LIST OF TABLES
6.3. Results and discussion
6.3.5. Molecular docking of α-amylase
Docking simulation allowed us to understand the most potential interaction and binding affinity between ligands and the target receptor. The best docked poses of ligands with best binding affinity for HPA at the active site were selected and shown in Fig. 6.7. After post- docking analysis, best docked poses were selected on the basis of MolDock (MD) score and re-rank score as depicted in Table 6.5. Best MD score and re-rank score was obtained for II compared to I, where, the known inhibitor (acarbose) showed highest MD score and re-rank score among all the ligands under study. MD score adopted here basically depends on a
piecewise linear potential (PLP) introduced by Gehlhaar et al. (1995, 1998) which was further advanced by Yang and Chen (2004) in GEMDOCK. Furthermore, a heuristic algorithm was employed as scoring function which considers the directionality of hydrogen bonding with a re-ranking method to enhance the docking accuracy. Here, the re-rank score refers to a linear combination of E-inter (hydrogen bonding, electrostatic, steric, van der Waals) between the ligand and the receptor, and E-intra (hydrogen bonding, electrostatic, torsion, sp2-sp2, van der Waals) of the ligand weighted by pre-defined coefficients (Thomsen and Christensen 2006). Ligand-receptor interaction energy was calculated for each ligand and corresponding best poses were selected. The interaction energy is mainly based on both electrostatic and H-bond energy between ligand and receptor towards better understanding of the binding mode (orientation) of each ligand and possible structure-activity relationship with receptor.
In case of acarbose, the best binding confirmation was found inside the active site cleft of HPA (Fig. 6.8A) which makes 10 H-bonding interactions with various key residues of HPA as shown in Fig. 6.8B. Within the active side cleft of HPA, three important residues viz.
Asp300, Glu233 and Asp197 are present which are principally responsible for hydrolysis of glycosidic bonds in carbohydrates. Previous report also suggested the critical functions and interactions of these residues with other inhibitors like myricetin and ethyl caffeate (Williams et al. 2012). Here, H-bonding and electrostatic interaction of acarbose within HPA active site are shown in Fig 6.8B and 6.8C respectively.
Fig. 6.7 Superimposed confirmations of best docked poses for respective ligands in the HPA active site. Acarbose, compound I and II were shown in blue, green and yellow colour respectively.
Fig. 6.8 Ligand-protein interaction for the best pose at the major binding cleft and interactive nearby residues were depicted for acarbose. (A) Predicted bonded and non-bonded interactions between acarbose and amino acid residues present in the active site of HPA. (B) Predicted ten H-bonded interaction between acarbose and the residues at the active site region. Here, blue dotted lines represent the H-bonding interactions between the ligand molecules and HPA. (C) Predicted non-bonded electrostatic interaction between acarbose and the residues at the active site region.
A
B
C
Both the compounds (I and II) were also docked with HPA where compound II showed higher MD and re-rank score as compared to compound I (Table 6.5). The predicted interactions between both the compounds and active site residues of HPA are shown in Fig.
6.9A and 6.9B. Notably, in these bound conformation, both the compounds showed bonded and non-bonded interactions with various key residues in the active site of HPA having two H-bond interactions. The H-bonding interaction of both the compounds (I and II) with HPA is shown in Fig. 6.10A and Fig. 6.10B where II were bonded with Arg195 and Asn298 residues and I showed H-bonding with Lys200 and Ile235 of HPA active site. The overall H- bonding interaction energy of II was found less than I which might have resulted due to the interaction distance and the individual interaction energy between ligand and interacting residues respectively. However, the overall interaction energy between the ligands and receptor revealed that compound II has better binding affinity to HPA than I. This suggests, other non-bonding interaction also plays a critical role apart from H-bonding interaction towards the binding of ligand with receptor in an energetically favoured condition. The non- bonded electrostatic interactions of both the compounds within HPA active site is depicted in Fig. 6.11.
Fig. 6.9 Ligand-protein interaction for the best pose at the major binding cleft and interactive nearby residues were depicted for compound I and II. (A) and (B) Predicted bonded and non- bonded interactions between amino acid residues present in the active site of HPA and diterpene molecules (I and II respectively).
A
B
Fig. 6.10 Predicted H-bonded interaction of I (A) and II (B) with the residues at the active site region of HPA. Here, blue dotted lines represent the H-bonding interactions between the ligand molecules and HPA.
A
B
Fig. 6.11 Predicted non-bonded electrostatic interaction of I (A) and II (B) with the residues at the active site region of HPA.
A
B
Table 6.5 Theoritical affinity of best docked poses for standard inhibitor (acarbose) and diterpene compounds (I and II) with HPA
Ligand H-bonded residues
Interaction energy (Kcal/mol)
Interaction dist. (Å)
H-Bond energy (Kcal/mol)
Interaction energy (Kcal/mol)
MolDock Score
Rerank score Acarbose Asp300
Glu233 Lys200 Tyr151 Asp197 Thr163
His101 Gln63
-1.73 -1.72 -2.5 -2.5 -2.5 -2.5/-1.72, -2.5 -0.86 -2.5
4.10 3.20 2.90 2.96 2.99 2.71/3.26, 3.10 2.40 2.75
-18.8607 -162.532 -121.3 -117.711
Compound I Ile235 Lys200
-2.5 -2.5
2.91 2.99
-3.62972 -108.4 -102.342 -79.8406
Compound II Arg195 Asn298
-2.35 -2.5
2.94 3.07
-2.87389 -114.369 -108.034 -88.9548
LigPlot is known as the comprehensive tool for expressing the hydrogen bonding and hydrophobic interactions involving the ligand molecule and active site residues (Wallace et al. 1995). Here, we have also confirmed by generating LigPlots for all the best fit docked poses for each compound including acarbose with HPA (Fig. 6.12). LigPlots for compound I and II revealed the same interacting residues as obtained in docking studies and both the compounds showed two H-bonding interactions with respective active site residues. LigPlot analyses were precisely advantageous in identifying the hydrophobic interaction pattern. Here for I and II, majority of the active site residues actively participated in non-bonded hydrophobic interactions in the vicinity of the ligands (Fig. 6.12A and B).
Fig. 6.12 LigPlot generated for the best poses obtained with compound I (A) and compound II (B), against HPA crystal structure. Hydrogen bonding interactions were shown as green dotted lines. Besides the H-bonding residues, other surrounding amino acids (in the vicinity of ligand) describe the hydrophobic interactions.
A B C
Similarly, clear interactions of acarbose with Asp197, Glu233 and Asp300 were observed along with other H-bonding residues in HPA active site. It is important to mention here that Asp197 of HPA was reported previously as the catalytic nucleophile in hydrolysis reactions for any polymeric substrates like dietary starch (Rydberg et al. 2002; Zhang et al. 2009).
Similarly, Glu233 is known to act as an acid-base catalyst during substrate hydrolysis reactions (Williams et al. 2012). Asp300 was also previously identified as the key player for optimizing the orientation of substrate molecule using hydrogen bonding interaction and even as regulator of steric conflicts for a better binding conformation of substrate (Li et al. 2005a;
Williams et al. 2012).