Insilico Docking Study of the Compounds Identified from the leaves of Orthosiphon stamineus for Hepatoprotective activity

 

Dr C. Maheswari¹, Dr Abdul Mohsen Alrohimi², Dr Amany Tawfik³, Dr Vanathi Meenashi⁴

Dr Zafer Saad⁵

¹Asst Professor in Pharmacy Practice College of Pharmacy-Female Sector Shaqra University, Al-Dawadmi Kingdom of Saudi Arabia

²College of Pharmacy-Male Sector Shaqra University, Al-Dawadmi Kingdom of Saudi Arabia.

³Asst Professor in Pharmacy Practice College of Pharmacy-Male Sector Shaqra University, Al-Dawadmi

Kingdom of Saudi Arabia

⁴ Lecturer, College of Pharmacy- Female Sector Shaqra University, Al-Dawadmi Kingdom of Saudi Arabia

⁵ Dean, College of Pharmacy, Shaqra University, Al-Dawadmi Kingdom of Saudi Arabia.

 

ABSTRACT:

To evaluate the Insilico docking study of the compounds identified from the leaves of Orthosiphon stamieus for Hepatoprotective activity. In the present study, in-silico docking analysis was carried out for the compounds present in leaves of Orthosiphon stamineus in comparison with standard Silymarin against Pregnene X receptor and NF-Κb Receptor. Experiments were performed using the program GLIDE (Grid-based Ligand Docking with Energetic) module version 5.9, Schrödinger, LLC, New York, NY, 2013 (Schrodinger Inc.). Compounds 1,2 and 4 have shown significant Pregnene X receptor inhibitory activity. Of the 3 compounds, compounds 1 and 4 showed maximum Pregnene X receptor inhibition than compound 2 and standard drug Silymarin. Compounds 1,4 and 7 have shown significant NF-Κb Receptor inhibitory activity. Of the 3 compounds , compounds 1 and 4 showed maximum NF-Κb Receptor inhibition than the compound 2 and the standard drug Silymarin. The study showed that the leaves of Orthosiphon stamineus is potential for the hepatoprotective activity by inhibiting Pregnene X receptor( PXR) and Nuclear Factor kappalight- chain-enhancer of activated b cells ( NF-Κb Receptor)

 

 

INTRODUCTION:

Liver is the principal organ related to metabolism and excretion of a wide variety of environmental pollutants and also therapeutic agents. These xenobiotics either directly or indirectly (after bio activation) may exert deleterious effects upon the structural as well as functional integrity of this vital organ, which are exhibited clinically as inflammatory, non-inflammatory or degenerative hepatic disorders.

 

In modern medicine, symptomatic relief is achieved through immunosuppressive agents without any modification in the course of disease. On the other hand, herbal remedies supports natural healing phenomenon through blocking the progression of the degenerative pathological processes. A number of herbal remedies (with claimed liver protecting activity) are marketed throughout the world, however, the information’s regarding scientific validation of these claims with a few exception are not available. Testing the hepatoprotective activity of chemical constituents in leaves of Orthosiphon stamineus by In-silico docking study offer an economical way to get a valuable information on the pharmacological activities of a chemical entity in short duration of time.

 

The pregnane X receptor (PXR) is a Nuclear receptor whose primary function is to sense the presence of foreign toxic substances and in response up regulate the expression of proteins involved in the detoxification and clearance of these substances from the body. PXR plays a critical role in the regulation of human cytochrome P450 3A4 (CYP3A4) gene and is a member of the nuclear receptor (NR) super family, is expressed in liver and intestine, front line organs involved in the absorption, distribution, metabolism and elimination of xenobiotics and endobiotics, in all mammalian species examined to date [6,7] . PXR is mainly associated with the cellular response to xenobiotics, including induction of enzymes involved in drug oxidation and conjugation, as well as induction of xenobiotic and endobiotic transporters [8, 9].

 

The abnormal retention of lipids within the cells results in steatosis, which reflects an impairment of the normal processes of synthesis and breakdown of triglycerides. As the liver is the primary organ of lipid metabolism, steatosis most often occurs in this tissue. The danger of hepatic steatosis is the result of the sequelae, such as liver fibrosis, cirrhosis, and carcinoma. In humans, hepatic steatosis is commonly associated with alcohol abuse or metabolic syndrome (diabetes, hypertension and dyslipidemia), but may also be caused by drugs and certain toxins. Recently, PXR activators were proposed as risk factors for hepatic steatosis. Hepatic lipid accumulation was noted in PXR-humanized mice treated with the human PXR activator rifampicin.

 

 The transcription factor Nuclear Factor kappa-light-chain-enhancer of activated B cells (NF-Kb) is the major regulator of inflammation and cell death leading to liver fibrosis and liver cancer. NF-κB is also implicated in inflammatory bowel disease such as Crohn’s disease and ulcerative colitis. NF-κB activation is evident in biopsies from such patients and treatment of patients with steroids decreases NF-κB activity in biopsies as well as reducing the clinical symptoms of disease. NF-κB is involved in the pathophysiology of the autoimmune disorder rheumatoid arthritis (RA). NF-κB itself is upregulated in RA and cytokines such as TNFα that activate NF-κB are elevated in the synovial fluid of patients with RA. (1-5)

Orthosiphon stamineus Benth known as kidney tea or java tea, is a genus of plants in the Lamiaceae family native to Southeast Asia. It is an herbaceous shrub which grows to a height of 1.5 m (4.9 ft). Orthosiphon is a popular garden plant because of its unique flower, which is white and bluish with filaments resembling a cat's whiskers and leaves has been shown to be a mild diuretic, kidney and bladder stones and infections of the urinary tract . In folk medicine, it is used for the above conditions and also for gout and liver failure (10, 11). A Bioactive compounds has been already reported from the leaves of Orthosiphon stamineus [12,13] and based on the extensive medicinal claims of leaves of Orthosiphon stamineus for the Hepatoprotective activity, the aim of the present study was to investigate the inhibitory activity of the compounds on hepatotoxicity by molecular docking studies and to analyze the ADME/T properties of the compounds such as Eupatorin, Rosmarinic acid, Orthosiphol A, Sinenstein, Cichoric acid, Betulinic acid, 5-Hydroxy- 6,7,3,4-tetramethoxyflavone and β-Sitosterol were used for docking on NF-ΚB Receptor (Nuclear Factor kappalight- chain-enhancer of activated b cells), Pregnane X Receptor (PXR) to confirm the therapeutic effect of the leaves of this plant.

 

MATERIALS AND METHODS:

Software’s handled:

·      GLIDE module version 5.9., mastero 9.4​

·      Quik prop -3.6 -Schrödinger, LLC, New York, NY, 2013- docking (16,17,18)

·      Swiss PDB viewer-4.04 – protein viewer

·      Pymol viewer 1.3 – image viewer

·      Marveen sketch 5.5​ – drawing ligand structure

 

Ligand Structure:

The chemical structure of each ligand was drawn using build module

1. Eupatorin 5. Cichoric acid

2. Rosmarinic acid 6. Betulinic acid

3.Orthosiphol A 7. 5-Hydroxy- 6, 7, 3, 4-tetramethoxyflavone

4. Sinenstein 8. β-Sitosterol

 

 

 

 

 

Ligand Preparation:

In order to prepare high quality, all-atom 3D structures for large numbers of drug-like molecules, starting with the 3D structures in SD Maestro format, Lig Prep was used. Lig Prep produced a single, low-energy, 3D structure with corrected chiralities for each successfully processed input structure.

 

Preparation of protein:

The typical structure file from the PDB is not suitable for immediate use in molecular modelling calculations. A typical PDB structure file consists only of heavy atoms and may include a co-crystallized ligand, water molecules, metal ions, and cofactors. Some structures are multimeric, and may need to be reduced to a single unit. Because of the limited resolution of X-ray experiments, it can be difficult to distinguish between NH and O, and the placement of these groups must be checked. PDB structures may be missing information on connectivity, which must be assigned, along with bond orders and formal charges. This was done using the Protein Preparation Wizard.

 

Ligand Docking:

This is carried out using GLIDE DOCK. Glide searches for favourable interactions between one or more ligand molecules and a receptor molecule, usually a protein. Each ligand acts as single molecule, while the receptor may include more than one molecule, e.g., a protein and a cofactor. Glide was run in rigid or flexible docking modes; the latter automatically generated conformations for each input ligand. The combination of position and orientation of a ligand relative to the receptor, along with its conformation in flexible docking, is referred to as a ligand pose. The ligand poses that Glide generates pass through a series of hierarchical filters that evaluate the ligand’s interaction with the receptor. The initial filters test the spatial fit of the ligand to the defined active site, and examine the complementarily of ligand-receptor interactions using a grid-based method patterned after the empirical Chem-Score function. Poses that passed these initial screens entered the final stage of the algorithm, which involves evaluation and minimization of a grid approximation to the OPLS-AA non bonded ligand-receptor interaction energy. Final scoring is then carried out on the energy-minimized poses. (14)

 

Glide Extra-Precision Mode (XP):

The extra-precision (XP) mode of Glide combines a powerful sampling protocol with the use of a custom scoring function designed to identify ligand poses that would be expected to have unfavourable energies, based on well-known principles of physical chemistry. The presumption is that only active compounds will have available poses that avoid these penalties and also receive favourable scores for appropriate hydrophobic contact between the protein and the ligand, hydrogen-bonding interactions, and so on. The chief purposes of the XP method are to weed out false positives and to provide a better correlation between good poses and good scores. Extra-precision mode is a refinement tool designed for use only on good ligand poses. Finally, the minimized poses are re-scored using Schrödinger’s proprietary Glide-Score scoring function. Glide-Score is based on Chem-Score, but includes a steric-clash term and adds buried polar terms devised by Schrodinger to penalize electrostatic mismatches:

 

Glide Score = 0.065*vdW + 0.130*Coul + Lipo + Hbond + Metal + BuryP + RotB + Site

 

Docking Procedure:

Docking studies of 8 compounds were performed using 2 proteins obtained from the RCSB Protein Data Bank, http://www.rcsb.org/pdb.(15) Experiments were performed using the program GLIDE (Grid-based Ligand Docking with Energetic) module version 5.9, Schrödinger, LLC, New York, NY, 2013 (Schrodinger Inc.)^16,17,18. Coordinates of the full-length substrate-complexed dimmer were prepared for Glide 5.9 calculations by running the protein preparation wizard. The p-prep script produces a new receptor file in which all residues are neutralized except those that are relatively close to the ligand (if the protein is complexed with a ligand) or form salt bridges. The impref script runs a series of restrained impact energy minimizations using the Impact utility. Minimizations were run until the average root mean square deviation (rmsd) of the non-hydrogen atoms reached 0.3Å. Glide uses two boxes that share a common centre to organize its calculations: a larger enclosing box and a smaller binding box. The grids themselves are calculated within the space defined by the enclosing box. The binding box defines the space through which the centre of the defined ligand will be allowed to move during docking calculations. It provides a measure of the effective size of the search space. The only requirement on the enclosing box is that it be large enough to contain all ligand atoms, even when the ligand centre is placed at an edge or vertex of the binding box. Grid files were generated using the co-crystallized ligand at the centre of the two boxes.

 

RESULTS AND DISCUSSION:

ADME properties of the ligands:

The ADME properties of the ligands were predicted using QikProp The compounds prepared were subjected to drug-likeness filter. The acceptance criteria of the filter includes Molecular weight (< 500), QPPMDCK (500 good , Q Plog BB (-3.0 to 1.2), Donar HB (0-6), Metabolism (1-8), Accept HB (2-20), Log P Value o/w (-2.0 to 6.5), CNS (-2 to +2), % Human Oral Absorption ( 80% high < 25-poor, > 500-Good), PSA (7-200), Q P log S (-6.5 to 0.5), Violation of Rule Of Three ( Max 3). All the ligands confirmed to the above mentioned acceptance criteria and they were evaluated for docking using GLIDE software and the results are presented in table 3.

 

Pregnene X Receptor and NF-Kb Receptor:

In the present study, in-silico docking was carried out for the compounds present in Orthosiphon stamineus in comparison with standard Silymarin against Pregnene X receptor and NF-Kb receptor. Compound 1,2 and 4 have shown significant Pregnene X receptor inhibitory activity. Of the 3 compounds , compounds 1 ( Figure 1 a and b ) and 4 ( Figure 2a and b) showed maximum Pregnene X receptor inhibition than compound 2 and std drug silymarin ( Figure 3a and b). Compounds 1,4 and 7 ( Figure 4a and b, 5a and b, 6a and b ) have shown significant NF-Κb Receptor inhibitory activity. Of the 3 compounds, compounds 1 and 4 showed maximum NF-Κb Receptor inhibition than the compound 2 and the standard drug silymarin (Figure 7a and b) and the docking results are presented in table 1,2. However, the study showed that the leaves of Orthosiphon stamineus is potential for the hepatoprotective activity by inhibiting Pregnene X receptor

 

Docking images (a) and 3D images (b) of designed compounds showing maximum activity against Pregnene X Receptor (PXR)

 

 

Compound 1-a (PXR)

 

 

Compound 1-b( PXR)

FIGURE-1

StdSilymarin-b)(NF-Kb)

FIGURE 7

Table 1. Summary of GLIDE result of ligands against pregnene X receptor for Hepatoprotective activity

 

Table 2. Summary of GLIDE result of ligands against NF-κB for Hepatoprotective activity

 

Table 2. Cont..

 

Table-3. Pharmacokinetic Parameters with their Optimum Range Important for CNS Activity and oral Bioavailability Obtained by qikprop tool

 

 

Table 3. Cont..

 

 

 

 

CONCLUSION:

Based on the docking analysis, the study concluded that the leaves of Orthosiphon stamineus is potential for the Hepatoprotective activity by inhibiting Pregnene X Receptor and NF-Kb receptor with better ADMET profiles.

 

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Received on 26.12.2015         Modified on 20.05.2016

Accepted on 20.06.2017      © RJPT All right reserved