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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 blocks HCV cell-to-cell transmission.Huh-7 cells were seeded on coverslips and infected with HCVcc for 2 hr at 37°C. Cells were then washed and cultured for 72 hr at 37°C in culture medium containing the 3/11 neutralizing antibody (50 µg/ml) in presence or in absence of 281816 at indicated concentrations. Cells cultured in presence of DMSO or EGCG at 50 µM were used as controls. The number of infected cells per focus was determined by A4 indirect immunofluorescence. The results show treatment with 281816 significantly reduces the number of infected cells per focus in a dose-dependent manner. Mean p values were below 0.001 for 1 µM 281816 and below 0.0001 for the 10 µM 281816 and EGCG treatment groups.
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pone-0111333-g013: 281816 blocks HCV cell-to-cell transmission.Huh-7 cells were seeded on coverslips and infected with HCVcc for 2 hr at 37°C. Cells were then washed and cultured for 72 hr at 37°C in culture medium containing the 3/11 neutralizing antibody (50 µg/ml) in presence or in absence of 281816 at indicated concentrations. Cells cultured in presence of DMSO or EGCG at 50 µM were used as controls. The number of infected cells per focus was determined by A4 indirect immunofluorescence. The results show treatment with 281816 significantly reduces the number of infected cells per focus in a dose-dependent manner. Mean p values were below 0.001 for 1 µM 281816 and below 0.0001 for the 10 µM 281816 and EGCG treatment groups.

Mentions: In addition to cell-free infection, HCV can also be transmitted to neighboring cells via cell-to-cell contact by a mechanism that is not completely understood [41], [42], [62]. Indeed, HCV is transmitted in the presence of monoclonal antibodies, such as the anti-E2 antibody 3/11, or patient-derived antibodies that are able to neutralize virus-free infectivity [42], [62]. Since cell-to-cell transmission has been suggested to be a major route of transmission for HCV [41], we next analyzed the effect of 281816 on this process. For this purpose, Huh-7 cells were infected at low multiplicity of infection with HCVcc for 2 hr and then cultured with neutralizing anti-E2 antibody (3/11), which blocks infection by free particles as shown in Figure 12[41], and in the presence of 281816 (1 µM and 10 µM). Cells cultured in the presence of 3/11 and solvent (DMSO) or Epigallocatechin-3-gallate (EGCG, 50 µM) [42] were used as negative and positive controls of inhibition, respectively. Three days post-infection, cells were fixed and foci of infected cells were visualized by immunofluorescence. Cell-to-cell transmission was measured by counting the number of infected cells per focus. The results showed that 281816 led to a significant reduction of the number of infected cells per focus in a dose-dependent manner (Figure 13). Together, these results indicate that 281816 also inhibits cell-to-cell transmission of HCV.


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 blocks HCV cell-to-cell transmission.Huh-7 cells were seeded on coverslips and infected with HCVcc for 2 hr at 37°C. Cells were then washed and cultured for 72 hr at 37°C in culture medium containing the 3/11 neutralizing antibody (50 µg/ml) in presence or in absence of 281816 at indicated concentrations. Cells cultured in presence of DMSO or EGCG at 50 µM were used as controls. The number of infected cells per focus was determined by A4 indirect immunofluorescence. The results show treatment with 281816 significantly reduces the number of infected cells per focus in a dose-dependent manner. Mean p values were below 0.001 for 1 µM 281816 and below 0.0001 for the 10 µM 281816 and EGCG treatment groups.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4214736&req=5

pone-0111333-g013: 281816 blocks HCV cell-to-cell transmission.Huh-7 cells were seeded on coverslips and infected with HCVcc for 2 hr at 37°C. Cells were then washed and cultured for 72 hr at 37°C in culture medium containing the 3/11 neutralizing antibody (50 µg/ml) in presence or in absence of 281816 at indicated concentrations. Cells cultured in presence of DMSO or EGCG at 50 µM were used as controls. The number of infected cells per focus was determined by A4 indirect immunofluorescence. The results show treatment with 281816 significantly reduces the number of infected cells per focus in a dose-dependent manner. Mean p values were below 0.001 for 1 µM 281816 and below 0.0001 for the 10 µM 281816 and EGCG treatment groups.
Mentions: In addition to cell-free infection, HCV can also be transmitted to neighboring cells via cell-to-cell contact by a mechanism that is not completely understood [41], [42], [62]. Indeed, HCV is transmitted in the presence of monoclonal antibodies, such as the anti-E2 antibody 3/11, or patient-derived antibodies that are able to neutralize virus-free infectivity [42], [62]. Since cell-to-cell transmission has been suggested to be a major route of transmission for HCV [41], we next analyzed the effect of 281816 on this process. For this purpose, Huh-7 cells were infected at low multiplicity of infection with HCVcc for 2 hr and then cultured with neutralizing anti-E2 antibody (3/11), which blocks infection by free particles as shown in Figure 12[41], and in the presence of 281816 (1 µM and 10 µM). Cells cultured in the presence of 3/11 and solvent (DMSO) or Epigallocatechin-3-gallate (EGCG, 50 µM) [42] were used as negative and positive controls of inhibition, respectively. Three days post-infection, cells were fixed and foci of infected cells were visualized by immunofluorescence. Cell-to-cell transmission was measured by counting the number of infected cells per focus. The results showed that 281816 led to a significant reduction of the number of infected cells per focus in a dose-dependent manner (Figure 13). Together, these results indicate that 281816 also inhibits cell-to-cell transmission of HCV.

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