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Identification of a novel drug lead that inhibits HCV infection and cell-to-cell transmission by targeting the HCV E2 glycoprotein.

Al Olaby RR, Cocquerel L, Zemla A, Saas L, Dubuisson J, Vielmetter J, Marcotrigiano J, Khan AG, Vences Catalan F, Perryman AL, Freundlich JS, Forli S, Levy S, Balhorn R, Azzazy HM - PLoS ONE (2014)

Bottom Line: Although several FDA approved drugs targeting the HCV serine protease and polymerase have shown promising results, there is a need for better drugs that are effective in treating a broader range of HCV genotypes and subtypes without being used in combination with interferon and/or ribavirin.Computational docking of a diverse library of 1,715 small molecules to this model led to the identification of a set of 34 ligands predicted to bind near conserved amino acid residues involved in the HCV E2: CD81 interaction.Collectively the results obtained with this new structural model of E2c suggest the development of small molecule inhibitors such as 281816 that target E2 and disrupt its interaction with CD81 may provide a new paradigm for HCV treatment.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry, The American University in Cairo, New Cairo, Egypt.

ABSTRACT
Hepatitis C Virus (HCV) infects 200 million individuals worldwide. Although several FDA approved drugs targeting the HCV serine protease and polymerase have shown promising results, there is a need for better drugs that are effective in treating a broader range of HCV genotypes and subtypes without being used in combination with interferon and/or ribavirin. Recently, two crystal structures of the core of the HCV E2 protein (E2c) have been determined, providing structural information that can now be used to target the E2 protein and develop drugs that disrupt the early stages of HCV infection by blocking E2's interaction with different host factors. Using the E2c structure as a template, we have created a structural model of the E2 protein core (residues 421-645) that contains the three amino acid segments that are not present in either structure. Computational docking of a diverse library of 1,715 small molecules to this model led to the identification of a set of 34 ligands predicted to bind near conserved amino acid residues involved in the HCV E2: CD81 interaction. Surface plasmon resonance detection was used to screen the ligand set for binding to recombinant E2 protein, and the best binders were subsequently tested to identify compounds that inhibit the infection of Huh-7 cells by HCV. One compound, 281816, blocked E2 binding to CD81 and inhibited HCV infection in a genotype-independent manner with IC50's ranging from 2.2 µM to 4.6 µM. 281816 blocked the early and late steps of cell-free HCV entry and also abrogated the cell-to-cell transmission of HCV. Collectively the results obtained with this new structural model of E2c suggest the development of small molecule inhibitors such as 281816 that target E2 and disrupt its interaction with CD81 may provide a new paradigm for HCV treatment.

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Ligand 281816 inhibits HCV-E2 binding to recombinant CD81-LEL.Binding of recombinant E2 protein to GST-tagged human CD81-LEL immobilized on a 96 well plate was determined using an ELISA assay. The plate was coated with GST-tagged human CD81-LEL (5 µg/ml) overnight as previously described [10], HCV E2 protein (5 µg/ml) was pre-incubated with different concentrations of 281816 for 30 min before adding to the plate, and the HCV-E2 protein (with or without the ligand) was then added to the GST-tagged human CD81-LEL coated plate and incubated for 1 hr. HCV-E2 binding was detected using a primary mouse anti-E2 antibody (H53 clone) and a secondary goat anti-mouse-HRP antibody by measuring the absorbance at 405 nm. The results, which are plotted as percent of E2 protein bound to CD81-LEL relative to E2 binding observed in the absence of the ligand (buffer control), show a dose-dependent effect of 281816 on the inhibition of the E2 protein binding to immobilized recombinant CD81-LEL. P values for the 0.05, 0.2, 0.5 and 1.0 µM 281816 samples are 0.0069, 0.0195, 0.0006 and 0.0009 respectively.
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pone-0111333-g009: Ligand 281816 inhibits HCV-E2 binding to recombinant CD81-LEL.Binding of recombinant E2 protein to GST-tagged human CD81-LEL immobilized on a 96 well plate was determined using an ELISA assay. The plate was coated with GST-tagged human CD81-LEL (5 µg/ml) overnight as previously described [10], HCV E2 protein (5 µg/ml) was pre-incubated with different concentrations of 281816 for 30 min before adding to the plate, and the HCV-E2 protein (with or without the ligand) was then added to the GST-tagged human CD81-LEL coated plate and incubated for 1 hr. HCV-E2 binding was detected using a primary mouse anti-E2 antibody (H53 clone) and a secondary goat anti-mouse-HRP antibody by measuring the absorbance at 405 nm. The results, which are plotted as percent of E2 protein bound to CD81-LEL relative to E2 binding observed in the absence of the ligand (buffer control), show a dose-dependent effect of 281816 on the inhibition of the E2 protein binding to immobilized recombinant CD81-LEL. P values for the 0.05, 0.2, 0.5 and 1.0 µM 281816 samples are 0.0069, 0.0195, 0.0006 and 0.0009 respectively.

Mentions: Ligand 281816 was originally selected for testing based on the prediction by docking that it would bind to a site on the HCV E2 protein where CD81 binds. The infection assay conducted with Huh-7 cells demonstrated 281816 is effective in inhibiting the entry step in the HCV life cycle. To confirm that the binding of 281816 to E2 inhibits the HCV E2-CD81 interaction, flow cytometry was used to monitor the binding of a recombinant form of the E2 protein to native CD81 overexpressed on Raji cells as a function of 281816 concentration. The results in Figure 8 show binding of the E2 protein to Raji cells is inhibited by 281816 in a dose dependent manner. Using a second technique (an ELISA-based assay), we observed a similar dose-dependent effect of 281816 on the inhibition of the E2 protein binding to recombinant CD81-LEL immobilized on micro titer plates (Figure 9). While an IC50 for 281816 blocking the binding of E2 to CD81 could not be determined from the flow cytometry data, the ELISA results indicate the IC50 is in the range of 0.2–0.5 µM.


Identification of a novel drug lead that inhibits HCV infection and cell-to-cell transmission by targeting the HCV E2 glycoprotein.

Al Olaby RR, Cocquerel L, Zemla A, Saas L, Dubuisson J, Vielmetter J, Marcotrigiano J, Khan AG, Vences Catalan F, Perryman AL, Freundlich JS, Forli S, Levy S, Balhorn R, Azzazy HM - PLoS ONE (2014)

Ligand 281816 inhibits HCV-E2 binding to recombinant CD81-LEL.Binding of recombinant E2 protein to GST-tagged human CD81-LEL immobilized on a 96 well plate was determined using an ELISA assay. The plate was coated with GST-tagged human CD81-LEL (5 µg/ml) overnight as previously described [10], HCV E2 protein (5 µg/ml) was pre-incubated with different concentrations of 281816 for 30 min before adding to the plate, and the HCV-E2 protein (with or without the ligand) was then added to the GST-tagged human CD81-LEL coated plate and incubated for 1 hr. HCV-E2 binding was detected using a primary mouse anti-E2 antibody (H53 clone) and a secondary goat anti-mouse-HRP antibody by measuring the absorbance at 405 nm. The results, which are plotted as percent of E2 protein bound to CD81-LEL relative to E2 binding observed in the absence of the ligand (buffer control), show a dose-dependent effect of 281816 on the inhibition of the E2 protein binding to immobilized recombinant CD81-LEL. P values for the 0.05, 0.2, 0.5 and 1.0 µM 281816 samples are 0.0069, 0.0195, 0.0006 and 0.0009 respectively.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4214736&req=5

pone-0111333-g009: Ligand 281816 inhibits HCV-E2 binding to recombinant CD81-LEL.Binding of recombinant E2 protein to GST-tagged human CD81-LEL immobilized on a 96 well plate was determined using an ELISA assay. The plate was coated with GST-tagged human CD81-LEL (5 µg/ml) overnight as previously described [10], HCV E2 protein (5 µg/ml) was pre-incubated with different concentrations of 281816 for 30 min before adding to the plate, and the HCV-E2 protein (with or without the ligand) was then added to the GST-tagged human CD81-LEL coated plate and incubated for 1 hr. HCV-E2 binding was detected using a primary mouse anti-E2 antibody (H53 clone) and a secondary goat anti-mouse-HRP antibody by measuring the absorbance at 405 nm. The results, which are plotted as percent of E2 protein bound to CD81-LEL relative to E2 binding observed in the absence of the ligand (buffer control), show a dose-dependent effect of 281816 on the inhibition of the E2 protein binding to immobilized recombinant CD81-LEL. P values for the 0.05, 0.2, 0.5 and 1.0 µM 281816 samples are 0.0069, 0.0195, 0.0006 and 0.0009 respectively.
Mentions: Ligand 281816 was originally selected for testing based on the prediction by docking that it would bind to a site on the HCV E2 protein where CD81 binds. The infection assay conducted with Huh-7 cells demonstrated 281816 is effective in inhibiting the entry step in the HCV life cycle. To confirm that the binding of 281816 to E2 inhibits the HCV E2-CD81 interaction, flow cytometry was used to monitor the binding of a recombinant form of the E2 protein to native CD81 overexpressed on Raji cells as a function of 281816 concentration. The results in Figure 8 show binding of the E2 protein to Raji cells is inhibited by 281816 in a dose dependent manner. Using a second technique (an ELISA-based assay), we observed a similar dose-dependent effect of 281816 on the inhibition of the E2 protein binding to recombinant CD81-LEL immobilized on micro titer plates (Figure 9). While an IC50 for 281816 blocking the binding of E2 to CD81 could not be determined from the flow cytometry data, the ELISA results indicate the IC50 is in the range of 0.2–0.5 µM.

Bottom Line: Although several FDA approved drugs targeting the HCV serine protease and polymerase have shown promising results, there is a need for better drugs that are effective in treating a broader range of HCV genotypes and subtypes without being used in combination with interferon and/or ribavirin.Computational docking of a diverse library of 1,715 small molecules to this model led to the identification of a set of 34 ligands predicted to bind near conserved amino acid residues involved in the HCV E2: CD81 interaction.Collectively the results obtained with this new structural model of E2c suggest the development of small molecule inhibitors such as 281816 that target E2 and disrupt its interaction with CD81 may provide a new paradigm for HCV treatment.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry, The American University in Cairo, New Cairo, Egypt.

ABSTRACT
Hepatitis C Virus (HCV) infects 200 million individuals worldwide. Although several FDA approved drugs targeting the HCV serine protease and polymerase have shown promising results, there is a need for better drugs that are effective in treating a broader range of HCV genotypes and subtypes without being used in combination with interferon and/or ribavirin. Recently, two crystal structures of the core of the HCV E2 protein (E2c) have been determined, providing structural information that can now be used to target the E2 protein and develop drugs that disrupt the early stages of HCV infection by blocking E2's interaction with different host factors. Using the E2c structure as a template, we have created a structural model of the E2 protein core (residues 421-645) that contains the three amino acid segments that are not present in either structure. Computational docking of a diverse library of 1,715 small molecules to this model led to the identification of a set of 34 ligands predicted to bind near conserved amino acid residues involved in the HCV E2: CD81 interaction. Surface plasmon resonance detection was used to screen the ligand set for binding to recombinant E2 protein, and the best binders were subsequently tested to identify compounds that inhibit the infection of Huh-7 cells by HCV. One compound, 281816, blocked E2 binding to CD81 and inhibited HCV infection in a genotype-independent manner with IC50's ranging from 2.2 µM to 4.6 µM. 281816 blocked the early and late steps of cell-free HCV entry and also abrogated the cell-to-cell transmission of HCV. Collectively the results obtained with this new structural model of E2c suggest the development of small molecule inhibitors such as 281816 that target E2 and disrupt its interaction with CD81 may provide a new paradigm for HCV treatment.

Show MeSH
Related in: MedlinePlus