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      Ethers and esters derived from apocynin avoid the interaction between p47 phox and p22 phox subunits of NADPH oxidase: evaluation in vitro and in silico

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      * , , , * , § , , * , * , 1
      Bioscience Reports
      Portland Press Ltd.
      apocynin derivatives, drug design, inhibitory activity, molecular modelling, NADPH oxidase, 5-ASA, 5-aminosalicylic acid, AIR, autoinhibitory region, AUC, area under the curve, CM-H, 1-hydroxy-3-methoxycarbonyl-2,2,5,5-tetramethylpyrrolidine, DME, dimethoxyethane, DPPH, 1,1-diphenyl-2-picrylhydrazyl, EC, endothelial cell, EPR, electron paramagnetic resonance, MPO, myeloperoxidase, NO, nitric oxide, NOX, NADPH oxidase, PBR, polybasic region, PRR, proline-rich region, SOD, superoxide dismutase, TEA, triethanolamine

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          Abstract

          NOX (NADPH oxidase) plays an important role during several pathologies because it produces the superoxide anion (O 2 •−), which reacts with NO (nitric oxide), diminishing its vasodilator effect. Although different isoforms of NOX are expressed in ECs (endothelial cells) of blood vessels, the NOX2 isoform has been considered the principal therapeutic target for vascular diseases because it can be up-regulated by inhibiting the interaction between its p47 phox (cytosolic protein) and p22 phox (transmembrane protein) subunits. In this research, two ethers, 4-(4-acetyl-2-methoxy-phenoxy)-acetic acid ( 1) and 4-(4-acetyl-2-methoxy-phenoxy)-butyric acid ( 2) and two esters, pentanedioic acid mono-(4-acetyl-2-methoxy-phenyl) ester ( 3) and heptanedioic acid mono-(4-acetyl-2-methoxy-phenyl) ester ( 4), which are apocynin derivatives were designed, synthesized and evaluated as NOX inhibitors by quantifying O 2 •− production using EPR (electron paramagnetic resonance) measurements. In addition, the antioxidant activity of apocynin and its derivatives were determined. A docking study was used to identify the interactions between the NOX2′s p47 phox subunit and apocynin or its derivatives. The results showed that all of the compounds exhibit inhibitory activity on NOX, being 4 the best derivative. However, neither apocynin nor its derivatives were free radical scavengers. On the other hand, the in silico studies demonstrated that the apocynin and its derivatives were recognized by the polybasic SH3A and SH3B domains, which are regions of p47 phox that interact with p22 phox. Therefore this experimental and theoretical study suggests that compound 4 could prevent the formation of the complex between p47 phox and p22 phox without needing to be activated by MPO (myeloperoxidase), this being an advantage over apocynin.

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          Apocynin is not an inhibitor of vascular NADPH oxidases but an antioxidant.

          A large body of literature suggest that vascular reduced nicotinamide-adenine dinucleotide phosphate (NADPH) oxidases are important sources of reactive oxygen species. Many studies, however, relied on data obtained with the inhibitor apocynin (4'-hydroxy-3'methoxyacetophenone). Because the mode of action of apocynin, however, is elusive, we determined its mechanism of inhibition on vascular NADPH oxidases. In HEK293 cells overexpressing NADPH oxidase isoforms (Nox1, Nox2, or Nox4), apocynin failed to inhibit superoxide anion generation detected by lucigenin chemiluminescence. In contrast, apocynin interfered with the detection of reactive oxygen species in assay systems selective for hydrogen peroxide or hydroxyl radicals. Importantly, apocynin interfered directly with the detection of peroxides but not superoxide, if generated by xanthine/xanthine oxidase or nonenzymatic systems. In leukocytes, apocynin is a prodrug that is activated by myeloperoxidase, a process that results in the formation of apocynin dimers. Endothelial cells and smooth muscle cells failed to form these dimers and, therefore, are not able to activate apocynin. Dimer formation was, however, observed in Nox-overexpressing HEK293 cells when myeloperoxidase was supplemented. As a consequence, apocynin should only inhibit NADPH oxidase in leukocytes, whereas in vascular cells, the compound could act as an antioxidant. Indeed, in vascular smooth muscle cells, the activation of the redox-sensitive kinases p38-mitogen-activate protein kinase, Akt, and extracellular signal-regulated kinase 1/2 by hydrogen peroxide and by the intracellular radical generator menadione was prevented in the presence of apocynin. These observations indicate that apocynin predominantly acts as an antioxidant in endothelial cells and vascular smooth muscle cells and should not be used as an NADPH oxidase inhibitor in vascular systems.
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            Vascular NADPH oxidases: molecular mechanisms of activation.

            Oxygen-derived free radicals are thought to contribute to the initiation and progression of cardiovascular disease via several different mechanisms, such as consumption of nitric oxide, oxidation of proteins and lipids, and activation of redox-sensitive signalling cascades. Vascular NADPH oxidases are important sources of vascular radical formation. The activities of these enzymes, which in some aspects are similar to the leukocyte NADPH oxidase, are controlled on the expression level and complex activation mechanisms. As a plethora of vascular stimuli, such as growth factors, cytokines, physical stimuli, and lipids elicits radical formation by these enzymes, a careful analysis is required for the understanding of the activation of the NADPH oxidases. This article reviews the components of the NADPH oxidases in leukocytes and vascular tissue. Emphasis is put on the activation of the oxidases, including upstream signalling events and molecular modes of interaction between the subunits.
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              A gp91phox containing NADPH oxidase selectively expressed in endothelial cells is a major source of oxygen radical generation in the arterial wall.

              Reactive oxygen species (ROS) play an important role in regulating vascular tone and intracellular signaling; the enzymes producing ROS in the vascular wall are, however, poorly characterized. We investigated whether a functionally active NADPH oxidase similar to the leukocyte enzyme, ie, containing the subunits p22phox and gp91phox, is expressed in endothelial cells (ECs) and smooth muscle cells (SMCs). Phorbol 12-myristate 13-acetate (PMA), a stimulus for leukocyte NADPH oxidase, increased ROS generation in cultured ECs and endothelium-intact rat aortic segments, but not in SMCs or endothelium-denuded arteries. NADPH enhanced chemiluminescence in all preparations. p22phox mRNA and protein was detected in ECs and SMCs, whereas the expression of gp91phox was confined to ECs. Endothelial gp91phox was identical to the leukocyte form as determined by sequence analysis. In contrast, mitogenic oxidase-1 (mox1) was expressed in SMCs, but not in ECs. To determine the functional relevance of gp91phox expression, experiments were performed in aortic segments from wild-type, gp91phox(-/-), and endothelial NO synthase (eNOS)(-/-) mice. PMA-induced ROS generation was comparable in aortae from wild-type and eNOS(-/-) mice, but was attenuated in segments from gp91phox(-/-) mice. Endothelium-dependent relaxation was greater in aortae from gp91phox(-/-) than from wild-type mice. The ROS scavenger tiron increased endothelium-dependent relaxation in segments from wild-type, but not from gp91phox(-/-) mice. These data demonstrate that ECs, in contrast to SMCs, express a gp91phox-containing leukocyte-type NADPH oxidase. This enzyme is a major source for arterial ROS generation and affects the bioavailability of endothelium-derived NO.
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                Author and article information

                Journal
                Biosci Rep
                Biosci. Rep
                bsr
                BSR
                Bioscience Reports
                Portland Press Ltd.
                0144-8463
                1573-4935
                26 June 2013
                2 August 2013
                2013
                : 33
                : 4
                : e00055
                Affiliations
                *Laboratorio de Biofísica y Biocatálisis, Sección de Estudios de Posgrado e Investigación de la Escuela Superior de Medicina del Instituto Politécnico Nacional, Plan de San Luis y Díaz Mirón s/n, Casco de Santo Tomás, México, D.F. 11340, México
                †Laboratorio de Química Inorgánica de la Escuela Nacional de Ciencias Biológicas del Instituto Politécnico Nacional, Prolongación de Carpio y Plan de Ayala s/n, Casco de Santo Tomas, México, D.F. 11340, México
                ‡Laboratorio de Química Orgánica, Departamento de Ciencias Básicas, Unidad Profesional Interdisciplinaria de Biotecnología del Instituto Politécnico Nacional, Av. Acueducto s/n., Barrio La Laguna, Ticomán, México, D.F. 07340, México
                §Laboratorio de Modelado Molecular y Bioinformática, Sección de Estudios de Posgrado e Investigación de la Escuela Superior de Medicina del Instituto Politécnico Nacional, Plan de San Luis y Díaz Mirón s/n, Casco de Santo Tomás, México, D.F. 11340, México
                ∥Department of Chemistry, 250 Hackberry Lane, The University of Alabama, Tuscaloosa, AL 35487-0336, U.S.A.
                Author notes
                1To whom correspondence should be addressed (email marcrh2002@ 123456yahoo.com.mx ).
                Article
                e00055
                10.1042/BSR20130029
                3731894
                23802190
                8ad41164-0917-463f-a912-2d95a5138a7c
                © 2013 The author(s) has paid for this article to be freely available under the terms of the Creative Commons Attribution Licence (CC-BY)(http://creativecommons.org/licenses/by/3.0/) which permits unrestricted use, distribution and reproduction in any medium, provided the original work is properly cited.

                This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution and reproduction in any medium, provided the original work is properly cited.

                History
                : 27 February 2013
                : 12 April 2013
                : 10 May 2013
                Page count
                Figures: 7, Tables: 1, References: 41, Pages: 12
                Categories
                Original Paper
                S2

                Life sciences
                apocynin derivatives,drug design,inhibitory activity,molecular modelling,nadph oxidase,5-asa, 5-aminosalicylic acid,air, autoinhibitory region,auc, area under the curve,cm-h, 1-hydroxy-3-methoxycarbonyl-2,2,5,5-tetramethylpyrrolidine,dme, dimethoxyethane,dpph, 1,1-diphenyl-2-picrylhydrazyl,ec, endothelial cell,epr, electron paramagnetic resonance,mpo, myeloperoxidase,no, nitric oxide,nox, nadph oxidase,pbr, polybasic region,prr, proline-rich region,sod, superoxide dismutase,tea, triethanolamine

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