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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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Comparison of structural templates used for modeling the HCV E2c protein.(A) Bar representation of E2 sequence showing the structural similarities between crystal structures 4MWF chains C and D (E2c structure, genotype 1a), and 4NX3 chain D (genotype 2a). Regions reported in the coordinates span amino acid residues from H421 to N645. The percent sequence identities between amino acid sequences taken from coordinates and corresponding sequence fragments from HCV E2 protein of genotype 1a are shown in the column Seq_ID. In green are colored regions where structural deviations are below 3 Ångstroms measured as Cα-Cα distances between corresponding residues from the superimposed structures. In red are regions where structural data is missing or deviations are greater than 3 Angstroms. The locations of amino acid residues that have been reported to be important for E2 binding to CD81 are marked with yellow stars. (B) Structural superposition of 4mwf_C and 4nx3_D shows strong conformational similarities between experimentally solved structures of E2 proteins for which the level of sequence identity is 69%. In blue and purple are colored structural fragments where two structures 4mwf_C (566–601; Blue: light-dark) and 4nx3_D (568–605; Purple: light-dark) significantly differ. (C) Surface presentation of the 4mwf_D structure showing the amino acid residues identified to be important for E2 binding to CD81 (yellow). The other amino acid residues are color coded with the most hydrophilic residues being colored blue, the most hydrophobic residues colored red orange, and intermediate residues colored white.
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pone-0111333-g001: Comparison of structural templates used for modeling the HCV E2c protein.(A) Bar representation of E2 sequence showing the structural similarities between crystal structures 4MWF chains C and D (E2c structure, genotype 1a), and 4NX3 chain D (genotype 2a). Regions reported in the coordinates span amino acid residues from H421 to N645. The percent sequence identities between amino acid sequences taken from coordinates and corresponding sequence fragments from HCV E2 protein of genotype 1a are shown in the column Seq_ID. In green are colored regions where structural deviations are below 3 Ångstroms measured as Cα-Cα distances between corresponding residues from the superimposed structures. In red are regions where structural data is missing or deviations are greater than 3 Angstroms. The locations of amino acid residues that have been reported to be important for E2 binding to CD81 are marked with yellow stars. (B) Structural superposition of 4mwf_C and 4nx3_D shows strong conformational similarities between experimentally solved structures of E2 proteins for which the level of sequence identity is 69%. In blue and purple are colored structural fragments where two structures 4mwf_C (566–601; Blue: light-dark) and 4nx3_D (568–605; Purple: light-dark) significantly differ. (C) Surface presentation of the 4mwf_D structure showing the amino acid residues identified to be important for E2 binding to CD81 (yellow). The other amino acid residues are color coded with the most hydrophilic residues being colored blue, the most hydrophobic residues colored red orange, and intermediate residues colored white.

Mentions: In order to maximize the likelihood that these experiments would lead to the discovery of small molecules that bind to E2 and block E2’s binding to CD81, we created a homology model of the core of the E2 protein to use as our docking target. This model was created using the HCV genotype 1a protein sequence NP_751921.1, which corresponds to isolate H77, and the crystal structure of E2c as the primary template (PDB entry: 4MWF). Using a model, rather than the E2c crystal structure, was important because the reported crystal structure of E2c has three large gaps in which atom coordinates for 57 amino acids, or one quarter of the E2c structure, is missing. The coordinates listed in PDB chains 4mwf_C and 4mwf_D provide structural information for only 169 and 171 residues respectively out of the 363 amino acids present in the full-length E2 protein. Within each of the deposited PDB chains, three stretches of amino acid sequence (large loop P453-P491 containing 39 amino acids, T542-G547 or V574-N577, and F586-R596) are missing from the structure (Figure 1A). Docking to structures lacking such a large proportion of their amino acids can be problematic because the missing peptide segments are usually located on the protein’s surface, and the underlying amino acid residues packed in the interior of the protein are exposed and incorrectly presented as the surface during the docking. Unfortunately, similar regions are also not present in the crystal structure of the genotype 2a HCV E2c protein (PDB chain: 4nx3_D) reported by Kahn et al. [19] which provides atom coordinates for only 119 amino acids. Structural superposition of 4mwf_C and 4nx3_D (Figure 1B) shows strong conformational similarities between the experimentally solved structures of the E2 proteins with a root mean square deviation of 1.07 Angstroms measured on 98 residues for which distances between corresponding Cα atoms are under 3 Angstroms. The most significant structural deviations are observed in the region 566–601 (numbering from 4mwf_C) which corresponds to the region that also exhibits the greatest variation in sequence (see sequence alignment in Figure 1A).


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)

Comparison of structural templates used for modeling the HCV E2c protein.(A) Bar representation of E2 sequence showing the structural similarities between crystal structures 4MWF chains C and D (E2c structure, genotype 1a), and 4NX3 chain D (genotype 2a). Regions reported in the coordinates span amino acid residues from H421 to N645. The percent sequence identities between amino acid sequences taken from coordinates and corresponding sequence fragments from HCV E2 protein of genotype 1a are shown in the column Seq_ID. In green are colored regions where structural deviations are below 3 Ångstroms measured as Cα-Cα distances between corresponding residues from the superimposed structures. In red are regions where structural data is missing or deviations are greater than 3 Angstroms. The locations of amino acid residues that have been reported to be important for E2 binding to CD81 are marked with yellow stars. (B) Structural superposition of 4mwf_C and 4nx3_D shows strong conformational similarities between experimentally solved structures of E2 proteins for which the level of sequence identity is 69%. In blue and purple are colored structural fragments where two structures 4mwf_C (566–601; Blue: light-dark) and 4nx3_D (568–605; Purple: light-dark) significantly differ. (C) Surface presentation of the 4mwf_D structure showing the amino acid residues identified to be important for E2 binding to CD81 (yellow). The other amino acid residues are color coded with the most hydrophilic residues being colored blue, the most hydrophobic residues colored red orange, and intermediate residues colored white.
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Related In: Results  -  Collection

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

pone-0111333-g001: Comparison of structural templates used for modeling the HCV E2c protein.(A) Bar representation of E2 sequence showing the structural similarities between crystal structures 4MWF chains C and D (E2c structure, genotype 1a), and 4NX3 chain D (genotype 2a). Regions reported in the coordinates span amino acid residues from H421 to N645. The percent sequence identities between amino acid sequences taken from coordinates and corresponding sequence fragments from HCV E2 protein of genotype 1a are shown in the column Seq_ID. In green are colored regions where structural deviations are below 3 Ångstroms measured as Cα-Cα distances between corresponding residues from the superimposed structures. In red are regions where structural data is missing or deviations are greater than 3 Angstroms. The locations of amino acid residues that have been reported to be important for E2 binding to CD81 are marked with yellow stars. (B) Structural superposition of 4mwf_C and 4nx3_D shows strong conformational similarities between experimentally solved structures of E2 proteins for which the level of sequence identity is 69%. In blue and purple are colored structural fragments where two structures 4mwf_C (566–601; Blue: light-dark) and 4nx3_D (568–605; Purple: light-dark) significantly differ. (C) Surface presentation of the 4mwf_D structure showing the amino acid residues identified to be important for E2 binding to CD81 (yellow). The other amino acid residues are color coded with the most hydrophilic residues being colored blue, the most hydrophobic residues colored red orange, and intermediate residues colored white.
Mentions: In order to maximize the likelihood that these experiments would lead to the discovery of small molecules that bind to E2 and block E2’s binding to CD81, we created a homology model of the core of the E2 protein to use as our docking target. This model was created using the HCV genotype 1a protein sequence NP_751921.1, which corresponds to isolate H77, and the crystal structure of E2c as the primary template (PDB entry: 4MWF). Using a model, rather than the E2c crystal structure, was important because the reported crystal structure of E2c has three large gaps in which atom coordinates for 57 amino acids, or one quarter of the E2c structure, is missing. The coordinates listed in PDB chains 4mwf_C and 4mwf_D provide structural information for only 169 and 171 residues respectively out of the 363 amino acids present in the full-length E2 protein. Within each of the deposited PDB chains, three stretches of amino acid sequence (large loop P453-P491 containing 39 amino acids, T542-G547 or V574-N577, and F586-R596) are missing from the structure (Figure 1A). Docking to structures lacking such a large proportion of their amino acids can be problematic because the missing peptide segments are usually located on the protein’s surface, and the underlying amino acid residues packed in the interior of the protein are exposed and incorrectly presented as the surface during the docking. Unfortunately, similar regions are also not present in the crystal structure of the genotype 2a HCV E2c protein (PDB chain: 4nx3_D) reported by Kahn et al. [19] which provides atom coordinates for only 119 amino acids. Structural superposition of 4mwf_C and 4nx3_D (Figure 1B) shows strong conformational similarities between the experimentally solved structures of the E2 proteins with a root mean square deviation of 1.07 Angstroms measured on 98 residues for which distances between corresponding Cα atoms are under 3 Angstroms. The most significant structural deviations are observed in the region 566–601 (numbering from 4mwf_C) which corresponds to the region that also exhibits the greatest variation in sequence (see sequence alignment in Figure 1A).

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