In silico studies on pancreatic lipase and cholesterol esterase inhibitor 2,6-di-tert-butyl phenol: A Novel molecule for Antiobesity

 

Srirekha Pandian¹, S. Narendar Sivaswamy², Waheeta Hopper¹*

¹Department of Biotechnology, School of Bioengineering, Faculty of Engineering and Technology,

SRM Institute of Science and Technology, Kattankulathur 603203, Tamil Nadu, India.

²Synkromax Biotech Pvt. Ltd; 2^nd Floor, SIDCO Multi Storeyed Complex, Thirumazhisai,

Chennai 600124, Tamil Nadu, India.

 

ABSTRACT:

Obesity is considered as one of the most important risk factors for atherosclerosis and associated cardiovascular diseases. Bioactive compounds extracted from plants and microbial sources are increasingly became attractive alternatives to combat these conditions. The present study is an attempt to evaluate the inhibitory activity of 2,6-di-tert-butylphenol towards pancreatic lipase and cholesterol esterase using in silico docking studies. In silico work was carried out using Autodock 4.2, based on the Lamarckian genetic algorithm principle. The inhibitor has a Binding energy of –4.28Kcal/mol, Inhibition constant 727.08uM and Intermolecular energy as -5.04 Kcal/mol towards pancreatic lipase. Towards cholesterol esterase, the Binding energy of the inhibitor was –5.21 Kcal/mol, Inhibition constant 150.59uM and Intermolecular energy as – 6.0Kcal/mol. So, 2,6-di-tert-butyl phenol could be a potent inhibitor for both pancreatic lipase and cholesterol esterase.

 

 

 

INTRODUCTION:

Obesity is considered to be an epidemic disorder leading to many chronic health problems related to cardiovascular diseases. The increasing rates of obesity are associated with diabetes mellitus, liver malfunctioning, reproductive and gastrointestinal diseases¹. Obesity is primarily considered to be a disorder of lipid metabolism and the enzymes involved in this process could be selectively targeted to develop antiobesity drugs². The pivotal enzymes are pancreatic lipase and cholesterol esterase, which are involved in the digestion and absorption of lipids as well as dietary cholesterol. Pancreatic lipase is a crucial enzyme which is synthesized and secreted by the pancreas. It plays an important role in the effective digestion of triglycerides into unesterified fatty acids and monoglycerides. The unesterified fatty acids and monoglycerides produced, will combine with bile salt, cholesterol, and lysophosphatidic acid to form micelles.

 

Once absorbed by the intestine, they are resynthesized into triacylglycerides and gets stored in the adipose tissues as a major source of energy for the human body³. Inhibition of pancreatic lipase and cholesterol esterase is one of the most important mechanisms in determining the potential efficacy of natural products as anti-obesity agents. Natural products provide a vast pool of inhibitors that can possibly be developed into clinical products. Tetrahydrolipstatin, a hydrogenated analogue of lipstatins isolated from Streptomyces, an actinomycetes, is an inhibitor of gastrointestinal lipases and is commercially marketed as orlistat. This enzyme acts on the nonpolar triacylglycerols in the lumen of the intestine and thereby plays a major role in the absorption of dietary fats⁴. Human pancreatic cholesterol esterase also known as bile salt-activated lipase is involved in the digestion of broad spectrum of substrates including triacylglycerides, phospholipids, cholesterol esters, esters of lipid-soluble vitamins and fatty acids. Cholesterol esterase belongs to the family of α/β-hydrolyase fold family. They share common secondary and tertiary structural characteristics and utilizes Ser194, Asp320, and His435 catalytic triad mechanism. The cholesterol esterase, ser­ine lipases and serine proteases possess a conserved Ser-Asp-His catalytic triad and share the same catalytic mechanism. So, these enzymes are considered to be prime enzymes that could be targeted towards the development of anti-obesity therapeutics. The phenolic compounds and its derivatives especially branched tert butyl groups are rare in its occurrence, due to the presence of functional group on the aromatic ring, they are considered to be natural products of plants, fungi, algae and bacteria possessing bioactivities which include antitumor, anticancer, antioxidant and antibacterial activity⁵. 2,6-di-tert-butylphenol is an organic compound with the structural formula 2,6-((CH₃)₃C)₂C₆H₃OH. It is a colorless solid alkylated phenol and its derivatives are used in industries as UV stabilizers and antioxidants⁶. Also, they are used as inhibitors of free radicals’ formation in the oxidative destruction of natural and synthetic substrates, their mode of action is associated with the stable phenoxyl radical formation⁷. Drug designing or bioinformatics is an inventive process of finding new medications based on the knowledge of the biological target⁸. It plays a key role in all dimensions of drug discovery, drug assessment and drug development. It has been intensively utilized for the development of new drugs against several dreadful diseases⁹. So the main aim of the present investigation is to study the in silico inhibitory effect of 2,6-di-tert-butyl phenol on pancreatic lipase and cholesterol esterase activity using molecular docking studies, Also the binding affinity of commercial drug lipstatin with the targeted enzymes, pancreatic lipase and cholesterol esterase has been compared. Molecular docking is a way to predict the preferred orientation of one molecule to another when bound to each other to form a stable complex¹⁰. The current research is an attempt to develop and design enzyme inhibitors as a target for antiobesity drug¹¹. In this study Molecular dynamics (MD) simulations were performed to obtain the conformational changes upon the binding of the ligand that are structurally compatible with the active site of the enzyme. Conformational analysis of molecule is based on molecular mechanics, it is a method for the calculation of molecular structures, conformational energies and other molecular properties using concept from classical mechanics¹². Molecular docking results were also used as final filter to assess the binding behavior of the identified compound.

 

MATERIAL AND METHODS:

Dataset:

The Protein- Ligand interaction plays an important role in structural based drug designing¹³. The datasets used for the current study on inhibition of query ligand are two drug targets namely the enzyme cholesterol esterase from human and Pancreatic lipase from pig. Three-dimensional structure of cholesterol esterase from human and Pancreatic lipase from pig was downloaded from PDB with PDB code 1F6W and 1ETH respectively (Fig.1 a, b). In silico analysis of the protein structure of drug targets were prepared by using chain A and other chains of water molecules and hetero atoms were removed from the structure. Both ligands, Lipstatin as well as its analogue tetrahydrolipstatin (Fig. 2 a, b), are commercially available drug for antiobesity, they are compared with the identified compound 2,6-di-tert-butylphenol for its inhibitory activity against Pancreatic lipase and cholesterol esterase (Fig. 3 a, b) the structures were downloaded from the Pubchem database in SDF and converted into PDB file using Open Babel software. The structure was optimized using ACD- Chemsketch software.

 

Molecular Docking:

The software AutoDock 4.2 was used for molecular docking of target ligand in order to explore the binding conformation and hydrogen bond interactions of the ligand with the target proteins. For the molecular docking, a grid spacing of 0.375 Å, Grid box center: X=63.996, Y= 29.156, Z= 125.427 and Grid box size: X=126, Y=126, Z= 124 was used. Autodock has generated 10 possible binding conformations, i.e. 10 runs for each docking by using Genetic Algorithm (GA-LS) searches. A default protocol was applied, with population size of 150 randomly placed individuals, a maximum number of 2.5 x 105 energy evaluations, and a maximum number of 2.7 x 104 generations, gene mutation rate of 0.02 and crossover rate of 0.8 were used. The docking results of target ligands were evaluated using binding energy, inhibition constant and hydrogen bond interactions. All visualization of docking results was analyzed using Discovery Studio.

 

Fig. 1: 3D structure of drug targets (a) cholesterol esterase from human PDB code 1F6W and (b) Pancreatic lipase from pig PDB code: 1ETH

 

Fig. 2: Commercial drug lipstatin (a) and its derivative (b) tertahydrolipstatin

Fig. 3: Ligand 2,6 diterbutyl phenol (a) 3D structure (b) 2D structure

 

RESULT:

Docking of commercial drug lipstatin with Pancreatic lipase:

The docking results have shown that there are three hydrogen bond interactions between the amino acid residues HIS152, GLY77 and SER153 of the pancreatic lipase to the ligand lipstatin. The atom ND1 of HIS152 and O3 of lipstatin was bonded through hydrogen bond with a length range of 2.94 Å. N atom of GLY77 and O3 atom of drug lipstatin bonded through hydrogen bond with length range of 2.99 Å and atom CB of SER153 and O4 of drug lipstatin was bonded through hydrogen bond with length range 2.90 Å. Thus, the three hydrogen bond interactions between pancreatic lipase and lipstatin favored the inhibition of the enzyme. In addition, hydrophobic interaction were also observed between lipase and the drug through amino acid residues, which include LA179, PHE78, TYR115, PRO181, PHE216, LEU265, ARG257, HIS264, HIS76, ASP80, VAL260, ALA261, ILE79, TRP253 (Fig 4 a).

 

Docking of Tetrahydrolipstatin with Pancreatic lipase:

The docking results showed that three amino acid residues HIS152, GLY77 and SER153 of pancreatic lipase are involved in hydrogen bond interactions with tetrahydrolipstatin. The atom ND1 of HIS152 and O3 of drug was bonded through hydrogen bond with length range 3.05 Å. N atom of GLY77 and O3 atom of drug tetrahydrolipstatin. was bonded through hydrogen bond with length range 3.09 Å and atom O6 of SER153 and O4 of drug was bonded through hydrogen bond with length range 2.70 Å. Thus, the three hydrogen bond interactions between pancreatic lipase and tetrahydrolipststin favored the inhibition. In addition, hydrophobic interactions were also observed between lipase and tetrahydrolipstatin through amino acid residues, ILE210, ALA179, PHE78, TYR115, PRO181, PHE216, LEU265, ARG257, HIS264, HIS76, ASP80, VAL260, ALA261, ILE79, TRP253 (Fig.4b).

 

Docking results of commercial drug lipstatin with Cholesterol esterase:

The docking results have shown that one amino acid THR354 residue of cholesterol esterase was involved in the hydrogen bond interaction, and the bond is between the atom N of THR354 and O2 of lipstatin with a bond length range of 3.15 Å. Thus, the one hydrogen bond interaction between esterase and lipstatin has favoured the inhibition through potential weak interaction. In addition, hydrophobic interactions were also observed between esterase and lipstatin through amino acid residues, TYR256, TRP522, ILE391, LEU351, ILE395, PRO396, LEU392, VAL353, PRO280, THR352, PRO226, HIS283, LEU282, TYR279, GLY350, TRP227, GLU230, ILE229, LEU392 (Fig.5a).

 

Docking results of Cholesterol esterase with tetrahydrolipstatin:

There was no hydrogen bond interactions observed between cholesterol esterase and tetrahydrolipstatin. Thus, esterase was not inhibited by the lipstatin derivative tetrahydrolipstatin. But some hydrophobic interactions were observed which favored the inhibition of esterase by certain amino acid residues, LEU392, TRP227, LEU282, LEU351, TYR526, LEU224, PRO226, ILE391, LYS355, ILE395, GLU527, and PRO396 (Fig.5b).

 

Fig. 4: and 5 Molecular Docking results of pancreatic lipase 4(a)and(b) above  and cholesterol esterase 5(a) and (b) below with Lipstatin and its derivative Tetrahydrolipstatin.

 

Molecular docking result of pancreatic lipase with novel inhibitor 2,6- di- tert-butylphenol:

Inhibition of pancreatic lipase by query ligand 2,6- di- tert-butylphenol was studied using molecular docking based on active site docking. The pancreatic lipase from pig was inhibited by 2,6 di tertbutyl phenol with least binding energy -4.28kcal/mol and other favorable energy terms. The active site pocket of the enzyme was well covered by the ligand. The docking results showed that one hydrogen bond interaction was involved using amino acid residue SER334. The atom O of SER334 and O1 of the inhibitor was bonded through hydrogen bond with a length range of 2.61 Å. Thus, the single hydrogen bond interaction between lipase and the inhibitor favored moderate inhibition. In addition, hydrophobic residues like PHE336, ASP273, ASN89, ALA272, ASN93 and LYS269 were also observed between lipase and the inhibitor, 2,6- di- tert-butylphenol. Also, the inhibition constant was low to favor the inhibition of target protein with 727.08 µM. Thus, the target pancreatic lipase was moderately inhibited by the query ligand (Fig.6).

 

Fig. 6: Molecular docking result of pancreatic lipase with novel inhibitor 2,6 di tertbutyl phenol

 

Binding energy: -4.28 kcal/mol, Inhibition constant: 727.08 µM, Intermolecular energy: -5.04 kcal/mol, Vdw_hb_desolvation energy: -4.93 kcal/mol, Electrostatic energy: -0.11 kcal/mol

 

Molecular docking of cholesterol esterase with 2,6 –di- tert-butyl phenol:

The inhibition of Cholesterol esterase was studied using molecular docking based on active site docking. The cholesterol lipase from human was inhibited by 2,6 di tertbutyl phenol with least binding energy -5.21kcal/mol and other favorable energy terms. The active site pocket was well covered by the ligand. The docking results showed that two hydrogen bond interactions were involved between HIS435 and SER194 residues of cholesterol esterase and ligand. The atom NE2 of HIS435 and O1 of ligand was bonded through hydrogen bond with length range 3.21 Å and atom OG of SER194 and O1 of ligand was bonded through hydrogen bond with length range of 2.52 Å. Thus, the two-hydrogen bond favored the inhibition between cholesterol esterase and 2,6- di tert-butylphenol. In addition, hydrophobic interactions were also observed between esterase and the ligand molecule through amino acid residues GLY107, PHE324, ALA108, TRP227, PHE393 and ALA195. The inhibition constant was found to be 727.08µM (Fig.7).

 

Fig. 7: Molecular docking of cholesterol esterase with 2,6 di tertbutyl phenol:

 

Grid boxcenter: X=1.581, Y= 8.301, Z= 12.885 Å, Grid box size: X=126, Y=126, Z= 124

 

Binding energy: -5.21 kcal/mol, Inhibition constant: 150.59 µM, Intermolecular energy: -6.0kcal/mol, Vdw_hb_desolvation energy: -6.0 kcal/mol, Electrostatic energy: 0kcal/mol

 

DISCUSSION:

The advancement of Computer-assisted drug design (CADD), also called computer-assisted molecular design (CAMD), represents the most recent applications of computer-based tools in the drug designing process. Design and development of new drugs are simplified and made more cost-effective because of these advances¹⁴. The applications of CADD, are to find a ligand (the putative drug) that will interact favorably with a receptor that represents the target protein, Binding of ligand to the receptor may involve hydrophobic, electrostatic, and hydrogen-bonding interactions.^15,16 Using this computer simulation techniques, it is now possible to study the interaction of the ligand and enzyme for elucidating the binding energy. This process provides in-depth understanding about the inhibition strength of various ligands¹⁷. Virtual screening (VS) is also one of the prime method followed for hit identification and lead optimization steps^18,19. In comparison to other methods VS found to be more direct, is a rational approach, economic and effective drug screening methodology than the experimental high-throughput screening (HTS)^20,21. Molecular docking is a powerful in-silico tool in structural molecular biology and computer-assisted drug design. The goal of ligand—protein docking is to predict the predominant binding mode (s) of a ligand with a protein of known three-dimensional structure²². Molecular docking requires the information of binding cavity or active site²³. In case, if the binding cavity information is missing, the other alternative is blind docking approach, which is implemented for the identification of same with the use of “reverse docking” protocol²⁴. The two drug targets namely Cholesterol esterase (Human) and Pancreatic lipase (Pig) were selected for the in-silico study. In case of Human cholesterol esterase, the experimental structure determined the catalytic domain and structure, which was well characterized with the catalytic triad, hydrolase fold and loops containing substrate binding cavity²⁵. In case of Pig Pancreatic lipase, the experimental structure determined the lipase active site and deciphered the functional aspects of the protein conformation²⁶. The identified compound 2,6-di-tert-butylphenol was used for the molecular docking analysis. The molecular docking results for the drug targets, Cholesterol esterase (Human) and Pancreatic lipase (Pig) were performed and analyzed by binding energy and weak interactions. In chemistry, the weak intermolecular interactions namely hydrogen bond interactions and hydrophobic interactions play a critical role in key process like formation of protein-ligand (drug) complexes and its stabilization²⁷. Due to weak interactions between protein-ligand binding, the drug efficacy has improved in case of complex²⁸. Hydrogen bond is defined as the bond between H-donor and H-acceptors from both protein and ligands^29,30. In case of Human cholesterol esterase, the results showed the protein-ligand complex formation with two hydrogen bonds acts as critical binding and interaction favored the inhibition mechanism. Two residues SER194 and HIS435 in the substrate binding cavity of the cholesterol esterase confirmed as key residues in the complex formation. Also, more number of residues involved in hydrophobic interactions favored the complex with further strong binding between protein and ligand. In case of pig pancreatic lipase, the results showed the protein-ligand complex formation with one hydrogen bond, which acts as critical binding and interaction favored the inhibition mechanism. Residues SER334 in the substrate binding cavity of the pancreatic lipase confirmed as key residue in the complex formation. Also, hydrophobic interactions with few residues favored the complex with further strong binding between protein and ligand. Hence, the screened phenolic compound 2,6-di-tert-butylphenol confirmed the effective inhibition of the target protein pancreatic lipase and cholesterol esterase and can acts as a novel lead compound for the development of antiobesity drugs.

 

ACKNOWLEDGMENT:

The authors are thankful to the Head, Department of Bioinformatics, Vels University Chennai, for providing the necessary facilities and equipment for conducting the purpose of the study.

CONFLICT OF INTEREST:

There is no conflict to declare.

 

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Received on 09.03.2020          Modified on 21.04.2020

Accepted on 28.05.2020         © RJPT All right reserved