Diseño y Evaluación Teórica y Experimental in vitro de Compuestos Derivados de Apocinina como Posibles Inhibidores de NADPH oxidasa

Abstract

The oxidative stress produced by high concentrations of superoxide anion O2•- derived from NADPH oxidase 2 (Nox2) in the vascular endothelium has been linked to cardiovascular diseases such as hypertension and atherosclerosis observed a decrease in nitric oxide (ON) by binding with O2•-. It has been proposed to inhibit Nox2 to decrease the concentration of O2•- and increase the bioavailability of NO to relax the vascular endothelium. Today has been reported that the dimeric form of apocynin, activated by the enzyme myeloperoxidase (MPO), inhibits Nox2 preventing the assembly of its subunits, however, apocynin has not been used as a drug because the affinity of MPO by this is low and not all is active at its dimeric form, furthermore, even it is not clear the mechanism by which NADPH oxidase inhibits apocynin. For these reasons were designed, synthesized and evaluated compounds derived from apocynin, with the aim of these do not require to be activated by MPO and its affinity for NADPH oxidase could be better than apocynin. The compounds purity was tested by chromatography and their structure were characterized by nuclear magnetic resonance (NMR) of proton (1H) and carbon (13C). In order to determinate if the compounds have antioxidant properties a DPPH assay was done. The activity of NADPH oxidase in the presence of these compounds was evaluated by electron paramagnetic resonance (EPR) in aorta homogenate of male wistar rat, using PP-H as a spin trap. The EPR data obtained were quantified by the area under the curve which represent the O2•- production and were analyzed by a method of analysis of variance (ANOVA). A value of p<0.05 was considered significant difference between the groups. We performed docking of the compounds previously designed, first these were optimized with Gaussian 98 and after these were docked on the crystallized fragment of the protein p47phox that contain the segment that it binds to p22phox and is mentioned bound the apocynin dimer. We compared the protein homology between rat and human in ClustalW2 to validate if the results obtained in homogenates of rat aortas are reproducible in humans. Were found that these compounds not are trapping of radicals and not reduced the DPPH, but decreased the area under the curve of O2•- according to the enzymatic activity of NADPH oxidase measured by EPR having the best activity as inhibitor the compound CINP2B. In the studies in silico was observed that the compounds are recognized in the p47phox region that binds to p22phox, and the compounds that showing higher affinity are CINP1B and CINP2B. Therefore, the best inhibitor is CINP2B and possibly its mechanism of action is preventing the assembly of the subunits p47phox and p22phox of Nox2.