IN SILICO MOLECULAR DOCKING AND ADME/T STUDIES OF SELECTED COMPOUNDS OF SWERTIA CHIRATA (GENTIANACEAE) AGAINST TWO RECEPTORS OF TYPE 2 DIABETES

Md. Nazmul Hasan 1 , Arkajyoti Paul 2,3 , Ramiz Ahmed Sultan 1 , Md. Tanveer Ahsan 1 And Mohammed Kamrul Hossain 1 . 1. Department of Pharmacy, Faculty of Biological Sciences, University of Chittagong, Chittagong-4331, Bangladesh. 2. Department of Pharmacy, Atish Dipankar University of Science and Technology, Dhaka-1230, Bangladesh. 3. Drug Discovery, GUSTO A Research Group, Chittagong, Bangladesh. ...................................................................................................................... Manuscript Info Abstract ......................... ........................................................................ Manuscript History Received: 12 April 2019 Final Accepted: 14 May 2019 Published: June 2019


ISSN: 2320-5407
Int. J. Adv. Res. 7 (6), 589-596 590 generation, or the body cells don't react legitimately to insulin to lower blood glucose, or the both (Boulton et al., 2005;Woodrow, 2011). Improper insulin level in the body results problems in glucose metabolism which in turn badly affects the body system, particularly blood vessels and nerves. Globally around 422 million individuals (roughly 8.5% of adults) are currently experiencing this disease and the number is on the rise with time (Roglic & World Health Organization, 2016). Diabetes has become an intense medical issue with a substantial financial weight to every nation. Several groups of oral hypoglycemic agents are on the use to manage diabetes which are not devoid of characteristic profiles of side effects (Li et al., 2012;Pickup & Williams, 1991). Thus management of diabetes with fewer side effects is still a big arena where challenges are huge to the medical system. This leads to emerging demand for natural products with anti-diabetic activity as historically they have less side effects. Among the many ethno-pharmacologically used anti-diabetic plants Swertia chirata (Family: Gentianaceae) is a good choice as it has been used to control diabetes for a long time (Chandrasekar, Bajpai, & Mukherjee, 1990). Also this plant has hepato-protective (Karan, Vasisht, & Handa, 1999), anti-oxidant (Balasundari, Singh, & Kavimani, 2005), anticarcinogenic (Saha & Das, 2010) and anti-inflammatory activities (Shivaji, Tapas, Suvra, Prabhash, & Sridhar, 2000). Several phytochemicals have been isolated from S. chirata which includes Mangiferin, Swertiamarin, Chiratenol, Episwertenol, Swertanone, Swertenol, Taraxerone, Amarogentin, Amaroswerin and Sweroside.
Pancreatic Alpha-amylase (PDB ID: 1PPI) is the main amylase enzyme found in humans as well as other mammals. This enzyme is involved in the hydrolysis of alpha bonds of large, alpha-linked polysaccharides, such as starch and glycogen, yielding glucose and maltose. It catalyzes the starting step of starch hydrolysis to glucose production and thus is a key enzyme for energy production. For this reason, alpha-amylase has become the target molecule of drug development for type 2 diabetes mellitus (T2DM) treatment. Also its inhibitors and its relationship with the disease have been extensively investigated (Nickavar & Abolhasani, 2013;Yadav, Bhartiya, Verma, & Nandkeoliar, 2013). Fructose-1,6-bisphosphatase (PDB ID: 2JJK) is a rate controlling enzyme which play major role in gluconeogenesis process (Erion et al., 2005;Hebeisen et al., 2008) and has been the prime target for developing new medicines for T2DM treatment. Endogenous glucose production can be directly influenced by controlling the Fructose 1,6bisphosphatase enzyme which exerts a unique strategy in treatment of diabetes (Aicher, Boyd, McVean, & Celeste, 2010).
Molecular docking is a fundamental instrument in the virtual screening of new novel compounds that can be potential for treatment of complex disorders. Docking method is a more extensive way of searching novel therapeutically active compounds as it gives molecular level data of ligand-receptor interactions. This is an accurate, fast and cost effective method in the process of drug discovery minimizing the risks of animal trials. Thus the aim of current study is to discover novel compounds against type 2 diabetes through molecular docking interactions and to analyze their ADME/T properties for a safer and effective anti-diabetic medication.

Materials and methods:-Preparation of receptors
Three dimensional crystal structures of Alpha amylase (PDB ID: 1PPI) and Fructose-1,6-bisphosphatase (PDB ID: 2JJK) involved in T2DM were downloaded in pdb format from the RCSB Protein Data Bank (www.rcsb.org). Structures of the protein targets were prepared and refined using the Protein Preparation Wizard of Schrödinger-Maestro v10.1. Charges and bond orders were assigned, hydrogens were added to the heavy atoms, zero order bonds were created to metals, disulfide bonds were created, selenomethionines were changed to methionines, missing side chains and missing loops were filled using Prime and all waters were eradicated that were beyond 5Å from het groups. Using force field OPLS_2005, minimization was finished converging heavy atoms to 0.30 Å RMSD (rootmean-square-deviation).

Molecular properties and ligand based ADME/T analysis
The molecular properties of compounds play crucial role on the selection of these agents as potential drug candidates. Lipinski's rule of five (RO5) is a useful parameter to evaluate molecular properties of drug compounds for estimation of important pharmacokinetic parameters for drug design and development (Ertl, (RO5) filter was applied to screen the ligands as drug candidates, which states that the compound has more permeability and passive absorption if it does not violate more than one of the following conditions: (i) molecular weight (acceptable range: <500); (ii) hydrogen bond donor (acceptable range: ≤5); (iii) hydrogen bond acceptor (acceptable range: ≤10); (iv) high lipophilicity (expressed as log P o/w , acceptable range: <5); and (v) molar refractivity should be between 40 and 130. The canonical SMILES for each compound is retrieved from PubChem and analysis was done through swissADME online database (Daina, Michielin, & Zoete, 2017).

Receptor grid generation
As each ligand should bind to a specific active site of the receptor, it is essential to have a computed receptor grid of arranged amino acid residues of proteins for fitting different ligands inside the anticipated active site during docking. Grids were created keeping the default parameters of van der Waals scaling factor 1.00 and charge cutoff 0.25 subjected to OPLS_2005 force field. A cubic box of was generated around the active site of receptor which was defined by the co-crystallized ligand. The box was generated to each direction with a measurement of 14 Å × 14 Å

Glide standard precision (SP) ligand docking
Glide standard precision (SP) ligand docking was performed to analyze the ligand bindings with the receptors. SP docking was performed in Glide of Schrödinger-Maestro v10.1, inside which penalties were connected to noncis/trans amide bonds. In this docking protocol the ligands are allowed to be flexible while receptor is fixed. Van der Waals scaling component and fractional charge cutoff was chosen to be 0.80 and 0.15, respectively for ligand atoms. Epik state penalties were added to docking scores and post docking minimization was performed. Final scoring was performed on energy minimized poses and showed as Glide score. Ligand poses having the least Glide score was considered as the best docked mode and recorded for each ligand. Finally the best docked poses were further analyzed for 3D binding interactions with amino acid residues using Biovia Accelrys Discovery Studio Visualizer (BIOVIA, 2017) software.

Results:-
This current study was performed to evaluate the binding affinity and binding pose of compounds isolated from the traditionally used medicinal plant S. chirata, for finding compounds that are potential for therapeutic activity in diabetes. Molecular docking approach of Schrodinger suite v10.1 was used to find docking score and interactions with the binding site residues. Molecules were screened for their molecular and ADME/T properties prior to docking study to validate them as potential therapeutic targets. Molecular properties of all the ligands screened through Lipinski's RO5 revealed that all compounds were within the acceptence range except Amarogentin, Amaroswerin and Mangiferin (Table 1) and these compounds were discarded from further docking studies.  Among the compounds Swertiamarin and Sweroside showed highest docking scores against 1PPI and 2JJK respectively ( Table 2). Swertiamarin showed best docking score (-6.803) among the ligands against 1PPI which showed both hydrogen bond interaction (GLN63, HIS299, ARG195, ASP197, ASP300, GLU233, HIS305) and hydrophobic interaction (HIS305) with the Pancreatic Alpha-Amylase enzyme (Figure 1). Sweroside showed highest score (-5.161) against 2JJK with hydrogen bond interaction (GLY26, GLY28, LYS112, GLU29, TYR113, ALA24) and hydrophobic interaction (LEU30, ALA24) with Fructose-1,6-bisphosphatase enzyme ( Figure 2). Other compounds showed docking scores ranging from -4.593 to -3.359 for Pancreatic Alpha-Amylase and -5.055 to -3.077 for Fructose-1,6-bisphosphatase enzyme.

Conclusion:-
Finding newer compounds with less side effects and optimum therapeutic activity is an urgent need in drug development process. The present study expresses molecular docking of important compounds from S. chirata, a traditionally used anti-diabetic medicinal plant against two receptors involved in diabetes (Pancreatic Alpha-Amylase and Fructose-1,6-bisphosphatase). The docking scores and binding poses of Swertiamarin and Sweroside evaluates them as suitable lead molecules for anti-diabetic drugs. Further, extensive quantitative structure activity relationship model is required to ensure its efficacy and safety inside animal models. The results suggest that this plant may be an emerging source for the development of new anti-diabetic drugs.