PPARγ is very important drug target in type 2 DM. Thiazolidinediones (TZDs) have high affinity for PPAR𝛾 and used as potent insulin sensitizers. The first TZD introduced in early1997 was Troglitazone and subsequently Pioglitazone and Rosiglitazone was introduced in 1999. Our age long traditional practices utilizes Ptercarpus marsupium (local name-Vijaysar) to lower down blood sugar and in vivo experimental studies by others also validated its antidiabetic potential. Isoliquiritigenin is an important constituents of P. Marsupium and chosen for protein ligand interaction study with PPARγ using molecular docking and dynamic simulation studies. PPARγ protein was taken as target since it plays an important role in glucose metabolism.
3.1 Molecular Docking
SiteMap study analysed five sites based on site score that considered various parameters such as amino acid exposure, hydrophobicity, hydrophilicity, donor/acceptor ratio, contact, size and volume. The amino acids in proposed binding active site having site score more than 1.0 and residues identified as 226, 227, 228, 247, 249, 255, 256, 258,259, 260, 280, 282, 288, 291, 292, 295, 296, 323, 326, 327, 329, 330, 333, 340, 341, 342, 343, 344, 345, 346, 348, 363, 364, 449, 469, 473 and 601 . PPARγ protein has ligand binding pocket in the centre of the ligand binding domain Pocket contains many polar residues such as Cys285, Ser289, His323, Tyr327, His449, and Tyr473. The central region of the ligand-binding pocket is surrounded mainly by nonpolar residues such as Leu330, Leu339, Leu353, and Met364. The Ω-pocket mainly contains hydrophobic residues Ile249, Met348, and Ile341, Leu255, Gly258, Ile262, Ile281 with few polar residues such as Glu259, Arg280, and Ser342 [41,42,43,44].
Isoliquiritigenin and Troglitazone (known agonist) docked into a PPARγ target structure in a different positions, conformations and orientations and ligand-protein pairwise interaction energies were calculated. The best possible binding modes of the Isoliquiritigenin and Troglitazone, at targeted protein’s active sites are displayed in Fig. 1a& b. In our study, H-bonds were formed between Tyr327, and Tyr473 of the PPARγ and Isoliquiritigenin with binding energy of -7.46 kcal/mol. The van der waals interaction were seen with Phe282, Gln286, Ser289, His323, Leu333, Phe363, Met364, Lys367, His449, Glu453, Leu465 and Leu469. Similarily, the pi-Sigma interaction with Ile326, pi-Alkyl with Ala292, leu330 and Pi-sulphur interaction with Cys285. Molecular docking results with Troglitazone showed H bond with Tyr327, Ser289 with binding energy of -11.01 kcal/mol.
The van der waals interactions was seen with Phe282, Gly284, Gln286, His323, Leu333, Phe363, Lys367, His449, Glu453, Leu465, Leu469 and Tyr473. Similarly, the pi-Alkyl with Cys285, Arg288, Met364, Leu330, Leu333, Val339, Ile341 and pi-sulphur interaction with His323. Both the ligands Isoliquiritigenin and Troglitazone were further evaluated with another docking software Glide with Extra precision (XP) mode to find out accurate binding during PPAR interaction with different poses of ligand. In case of Isoliquiritigenin, the binding energy in XP was -6.74 kcal/mol. The important residues in H bond were Leu340 and Tyr473, in van der waals interactions was with Phe282, Gln286, Ser289, His323, Tyr327, Leu333, Phe363, Lys367, His449, Glu453, Leu465 and Leu469 (Figure 2a). The pi-Alkyl interaction with Arg288, Leu330, Val339 and pi-sulphur interaction with Met364 (Figure 2a). Troglitazone docking, gave binding energy in XP mode as -9.59 kcal/mol. The important residues in H bond were His323 and Ser289, while Phe282, Gly284, Gln286, Leu333, Phe363, Lys367, His449, Glu453, Leu465, Leu469, and Tyr473 showed van der waals interactions. The pi-Alkyl interaction with Cys285, Arg288, Leu330, Leu333, Val339, Ile341and pi-sulphur interaction with Met364 (Figure 2b).
Induced fit docking was done to evaluate the conformational changes in PPARγ induced by Isoliquiritigenin binding. This method utilizes different poses, by using reduced van der waals radii and an increased coulomb-vdw cut off. Highly flexible side chains were ignored while doing energy minimization to predict structure with different poses . The best Induced fit docking score of the optimised protein–ligand complexes was -9.39 Kcal/mol. The important residues in H bond were Cys285, Arg288, Tyr327 and Leu340. The van der waals interactions were observed with Gln286, Ser289, His323, Tyr327, Leu333, Val339, Ile341, Ser342, Phe363, His449 and pi-Alkyl interaction with Ile326 and leu330 (Figure 3).
There were several classes of scaffolds developed for PPARγ agonists. The important ones are TZDs, Indoles and Benzimidazoles. It was observed that Tyr327, Arg288, and His323 plays critical role in activity of PPARγ. Some of the first partial agonists developed for PPAR𝛾 uses Indoles which interacts with residue Ser342 and van der waals interaction with Cys285 and Arg288. These compounds stabilize secondary structure by interacting with Ile341, also by hydrophobic contacts with Leu330 and Leu333. Side chain of Ser289, Tyr327 and Tyr473 forms hydrogen bonds with Benzimidazoles. They also make hydrophobic contacts with Leu469 [46, 47, 48]. The present study as reflected in Figure 1a, 2a and 3 showed similar pattern of interaction between PPARγ and Isoiquiritigenin.
3.2 Molecular dynamics simulation (MD)
MD simulations utilizes theoretical models to study the spatial and energetic dynamics of PPARγ and Isoiquiritigenin at an atomic level. Binding free energy changes of a complex was examined to see the energetics and mechanisms of conformational change. The force between atoms is combination of bonded (Bond angle, bond length, Torsion) and non-bonded interactions (Coulomb & Leonard-jones). During simulation, time is divided into discreet time steps e.g. 1 fs (10−15) and the forces were calculated for each time steps while adjusting the position. Isoiquiritigenin collides with residues of PPARγ protein with preference for binding sites during 100 ns MD simulations and changes in structure and stability in water model was evaluated using GROMACS 4.6.7. This interaction in time dependent MD trajectories was recorded as RMSD, RMSF and radius of gyration. The conformation results obtained after simulation are more significant and stable than the docked conformation. Therefore, the binding orientation of Isoiquiritigenin with PPARγ predicted through MD simulation showed better correlation to their biological activity.
The structural variations in the PPARγ were analysed by root mean square deviation (RMSD) and the radius of gyration (Rg). RMSD of complex showed that after small rearrangement from the early conformation, the complex was fairly stable during complete MD simulation period. RMSD for the complexes of PPARγ with isoliquiritigenin showed that the structures of the systems equilibrated well after 6 ns of MD simulation (Fig. 4a). It showed that the RMSD profiles were always less than 0.4 nm for complex and PPARγ alone during the entire 100 ns simulation. Thus, the stability of PPARγ in Isoliquiritigenin bound state found as suitable candidate for post analysis. RMSD of Isoliquiritigenin showed that it stably bounded to PPARγ pocket. It showed stable profile during the simulation (Fig. 4b).
The radius of gyration was calculated to analyse structural changes of PPARγ, when the Isoliquiritigenin was bound. The plot of radius of gyration in simulation time for PPARγ is given in figure 5. The average Rg values throughout the simulation time was 19.17 Å for the complex (Fig. 5) and 18.79 Å for PPARγ alone. Comparative analysis of final pose of PPARγ - isoliquiritigenin complex after 10 ns molecular dynamics simulation with crystal structure of PPARγ revealed that binding of the Isoliquiritigenin did not bring significant conformational changes in the PPARγ structure and structure of protein was compact.
Root mean square fluctuation (RMSF) of PPAR γ around its average conformations play an important indicator of protein activity in complex formations. Significant change in complex fluctuation occur around residues 240, 243, 253, 257,477 of N and C terminal respectively. Protein residue 357, 443, 447, 448, 457, 463 got stabilized on ligand binding. Residues critical in interaction with ligand were Arg 288, Tyr 327, Ser 289, His 323, Leu333, His449 and Tyr 473 (Figure 6).
The H-bond between Isoliquiritigenin and PPARγ was analyzed and average of all H-bond candidates was calculated as 2.71 H-bonds as shown in Fig. 7.
The binding of the Isoliquiritigenin to PPARγ did not induce a large change in the solvent accessible surface area (SASA) of the complexes. SASA of PPARγ and PPARγ with Isoliquiritigenin was 139.53 and 143.12 respectively. The above results indicate the stability of the overall structure of protein when the inhibitor was bound.
Comparative analysis of different poses 100 ns simulation of PPARγ bound with Isoliquiritigenin was done. Two dimensional (2D) plots of final pose were generated and compared with initial structure. The interaction of PPARγ with ligand Isoliquiritigenin was stable and remain in the same binding pocket. The important residues in H bond were Lys367, in van der waals interactions with Phe282, Gln 286, Ser 289, His 323, Tyr 327, Leu 333 and Phe 363 during entire simulation (Fig. 8a,b,c,d).
3.4 Drug-likeness of Isoliquiritigenin
Isoliquiritigenin was studied to predict its drug-likeness properties by considering following molecular descriptors: logP (partition coefficient), molecular weight (MW), topological polar surface area (TPSA), hydrogen bond acceptors and donors count in a molecule. Isoliquiritigenin was found to have logP as 2.77, TPSA as 77.75, total number of atoms (n Atoms) : 19, Mol .Wt : 256.26, number of hydrogen bond acceptors was 4, number of hydrogen bond donors: 3, and 0 violation of the Lipinsky’s rule. Lipinski, Ghose and Veber rules states that membrane permeability must possess logP ≤ 5, number of hydrogen bond acceptors ≤10, number of hydrogen bond donors ≤ 5, molecular weight ≤500, topological polar surface area (TPSA) < 140 Ǻ and number of rotatable bonds (n rotb) < 10 (measures molecular flexibility) and also, the total number of atoms between (n Atoms) 20 and 70. This was used as filter for drug-like properties [38,39,40,41,42,43,44,45,46,47,48, 49].
PPRs acts as ligand inducible transcription factors belongs to members of the nuclear receptor superfamily. They are mainly involved in energy, carbohydrate and lipid metabolism. Lee et al., (2017) showed binding modes of Pioglitazones and Lobeglitazones with PPAR γ. The head groups of TZDs forms H bonds with Tyr 473 of PPAR γ in active conformations, which correlates with full agonism of the drugs. Similar binding mode was observed in molecular docking results . The pi interactions with Phe 264, Phe 363 and h bond with Leu 340, Tyr 473. in silico identification of PPAR γ agonist from Chinese medicine showed similar interactions as H bond with residue Tyr 327, Lys 367 etc . Quantitative parameters such as RMSD showed structural stability of the protein ligand complex. Profile of the protein and complex was found to be relatively stable about 0.34nm and 0.44nm similar which in acceptable range . RMSF of given protein in MD trajectories showed fluctuations more in N and C terminal ends whereas very low fluctuations in the area where amino acid and ligand were interacting. Lower RMSD and small fluctuations in RMSF and Rg of docking complex are good indication of system stability. MMPBSA was used to calculate free energies of Isoliquiritigenin, PPAR γ and complex. All the ensemble averages the change energy of bonded, nonbonded, polar and non-polar interactions for a binding affinity calculation, led to very stable binding pattern between protein and ligand under study as shown in Table 1 [11, 34]. Drug likeliness property showed Isoliquiritigenin as an ideal candidate as it did not violate criteria given by Lipinsky. Similar reports on virtual screening and ADMET analysis showed by Liu et al., 2017 .