الفهرس 
1.3. Catalytic MechanismOnly 14 pages are availabe for public view
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Abstract
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reaction for a period of 10-100 s. The CO2 concentrations ranged from 1.7 to 17 mM for the determination of the kinetic parameters and inhibition constants. For each inhibitor at least six traces of the initial 5-10% of the reaction have been used for determining the initial velocity. The uncatalyzed rates were determined in the same manner and subtracted from the total observed rates. Stock solutions of inhibitor (0.1 mM) were prepared in distilled-deionized water and dilutions up to 0.01 nM were done thereafter with the assay buffer. Inhibitor and enzyme solutions were preincubated together for 15 min at room temperature prior to assay, in order to allow for the formation of the E-I complex. The inhibition constants were obtained by non-linear least-squares methods using PRISM 3 and the Cheng-Prusoff equation, and represent the mean from at least three different determinations. [111]
5.2.2. In Vitro Antiproliferative Activity:
The two examined human cell lines (MCF-7 and MCF-10A) have been obtained from American Type Culture Collection (ATCC). Cells lines were maintained as monolayers in Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 10% FBS, 2 mM Lglutamine, 100 U/ml penicillin and 100μg/ml streptomycin sulfate. Cobalt (II) chloride (CoCl2) was utilized as an inducer of HIF-1α to furnish a chemically-induced hypoxia. Cells were sub- 3 cultured with trypsine /EDTA solution, counted with haemocytometer and plated onto 96-well plates (5000 cells/well) and left overnight to form a semi-confluent monolayer. Cell monolayers were treated in quadrates with vehicle (DMSO, 0.1% v/v), test samples or Adriamycin as positive control for an exposure time of 48 h. At the end of exposure, MTT solution in PBS (5 mg/ml) was then added to all well including no cell blank and left to incubate for 90 min. The formation of formazan crystals was visually confirmed using phase contract microscopy. DMSO (100 μl/well) was added to dissolve the formazan crystals with shaking for 10 min after which the absorbance
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was read at 590 nm against no cell blanks on a FLuo Star Optima microplate reader (BMG technologies, Germany). Cell proliferation was calculated comparing the OD values of the DMSO control wells and those of the samples represented as % proliferation to the control. Dose-response experiment was performed on samples producing > or =50% loss of cell proliferation using five serial 2-fold dilutions (50, 25, 12.5, 6.25 and 3.125 μM) of the sample. IC50 values (concentration of sample causing 50% loss of cell proliferation of the vehicle control) were calculated using non-linear regression curve fitting of the dose response plots on GraphPad Prism V.6.0 software. [115-129]
5.3. Molecular docking study:
The molecular simulation studies were performed by docking the final targeted compounds (IXa, Va, Ve, XIIIa, XIVa and XVIa) in hCA I and hCA II. The crystal structure of hCA I (PDB 3K34) and II (PDB 3K34) was prepared using the Protein Preparation Wizard tool implemented in the Schrödinger suite. The energy minimization protocol with a root mean square deviation (RMSD) value of 0.30 Å was applied using force field OPLS3e. The 3D ligand structures were prepared by Maestro (v.11.9) and evaluated for their ionization states at pH 7.4 ± 0.5 with Epik (v.4.7). The conjugate gradient method in Macro model (v.12.3) was used for energy minimization (maximum iteration number: 2500; convergence criterion: 0.05 Kcal/mol/Å2). Grids were centered on the centroids of the zinc-coordinating residues and for molecular docking studies the software Glide SP (v.8.2; default settings) was used. The standard precision (SP) mode of the Glide Score function was applied to evaluate the predicted binding poses.[130-136]