Molecular docking analysis of curcumin analogues with COX-2

Curcumin analogues were evaluated for COX-2 inhibitory as anti-inflammatory activities. The designed analogues significantly enhance COX-2 selectivity. The three compounds could dock into the active site of COX-2 successfully. The binding energies of -8.2, - 7.6 and -7.5 kcal/mol were obtained for three analogues of curcumin respectively. Molecular docking study revealed the binding orientations of curcumin analogues in the active sites of COX-2 towards the design of potent inhibitors.

Although the genes of both isoforms are different, COX-1 and COX-2 have similar structures and catalytic activities. The amino acid sequences for the substrate binding and catalytic sites are almost identical, but COX-2 has valine substituted for isoleucine at positions 434 and 523 [10,11]. Valine is smaller than isoleucine by a methyl group. These substitutions result in a larger and more flexible substrate channel and a secondary internal pocket off the inhibitor-binding site of COX-2, which is not observed in COX-1.
Curcumin is found as a major pigment in the Indian spice turmeric (C. longa, Zingiberaceae). The rhizome of the C. longa has been used in indigenous medicine for the treatment of inflammatory disorders and its medicinal activity has been known since ancient times. Curcumin is reported to have antiinflammatory, antioxidant and anticancer properties [12]. From the literature it was found that curcumin was investigated for COX inhibitory activity using bovine seminal vesicles, microsomes and cytosol from homogenates of mouse epidermis showed IC50 value of 2 µM [13], 52 µM [14], and 5-10 µM [15], respectively.
Pharmachophore modification of the dienone functional group curcumin into monoketone and side chain of aromatic ring with symmetrical or asymmetrical substituents has been might give better activity and stability than the parent compound [16-18]. Robinson, et al. has proven that the change of β-diketone on the structure into α, β-unsaturated ketone did not change the activity of the curcumin analogue to inhibit the cancer cell. Even, in several cases such compound gave better activities than the curcumin itself [19].
Molecular docking is an efficient tool to get an insight into ligand-receptor interactions. All molecular docking calculations were performed on AutoDock software. The AutoDock Tools (ADT) graphical user interface was used to calculate Kollman charges for the protein and to add polar hydrogen. Molecular docking is a computational procedure that attempts to predict non-covalent binding of macromolecules or, more frequently, of a macromolecule (receptor) and a small molecule (ligand) efficiently, starting with their unbound structures, structures obtained from MD simulations, or homology modeling, etc. The goal is to predict the bound conformations and the binding affinity. In the present study, we describe binding properties of 15 curcumin analogues to the 6COX subdomains of COX-2, using molecular docking studies.

Methodology: Softwares Used:
The ligand preparation done by using ACD/ChemSketch 12.01 (Advanced Chemistry Development, Inc), geometries were optimized using Hyperchem 8.0.3 and for protein preparation Wizard of AutoDock tools 1.5.6 are used. Molecular docking calculation has done by AutoDock tools 1.5.6 and MGL tools 1.5.6 packages (The Scripps Research Institute, Molecular Graphics Laboratory, 10550 North Torrey Pines Road, CA, 92037).

Docking Procedure: Protein Preparation:
Three-dimensional coordinates COX-2 (pdb code 6-COX) were retrieved from Brookhaven Protein Data Bank. The pdb file was submitted to "Build/check/repair model" and "Prepare PDB file for docking programs" modules where missing side chains were modeled in, a small regularization was performed, water positions and symmetry were corrected, and hydrogen were added. Only chain A of the repaired pdb file was evaluated and passed to AutodockTools (ADT ver.1.5.6) for pdbqt file preparation. Thus, water molecules and non-standard residues were removed, only polar hydrogen was maintained, and Gasteiger charges were computed for protein atoms by ADT.

Ligands Preparation:
All the molecules were constructed with ChemSketch-12.01 program and these geometries were optimized using the Austin Model 1 to the corresponding mol2 file that was submitted to ADT for pdbqt file preparation and docking with AutoDock4. The geometry of built compound was optimized, partial charges were also calculated, and saved as mol2 files that was passed, as usual, to ADT for pdbqt file preparation.

Results & Discussion:
The level of COX-2 inhibitory and anti-inflammatory activities of 15 curcumin analogues (Table 1), prompted us to perform molecular docking studies to understand the ligand-protein interactions and COX-2 selectivity in detail. All the calculations were performed using Autodock Tools (ADT) ver.1.5.6. The crystal structures of COX-2 enzymes complexes with SC-558 [6COX.pdb] were used for docking. Extracting co-crystallized inhibitor from the protein and then docking the same tested the docking protocol. The docking protocol predicted the same conformation as was present in the crystal structure with RMSD value well within the allowed range of 2 Å [22].
The ADT program is an automated docking program, was used to dock compounds curcumin analogues on the active sites of COX-2 enzymes. For each compound the most stable docking model was selected according to the best scored conformation predicted by the Autodock scoring function. The complexes were energy-minimized with an Austin model 1 force field till the gradient convergence 0.01 kcal/mol was reached.
However, only one hydrogen bond was observed between the methoxy group and OH of Ser 530 (3.6 Å, O … O, 3.8 Å (O … H) (Figure 3). Based on this study, there are three curcumin analogues showed significant inhibition of the enzyme COX-2. It is clear that this compound has the potential to inhibit COX enzymes, however, they need to be confirmed from the biological evaluation and in vitro testing.

Conclusion:
Three curcumin analogues were investigated for COX-2 inhibitory activities. Pharmachophore modification of the dienone functional group into monoketone and side chain of aromatic rings with symmetrical or asymmetrical substituents give better activity and stability than the parent compound. Molecular docking studies further helps in understanding the various interactions between the ligands and enzyme active sites in detail and thereby helps to design novel potent inhibitors.