Current Practice in Drug Development

Result and discussion

In present work molecular docking approach has been applied to investigate binding affinities of selected compounds between three different proteins Xray01, 2AYL and 3PGH. In order to better understand docking results, it is necessary to understand geometrical and structural conformations of studied proteins. As shown in figure 1, the superimposition of selected pdbs 2AYL and 3PGH over reference one (Xray01); all proteins share high structural similarity in terms of folding pattern of alpha helix and beta plated sheets. In figure 1 all structures almost overlapped to each other with RMSD value not more than 6Å. Interestingly, the ligand binding site in 2AYL and 3PGH also appears to be in same position as that in Xray01. The graphical analysis selected proteins using MOLCAD module of sybylX 1.3 docking tool has further indicated the presence of various hits in different binding sites of Xray01, 2AYL and 3PGH. However, all proteins were found to bear three common hits including octyl beta D glucopyraniside (BOG), flurbiprofen (FLP), and 2 acetamido 2 deoxy beta D glucopyranise (NAG) with exception of Xray01 where FLP was replaced with Lig1 (Figure 2). The simplified 2D interaction diagrams are shown in figure 1, showing interaction patterns of OGP, FLP and MAN in 2AYL. In addition, MANGANESE PROTOPORPHYRIN IX (MNH) and GLYCEROL (GOL) were also found to be common hits bonded to all selected proteins. Furthermore, a glucosidal pentamer of alpha D mannopyranose (MAN) was found to be attached at surface of all selected proteins. However, the active inhibitor (Lig1) of Xray01 protein was found to be attached in a deep cleft near MNH binding site where it may induce several structural changes to interfere with MNH Xray01 binding. In order to compare the binding mode of Lig1 in studied system the 3D bioactive conformation of Lig1 was extracted from Xray01 to be used as initial conformation for docking analysis. The bioactive conformation of Lig1 (Figure 2) was docked into the ligand binding cleft of 2AYL and 3PGH.    

As shown in figure 1, apparently all three proteins share highly similar structures, however, minor sequential differences in protein structures particularly in active site may serve as key molecular basis for difference in ligand binding selectivity. The best docking poses of ligands were selected on the basis of cScore in sybylX 1.3. The graphical analysis has shown that Lig1 occupies the same binding site in 2AYL and 3PGH (Figure 1).

 (B) Lig1 3PGH (C) Lig1 2AYL where ligands are shown in slate color and H bond interactions are displayed by dashed lines. Simplified 2D interaction diagrams of: (D) Lig1 Xray01 (E) Lig1 3PGH (F) Lig1 2AYL where H bonds are shown in green color dashed lines.

As shown in figure 3A, in Lig1 Xray01 bonded system Lig1 is surrounded by V116, Q192, V349, L352, Y355, W387, L359, I517, F518,L531, M522 and A527 to establish several hydrophobic and van der waals (vdW) contacts. The –NH₂ group of phenyl sulfonamide moiety extends towards a smaller sub site to donate a pair of H bonds to the side chains of Q192 and L352. The phenyl moiety of Lig1 is positioned in such a way that it faces the phenyl moiety of Y355 to establish pi pi hydrophobic contacts. On the other side, Lig1 establishes only one H bond interaction with H90 of 3PGH. The methyl cyclo hexyl is extended A527, S530 and T355 to establish hydrophobic and vdW contacts. Unlike, Lig1 Xray01 system no H bond interaction was observed in Lig1 2AYL system. In 2AYL bonded system, Lig1 is settled in same binding site where it is surrounded by residues L93, M113, V116, L117, R120, I345, V349, S355, F518, M522, A527, S530, and L531 to make several interaction however, H bond remained to be missing in 2AYL bonded system.  Hence, these variations in binding interactions reflects that fact that the compound Lig1 is more tightly bonded to Xray01 than 3PGH and 2AYL. 

2. Docking analysis: You are provided with a set of calculations (see individual email for you) and have to analyse the interactions and draw conclusions regarding the inhibition capability of the provided ligands to COX enzymes.

You have to analyse the docking for three molecules (Ax, Bx, Cx), calculations are provided for COX 1 and COX 2.

Discuss and compare the results for the different enzymes.

In another attempt 44 hits derived from three different leads AX, BX and CX were docked into two proteins COX1 and COX2 to reveal variations in binding modes and fundamentals of ligand receptor complex formation. Docking results of ligand A01 06 bonded to COX 1 are displayed in figure 4A F. All six ligands occupied the same binding site in COX 1 predominantly surrounded by residues F86, H90, V349, L352, S353, F470, H513, F518, and S530.      

As depicted in figure 4A, ligand A01 establishes a couple of H bond interaction with nearby residues F470 and M472 whereas the trifluoro methyl moiety extends towards L115 to establish hydrophobic interaction. Ligand A02 04 establishes only one H bond interaction with residues R120, S530, and T385, respectively. However, no H bond interaction was observed in A05 and A06 bonded systems. These findings clearly suggest that ligand A01 is the most potent inhibitor of COX 1 in A01 series of inhibitors. Meanwhile, all six ligands were also docked into COX 2 enzyme to compare the binding modes AX series COX 1 and COX 2 enzymes.

As shown in figure 5A01 06, A01 and A02 were able to establish four and three H bond interactions, respectively, in their corresponding systems. In addition, ligand A05 also establishes a network of five H bond interactions with Q192, L352, and F518 residues of COX 2 enzyme. These findings suggest that ligands A01, A02 and A05 are more potent inhibitors of COX2 as compared to COX1 enzyme.  Similarly, eight derivatives of BX series were docked in COX1 and COX2 enzymes (Figure 6A P). Like AX enzyme systems compounds of BX series also showed promising binding potential towards COX2 as than COX 1. Ligands B03, B04, B05, and B07 are able to establish a pair H bond interaction with nearby residues in COX 2 binding site. In COX 1 BX bonded system B01. B02, and B08 were able to establish couple of H bond contacts while no other ligand establishes more than one H bond contact. Thus, on the basis of difference in interaction patterns one may conclude the ligands of BX series are more potent inhibitors of COX 2 than COX 1. 

1, (D) B04 COX 1, (E) B05 COX 1, (F) B06 COX 1, (G) B07 COX 1, (H) B08 COX 1, (I) B01 COX 2 (J) B02 COX 2 (K) B03 COX 2, (L) B04 COX 2 (M) B05 COX 2 (N) B06 COX 2 (O) B07 COX 2 (P) B08 COX 2where H bonds are shown in green color dashed lines.

Last but not least another series of docked ligands C01 08 bonded to COX 1 and COX 2 enzymes were viewed graphically analyzed.

1, (D) C04 COX 1, (E) C05 COX 1, (F) C06 COX 1, (G) C07 COX 1, (H) C08 COX 1, (I) C01 COX 2 (J) C02 COX 2 (K) C03 COX 2, (L) C04 COX 2 (M) C05 COX 2 (N) C06 COX 2 (O) C07 COX 2 (P) C08 COX 2 where H bonds are shown in green color dashed lines.

As shown in figure 7, in COX 1 bonded system ligand C08 establishes maximum number H bond interactions. C08 establishes three H bond interactions with R120 and T385 in donor acceptor motif. Conversely, ligand C06 establishes a pair of H bond interaction in COX 2 while showed not a single H bond interaction in COX 1 bonded system. No other ligand showed more than one H bond interaction in COX 1 or COX 2 bonded system, which. Since, C08 shows highest number of molecular interactions in COX 1 bonded system among all CX COX 1 and –COX 2 bonded system it could be speculated that C08 is most suitable inhibitor of COX 1 while C06 is the stronger inhibitor of COX 2. 

Conclusion

In present an exhaustive analysis of docking results has been performed. At first, a comparison of docked ligand from Xray01 protein into 2AYL and 3PGH was performed. During superimposition of protein structures, all three proteins were found to share similar structural folding and geometrical conformation. Graphical analysis of docking results reveal that the Lig1 was most active ligand for Xray01 protein, because it was able to establish two H bond interaction in Xray01 protein but only one H bond interaction in 3PGH. However, no H bond interaction in 2AYL system. Therefore, these minor differences in interaction pattern might be responsible for stronger binding affinity of Lig1 towards Xray01 than 3PGH and 2AYL. Furthermore, the docked conformations of 22 hits derived from three leads AX, BX and CX in COX 1 and COX 2 proteins are critically analyzed. Apparently, the molecules involved in making greater number of H bond interactions were found to be more potent. Among all selected systems ligand A01 and A05 were found to be more patent for COX 2 with four H bond interactions. Ligand B03, B04, B05 and B07 also displayed higher binding affinity towards COX 2 than COX 1. Conversely, compound CX series (C08) showed greater binding potential towards COX 1 with three H bond than all other ligands bonded to COX 1 and COX 2.      

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