Molecular modeling of Ruellia tuberosa L compounds as a-amylase inhibitor: an in silico comparation between human and rat enzyme model

Inhibition of α-amylase is an important strategy to control post-prandial hyperglycemia. The present study on Ruellia tuberosa, known as traditional anti-diabetic agent, is being provided in silico study to identify compounds inhibiting α-amylase in rat and human. Compounds were explored from PubChem database. Molecular docking was studied using the autodock4. The interactions were further visualized and analyzed using the Accelrys Discovery Studio version 3.5. Binding energy of compounds to α-amylase was varying between -1.92 to -6.66 kcal/mol in rat pancreatic alpha amylase and -3.06 to -8.42kcal/mol in human pancreatic alpha amylase, and inhibition konstanta (ki) was varying between 13.12- 39460µM in rat and 0.67-5600µM in human. The docking results verify that betulin is the most potential inhibitor of all towards rat model alpha amylase and human alpha amylase. Further analysis reveals that betulin could be a potential inhibitor with non-competitive pattern like betulinic acid. In comparison, betulin has smaller Ki (0.67µM) than acarbose (2.6 µM), which suggesting that betulin is more potential as inhibitor than acarbose, but this assumption must be verified in vitro.

amylase is an important strategy to control post-prandial hyperglycemia. The present study on diabetic agent, is being provided in silico study to identify compounds inhibiting α human. Compounds were explored from PubChem database. Molecular docking was studied using the interactions were further visualized and analyzed using the Accelrys Discovery Studio version 3.5.
1.92 to -6.66 kcal/mol in rat pancreatic alpha amylase and -, and inhibition konstanta (ki) was varying between 13.12-39460µM in rat and 0.67 y that betulin is the most potential inhibitor of all towards rat model alpha amylase and human . Further analysis reveals that betulin could be a potential inhibitor with non-competitive (0.67µM) than acarbose (2.6 µM), which suggesting that betulin is verified in vitro.
Inhibitor of carbohydrate digesting enzyme as alpha amylase is now actively searched for the medicine against diabetes, since it could control postprandial increase of blood glucose amylase, multidomain protein, catalitic triads of Asp197, Glu233, between ligand and catalytic domain can inhibit the enzyme activities.
This research points out the modeling on an interaction between Alpha-amylase and compounds of prandial hyperglycemia. The present study on Ruellia tuberosa, silico study to identify compounds inhibiting α-amylase in rat and human. Compounds were explored from PubChem database. Molecular docking was studied using the autodock4. The Accelrys Discovery Studio version 3.5. Binding energy of compounds -3.06 to -8.42kcal/mol in human 39460µM in rat and 0.67-5600µM in human. potential inhibitor of all towards rat model alpha amylase and human alpha competitive pattern like betulinic acid. In than acarbose (2.6 µM), which suggesting that betulin is more potential as inhibitor glucoronide, apigenin glucoside, apigenin rutinoside, luteolin glucoside, flavone glycoside were 5].
Inhibitor of carbohydrate digesting enzyme as alpha amylase is now actively searched for the medicine against diabetes, since it could control postprandial increase of blood glucose [6]. Αlphaamylase, multidomain protein, has a catalitic (β/α)8-barrel with Asp197, Glu233, and Asp300. The interactions catalytic domain can inhibit the enzyme the modeling on an interaction and compounds of R. tuberosa that has an anti-diabetic activity. The molecular modeling will show an energy binding afinity (Ea), and an inhibition constant (Ki) of the compound.
Alpha amylase inhibitor becomes a part of drug used for diabetes. Although the final target of inhibitor is human pancreatic alpha amylase, it is still common use in vitro or in vivo studies on rat. It would be interesting to see the interaction between inhibitor of rat pancreatic alpha amylase (RPA) and human pancreatic alpha amylase (HPA) using a molecular docking.

Model generation
The Swiss model program was used in order to make a RPA model. SWISS-MODEL workspace (http://swissmodel.expasy. org/) is a web-based integrated service dedicated to protein structure homology modeling [8]. To make a three dimensional protein model, the program uses the protein sequence (model), and a three dimensional structure (template) that has a high enough similarity to the sequence. In this case, porcine pancreatic alpha amylase (PDB ID: 1BVN) with 1.97 Å was used as a template. Their energy forms were minimized, geometrical structure were optimized semi empirically AM1 with conjugate direction algoritm using the HyperChem and were converted to PDB format by the Open Babel 2.3.1. All ligands were prepared to pdbqt format using the AutoDock Tools 1.5.6.

Docking ligand-receptor
All receptors (alpha amylase model and 3OLD.pdb) were prepared with the AudoDock Tools 1.5.6 for docking. Docking (rigid docking with genetic algorithm parameter) was performed with the autodock version 4.2.5.1 [9]. Additional molecules to alpha amylase, except cofactor (Ca 2+ , Cl -) and solvent were deleted prior to the docking using the Accelrys Discovery Studio version 3.5. The bonds in the ligands were set to be rotatable to maximize the flexibility of the ligand. The Autodock Tools is the graphical interface to assign gasteiger charge to reseptor and ligand molecule. The docking box was positioned at x = 8.458, y = -5.795, z = 15.737 with a size of 62x76x66 for 3OLD.pdbqt and x = 37.309, y = 31.28, z = 44.36 with a size of 60x72x74 for RPA model. To validate the docking method that was used, we calculate RMSD between actual pose of the co-crystallized ligand and the redocking co-crystallized ligand (pseudo-pentasaccharide of trestatin family) into their respective binding sites in HPA (ib2y.pdb).
Further interaction analysis was done using the autodock tools and was visualized using the Accelrys Discovery Studio version 3.5. The predicted binding energy (kcal/mol), which indicates how strongly a ligand binds to the receptor, was calculated based on the scoring function used in the AutoDock. A more negative binding affinity indicates stronger binding.
Discussion: Supplementary Figure 1 shows the result of multiple alignment between RPA and HPA sequences. It reveals that rat and human have a high identity (84%) and a similarity (92%). It means that the homology between the two species is very high. However, the rat sequences are shorter than the human sequences. There is a gap in the rat sequence at the position of amino acids 142-144 in HPA.
Since there is no crystal structure of rat enzymes, computer generated model was used in this study. Quality assesment of generated model indicated to be reliable. Identity more than 30% between template and target is sufficient to obtain a reliable model [10]. Rat pancreatic alpha amylase model has a high sequence identity 84,677%. Futhermore the good model show Z-score Q MEAN -0.723 and QMEAN 0.715 with a residual error < 1 Ǻ. The resulting QMEAN z-score provides an estimate of the 'degree of nativeness' of the structural features observed in a model and indicates whether the model is of comparable quality to experimental structures [11]. QMEAN is a scoring fuction consisting of a linear combination of structural descriptor: two distance-dependent interaction potentials of mean force based on C-β atoms and on all atom types are used to assess long-range interactions both are secondary structure dependent; a torsion angle potential; finally, the agreement of predicted and calculated secondary structure and solvent accessibility is included in the form of two agreement terms [12]. QMEAN and agreement terms range from 0 to 1 with higher values for more reliable candidates. Ramachandran plot of RPA model indicates that 96.5% of its residues are situated in the favoured and 3.15% in allowed region. According to this quality assessment results, we believe that this model could be considered to have enough accuracy and biological posibility for further ligand binding studies.
An RMSD of 0.0011 Å was obtained between the best pose obtained by redocking and the actual binding mode of ligand to ib2y.pdb. Futhermore, an RMSD of 0.0185 Å was obtained by redocking betulin to ib2y.pdb and the actual binding mode of ligand to ib2y.pdb. This is satisfactory with regard of less than 2 Å threshold was usually used to assess successful docking [13]. Binding energy and Ki of ligand to HPA and RPA model was shown in table 1 of the suplementary material. Binding energy is vary between -1.92 to -6.66 kcal/mol in RPA and -3.06 to -8.42kcal/mol in HPA. In general betulin is calculated to be the strongest binding to alpha amylase both in RPA (E binding -6.66 kcal/mol, Ki 13.12 µM) and HPA (E binding -8.42 kcal/mol, Ki 0.67µM). These docking results verify that betulin is more efficient ligand and more affinity of all towards alpha amylase in RPA model and HPA. Interestingly, betulin binds stronger in HPA than RPA.
Betulin-alpha amylase complex was shown bellow and B1). Further analysis shows that betulin has vanderwaals interaction with ASN 115, ASN 152, ARG 170, ASP 179, HIS 213 (Figure 1 B2) and hydrogen bond 2.22Å and 2.32 Å with ASP3 in RPA. This interaction is different between betulin and HPA In order to get an approximation of the possible effectiveness of betulin as potential inhibitor to alpha amylase, docking score was obtained for the betulinic acid. Betulin is betulinic acid. From the experimental study, betulinic acid, compound of aqueous extract S cumini's show 98% inhibitory activity to porcine pancreatic alpha amylase with non competitive manner [14]. Molecular docking of Betulin has smaller E binding and Ki value ((E binding -6.66 kcal/mol, Ki 13.12 µM to RPA and (E binding -8.42 kcal/mol, Ki 0.67µM to HPA) than betulinic acid (E binding -6.44 kcal/mol, Ki 18.97 µM to RPA and E binding -7.08 kcal/mol, Ki 6.48 µM to HPA). Futhermore, BIOINFORMATION 211 in RPA model and HPA. Interestingly, betulin binds stronger in alpha amylase complex was shown bellow (Figure 1. A1 that betulin has vanderwaals interaction with ASN 115, ASN 152, ARG 170, ASP 179, HIS 213 ) and hydrogen bond 2.22Å and 2.32 Å with ASP368 different between betulin and HPA (vanderwaals interaction with ASN 100, ASN137, ARG 158, ASP167, ASP 197, HIS201, and hydrogen bond 2.44Å with ASP 300) (Figure 1 A2). Betulin has site both in HPA and RPA. It means that betulin could be a potential inhibitor of alpha amylase. The ligan diferences in alpha amylase are RPA and HPA sequence.

Conclusion:
Overall, betulin is the most compound in Ruellia tuberosa. ). Betulin has an interaction with the catalytic n HPA and RPA. It means that betulin could be a potential inhibitor of alpha amylase. The ligand position are due to gab presence between rat pancreatic alpha amylase complex; B1) Two dimensional diagram shows van der waals interaction between betulin (plus sign) and ASN100 (1. betulinic acid and betulin shows the same interaction to amino mylase Table 1 (see supplementary that betulin could be potential inhibitor competitive pattern like betulinic acid. In comparison, acarbose had Ki around 2.6 µM [15], which suggesting that betulin could be potentially better than acarbose, but this remains to be verified.
most potential α-amylase inhibitor . It suggests the inhibition of pancreatic alpha amylase both in rat and human. The shortening of α-amylase residue in rat enzyme should be highlighted, as it may produce effect in the case of ki, Ebinding, and the interaction between ligand and enzyme. The approximity based on the ki suggesting that betulin is more potential as a inhibitor rather than acarbose with noncompetitive pattern inhibition, but this assumption must be verified in vitro.
Supplementary material: Figure 1: Results of the online blastP alignment for the pancreatic alpha enzymes. Conserved regions are shown in yellow, gap are shown in pink