Limits...
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.

Show MeSH

Related in: MedlinePlus

Viability of Huh-7 cells treated with 281816 in the HCV entry experiments.An MTS assay [38] was used to determine the viability of cells treated with 10 µM 281816 in DMSO (and DMSO alone, as a control) for 1 hr, 2 hr, or 3 hr under the same conditions used in the HCV entry experiments. 281816 is not toxic under any of the conditions used in this assay. There were no significant differences between the 281816 treated and control samples (p values <0.05).
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC4214736&req=5

pone-0111333-g007: Viability of Huh-7 cells treated with 281816 in the HCV entry experiments.An MTS assay [38] was used to determine the viability of cells treated with 10 µM 281816 in DMSO (and DMSO alone, as a control) for 1 hr, 2 hr, or 3 hr under the same conditions used in the HCV entry experiments. 281816 is not toxic under any of the conditions used in this assay. There were no significant differences between the 281816 treated and control samples (p values <0.05).

Mentions: After attachment to the cell surface and binding to entry factors, HCV virions are internalized by clathrin-mediated endocytosis [54], [55]. Following internalization, HCV is transported to early endosomes along actin stress fibers, where fusion seems to take place [55], [56]. To determine which step in HCV entry is impaired by 281816, we administered the ligand at different intervals during the early phase of infection. Virus attachment and binding were performed at 4°C (Figure 6B, Steps 1 and 2), Then, cells were shifted to 37°C to allow endocytosis and fusion (Figure 6B, Step 3). Cells treated with JS-81 were used as controls. The addition of 281816 during step 2 and step 3 led to the strongest inhibition of HCV infection, as strong as the one observed when 281816 was present during all three steps. We also observed a significant inhibition of HCV infection when 281816 was added during the early attachment/binding steps (Figure 6B, Step 1). An MTS assay performed with 10 µM 281816 for each length of time the cells were treated with 281816 (1 hr, 2 hr, and 3 hr) showed the compound was not cytotoxic to the cells under the conditions used in the assay (Figure 7). Together, these results indicate that 281816 inhibits HCV infection by acting on more than the first (attachment/binding) step of viral entry. These data suggest the ligand also affects interactions during HCV internalization and fusion.


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)

Viability of Huh-7 cells treated with 281816 in the HCV entry experiments.An MTS assay [38] was used to determine the viability of cells treated with 10 µM 281816 in DMSO (and DMSO alone, as a control) for 1 hr, 2 hr, or 3 hr under the same conditions used in the HCV entry experiments. 281816 is not toxic under any of the conditions used in this assay. There were no significant differences between the 281816 treated and control samples (p values <0.05).
© Copyright Policy
Related In: Results  -  Collection

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

pone-0111333-g007: Viability of Huh-7 cells treated with 281816 in the HCV entry experiments.An MTS assay [38] was used to determine the viability of cells treated with 10 µM 281816 in DMSO (and DMSO alone, as a control) for 1 hr, 2 hr, or 3 hr under the same conditions used in the HCV entry experiments. 281816 is not toxic under any of the conditions used in this assay. There were no significant differences between the 281816 treated and control samples (p values <0.05).
Mentions: After attachment to the cell surface and binding to entry factors, HCV virions are internalized by clathrin-mediated endocytosis [54], [55]. Following internalization, HCV is transported to early endosomes along actin stress fibers, where fusion seems to take place [55], [56]. To determine which step in HCV entry is impaired by 281816, we administered the ligand at different intervals during the early phase of infection. Virus attachment and binding were performed at 4°C (Figure 6B, Steps 1 and 2), Then, cells were shifted to 37°C to allow endocytosis and fusion (Figure 6B, Step 3). Cells treated with JS-81 were used as controls. The addition of 281816 during step 2 and step 3 led to the strongest inhibition of HCV infection, as strong as the one observed when 281816 was present during all three steps. We also observed a significant inhibition of HCV infection when 281816 was added during the early attachment/binding steps (Figure 6B, Step 1). An MTS assay performed with 10 µM 281816 for each length of time the cells were treated with 281816 (1 hr, 2 hr, and 3 hr) showed the compound was not cytotoxic to the cells under the conditions used in the assay (Figure 7). Together, these results indicate that 281816 inhibits HCV infection by acting on more than the first (attachment/binding) step of viral entry. These data suggest the ligand also affects interactions during HCV internalization and fusion.

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