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

Ligand 281816 does not compete with binding of an anti-CD81 antibody to human CD81-LEL.Binding of anti-CD81 monoclonal antibody 5A6 to recombinant GST-tagged human CD81-LEL protein was determined by ELISA. Serial dilutions of 5A6 antibody were incubated with the CD81-LEL protein in the presence of 1 µM ligand 281816 (black squares) or PBS as a control (open circles). The amount of 5A6 antibody bound to CD81 remained the same in the presence and absence of the ligand, demonstrating that 281816 does not bind sufficiently well to the E2 binding site on CD81 to block 5A6 binding, even when the antibody concentration was reduced to 1/2000th the concentration of the ligand.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC4214736&req=5

pone-0111333-g011: Ligand 281816 does not compete with binding of an anti-CD81 antibody to human CD81-LEL.Binding of anti-CD81 monoclonal antibody 5A6 to recombinant GST-tagged human CD81-LEL protein was determined by ELISA. Serial dilutions of 5A6 antibody were incubated with the CD81-LEL protein in the presence of 1 µM ligand 281816 (black squares) or PBS as a control (open circles). The amount of 5A6 antibody bound to CD81 remained the same in the presence and absence of the ligand, demonstrating that 281816 does not bind sufficiently well to the E2 binding site on CD81 to block 5A6 binding, even when the antibody concentration was reduced to 1/2000th the concentration of the ligand.

Mentions: To determine if ligand 281816 also binds to CD81, surface plasmon resonance was used to test for 281816 binding to recombinant CD81-LEL protein immobilized on a chip. As shown in Figure 10, 281816 does bind to CD81-LEL. A kinetic analysis of this binding has shown that the ligand binds to CD81-LEL (Kd = 57 µM) almost as well as it binds to E2 (Kd = 41 µM). However, a competition ELISA experiment that used the anti-CD81 antibody, 5A6, that blocks E2 binding to CD81 (Figure 11) revealed that 281816 does not bind to the E2 binding site on CD81. In this experiment, CD81-LEL was immobilized on a micro titer plate and the binding of the 5A6 antibody was monitored in the presence of 1 µM 281816 as a function of antibody dilution. The antibody 5A6 has been shown previously to bind to the same site on CD81 recognized by E2 [57], [58] with an affinity (75 nM) [59] about 1/10th that of E2 (4–10 nM) [60], [61]. Working at the same concentration of 281816 (1 µM) that provided the best inhibition of E2 binding to CD81-LEL, 281816 did not inhibit the 5A6 antibody binding to CD81 even when the antibody concentration was reduced to 1/2000th the concentration of the ligand (Figure 11). The amount of 5A6 antibody bound to CD81 remained the same in the presence and absence of the ligand, demonstrating that 281816 does not bind sufficiently well to the E2 binding site on CD81 to block 5A6 binding.


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 does not compete with binding of an anti-CD81 antibody to human CD81-LEL.Binding of anti-CD81 monoclonal antibody 5A6 to recombinant GST-tagged human CD81-LEL protein was determined by ELISA. Serial dilutions of 5A6 antibody were incubated with the CD81-LEL protein in the presence of 1 µM ligand 281816 (black squares) or PBS as a control (open circles). The amount of 5A6 antibody bound to CD81 remained the same in the presence and absence of the ligand, demonstrating that 281816 does not bind sufficiently well to the E2 binding site on CD81 to block 5A6 binding, even when the antibody concentration was reduced to 1/2000th the concentration of the ligand.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0111333-g011: Ligand 281816 does not compete with binding of an anti-CD81 antibody to human CD81-LEL.Binding of anti-CD81 monoclonal antibody 5A6 to recombinant GST-tagged human CD81-LEL protein was determined by ELISA. Serial dilutions of 5A6 antibody were incubated with the CD81-LEL protein in the presence of 1 µM ligand 281816 (black squares) or PBS as a control (open circles). The amount of 5A6 antibody bound to CD81 remained the same in the presence and absence of the ligand, demonstrating that 281816 does not bind sufficiently well to the E2 binding site on CD81 to block 5A6 binding, even when the antibody concentration was reduced to 1/2000th the concentration of the ligand.
Mentions: To determine if ligand 281816 also binds to CD81, surface plasmon resonance was used to test for 281816 binding to recombinant CD81-LEL protein immobilized on a chip. As shown in Figure 10, 281816 does bind to CD81-LEL. A kinetic analysis of this binding has shown that the ligand binds to CD81-LEL (Kd = 57 µM) almost as well as it binds to E2 (Kd = 41 µM). However, a competition ELISA experiment that used the anti-CD81 antibody, 5A6, that blocks E2 binding to CD81 (Figure 11) revealed that 281816 does not bind to the E2 binding site on CD81. In this experiment, CD81-LEL was immobilized on a micro titer plate and the binding of the 5A6 antibody was monitored in the presence of 1 µM 281816 as a function of antibody dilution. The antibody 5A6 has been shown previously to bind to the same site on CD81 recognized by E2 [57], [58] with an affinity (75 nM) [59] about 1/10th that of E2 (4–10 nM) [60], [61]. Working at the same concentration of 281816 (1 µM) that provided the best inhibition of E2 binding to CD81-LEL, 281816 did not inhibit the 5A6 antibody binding to CD81 even when the antibody concentration was reduced to 1/2000th the concentration of the ligand (Figure 11). The amount of 5A6 antibody bound to CD81 remained the same in the presence and absence of the ligand, demonstrating that 281816 does not bind sufficiently well to the E2 binding site on CD81 to block 5A6 binding.

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