The inhibition of NSAIDs towards GLOI provides a direction f

The inhibition of NSAIDs towards GLOI provides a direction for the development of novel effective GLOI inhibitors. Although the inhibitory activity of indomethacin was determined and the interactions between it Etomoxir mg and GLOI were probed by NMR titration experiments, no detailed inhibitory mechanism is provided at a structural level. In the present work, a parallel study on indomethacin and its analogues is performed both experimentally and theoretically to better understand the inhibitory mechanisms of NSAIDs towards GLOI. The main objective herein is to study the quantitative relationship between the chemical structures of representative indomethacin-type NSAIDs and two structurally related inhibitors (curcumin and bisdemethoxycurcumin we studied previously) and their inhibitory affinities against GLOI. The quantitative relationship is explained in terms of the physicochemical interactions between the NSAID ligands and GLOI. The present study would aid in discovery of novel GLOI inhibitors based on the chemical skeleton of indomethacin. Kinetic measurement of GLOI activity was performed similarly as described in our recent work with minor modification. The formation of --lactoylglutathione was measured by monitoring the increment in absorbance at 240nm at 30°C with a thermostated spectrophotometer. Reaction buffer containing 0.10M sodium phosphate (pH=7.1) and hemithioacetal (MG-SG) was pre-incubated for 20 minutes at 30°C. Concentrations of MG and GSH were calculated and varied by using the equilibrium constant (3.0mM) to obtain desired concentration of MG-SG. Excess free GSH in the assay was kept at 0.10mM. Reaction was initiated by the addition of recombinant 6His-tagged human His-GLOI (3.0 nM) to the reaction buffer. The NSAIDs inhibitors indomethacin, zomepirac, fenoprofen, and ketoprofen were purchased from Sigma. values of these inhibitors were determined by Dixon plot. All model calculations of this work were carried out based on the X-ray crystal structure of human GLOI with bound -(-hydroxy---iodophenylcarbamoyl) glutathione (HIPC-GSH, GSH refers to glutathione) (PDB code: 1QIN). Hydrogen atoms were added according to the default protonation states of all residues at neutral pH. Residue His126 adjacent to the zinc ion is set to HID (Histidine, delta H). The partial atomic charge of the zinc ion was set to +2. Various docking methods (CDOCKER, ligandfit, and Surflex-dock) were compared to verify their credibility to the GLOI system by re-docking the substrate HIPC-GSH into the active site pocket of GLOI. As a result, the optimal docking approach CDOCKER for this system gave an RMSD value of 0.83Å for HIPC-GSH to the X-ray crystal counterpart, which confirms the reliability of this docking protocol. Details of the docking procedure are described in . From the top ten poses with the most negative CDOCKER-Interaction-Energy, the optimal one based on both the docking energy and cluster popularity for each inhibitor was chosen. Each system was prepared by using GLOI in complex with the optimal docking pose of each inhibitor. The AMBER version 10 program suite was used to perform all the MD (molecular dynamics) simulations. The AMBER ff03 force field was applied as the parameters of the target protein GLOI, while the general AMBER force field (GAFF) was used for the parameterization of inhibitors. The partial atomic charges of the inhibitors were calculated at the level by Gaussian 03 to generate the electrostatic potential (ESP), which was then fitted to the restricted electrostatic potential (RESP) by the Antechamber module in the AMBER package. The Zn ion was treated by the ‘non-bonded model’ method., , All initial models after docking were completed using the XleaP module included in AMBER. The complexes were neutralized by adding eight sodium counter ions which were outside the active site pocket, and solvated in a rectangular box of water molecules with solvent layers of 10Å from the box edges to solute surface.