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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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281816 inhibition of HCV E2 protein binding to native CD81 on Raji cells.Flow cytometry was used to quantify recombinant HCV E2 protein binding to native CD81 over-expressed on Raji cells. Binding of the recombinant E2 protein to native CD81 on the surface of Raji cells was detected using the mouse monoclonal E2 antibody clone H53 followed by staining with a secondary FITC anti-mouse antibody. E2 binding is inhibited by 281816 in a dose-dependent manner up to 100 µM.
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pone-0111333-g008: 281816 inhibition of HCV E2 protein binding to native CD81 on Raji cells.Flow cytometry was used to quantify recombinant HCV E2 protein binding to native CD81 over-expressed on Raji cells. Binding of the recombinant E2 protein to native CD81 on the surface of Raji cells was detected using the mouse monoclonal E2 antibody clone H53 followed by staining with a secondary FITC anti-mouse antibody. E2 binding is inhibited by 281816 in a dose-dependent manner up to 100 µM.

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)

281816 inhibition of HCV E2 protein binding to native CD81 on Raji cells.Flow cytometry was used to quantify recombinant HCV E2 protein binding to native CD81 over-expressed on Raji cells. Binding of the recombinant E2 protein to native CD81 on the surface of Raji cells was detected using the mouse monoclonal E2 antibody clone H53 followed by staining with a secondary FITC anti-mouse antibody. E2 binding is inhibited by 281816 in a dose-dependent manner up to 100 µM.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC4214736&req=5

pone-0111333-g008: 281816 inhibition of HCV E2 protein binding to native CD81 on Raji cells.Flow cytometry was used to quantify recombinant HCV E2 protein binding to native CD81 over-expressed on Raji cells. Binding of the recombinant E2 protein to native CD81 on the surface of Raji cells was detected using the mouse monoclonal E2 antibody clone H53 followed by staining with a secondary FITC anti-mouse antibody. E2 binding is inhibited by 281816 in a dose-dependent manner up to 100 µM.
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