line: Coumestan interference at the step of NS5B-RNA binary complex formation was confirmed by cross-linking experiments.Molecular docking of coumestans within the allosteric site of NS5B yielded significant correlation between their calculated binding energies and IC(50) values.Coumestans thus add to the diversifying pool of anti-NS5B agents and provide a novel scaffold for structural refinement and development of potent NS5B inhibitors.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Molecular Biology, UMDNJ-New Jersey Medical School, 185 South Orange Avenue, Newark, NJ 07103, USA. kaushik@umdnj.edu

ABSTRACT
The hepatitis C virus (HCV) NS5B is essential for viral RNA replication and is therefore a prime target for development of HCV replication inhibitors. Here, we report the identification of a new class of HCV NS5B inhibitors belonging to the coumestan family of phytoestrogens. Based on the in vitro NS5B RNA-dependent RNA polymerase (RdRp) inhibition in the low micromolar range by wedelolactone, a naturally occurring coumestan, we evaluated the anti-NS5B activity of four synthetic coumestan analogues bearing different patterns of substitutions in their A and D rings, and observed a good structure-activity correlation. Kinetic characterization of coumestans revealed a noncompetitive mode of inhibition with respect to nucleoside triphosphate (rNTP) substrate and a mixed mode of inhibition towards the nucleic acid template, with a major competitive component. The modified order of addition experiments with coumestans and nucleic acid substrates affected the potencies of the coumestan inhibitors. Coumestan interference at the step of NS5B-RNA binary complex formation was confirmed by cross-linking experiments. Molecular docking of coumestans within the allosteric site of NS5B yielded significant correlation between their calculated binding energies and IC(50) values. Coumestans thus add to the diversifying pool of anti-NS5B agents and provide a novel scaffold for structural refinement and development of potent NS5B inhibitors.

Show MeSH

Related in: MedlinePlus

Coumestans inhibit NS5B-RNA binary complex formation. Binding of NS5B to RNA as a function of individual coumestan derivative was evaluated by UV-mediated cross-linking. NS5BCΔ21 (1.5 μg) was incubated with 30 nM [5′-32P] labeled rA20/U12 template/primer (200K Cerenkov c.p.m.) in a standard irradiation mixture in the absence or presence of increasing concentrations of the indicated coumestan compound and exposed to UV radiation. The cross-linked species were resolved by SDS-PAGE and visualized on a phosphorimager. The extent of NS5B-RNA cross-linked species formed was quantified using ImageQuant software (Molecular Dynamics). Lane 1 in each set represents the control reaction carried out in the absence of the inhibitor. Lanes 2–9 represent indicated compounds at concentrations of 1, 5, 10, 15, 30, 50, 100 and 200 μM, respectively.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC2275130&req=5

Figure 6: Coumestans inhibit NS5B-RNA binary complex formation. Binding of NS5B to RNA as a function of individual coumestan derivative was evaluated by UV-mediated cross-linking. NS5BCΔ21 (1.5 μg) was incubated with 30 nM [5′-32P] labeled rA20/U12 template/primer (200K Cerenkov c.p.m.) in a standard irradiation mixture in the absence or presence of increasing concentrations of the indicated coumestan compound and exposed to UV radiation. The cross-linked species were resolved by SDS-PAGE and visualized on a phosphorimager. The extent of NS5B-RNA cross-linked species formed was quantified using ImageQuant software (Molecular Dynamics). Lane 1 in each set represents the control reaction carried out in the absence of the inhibitor. Lanes 2–9 represent indicated compounds at concentrations of 1, 5, 10, 15, 30, 50, 100 and 200 μM, respectively.

Mentions: Similar to other polymerases, NS5B requires multiple substrates, including a TP or a template-initiation nucleotide complex in a sequential fashion, in which the first step is formation of the binary complex involving binding of NS5B to the TP (62). We have earlier demonstrated the formation of this binary complex by polymerase-TP cross-linking experiments for the Klenow fragment of E. coli DNA polymerase I (47), MuLV-RT (63) and HIV-1 RT (48). The observation that pre-incubation of NS5B-RNA protected the enzyme from being inhibited by the coumestans, prompted us to ascertain whether the coumestans adversely affect the binding of the TP to NS5B. A direct photochemical cross-linking of 5′-32P-labeled U12/rA20 to NS5B was performed by UV irradiation of the NS5B-TP complexes in the presence of increasing amounts of coumestans under standard RdRp assay conditions. The extent of NS5B-TP covalent complex formed as a function of inhibitor concentration was analysed by SDS-PAGE. Results shown in Figure 6 indicate a reduction in the E-TP binary complex formation in the presence of the inhibitor. This reduction directly correlated with the inhibitor concentration (Figure 6, lanes 2–9) and is in agreement with the competitive mode of TP binding as well as the protective effect of RNA TP against coumestan-mediated inhibition of NS5B. Further, the reduction of NS5B-RNA binary complex species was higher in case of LQB34 compared to wedelolactone and correlated well with their respective IC50 values. The binary complex formation data substantiates our hypothesis that inhibition may be mediated at the RNA binding step among others.Figure 6.


Identification and characterization of coumestans as novel HCV NS5B polymerase inhibitors.

Kaushik-Basu N, Bopda-Waffo A, Talele TT, Basu A, Costa PR, da Silva AJ, Sarafianos SG, Noël F - Nucleic Acids Res. (2008)

Coumestans inhibit NS5B-RNA binary complex formation. Binding of NS5B to RNA as a function of individual coumestan derivative was evaluated by UV-mediated cross-linking. NS5BCΔ21 (1.5 μg) was incubated with 30 nM [5′-32P] labeled rA20/U12 template/primer (200K Cerenkov c.p.m.) in a standard irradiation mixture in the absence or presence of increasing concentrations of the indicated coumestan compound and exposed to UV radiation. The cross-linked species were resolved by SDS-PAGE and visualized on a phosphorimager. The extent of NS5B-RNA cross-linked species formed was quantified using ImageQuant software (Molecular Dynamics). Lane 1 in each set represents the control reaction carried out in the absence of the inhibitor. Lanes 2–9 represent indicated compounds at concentrations of 1, 5, 10, 15, 30, 50, 100 and 200 μM, respectively.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2275130&req=5

Figure 6: Coumestans inhibit NS5B-RNA binary complex formation. Binding of NS5B to RNA as a function of individual coumestan derivative was evaluated by UV-mediated cross-linking. NS5BCΔ21 (1.5 μg) was incubated with 30 nM [5′-32P] labeled rA20/U12 template/primer (200K Cerenkov c.p.m.) in a standard irradiation mixture in the absence or presence of increasing concentrations of the indicated coumestan compound and exposed to UV radiation. The cross-linked species were resolved by SDS-PAGE and visualized on a phosphorimager. The extent of NS5B-RNA cross-linked species formed was quantified using ImageQuant software (Molecular Dynamics). Lane 1 in each set represents the control reaction carried out in the absence of the inhibitor. Lanes 2–9 represent indicated compounds at concentrations of 1, 5, 10, 15, 30, 50, 100 and 200 μM, respectively.
Mentions: Similar to other polymerases, NS5B requires multiple substrates, including a TP or a template-initiation nucleotide complex in a sequential fashion, in which the first step is formation of the binary complex involving binding of NS5B to the TP (62). We have earlier demonstrated the formation of this binary complex by polymerase-TP cross-linking experiments for the Klenow fragment of E. coli DNA polymerase I (47), MuLV-RT (63) and HIV-1 RT (48). The observation that pre-incubation of NS5B-RNA protected the enzyme from being inhibited by the coumestans, prompted us to ascertain whether the coumestans adversely affect the binding of the TP to NS5B. A direct photochemical cross-linking of 5′-32P-labeled U12/rA20 to NS5B was performed by UV irradiation of the NS5B-TP complexes in the presence of increasing amounts of coumestans under standard RdRp assay conditions. The extent of NS5B-TP covalent complex formed as a function of inhibitor concentration was analysed by SDS-PAGE. Results shown in Figure 6 indicate a reduction in the E-TP binary complex formation in the presence of the inhibitor. This reduction directly correlated with the inhibitor concentration (Figure 6, lanes 2–9) and is in agreement with the competitive mode of TP binding as well as the protective effect of RNA TP against coumestan-mediated inhibition of NS5B. Further, the reduction of NS5B-RNA binary complex species was higher in case of LQB34 compared to wedelolactone and correlated well with their respective IC50 values. The binary complex formation data substantiates our hypothesis that inhibition may be mediated at the RNA binding step among others.Figure 6.

Bottom Line: Coumestan interference at the step of NS5B-RNA binary complex formation was confirmed by cross-linking experiments.Molecular docking of coumestans within the allosteric site of NS5B yielded significant correlation between their calculated binding energies and IC(50) values.Coumestans thus add to the diversifying pool of anti-NS5B agents and provide a novel scaffold for structural refinement and development of potent NS5B inhibitors.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Molecular Biology, UMDNJ-New Jersey Medical School, 185 South Orange Avenue, Newark, NJ 07103, USA. kaushik@umdnj.edu

ABSTRACT
The hepatitis C virus (HCV) NS5B is essential for viral RNA replication and is therefore a prime target for development of HCV replication inhibitors. Here, we report the identification of a new class of HCV NS5B inhibitors belonging to the coumestan family of phytoestrogens. Based on the in vitro NS5B RNA-dependent RNA polymerase (RdRp) inhibition in the low micromolar range by wedelolactone, a naturally occurring coumestan, we evaluated the anti-NS5B activity of four synthetic coumestan analogues bearing different patterns of substitutions in their A and D rings, and observed a good structure-activity correlation. Kinetic characterization of coumestans revealed a noncompetitive mode of inhibition with respect to nucleoside triphosphate (rNTP) substrate and a mixed mode of inhibition towards the nucleic acid template, with a major competitive component. The modified order of addition experiments with coumestans and nucleic acid substrates affected the potencies of the coumestan inhibitors. Coumestan interference at the step of NS5B-RNA binary complex formation was confirmed by cross-linking experiments. Molecular docking of coumestans within the allosteric site of NS5B yielded significant correlation between their calculated binding energies and IC(50) values. Coumestans thus add to the diversifying pool of anti-NS5B agents and provide a novel scaffold for structural refinement and development of potent NS5B inhibitors.

Show MeSH
Related in: MedlinePlus