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Structure-based drug design of novel peptidomimetic cellulose derivatives as HCV-NS3 protease inhibitors
Life Sciences 187 (2017) 58–63
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Structure-based drug design of novel peptidomimetic cellulose derivatives as HCV-NS3 protease inhibitors
MARK
Noha A. Saleh, Wael M. Elshemey⁎ Biophysic Department, Faculty of Science, Cairo University, Giza 12613, Egypt,
A R T I C L E I N F O
A B S T R A C T
Keywords: Cellulose Docking HCV Hexa-peptide NS3 protease NS3 inhibitors Structure-based drug design
Hepatitis C Virus (HCV) represents a global health threat not only due to the large number of reported worldwide HCV infections, but also due to the absence of a reliable vaccine for its prevention. HCV NS3 protease is one of the most important targets for drug design aiming at the deactivation of HCV. In the present work, molecular docking simulations are carried out for suggested novel NS3 protease inhibitors applied to the Egyptian genotype 4. These inhibitors are modifications of dimer cellulose by adding a hexa-peptide to the cellulose at one of the positions 2, 3, 6, 2′, 3′ or 6′. Results show that the inhibitor compound with the hexa-peptide at position 6 shows significantly higher simulation docking score with HCV NS3 protease active site. This is supported by low total energy value of docking system, formation of two H-bonds with HCV NS3 protease active site residues, high binding affinity and increased stability in the interaction system.
1. Introduction Hepatitis C is a liver disease caused by Hepatitis C Virus (HCV). It was first discovered in 1970 and was given remarkable interest as an unidentified third type of virus other than Hepatitis A and B. In 1989, HCV was identified and its genome cloned [1]. Research efforts in the field of HCV has been continuously progressing due to the large number of reported worldwide HCV infections (∼ 3% of the world's population, 150–200 million people, with specially high prevalence in Asia and North of Africa) [2,3]. The fact that over 85% of HCV patients will probably develop chronic hepatitis and that 20% of the chronic infections will progress to liver cirrhosis and Hepatocellular Carcinoma (HCC) [4,5] represented a strong motivation to conduct extensive research in this field. HCV is a member of the Flaviviridae family with a single positivestrand RNA encoding a poly-protein that undergoes proteolytic cleavage to 10 polypeptides, each with a distinct function. The structural proteins consist of a nucleocapside (C protein) which interacts with progeny viral genomes for virus assembly, in addition to two envelope glycoproteins (E1, E2) both of which are targets of the host antibody response and possibly ion channels (p7). The nonstructural proteins NS2, NS3, NS4A, NS4B, NS5A, and NS5B form a complex with viral RNA to initiate viral replication in a cytoplasmic membranous structure [6–12]. Unlike Hepatitis A and B, there is no vaccine for prevention of HCV infection. The current recommended therapy for HCV infection relies
⁎
only on antiviral drugs. Before 2011, the treatment of choice was a combination of Pegylated Interferon (IFN) alpha with the antiviral nucleoside analogue ribavirin [13,14]. This therapy is effective in 50–80% of patients, depending on the HCV genotype. However, it is expensive and often associated with side effects that force discontinuation of the therapy [15,16]. Recently, several inhibitors targeting the viral NS3 protease, NS5B or NS5A have been approved for the treatment of HCV [16–18]. This is in addition to several other proposed inhibitor compounds that are suggested via theoretical and molecular modeling studies on HCV [19–23]. Similar to RNA viruses, HCV has an error-prone RNA-dependent RNA polymerase (RdRp) that allowed an original HCV ancestor to evolve into seven genotypes and more than 80 subtypes of HCV, currently described according to the nucleotide variation found among different HCV isolates [24]. Egypt has the highest prevalence of HCV worldwide with almost 20% of the population being infected. The HCV subtype 4a belonging to genotype 4 (HCV-4) is the most common genotype in Egypt [25–28]. The sequence of the NS5A/NS5B junction for the Egyptian genotype 4 is Glu-Asp-Val-Val-Cys-Cys consequently, the hexa-peptide NH2-P6-P5P4-P3-P2-P1-OH represents a natural substrate [27,29]. Computer-Aided Drug Design (CADD) technology can be divided into three categories: structure-based drug design, ligand-based drug design, and validation by molecular dynamics simulation. The structure-based drug design is an essential field in CADD involving docking of small molecules into macromolecules, especially protein targets.
Corresponding author at: Biophysics Department, Faculty of Science, Cairo University, P.O. Box 12613, Dokky, Giza, Egypt. E-mail address: [email protected] (W.M. Elshemey).
http://dx.doi.org/10.1016/j.lfs.2017.08.021 Received 30 April 2017; Received in revised form 15 August 2017; Accepted 21 August 2017 Available online 24 August 2017 0024-3205/ © 2017 Elsevier Inc. All rights reserved.
Life Sciences 187 (2017) 58–63
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inhibition activity and interaction modes of these selected compounds with the HCV NS3 protease active site and are compared with a natural substrate. The selected and investigated compounds are built up from dimer cellulose and the hexa-peptide sequences Glu-Asp-Val-Val-CysCys. These hexa-peptide sequences with dimer cellulose are modeled at positions 2, 3, 6, 2′, 3′ or 6′ (Scheme 2). Fig. 1 shows the general orientation mode of interactions between the suggested peptidomimetic inhibitors and the HCV-NS3 protease active site (His57, Asp81, Ser139) and Gly137. The docking simulation systems and detailed mode of interaction between HCV NS3 protease and studied compounds as well as the natural substrate were calculated through docking simulation and presented in Fig. 2. The red dashed lines in Fig. 2 represent the hydrogen bonds (H-bonds) formed within the investigated compounds or ligands, while the yellow dashed lines represent the hydrogen bonds (H-bonds) formed between the investigated compounds or ligands and the HCV NS3 protease residues. The number of H-bonds which are formed in docking systems either between the investigated compounds and HCV NS3 protease or in investigated compounds are listed in Table 1. The number of hydrogen bonds within the studied ligands or compounds is assumed to provide an indication of the stability of the ligand through the interaction with the HCV NS4 protease. Increases in hydrogen bonds are likely to reflect more stability in the docking system. Table 1 presents the total energy of docking systems, a final docking score and the binding energy in Kcal/mol. Base on Fig. 2, the mode of binding and the interaction of substrate and introduced compounds within the binding site residues in HCV NS3 protease are now described in detail.
Protein–ligand or protein–protein docking is a computational technology used to predict the orientation of a ligand when it is bound to a protein receptor or enzyme. The process involves applying a scoring function to estimate the likelihood that a ligand will bind to protein with high affinity for further biochemical experiments and developments [30–34]. Cellulose is a linear polymer that has the same β-(1-4)-D-glucopyranose units backbone as chitosan, except that the acetamide is replaced by 2-hydroxy group. It forms crystals, where intra-molecular and intrastrand hydrogen bonds hold the network flat allowing the more hydrophobic ribbon faces to stack [35]. The similarity of cellulose and chitosan in primary structures suggests that they may be sufficiently similar to facilitate the formation of a homogeneous composite [36]. The present work investigates the potential antiviral properties of compounds characterized by a cellulose base with hexa-peptide functionalities at different positions. This design is based on previous experimental work making use of simple sugars (e.g. β-glucose, where cellulose is built of units of glucose) as a scaffold in many suggested antiviral inhibitors (especially HCV protease inhibitors) [37–42]. On the other hand, experimental work on peptide inhibitors (such as tri, tetra or hexa-peptides) demonstrated HCV NS3 protease inhibition [43–47]. The design of HCV inhibitor compounds based on hexa-peptides benefits from being peptidomimetic and acting as competitive inhibitors in the same time. Structure-based drug design is utilized in the investigation of the mode of interaction between each of the proposed inhibitor compounds and the NS5A/NS5B junction (Glu-Asp-Val-ValCys-Cys) of HCV NS3 protease of the Egyptian genotype 4.
2.1. Binding mode analysis of natural substrate 1.1. Computational methods The natural substrate (NS5A/NS5B junction) is used as a reference compound to be compared with the binding affinity of other suggested compounds. Through docking calculations, the natural substrate (GluAsp-Val-Val-Cys-Cys) forms only one H-bond with Gln41 amino acid of HCV NS3 protease as shown in Fig. 2. There are two H-bonds that are formed with the natural substrate in the docking system. From Table 1, the total energy of docking system with natural substrate is 400.38 Kcal/mol, the docking score is − 68.04 Kcal/mol and the binding energy is −17.65 Kcal/mol.
From the Protein Data Bank, the origin of the HCV-NS3 protease used in the present calculations is PDB code:3LOX [48]. Addition of hydrogen atoms and optimized geometry calculations of HCV-NS3 protease are performed in order to prepare the protein structure for molecular docking calculations. These are carried out using MM3 force field method [49]. Molecular docking simulations of the investigated compounds are performed with the help of SCIGRESS 3.0 software [50]. The conserved catalytic triad residues of HCV NS3 protease active site (His57, Asp81 and Ser139) and Gly137 are selected as a group to perform the docking calculations and find the best score for the mode of interaction with the studied compounds. FASTDOCK and PMF04 scoring functions utilize a genetic algorithm in order to dock the ligand into the selected HCV NS3 protease active site residues [51–55]. The docking systems yielding best scores are re-optimized using MM3 force field. The binding free energy (thermodynamic quantity) of the bestdocked compounds in the complex with HCV-NS3 protease is calculated using MM3 method according to the following equation:
2.2. Binding mode analysis of compound with position 2 The compound with position 2 forms two H-bonds with HCV NS3 protease and forms five H-bonds within the compound as shown in Fig. 2. One of the two H-bonds formed with HCV NS3 protease is located between the OH group of dimer cellulose at position 3 and Asp81 active site of HCV NS3 protease, the other one is formed between the OH group of Glu of hexa-peptide residue and Ser139 active site of HCV NS3 protease. The five H-bonds are formed within the compound with position 2. Two of them are formed within dimer cellulose. The other two bonds are formed between the dimer cellulose and hexa-peptide residues. The fifth bond is formed within the hexa-peptide residues. The addition of natural substrate to cellulose at position 2 increases the total energy of the docking system but decreases both docking score and binding energy. The total energy is 519.77 Kcal/mol, docking score is − 118.74 Kcal/mol and binding energy is − 68.33 Kcal/mol (Table 1).
ΔGBinding = GComplex − (GLigand + GReceptor ) 2. Results and discussion The theoretical electronic and QSAR properties of some novel peptidomimetic HCV NS3 protease inhibitors are presented. These suggested inhibitors are targeting the most common genotype (genotype 4) in Egypt [56–58]. The NS5A/NS5B junction is represents one of the four natural substrate cleavage sites for HCV NS3 protease. The sequence of the NS5A/NS5B junction for Egyptian genotype 4 is GluAsp-Val-Val-Cys-Cys and is shown in Scheme 1. The contemplated target compounds contain a hexa-peptide functionality of cellulose as competitive peptidomimetic inhibitors for HCV NS3 protease, especially genotype 4. From previous studies, the suggested inhibitor compounds with dimer cellulose have QSAR properties better than those consisting of monomer cellulose [57,58]. The protein–ligand docking calculations are simulated in this study to investigate the
2.3. Binding mode analysis of compound with position 2′ In case of compound with position 2′, there are two H-bonds with the binding pocket of HCV NS3 protease (similar to the compound with position 2); one bond is between the OH group of dimer cellulose at position 1′ and His57 active site residue and the other is between the OH terminal of hexa-peptide at Cys residue and Arg155 of NS3 protease. There are also five H-bonds within the compound with position 2′ distributed as; one H-bond in dimer cellulose, two H-bonds in hexa59
Life Sciences 187 (2017) 58–63
N.A. Saleh, W.M. Elshemey
HO
Scheme 1. The chemical structure of hexa-peptide natural substrate (NS5A/NS5B junction) of NS3 protease (Glu-Asp-Val-Val-Cys-Cys).
O
HS
O
O
O
H N
H2N
H N
N H
OH
N H
N H
O
O
O SH
O OH
H
OH
6'
6 HO
H
4
O
H
3
H
O
OH
1'
H H
2
Scheme 2. The chemical structure of investigated HCV-NS3 protease inhibitors (cellulose dimer binding with hexa-peptide [GulAsp-Val-Val-Cys-Cys] at position 2, 3, 6, 2′, 3′ or 6′). Hexa-peptide located at the 2′ position is indicated.
4'
O
1
H
OH
H
3'
OH OH
H
H
2'
-Hexapeptide
OH
Fig. 1. The general orientation mode of interaction between the suggested compounds and HCV-NS3 protease. The space filling residues represent amino acids present in the NS3 protease active site (His57, Asp81, Ser139) and Gly 137.
dimer cellulose at positions 2 and 2′. It also forms one H-bond between Ser139 active site residue and the OH group of dimer cellulose at position 3′. Within the compound, one H-bond is formed within the dimer cellulose and three are formed within the hexa-peptide residues. This mode of interaction exhibits the highest total energy of docking system and binding energy values in this study. Moreover, it provides the lowest docking score value in this study. The total energy and binding energy values are 649.45 Kcal/mol and 56.95 Kcal/mol, respectively, while the docking score value is − 137.99 Kcal/mol.
peptide residues and two H-bonds between dimer cellulose and hexapeptide residues. As listed in Table 1, the docking system of compound 2′ has higher total energy value (531.77 Kcal/mol) and docking score (− 52.06 Kcal/mol) and lower binding energy (− 73.26 Kcal/mol) than natural substrate. 2.4. Binding mode analysis of compound with position 3 Fig. 2 shows three intermolecular H-bond interactions between the compounds with position 3 and HCV NS3 protease binding site, as well as four H-bonds within the compound. The compound with position 3 forms two H-bonds between His57 active site residue and OH group of 60
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N.A. Saleh, W.M. Elshemey
GLN 41
Substrate
SER 139
ARG 155 ASP 81
HIS 57
Position 2
Position 2'
HIS 57
HIS 57 SER 139
Position 3
Position 3'
ASP 168
ASP 81
HIS 57 LYS 20
ALA 157
Position 6
Position 6'
Fig. 2. The docking systems and mode of interaction between the ligands and HCV-NS3 protease. The cylindrical molecules represent the studied compounds and the ball & cylinder molecules represent the amino acid residues of protein (The cyan, white, red and blue balls represent the carbon, hydrogen, oxygen and nitrogen atoms, respectively). The yellow dashed lines represent the hydrogen bonds between the ligands and amino acid residues of protein, while the red dashed lines represent the hydrogen bonds within the ligands. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
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cellulose leads to an increase in the total energy of interaction systems. The total energy varies from 479.75 Kcal/mol for the compound with position 6 to 649.45 Kcal/mol for the compound with position 3. The docking score values of the docking systems are listed in Table 1. The docking score of the docking system with natural substrate is − 68.04 Kcal/mol. The addition of natural substrate to dimer cellulose leads to a decrease in the docking score values of all interaction systems except for the compound with position 2.
Table 1 The total energy, docking score, binding energy (in Kcal/mol) and hydrogen bonds number of docking (interaction) systems via force field MM3 molecular mechanics method.
Substrate 2 2′ 3 3′ 6 6′
Total energy (Kcal/ mol)
Docking score (Kcal/mol)
Binding energy (Kcal/mol)
No. of Hbonds (ligand and protein)
No. of Hbonds (ligand)
400.38 519.77 531.77 649.45 523.68 479.75 496.11
− 68.04 − 118.74 − 52.06 − 137.99 − 134.66 − 101.17 − 86.91
− 17.65 − 68.33 − 73.26 56.95 − 14.44 − 114.93 − 30.85
1 2 2 3 1 2 3
2 5 5 4 4 5 4
3. Conclusion The addition of cellulose to natural substrate increases the number of H-bonds which indicates an increase in the stability of the docking systems. The investigated compounds are more stable than natural substrate in docking systems, where the natural substrate has two Hbonds while the investigated compounds have four or five H-bonds. The binding energy of suggested compounds is lower than that for natural substrate except for the compound with position 3. The functionality of dimer cellulose with the natural substrate resulted in an increase in the total energy values of the final docking system. The total energy values of docking system for the compound with position 6 and position 6′ (479.75 Kcal/mol and 496.11 Kcal/mol respectively) are close to the total energy value of the docking system of natural substrate (400.38 Kcal/mol). On the other hand, the total energy values of the rest of the docking systems are increased from the total energy value of the docking system for the natural substrate. The investigated compounds (except the compound with position 3′) form more than one Hbond with HCV NS3 protease. The compound with position 3′ and the natural substrate form only one H-bond with HCV NS3 protease. Cellulose plays an important role in the mode of interaction with the HCV NS3 protease. Generally, the compounds with the cellulose modified by adding the natural substrate at positions 2, 2′, 3, 3′, 6 or 6′ enhance the docking activity and binding affinity of suggested compounds and are predicted to have enhanced HCV NS3 inhibitory activity than the natural substrate. Specially, the compound with position 6, can be considered a promising NS3 protease inhibitor. It has the lowest total energy value of docking system (479.75 Kcal/mol), forms two H-bonds with HCV NS3 protease active site residues and is more stable in interaction system (five H-bonds within itself). Finally, in the docking system, the compound with position 6 has the lowest binding energy (− 114.93 Kcal/mol). Although this compound forms only Hbonds, its binding energy is the lowest and the binding affinity is the highest in this study. This means that there are possibly hydrophobic interactions as well as hydrophilic interactions (H-bonds) with the residues in HCV NS3 protease active site which increase the binding affinity. In the near future, the best compound in this study (compound with position 6) will be subject to an extensive study in order to either modify it to increase its inhibition activity for HCV NS3 protease or to increase the oral bioavailability of the compound by decreasing the peptic nature. The optimality of the compound should be confirmed experimentally and its binding modes compared with a number of HCV NS3 protease mutants. Compliance with ethical standards
2.5. Binding mode analysis of compound with position 3′ The compound with position 3′ forms only one H-bond with HCV NS3 protease in the docking system. This H-bond binds to the OH group of dimer cellulose at position 1′ with His57 active site residue. This compound forms four H-bonds within itself. One of them forms in the dimer cellulose. The other one forms between dimer cellulose and hexapeptide residues. The left two H-bonds forms within the hexa-peptide residues. The docking system total energy is 523.68 Kcal/mol, while the docking score is −134.66 Kcal/mol, which is lower than that of the substrate system. The binding energy value for this interaction is − 14.44 Kcal/mol. This is still higher than that for the substrate system. 2.6. Binding mode analysis of compound with position 6 The compound with position 6 is similar to the compounds with position 2 and 2′. It forms two H-bonds with the HCV NS3 protease and five H-bonds within the compound. The two H-bonds that form with NS3 protease are between the Asp81 active site residue and the OH of dimer cellulose at position 2, and between the Ala157 residue and the OH terminal of hexa-peptide at the Cys residue. The five H-bonds that form within the compound with position 6 consist of three within dimer cellulose and two within the hexa-peptides residues. Although the compound with position 6 forms the same number of H-bonds as the compounds with position 2 and 2′, the compound with position 6 has total energy in the docking system (479.75 Kcal/mol) and binding energy (− 114.93 Kcal/mol) lower than that of the compounds with position 2 and 2′. The binding energy value of the docking system of the compound with position 6 is the lowest value in this study. The docking score of interaction of the compounds with position 6 is −101.17 Kcal/ mol. 2.7. Binding mode analysis of compound with position 6′ Similar to the compound with position 3, the compound with position 6′ forms four H-bonds within the compound and forms three Hbonds with HCV NS3 protease. The three H-bonds with the HCV NS3 protease pocket site are; one bond between the His57 active site residue and the OH group of dimer cellulose at position 1′,one bond between the Asp168 residue and the OH group of dimer cellulose at position 2, and one third bond between the Lys20 and the OH terminal of hexapeptide at the Cys residue. Within the compound with position 6′, there are three H-bonds in dimer cellulose and one between the dimer cellulose and the hexa-peptide residues. This docking system has a total energy value equal to 496.11 Kcal/mol. This is lower than that of the compound with position 3 and higher compared to natural substrate. The modification of cellulose by attaching the substrate to position 6′ decreases the docking score (− 86.91 Kcal/mol) and binding energy (− 30.85 Kcal/mol) than that of the substrate alone. The modification of natural substrate through the addition of dimer
Funding This study received no fund and was not funded by any one.
Conflict of interest Author Wael M Elshemey declares that he has no conflict of interest. Author Noha A Saleh declares that she has no conflict of interest. 62
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Full length article
Structure-based drug design of novel peptidomimetic cellulose derivatives as HCV-NS3 protease inhibitors
MARK
Noha A. Saleh, Wael M. Elshemey⁎ Biophysic Department, Faculty of Science, Cairo University, Giza 12613, Egypt,
A R T I C L E I N F O
A B S T R A C T
Keywords: Cellulose Docking HCV Hexa-peptide NS3 protease NS3 inhibitors Structure-based drug design
Hepatitis C Virus (HCV) represents a global health threat not only due to the large number of reported worldwide HCV infections, but also due to the absence of a reliable vaccine for its prevention. HCV NS3 protease is one of the most important targets for drug design aiming at the deactivation of HCV. In the present work, molecular docking simulations are carried out for suggested novel NS3 protease inhibitors applied to the Egyptian genotype 4. These inhibitors are modifications of dimer cellulose by adding a hexa-peptide to the cellulose at one of the positions 2, 3, 6, 2′, 3′ or 6′. Results show that the inhibitor compound with the hexa-peptide at position 6 shows significantly higher simulation docking score with HCV NS3 protease active site. This is supported by low total energy value of docking system, formation of two H-bonds with HCV NS3 protease active site residues, high binding affinity and increased stability in the interaction system.
1. Introduction Hepatitis C is a liver disease caused by Hepatitis C Virus (HCV). It was first discovered in 1970 and was given remarkable interest as an unidentified third type of virus other than Hepatitis A and B. In 1989, HCV was identified and its genome cloned [1]. Research efforts in the field of HCV has been continuously progressing due to the large number of reported worldwide HCV infections (∼ 3% of the world's population, 150–200 million people, with specially high prevalence in Asia and North of Africa) [2,3]. The fact that over 85% of HCV patients will probably develop chronic hepatitis and that 20% of the chronic infections will progress to liver cirrhosis and Hepatocellular Carcinoma (HCC) [4,5] represented a strong motivation to conduct extensive research in this field. HCV is a member of the Flaviviridae family with a single positivestrand RNA encoding a poly-protein that undergoes proteolytic cleavage to 10 polypeptides, each with a distinct function. The structural proteins consist of a nucleocapside (C protein) which interacts with progeny viral genomes for virus assembly, in addition to two envelope glycoproteins (E1, E2) both of which are targets of the host antibody response and possibly ion channels (p7). The nonstructural proteins NS2, NS3, NS4A, NS4B, NS5A, and NS5B form a complex with viral RNA to initiate viral replication in a cytoplasmic membranous structure [6–12]. Unlike Hepatitis A and B, there is no vaccine for prevention of HCV infection. The current recommended therapy for HCV infection relies
⁎
only on antiviral drugs. Before 2011, the treatment of choice was a combination of Pegylated Interferon (IFN) alpha with the antiviral nucleoside analogue ribavirin [13,14]. This therapy is effective in 50–80% of patients, depending on the HCV genotype. However, it is expensive and often associated with side effects that force discontinuation of the therapy [15,16]. Recently, several inhibitors targeting the viral NS3 protease, NS5B or NS5A have been approved for the treatment of HCV [16–18]. This is in addition to several other proposed inhibitor compounds that are suggested via theoretical and molecular modeling studies on HCV [19–23]. Similar to RNA viruses, HCV has an error-prone RNA-dependent RNA polymerase (RdRp) that allowed an original HCV ancestor to evolve into seven genotypes and more than 80 subtypes of HCV, currently described according to the nucleotide variation found among different HCV isolates [24]. Egypt has the highest prevalence of HCV worldwide with almost 20% of the population being infected. The HCV subtype 4a belonging to genotype 4 (HCV-4) is the most common genotype in Egypt [25–28]. The sequence of the NS5A/NS5B junction for the Egyptian genotype 4 is Glu-Asp-Val-Val-Cys-Cys consequently, the hexa-peptide NH2-P6-P5P4-P3-P2-P1-OH represents a natural substrate [27,29]. Computer-Aided Drug Design (CADD) technology can be divided into three categories: structure-based drug design, ligand-based drug design, and validation by molecular dynamics simulation. The structure-based drug design is an essential field in CADD involving docking of small molecules into macromolecules, especially protein targets.
Corresponding author at: Biophysics Department, Faculty of Science, Cairo University, P.O. Box 12613, Dokky, Giza, Egypt. E-mail address: [email protected] (W.M. Elshemey).
http://dx.doi.org/10.1016/j.lfs.2017.08.021 Received 30 April 2017; Received in revised form 15 August 2017; Accepted 21 August 2017 Available online 24 August 2017 0024-3205/ © 2017 Elsevier Inc. All rights reserved.
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inhibition activity and interaction modes of these selected compounds with the HCV NS3 protease active site and are compared with a natural substrate. The selected and investigated compounds are built up from dimer cellulose and the hexa-peptide sequences Glu-Asp-Val-Val-CysCys. These hexa-peptide sequences with dimer cellulose are modeled at positions 2, 3, 6, 2′, 3′ or 6′ (Scheme 2). Fig. 1 shows the general orientation mode of interactions between the suggested peptidomimetic inhibitors and the HCV-NS3 protease active site (His57, Asp81, Ser139) and Gly137. The docking simulation systems and detailed mode of interaction between HCV NS3 protease and studied compounds as well as the natural substrate were calculated through docking simulation and presented in Fig. 2. The red dashed lines in Fig. 2 represent the hydrogen bonds (H-bonds) formed within the investigated compounds or ligands, while the yellow dashed lines represent the hydrogen bonds (H-bonds) formed between the investigated compounds or ligands and the HCV NS3 protease residues. The number of H-bonds which are formed in docking systems either between the investigated compounds and HCV NS3 protease or in investigated compounds are listed in Table 1. The number of hydrogen bonds within the studied ligands or compounds is assumed to provide an indication of the stability of the ligand through the interaction with the HCV NS4 protease. Increases in hydrogen bonds are likely to reflect more stability in the docking system. Table 1 presents the total energy of docking systems, a final docking score and the binding energy in Kcal/mol. Base on Fig. 2, the mode of binding and the interaction of substrate and introduced compounds within the binding site residues in HCV NS3 protease are now described in detail.
Protein–ligand or protein–protein docking is a computational technology used to predict the orientation of a ligand when it is bound to a protein receptor or enzyme. The process involves applying a scoring function to estimate the likelihood that a ligand will bind to protein with high affinity for further biochemical experiments and developments [30–34]. Cellulose is a linear polymer that has the same β-(1-4)-D-glucopyranose units backbone as chitosan, except that the acetamide is replaced by 2-hydroxy group. It forms crystals, where intra-molecular and intrastrand hydrogen bonds hold the network flat allowing the more hydrophobic ribbon faces to stack [35]. The similarity of cellulose and chitosan in primary structures suggests that they may be sufficiently similar to facilitate the formation of a homogeneous composite [36]. The present work investigates the potential antiviral properties of compounds characterized by a cellulose base with hexa-peptide functionalities at different positions. This design is based on previous experimental work making use of simple sugars (e.g. β-glucose, where cellulose is built of units of glucose) as a scaffold in many suggested antiviral inhibitors (especially HCV protease inhibitors) [37–42]. On the other hand, experimental work on peptide inhibitors (such as tri, tetra or hexa-peptides) demonstrated HCV NS3 protease inhibition [43–47]. The design of HCV inhibitor compounds based on hexa-peptides benefits from being peptidomimetic and acting as competitive inhibitors in the same time. Structure-based drug design is utilized in the investigation of the mode of interaction between each of the proposed inhibitor compounds and the NS5A/NS5B junction (Glu-Asp-Val-ValCys-Cys) of HCV NS3 protease of the Egyptian genotype 4.
2.1. Binding mode analysis of natural substrate 1.1. Computational methods The natural substrate (NS5A/NS5B junction) is used as a reference compound to be compared with the binding affinity of other suggested compounds. Through docking calculations, the natural substrate (GluAsp-Val-Val-Cys-Cys) forms only one H-bond with Gln41 amino acid of HCV NS3 protease as shown in Fig. 2. There are two H-bonds that are formed with the natural substrate in the docking system. From Table 1, the total energy of docking system with natural substrate is 400.38 Kcal/mol, the docking score is − 68.04 Kcal/mol and the binding energy is −17.65 Kcal/mol.
From the Protein Data Bank, the origin of the HCV-NS3 protease used in the present calculations is PDB code:3LOX [48]. Addition of hydrogen atoms and optimized geometry calculations of HCV-NS3 protease are performed in order to prepare the protein structure for molecular docking calculations. These are carried out using MM3 force field method [49]. Molecular docking simulations of the investigated compounds are performed with the help of SCIGRESS 3.0 software [50]. The conserved catalytic triad residues of HCV NS3 protease active site (His57, Asp81 and Ser139) and Gly137 are selected as a group to perform the docking calculations and find the best score for the mode of interaction with the studied compounds. FASTDOCK and PMF04 scoring functions utilize a genetic algorithm in order to dock the ligand into the selected HCV NS3 protease active site residues [51–55]. The docking systems yielding best scores are re-optimized using MM3 force field. The binding free energy (thermodynamic quantity) of the bestdocked compounds in the complex with HCV-NS3 protease is calculated using MM3 method according to the following equation:
2.2. Binding mode analysis of compound with position 2 The compound with position 2 forms two H-bonds with HCV NS3 protease and forms five H-bonds within the compound as shown in Fig. 2. One of the two H-bonds formed with HCV NS3 protease is located between the OH group of dimer cellulose at position 3 and Asp81 active site of HCV NS3 protease, the other one is formed between the OH group of Glu of hexa-peptide residue and Ser139 active site of HCV NS3 protease. The five H-bonds are formed within the compound with position 2. Two of them are formed within dimer cellulose. The other two bonds are formed between the dimer cellulose and hexa-peptide residues. The fifth bond is formed within the hexa-peptide residues. The addition of natural substrate to cellulose at position 2 increases the total energy of the docking system but decreases both docking score and binding energy. The total energy is 519.77 Kcal/mol, docking score is − 118.74 Kcal/mol and binding energy is − 68.33 Kcal/mol (Table 1).
ΔGBinding = GComplex − (GLigand + GReceptor ) 2. Results and discussion The theoretical electronic and QSAR properties of some novel peptidomimetic HCV NS3 protease inhibitors are presented. These suggested inhibitors are targeting the most common genotype (genotype 4) in Egypt [56–58]. The NS5A/NS5B junction is represents one of the four natural substrate cleavage sites for HCV NS3 protease. The sequence of the NS5A/NS5B junction for Egyptian genotype 4 is GluAsp-Val-Val-Cys-Cys and is shown in Scheme 1. The contemplated target compounds contain a hexa-peptide functionality of cellulose as competitive peptidomimetic inhibitors for HCV NS3 protease, especially genotype 4. From previous studies, the suggested inhibitor compounds with dimer cellulose have QSAR properties better than those consisting of monomer cellulose [57,58]. The protein–ligand docking calculations are simulated in this study to investigate the
2.3. Binding mode analysis of compound with position 2′ In case of compound with position 2′, there are two H-bonds with the binding pocket of HCV NS3 protease (similar to the compound with position 2); one bond is between the OH group of dimer cellulose at position 1′ and His57 active site residue and the other is between the OH terminal of hexa-peptide at Cys residue and Arg155 of NS3 protease. There are also five H-bonds within the compound with position 2′ distributed as; one H-bond in dimer cellulose, two H-bonds in hexa59
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HO
Scheme 1. The chemical structure of hexa-peptide natural substrate (NS5A/NS5B junction) of NS3 protease (Glu-Asp-Val-Val-Cys-Cys).
O
HS
O
O
O
H N
H2N
H N
N H
OH
N H
N H
O
O
O SH
O OH
H
OH
6'
6 HO
H
4
O
H
3
H
O
OH
1'
H H
2
Scheme 2. The chemical structure of investigated HCV-NS3 protease inhibitors (cellulose dimer binding with hexa-peptide [GulAsp-Val-Val-Cys-Cys] at position 2, 3, 6, 2′, 3′ or 6′). Hexa-peptide located at the 2′ position is indicated.
4'
O
1
H
OH
H
3'
OH OH
H
H
2'
-Hexapeptide
OH
Fig. 1. The general orientation mode of interaction between the suggested compounds and HCV-NS3 protease. The space filling residues represent amino acids present in the NS3 protease active site (His57, Asp81, Ser139) and Gly 137.
dimer cellulose at positions 2 and 2′. It also forms one H-bond between Ser139 active site residue and the OH group of dimer cellulose at position 3′. Within the compound, one H-bond is formed within the dimer cellulose and three are formed within the hexa-peptide residues. This mode of interaction exhibits the highest total energy of docking system and binding energy values in this study. Moreover, it provides the lowest docking score value in this study. The total energy and binding energy values are 649.45 Kcal/mol and 56.95 Kcal/mol, respectively, while the docking score value is − 137.99 Kcal/mol.
peptide residues and two H-bonds between dimer cellulose and hexapeptide residues. As listed in Table 1, the docking system of compound 2′ has higher total energy value (531.77 Kcal/mol) and docking score (− 52.06 Kcal/mol) and lower binding energy (− 73.26 Kcal/mol) than natural substrate. 2.4. Binding mode analysis of compound with position 3 Fig. 2 shows three intermolecular H-bond interactions between the compounds with position 3 and HCV NS3 protease binding site, as well as four H-bonds within the compound. The compound with position 3 forms two H-bonds between His57 active site residue and OH group of 60
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GLN 41
Substrate
SER 139
ARG 155 ASP 81
HIS 57
Position 2
Position 2'
HIS 57
HIS 57 SER 139
Position 3
Position 3'
ASP 168
ASP 81
HIS 57 LYS 20
ALA 157
Position 6
Position 6'
Fig. 2. The docking systems and mode of interaction between the ligands and HCV-NS3 protease. The cylindrical molecules represent the studied compounds and the ball & cylinder molecules represent the amino acid residues of protein (The cyan, white, red and blue balls represent the carbon, hydrogen, oxygen and nitrogen atoms, respectively). The yellow dashed lines represent the hydrogen bonds between the ligands and amino acid residues of protein, while the red dashed lines represent the hydrogen bonds within the ligands. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
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cellulose leads to an increase in the total energy of interaction systems. The total energy varies from 479.75 Kcal/mol for the compound with position 6 to 649.45 Kcal/mol for the compound with position 3. The docking score values of the docking systems are listed in Table 1. The docking score of the docking system with natural substrate is − 68.04 Kcal/mol. The addition of natural substrate to dimer cellulose leads to a decrease in the docking score values of all interaction systems except for the compound with position 2.
Table 1 The total energy, docking score, binding energy (in Kcal/mol) and hydrogen bonds number of docking (interaction) systems via force field MM3 molecular mechanics method.
Substrate 2 2′ 3 3′ 6 6′
Total energy (Kcal/ mol)
Docking score (Kcal/mol)
Binding energy (Kcal/mol)
No. of Hbonds (ligand and protein)
No. of Hbonds (ligand)
400.38 519.77 531.77 649.45 523.68 479.75 496.11
− 68.04 − 118.74 − 52.06 − 137.99 − 134.66 − 101.17 − 86.91
− 17.65 − 68.33 − 73.26 56.95 − 14.44 − 114.93 − 30.85
1 2 2 3 1 2 3
2 5 5 4 4 5 4
3. Conclusion The addition of cellulose to natural substrate increases the number of H-bonds which indicates an increase in the stability of the docking systems. The investigated compounds are more stable than natural substrate in docking systems, where the natural substrate has two Hbonds while the investigated compounds have four or five H-bonds. The binding energy of suggested compounds is lower than that for natural substrate except for the compound with position 3. The functionality of dimer cellulose with the natural substrate resulted in an increase in the total energy values of the final docking system. The total energy values of docking system for the compound with position 6 and position 6′ (479.75 Kcal/mol and 496.11 Kcal/mol respectively) are close to the total energy value of the docking system of natural substrate (400.38 Kcal/mol). On the other hand, the total energy values of the rest of the docking systems are increased from the total energy value of the docking system for the natural substrate. The investigated compounds (except the compound with position 3′) form more than one Hbond with HCV NS3 protease. The compound with position 3′ and the natural substrate form only one H-bond with HCV NS3 protease. Cellulose plays an important role in the mode of interaction with the HCV NS3 protease. Generally, the compounds with the cellulose modified by adding the natural substrate at positions 2, 2′, 3, 3′, 6 or 6′ enhance the docking activity and binding affinity of suggested compounds and are predicted to have enhanced HCV NS3 inhibitory activity than the natural substrate. Specially, the compound with position 6, can be considered a promising NS3 protease inhibitor. It has the lowest total energy value of docking system (479.75 Kcal/mol), forms two H-bonds with HCV NS3 protease active site residues and is more stable in interaction system (five H-bonds within itself). Finally, in the docking system, the compound with position 6 has the lowest binding energy (− 114.93 Kcal/mol). Although this compound forms only Hbonds, its binding energy is the lowest and the binding affinity is the highest in this study. This means that there are possibly hydrophobic interactions as well as hydrophilic interactions (H-bonds) with the residues in HCV NS3 protease active site which increase the binding affinity. In the near future, the best compound in this study (compound with position 6) will be subject to an extensive study in order to either modify it to increase its inhibition activity for HCV NS3 protease or to increase the oral bioavailability of the compound by decreasing the peptic nature. The optimality of the compound should be confirmed experimentally and its binding modes compared with a number of HCV NS3 protease mutants. Compliance with ethical standards
2.5. Binding mode analysis of compound with position 3′ The compound with position 3′ forms only one H-bond with HCV NS3 protease in the docking system. This H-bond binds to the OH group of dimer cellulose at position 1′ with His57 active site residue. This compound forms four H-bonds within itself. One of them forms in the dimer cellulose. The other one forms between dimer cellulose and hexapeptide residues. The left two H-bonds forms within the hexa-peptide residues. The docking system total energy is 523.68 Kcal/mol, while the docking score is −134.66 Kcal/mol, which is lower than that of the substrate system. The binding energy value for this interaction is − 14.44 Kcal/mol. This is still higher than that for the substrate system. 2.6. Binding mode analysis of compound with position 6 The compound with position 6 is similar to the compounds with position 2 and 2′. It forms two H-bonds with the HCV NS3 protease and five H-bonds within the compound. The two H-bonds that form with NS3 protease are between the Asp81 active site residue and the OH of dimer cellulose at position 2, and between the Ala157 residue and the OH terminal of hexa-peptide at the Cys residue. The five H-bonds that form within the compound with position 6 consist of three within dimer cellulose and two within the hexa-peptides residues. Although the compound with position 6 forms the same number of H-bonds as the compounds with position 2 and 2′, the compound with position 6 has total energy in the docking system (479.75 Kcal/mol) and binding energy (− 114.93 Kcal/mol) lower than that of the compounds with position 2 and 2′. The binding energy value of the docking system of the compound with position 6 is the lowest value in this study. The docking score of interaction of the compounds with position 6 is −101.17 Kcal/ mol. 2.7. Binding mode analysis of compound with position 6′ Similar to the compound with position 3, the compound with position 6′ forms four H-bonds within the compound and forms three Hbonds with HCV NS3 protease. The three H-bonds with the HCV NS3 protease pocket site are; one bond between the His57 active site residue and the OH group of dimer cellulose at position 1′,one bond between the Asp168 residue and the OH group of dimer cellulose at position 2, and one third bond between the Lys20 and the OH terminal of hexapeptide at the Cys residue. Within the compound with position 6′, there are three H-bonds in dimer cellulose and one between the dimer cellulose and the hexa-peptide residues. This docking system has a total energy value equal to 496.11 Kcal/mol. This is lower than that of the compound with position 3 and higher compared to natural substrate. The modification of cellulose by attaching the substrate to position 6′ decreases the docking score (− 86.91 Kcal/mol) and binding energy (− 30.85 Kcal/mol) than that of the substrate alone. The modification of natural substrate through the addition of dimer
Funding This study received no fund and was not funded by any one.
Conflict of interest Author Wael M Elshemey declares that he has no conflict of interest. Author Noha A Saleh declares that she has no conflict of interest. 62
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