Molecular docking studies of bioactive compounds from the leaves of Epiphyllum oxypetalum against Treponema pallidum, Zika virus and liver cirrhosis

Treponema pallidum

Syphilis is a chronic sexually transmitted disease caused by T. pallidum outbreak in Italy, Germany, United Kingdom, and France. This disease mainly gets benefited in the anus and vagina region or it will be most probably grown in mouth or in the rectum. This is motile Gram-negative spirochaete can be transmitted both sexually and from mother to child by suppressing the immune system. Treponema pallidum has long been regarded as a stealth pathogen because of its poorly antigenic and non-inflammatory surface (Fraser et al., 1998). There is now increasing evidence that the antigenic variation also contributes the ability of the spirochaete to evade host defences. The amino acids encoded with protein plays a vital role in enhancing the virulent factor of the disease by supplying sufficient nutrients and minerals that plays a major role in regulating cAMP (Deka et al., 2015; Schuppan et al., 1999).

Table 1. Gas chromatography mass spectroscopy analysis of leaves of E. oxypetalum. [Click here to view]

Liver cirrhosis

Liver is a major organ of the human body. Viral hepatitis increases the chances for cirrhosis by affecting the liver inducing alkaline phosphatase enzymes which will be responsible for inhibiting the immune response. Individuals suffering from hepatitis are required to get regular check-ups because it is easily transmittable from one person to the other. It carries out a wide range of functions, such as digestion, adsorption and processing of food, detoxification of alcohol and drugs, and filtering of blood from the digestive tract (Amitrano et al., 2004). Liver cirrhosis is one of the major diseases prevalent in India. Liver cirrhosis is a degenerative liver disease involving four steps that results due to irreversible tissue scarring leading to cell death (Tsochatzis et al., 2014). Cirrhosis affects the abdominal blood vessels and the huge portal vein which transports blood from the intestines and spleen through the liver. Thus, blood finds alternative pathway to return to heart adding pressure on the blood vessels rupturing it and rendering its original function. Liver losses it’s functioning capacity leading liver failure and eventually death in severe cases (Alberino et al., 2001; Dick et al., 1952).

Zika virus

Zika virus (ZIKV) is a flavivirus first isolated in 1948 from a senrinel rhesus monkey in the Zika forest of Uganda. Infection with this virus has been reported in Africa, India, Southeast Asia, and Micronesia. Recent phylogenetic analysis of reported ZIKV strains has suggested that strains from Africa and Asia have emerged as two distinct virus lineages. ZIKV has been isolated from humans, nonhuman primates, and mosquitoes (Musso et al., 2015). This disease mainly relates with the blood cells of living organisms by resisting the antibody production and grows by benefiting itself. The clinical research confirmed that the ZIKV disease had titers of neutralizing antibodies against ZIKV that were at least four times as high as their titters of neutralizing antibodies against dengue virus (Faye et al., 2014; Lanciotti et al., 2008).

The current research work has been done to prove the inhibition potential of the novel drug compounds in the E. oxypetalum leaves by targeting the protein that is responsible for the T. pallidum, liver Cirrhosis and ZIKV disease.

Selection and retrieval of protein structure from database

In this study, syphilis protein, namely, flavin traffling protein [Protein Data Bank (PDB) ID: 4XDR], from Cirrhosis protein, namely, pyruvate dehydrogenase kinase 2 (PDHK2) (PDB ID: 3CRK) and Zika E protein (PDB ID: 5UHY) were chosen. The correct crystal structure for these Syphilis, Cirrhosis, and ZIKV target has been obtained from Research Collaboratory for Structural Bioinformatics Data Bank.

Construction or retrieval of ligand structure

The bioactive compounds are chosen from the GC-MS analysis of E. oxypetalum leaves were used in this present study. Chem sketch is a chemically advanced quick drawing interface freeware, designed, and develop to design the structure of most ligands. Using the “draw mode” and then it was saved in the format of molecular file format. The other ligand structures were retrieved from Pub chem compound database (Lagunin et al., 2014).

Preparation of protein target

The virulent enzymes were studied and retrieved from the PDB. These proteins were commonly selected based on the suppression factor of human antibody against the diseases. These proteins (4XDR, 3CRK, and 5UHY) are mainly responsible for inducing immuno suppression diseases, such as syphilis, liver cirrhosis, and ZIKV. The hydrophobic molecules were removed along with the heteroatomic molecules. Single chain was determined for the docking analysis and was confirmed by visualizing in visualize software.

Molecular docking analysis

Docking is used to find the best matching between two molecules: a receptor and ligand. The various steps involved in Docking are as follows:

• Ligand preparation
• Receptor Preparation
• Binding Site preparation
• Scoring/Energy Evaluation

Molecular docking studies were performed for knowing the inhibition potential of drug molecules against Syphilis, ZIKV, and liver cirrhosis with bioactive compounds from the E. oxypetalum leaves using Auto Dock 4.2. It is an automated docking tool which works by Lamarckian Genetic Algorithm. The binding of molecules to a receptor with known 3D structure is predicted by Auto Dock 4.2 (Pal et al., 2006). The interaction of the molecules with their molecular targets forms the basis for drug development process. To perform docking experiment, the original protein were taken from the pdb file and the ligand pdb files from PRODRG server were submitted to AutoDock 4.2 for docking studies (Meenambiga et al., 2018). The binding energy is obtained for each ligand and the visual analysis of the docked complexes was done using Discovery studio Visulaizer 3.1 (Moroy et al., 2012; Muegge and Rarey, 2001; Walters, 2012).

Grid parameters

The default grid size in autodock software was set at 20 Å with grid points per map are 64,000. The default grid spacing was 0.375 Å with center grid box size at x axis −16.302, y axis −23.34, and z axis −16.245, respectively.

Lipinski’s rule

All the compounds were selected on the basis of Lipinski’s rule of five and compounds with any Ro5 violations were eliminated. It includes properties, such as molecular weight, lipophilicity, molar refractivity, number of hydrogen bond donors and acceptors

Discovery Studio Visualizer 3.1

Discovery Studio Visualizer is free software used for simulating and molecular modeling experiments for small and macromolecule. It generates 3D and 2D protein-ligand interaction plots. The ligand binding patterns are analyzed between the receptor protein and the ligand of interest (Singh and Romanowski, 1999).

Active sites representation of proteins

Treponema pallidum (4XDR)

Crystal structure of T. pallidum TP0796 Flavin trafficking protein, a bifunctional flavin mononucleotide (FMN) transferase/flavin adenine dinucleotide (FAD) pyrophosphatase, D284A mutant, Area distribution node bound form. Treponema pallidum cause syphilis. Using modern molecular techniques, studying this species has become possible. TP0796 is a bacterial metal-dependent FAD pyrophosphatase of its kind (Giacani and Centurion-Lara, 2012). It hydrolyses FAD into AMP and FMN in the spirochete’s periplasm. Orthologs of Ftp Tp from other bacteria appear to lack this hydrolytic activity; rather, they bind and flavinylate subunits of a cytoplasmic membrane redox system (Nqr/Rnf) (Radolf and Desrosiers, 2009). A single amino acid change in T. pallidum converts it from magnesium dependent FAD pyrophosphatase to an FAD-binding protein. Mutation in a metal binding residue reduces T. pallidum dual activity, thereby reducing the role of magnesium in enzyme-catalyzed reaction. 4XDR interacts selectively using non-covalent bonds with metal ion. It is present on the membrane that enables ease of interaction. The selective inhibitor and the active site presentation were shown in Figures 1 and 2.

Liver cirrhosis (3CRK)

Crystal structure of the PDHK2-L2 complex was taken from the data bank of protein. The activity of Pyruvate dehydrogenase complex is regulated by PDHK. PDHK is a mitochondrial protein kinase carries out phosphorylation, which inactivates the entire compound. In humans, four closely-related protein kinases are present: PDHK1, PDHK2, PDHK3, and PDHK4, each have its unique function. PDHK2 is bound to the inner lipoyl-bearing domain of dihydrolipoamide transacetylase (L2) (Tsukamoto et al., 1995). PDHK2-L2 complex structure shows PDHK2 dimer bound to two L2 domains. R domain of PDHK-L2 complex composes of Ser12–Ala183, and the K domain encompasses residues His184–Tyr404. Residues of C-tail of the K domain contribute to the L2-bindind site of the PDHK2 molecule. The binding L2-site of PDHK2 is structured using amino acids of the R domain and one of PDHK2 dimer C-tail as well as amino acids of far carboxyl end present on the C-tail of the neighboring subunit of the dimer (Elbekai et al., 2004). PDHK2 and PDHK3 differ based on their lipoyllysine-binding cavities. These two structures display a novel type II potassium-binding site present on the PDHK2 interface consisting of L2 domain. Potassium ions binding at this interface plays a role in determining the strength of L2 binding. By comparing PDHK2-L2 complex structure with apo-PDHK2 it was found that the rearrangement within PDHK2 structure will affect the L2- and E1-binding sites (Marchette et al., 1969). The selective inhibitor and the active site presentation were shown in Figures 3 and 4.

Figure 1. FAD protein, PDB ID:4XDR with chain A in complex with selective inhibitor. [Click here to view]

Figure 2. FAD in complex with selective inhibitors showing good active site region in asparagines (Asn) and tyrosine (Try). [Click here to view]

Zika virus (5UHY)

Zika virus (ZIKV) is an epidemic disease linked with severe clinical manifestations, such as microcephaly in foetuses of infected pregnant women and Guillian–Barre syndrome in adults. Many antibodies have been synthesized to prevent and control ZIKV infection. This is an infectious ZIKV complex consisting of Fab fragment of a therapeutic and neutralizing monoclonal antibody (Dai et al., 2017). This antibody has shown to reduce fetal infection and demise in mice in which the ZIKV-117 Fabs cross-links the monomers with the surface of E glycoprotein dimers as well as neighboring dimers (Ioos et al., 2014). The ZIKV-Fab surface extends from the surface of ZIKV. Fab site has two-fold, three-fold, and five-fold axes are labeled. It has asymmetric unit and icosahedral two-, three-, and five-fold axes. ZIKV surface has Fab binding sites A, C, and E. The A chains are arranges as dimer surrounding the icosahedral two-fold axis. C and E chains are arranged as general dimer around quasi two-fold axis. Asp67, Gln89, and Lys118 are present on the Fab binding site by mutagenesis (Calvet et al., 2016; Deka et al., 2015). The selective inhibitor was only being notified as shown in Figure 5.

Figure 3. PDHK2 PDB ID: 3CRK with A and B chain in complex with selective inhibitor. [Click here to view]

Figure 4. PDHK2 in complex with selective inhibitors showing good active site region Histone (His) and Trypsin (Trp). [Click here to view]

Treponema pallidum (4XDR)

For the 4XDR active site the binding energy for each ligand, hydrogen bond contacts, and other interactions were tabulated in Table 3. The megastigmatrienone shows the highest binding energy with the active site of 4XDR and docked conformation was shown in Figure 6 in which megastigmatrienone forms hydrogen bonds with ASN 98, ALA 258, ILE 258, ASP 159, and EDO 411 at the active site region of 4XDR protein that is represented as surface image in Figure 7. The binding efficiency with active site 3CRK protein was found to be good and strong and it has the aromatic and anti-bacterial activity against syphilis. The phenolic compound (4-hydroxy-2-methylacetophenome, 4-((1E)-3-Hydroxy-1-propenyl)-2-methoxyphenol), terpenoid compound 6-octen-1-ol,3,7-dimethyl) have found to be more potent with 4XDR through comparative analysis in this docking experiment.

Liver cirrhosis (3CRK)

The energy obtained by docked interactions with bond contacts and alternative interactions were shown in Table 4. The megastigmatrienone compound has the highest binding energy with the active site 3CRK protein and the docked conformations were shown in Figures 7 and 8. The C13 nor-isoprenoid derived from carotenoids (terpene) compound Megastigmatrienone forms hydrogen bonds with TYR 336 at the active site region of 3CRK protein shown in Figures 8 and 9. The binding efficiency with active site 3CKR protein was found to be good and strong aromatic antiviral activity against liver cirrhosis. Thus, based on the molecular interaction analyzed and studied (Green et al., 2014). The phenolic compound (4-hydroxy-2-methylacetophenome, 4-((1E)-3-Hydroxy-1-propenyl)-2-methoxyphenol), terpenoid compound 6-octen-1-ol,3,7-dimethyl) have found to be more potent with 3CKR through comparative analysis in this docking experiment.

Figure 7. Surface structure of Megastigmatrienone. [Click here to view]

Table 2. Lipinski’s rule of five properties for the leaves of E. oxypetalum. [Click here to view]

Table 3. Molecular docking analysis for leaves of E. oxypetalum against syphilis FAD protein (4XDR). [Click here to view]

Table 4. Molecular docking analysis for leaves of E. oxypetalum against liver cirrhosis (3CRK). [Click here to view]

Table 5. Molecular docking analysis for leaves of E. oxypetalum against ZIKV E protein (5UHY). [Click here to view]

Figure 9. Surface structure of Megastigmatrienone. [Click here to view]

Zika virus (5UHY)

For the 5UHY active site, the binding energy for each ligand, hydrogen bond contacts, and other interactions was described in Table 5. The steroid compound Testerone cypionate has the highest binding energy with the active site of 5UHY and docked conformation is in the Figures 10 and 11. The property of the Testerone cypionate is replacing or supplementing the testosterone that is naturally made in the body. The binding efficiency of the 5UHY was found to be good with the bond interaction. Thus, this drug compounds shows better efficiency of the ZIKV with better molecular interactions (Cox et al., 2015). The phenolic compound 4-hydroxy-2-methylacetophenome, 4-((1E)-3-Hydroxy-1-propenyl)-2-methoxyphenol), terpenoid compound 6-octen-1-ol, 3, 7-dimethyl) have found to be more potent with 5UHY through comparative analysis in this docking experiment.

Figure 10. 3D and 2D residual interaction maps of Testeronecypionate against ZIKV. [Click here to view]

Figure 11. Docked confirmation of Testeronecypionate. [Click here to view]

In this current study, docking results revealed the activity of compounds 3CKR, 5UHY, and 4XDR activity against the diseases liver cirrhosis, ZIKV, and Syphilis, respectively. In 3CKR protein active site, three hydrogen bonds are formed that works efficiently against liver cirrhosis due to its aromatic and anti-viral activity against the disease. 5UHY binding worked efficiently along with bond interaction that against ZIKV effectively (Muegge and Rarey, 2001; Saif et al., 2017). Megastigmatrienone had the highest binding energy with the active site of 4XDR. Among all the docked compounds the phenolic compound (4-hydroxy-2-methylacetophenome, 4-((1E)-3-Hydroxy-1-propenyl)-2-methoxyphenol), terpenoid compound 6-octen-1-ol,3,7-dimethyl)) have found to be more potent with 5UHY through comparative analysis in this docking experiment (Abdellatif et al., 2015). Furthermore it satisfies Lipinski’s rule forming the base to use as an oral drug which can be initiated for further studies. In future, in vitro studies will be done for proof reading of the novel drug molecules against liver cirrhosis, ZIKV, and Syphilis.