Target Protein & Binding Site
Pancreatic alpha-amylase protein of human, co-crystallized with acarbose (PDB ID: 2QV4) was chosen as the protein drug target to perform this in-silico screening. Initial investigation of PDB crystal structure of the protein along with its known inhibitor, revealed the binding site / active site of the protein, that was used as the docking site to perform this investigation. The site where the acarbose was bound to, is composed of Ile-051, Trp-058, Trp-059, Glu-060, Tyr-062, Gln-063, His-101, Gly-104, Asn-105, Ala-106, Val-107, Tyr-151, Leu-162, Thr-163, Gly-164, Leu-165, Arg-195, Asp-197, Ala-198, Lys-200, His-201, Glu-233, Ile-235, Asn-298, His-299, Asp-300 and His-305. The position of acarbose molecule and conformation of the active site and its residues are shown in Figure.1.
Protein-Ligand Docking
Protein-ligand docking analysis was performed using AutoDock Vina algorithm, inbuilt within PyRx tool. A total of 37 ligands (Acarbose as reference ligand, 14 ligands from Coccinia indica & 22 ligands from Withania coagulans) were docked with the targeted protein within the highlighted binding site. Results of the protein-ligand docking analysis are tabulated in Table.1. Three-dimensional and two-dimensional analysis of protein-ligand interactions of the most significant ligands are represented in Figure.2 and Figure.3 respectively. The reference ligand Acarbose (41774) demonstrated a strong binding interaction with the target protein with a binding free energy of -8.6 Kcal/mol and forming 7 hydrogen bonds with Gln-063, Asn-105, Thr-163, Asp-197, Glu-233, His-299 and Asp-300. Among the 14 ligands of C. indica most significant interaction was demonstrated by Taraxerol (92097) with a binding free energy of -10.2 Kcal/mol without the formation of any hydrogen bonds. Among the 22 ligands of W. coagulans most significant interaction was demonstrated by Epoxywithanolide-I (10790456) with an exceptional binding free energy of -11.9 Kcal/mol and formation of 3 hydrogen bonds with Thr-233 and Glu-233. Based on the docking analysis, Taraxerol from C. indica and Epoxywithanolide-I of W. coagulans were identified as the most significant ligands and were further subjected for detailed analysis as potent alpha-amylase inhibitors in their respective plants.
Table.1
Protein-Ligand docking analysis of phytochemicals against alpha-amylase protein
Chemical | PubChem ID | Docking Binding Free Energy (Kcal/Mol) |
Acarbose | 41774 | -8.6 |
Coccinia indica |
Taraxerol | 92097 | -10.2 |
Daucosterol | 5742590 | -10.2 |
Beta-Carotene | 5280489 | -10 |
Beta-Amyrin | 73145 | -9.8 |
Karounidiol | 159490 | -9.8 |
Taraxerone | 92785 | -9.7 |
Lupeol | 259846 | -9.2 |
Delta-7-Stigmastenone-(3) | 5748344 | -9.2 |
Erythrodiol | 101761 | -9.1 |
Betulin | 72326 | -9 |
29-Hydroxylupeol | 489919 | -8.9 |
Caffeic-Acid | 689043 | -6.7 |
Citrulline | 9750 | -5.3 |
5-Methylcytosine | 65040 | -5.2 |
Withania coagulans |
14,15beta-Epoxywithanolide I | 10790456 | -11.9 |
20beta-Hydroxy-1-oxo-(22R)-I-2,5,24-trienolide | 637266 | -11.2 |
17beta-hydroxywithanolide K | 44562998 | -11.1 |
Coagulin C | 44562952 | -11 |
Withacoagulin | 12115994 | -10.9 |
24,25-dihydrowithanolide D | 23266167 | -10.9 |
Withacoagin G | 102190729 | -10.8 |
Withacoagin I | 102190731 | -10.5 |
Coagulin F | 24941991 | -10.4 |
Coagulin B | 101936450 | -10.3 |
3beta-Hydroxy-2,3-dihydrowithanolide F | 135887 | -10 |
Coagulin E | 15341418 | -10 |
Coagulanolide | 44562997 | -10 |
Sitosterol Glucoside | 70699351 | -10 |
Withacoagulin H | 71524298 | -9.9 |
Withacoagin H | 102190730 | -9.9 |
Coagulin D | 101936452 | -9.8 |
Coagulin R | 15977628 | -9.6 |
Coagulin M | 100920595 | -9.6 |
Coagulin J | 15968792 | -9.2 |
Coagulin I | 15969311 | -9.2 |
Coagulin G | 24941992 | -9.1 |
Molecular Dynamics Simulation
Molecular Dynamics Simulation (MDS) analysis for a time period of 150 ns was performed to study the stability of the protein-ligand complex and to understand the ability of the selected phytochemicals to be a potent inhibitor of alpha-amylase. A total of three different MDS (150ns each) were performed i.e., 1. Alpha-Amylase with Acarbose; 2. Alpha-Amylase with Taraxerol; 3. Alpha-Amylase with Epoxywithanolide-I. The results of all the MDS were compared to understand the ability of the tested phytochemicals to inhibit the alpha-amylase protein when in comparison to known inhibitor acarbose. The results of the MDS were analyzed by means of RMSD, RMSF, SASA, Potential Energy, Hydrogen Bonds and Protein Gyration.
Root Means Square Deviation (RMSD)
RMSD of the protein backbone structure for the 3 MDS combinations are graphically represented in Figure.4(A). Initial observation strongly suggests that, among the 3 combinations of MDS, the most stabilized protein structure with least and lowest fluctuation of backbone RMSD was demonstrated by alpha-amylase enzyme when in combination with Epoxywithanolide-I ligand. Taraxerol demonstrated good complex stability up to 70 ns of the MDS, after which the structure demonstrated very high fluctuation in the RMSD ranging up to 3.4 Å, which was highest among the 3 MDS complex. However, the stability of the complex increased over time and reached 2.2 Å at the end of 150 ns simulation. Epoxywithanolide-I demonstrated much significant complex stabilization with lowest RMSD of 1.8 Å towards the end of the 150 ns MDS, while, the reference molecule also demonstrated a lowest RMSD of 1.8 Å towards the end of 150 ns MDS. RMSD graph in Figure.3(A) strongly suggests that, among the 2 test ligands, Epoxywithanolide-I shows promising inhibition potential, on power with the reference molecule acarbose.
Root Mean Square Fluctuation (RMSF) of Residues
RMSF analysis of individual residues in the protein structure for all 3 MDS complexes are represented in Figure.4(B). It is evident that, Epoxywithanolide-I strongly reduces the RMSF of interacting residues (i.e., Asn-100 to Lys-200) than the reference ligand acarbose itself. This strongly suggests that the test ligand Epoxywithanolide-I is imposing great restraint on the flexibility of the interacting residues at the active site of the protein, there by conferring a much stronger protein-ligand complex stabilization than the reference ligand. The most significant restraint was imposed on Asn-152 residue, which demonstrated 2.8 Å with Acarbose, 3.2 Å with Taraxerol and 2.4 Å with Epoxywithanolide-I. However, the key active residue of this protein structure is annotated to be Asp-197, for which a RMSF 0.7 Å with Acarbose, 0.6 Å with Taraxerol and 0.7 Å with Epoxywithanolide-I. Highest fluctuation in this study was shown by residue 146 in when combination with Taraxerol, with a maximum of 6.7 Å. This RMSF analysis shows that, both the test ligands, Taraxerol and Epoxywithanolide-I instills rigidity to the protein structure, conferring a stronger protein-ligand complex and stability, when compared to the reference ligand Acarbose.
Solvent Accessible Surface Area (SASA)
SASA of a protein denotes the expansion of the protein surface which denotes functionality of the protein. Reduction in the SASA denotes restriction in the functionality of the protein, there by suggesting the inhibition of the protein. SASA analysis of the 3 MDS combinations are represented in Figure.5(A). Overall comparison shows that Taraxerol shows least SASA value ranging between 200 to 210 nm2 only upto 80 ns after which its increased to 210 to 220 nm2. Reference ligand Acarbose demonstrated highest SASA value, averaging between 210 to 220 nm2 throughout the 150 ns MDS. Epoxywithanolide-I demonstrated a SASA value, averaging between 200 to 210nm2 throughout the entire 150 ns MDS. Overall, the hydrophobicity of Taraxerol contributed towards reduced SASA values of the complex, but was not constant throughout the MDS. It is evident that, Epoxywithanolide-I demonstrated a stronger and much stable complex, as the SASA value ranged in a steady average and wide fluctuations were not observed. The SASA value demonstrated by Epoxywithanolide-I and Taraxerol were highly significant than the reference ligand Acarbose, suggesting them to be a potent inhibitor.
Potential Energy of the Complexes
Total potential energy of the protein-ligand complex determines the inhibition potential and its complex stability. Potential energy plot of the 3 MDS complexes are graphically represented in Figure.5(B). Among the 3 MDS complexes, the lowest potential energy was demonstrated by Epoxywithanolide-I, suggesting it to be the most potent inhibitor in this comparison. The reference ligand Acarbose complex demonstrated a lowest potential energy of – 1910036 Kj/mol at 59.2 ns. Both the test ligands demonstrated a much significant potential energy, lower than the reference molecule, suggesting an increased inhibition potential. Taraxerol complex with protein demonstrated a lowest potential energy of -1924605.2 Kj/mol at 50.4 ns, while Epoxywithanolide-I demonstrated a lowest potential energy of − 1964113.3 Kj/mol at 118.7 ns. This plot clearly indicates that, the test ligands Epoxywithanolide-I and Taraxerol demonstrated high potential as an inhibitor of alpha-amylase, that is better than the reference ligand Acarbose.
Hydrogen Bond Analysis
Total number of hydrogen bonds determines the strength of the polar interactions between protein-ligand complexes. Hydrogen Bond analysis plot of the 3 protein-ligand complexes is represented in Figure.6 (i.e., A: Acarbose; B: Taraxerol; C: Epoxywithanolide-I). It is evident from the graph that, Acarbose exhibited strong polar interactions with the amylase protein, with a maximum of 4 hydrogen bonds with a significant frequency throughout the 150 ns MDS. However, the 2 test ligands, (Taraxerol & Epoxywithanolide-I) demonstrated weak polar interactions with the amylase protein, where Taraxerol formed an average of one hydrogen bond with high frequency throughout the MDS, while Epoxywithanolide-I demonstrated very weak polar interactions, with formation of one hydrogen bond occasionally during the entire MDS. This provides insight into the nature of the test ligands, that, both Taraxerol & Epoxywithanolide-I are hydrophobic molecules, that do not involve formation of hydrogen bonds with the target amylase protein. However, these hydrophobic molecules are demonstrating a higher potential to inhibit the target protein, when compared to the contrasting reference ligand Acarbose.
Radius of Gyration (Rg)
Radii of Gyration (Rg) determines the rigidity and compactness of a system and hence indicates the inhibition of the target protein. Rg analysis of the 3 MDS complexes are represented in Figure.7. Reduced Rg values and fluctuation determines the rigidity of the protein and there by inhibition of the catalytic function. All 3 MDS complexes, exhibited an average Rg value of 2.3 nm with mild fluctuations. Among the 3 ligand complexes, Taraxerol demonstrated greater stability in regards to fluctuation of Rg values. Acarbose & Epoxywithanolide-I complexes however, demonstrated significant fluctuations throughout the MDS likewise. The test ligand Epoxywithanolide-I behavior was comparative to that of Acarbose and hence the Rg values could be justified to be significant. This plot also confirms that the 3 ligand complexes instill stability and rigidity to the target protein structure there by inhibiting the target protein from its catalytic function.
Potent Inhibitors of Alpha-Amylase
The MDS analysis of the test ligands confirms that, both Taraxerol of C. indica and Epoxywithanolide-I of W. coagulans are demonstrating significant potential as inhibitors of the target Alpha-Amylase protein. When compared to the known reference inhibitor, results of Epoxywithanolide-I are more promising than the Taraxerol, based on the RMSD & RMSF analysis. The results of MDS suggests that both the phytochemical test ligands are potent inhibitors of Alpha-Amylase enzyme, aiding to the anti-diabetic properties of their respective plant sources. Suggestive conclusion of this MDS analysis would however strongly support that, Epoxywithanolide-I has much stronger inhibition potential than the reference Acarbose molecule and could further be investigated in-vitro for further validation of this in-silico study.
ADMET & Drugability Analysis
Adsorption, Distribution, Metabolism, Excretion and Toxicity (ADMET) & Drugability of the two phytochemical test ligands were investigated using in-silico tools, by comparing these to the properties of reference molecule Acarbose. The results of the in-silico ADMET and Drugability analysis are summarized in Table.2. Molinspiration analysis of the ligands strongly suggest that, the 2 test ligands are more prominent enzyme inhibitors than that of the reference acarbose. The 2 test ligands show higher LogP values than the reference acarbose, aiding to their hydrophobic nature and poor solubility in water. However, all other physicochemical properties of Taraxerol and Epoxywithanolide-I are significantly better than the reference Acarbose ligand. Epoxywithanolide-I did not show any violation of Rule-of-Five (RoF), while Taraxerol showed 1 violation of the RoF, however this is a significant improvement compared to Acarbose that demonstrated 3 violations of RoF. The ADMET properties of Taraxerol and Epoxywithanolide-I are significantly better than Acarbose, in regards to their cellular metabolization, excretion and being non-toxic to host cells. The test ligands were observed to be substrates of Cytochrome P450 enzyme, suggesting their ability to be metabolized and detoxified in the liver. The test ligands also demonstrated low risk for hERG inhibition, when compared to the reference which demonstrated an uncertain potential for hERG inhibition. This suggests that there is very low potential for cardiotoxicity by the test ligands, however being natural products as part of the edible plant sources, the low risk can be neglected. Based on the data presented in Table.2, it is evident that, Taraxerol and Epoxywithanolide-I are promising drug like molecules that demonstrate significant physicochemical properties with great potential to inhibit the target enzyme Alpha-Amylase.