Polyamine-Conjugated Nitroxides Are Efficacious Inhibitors of Oxidative Reactions Catalyzed by Endothelial-Localized Myeloperoxidase

Abstract

The heme enzyme myeloperoxidase (MPO) is a key mediator of endothelial dysfunction and a therapeutic target in cardiovascular disease. During inflammation, MPO released by circulating leukocytes is internalized by endothelial cells and transcytosed into the subendothelial extracellular matrix of diseased vessels. At this site, MPO mediates endothelial dysfunction by catalytically consuming nitric oxide (NO) and producing reactive oxidants, hypochlorous acid (HOCl) and the nitrogen dioxide radical (•NO2). Accordingly, there is interest in developing MPO inhibitors that effectively target endothelial-localized MPO. Here we studied a series of piperidine nitroxides conjugated to polyamine moieties as novel endothelial-targeted MPO inhibitors. Electron paramagnetic resonance analysis of cell lysates showed that polyamine conjugated nitroxides were efficiently internalized into endothelial cells in a heparan sulfate dependent manner. Nitroxides effectively inhibited the consumption of MPO’s substrate hydrogen peroxide (H2O2) and formation of HOCl catalyzed by endothelial-localized MPO, with their efficacy dependent on both nitroxide and conjugated-polyamine structure. Nitroxides also differentially inhibited protein nitration catalyzed by both purified and endothelial-localized MPO, which was dependent on •NO2 scavenging rather than MPO inhibition. Finally, nitroxides uniformly inhibited the catalytic consumption of NO by MPO in human plasma. These studies show for the first time that nitroxides effectively inhibit local oxidative reactions catalyzed by endothelial-localized MPO. Novel polyamine-conjugated nitroxides, ethylenediamine-TEMPO and putrescine-TEMPO, emerged as efficacious nitroxides uniquely exhibiting high endothelial cell uptake and efficient inhibition of MPO-catalyzed HOCl production, protein nitration, and NO oxidation. Polyamine-conjugated nitroxides represent a versatile class of antioxidant drugs capable of targeting endothelial-localized MPO during vascular inflammation.

Multi-Oxidant Environment as a Suicidal Inhibitor of Myeloperoxidase

“…This formation of compound II contains a ferryl iron-oxygen moiety, and superoxide anion radicals can convert ferric MPO into compound III, a mixed ferric-superoxide/ferrousdioxygen complex. Interestingly, a negative feedback loop of H 2 O 2 on MPO was shown in vitro [13], and similar findings were observed in the presence of nitroxide [14,15]. During an immune response, a mixture of reactive species, including H 2 O 2 , is present [16,17], supporting the hypothesis of inhibiting and controlling MPO for homeostasis.…”

Polyamine–Drug Conjugates: Do They Boost Drug Activity?

“…Par-ticularly, TEMPO exerts a multifaceted antioxidant efficacy by inhibiting myeloperoxidase, a heme peroxidase enzyme impaired in inflammatory diseases, and directly counteracting oxidant species. In this view, Maiocchi et al exploited the TEMPO nucleus to attach different polyamines in order to increase its bioavailability and antioxidant potency [114]. Notably, Put-TEMPO hybrid 70 (Figure 20) achieved one of the highest cellular uptakes, which allowed it to retain efficient inhibition of cellular myeloperoxidase activities such as chlorination, H 2 O 2 consumption, HOCl production, protein nitration and NO oxidation [114].…”

“…In this view, Maiocchi et al exploited the TEMPO nucleus to attach different polyamines in order to increase its bioavailability and antioxidant potency [114]. Notably, Put-TEMPO hybrid 70 (Figure 20) achieved one of the highest cellular uptakes, which allowed it to retain efficient inhibition of cellular myeloperoxidase activities such as chlorination, H 2 O 2 consumption, HOCl production, protein nitration and NO oxidation [114]. Minoxidil belongs to a similar nitrone family and is an antihypertensive agent with antinflammatory properties.…”

“…Molecules 2023, 28, x FOR PEER REVIEW 23 of 31 such as chlorination, H2O2 consumption, HOCl production, protein nitration and NO oxidation [114]. Minoxidil belongs to a similar nitrone family and is an antihypertensive agent with antinflammatory properties.…”

Halogenation Activity of Mammalian Heme Peroxidases

“…MPO binds to lipoproteins such as LDL [148][149][150] (where apoB-100, the major apolipoprotein of LDL, and respective lipid species represent major targets for HOCl attack [151,152]) and high-density lipoprotein (HDL) [153][154][155] (quite heterogenous lipoprotein particles varying in density, size, electrophoretic mobility, and protein and lipid composition; for a review, see [156]). Although electron microscopy studies originally revealed limited binding and internalization of MPO (compared to EPO) to inflammatory cells [157], ongoing studies clearly underscore the adverse oxidative reactions of endothelial-localized MPO [158] as well as endothelial-transcytosed MPO [109] leading to targeted subendothelial matrix oxidation of proteins [159][160][161][162][163][164] via MPO-mediated nitration and/or chlorination; the latter reaction could by suppressed by SCN − and nitrite (NO 2 − ) [162].…”