Nitroarachidonic Acid, a Novel Peroxidase Inhibitor of Prostaglandin Endoperoxide H Synthases 1 and 2

Abstract

Prostaglandin endoperoxide H synthase (PGHS) catalyzes the oxidation of arachidonate to prostaglandin H2. We have previously synthesized and chemically characterized nitroarachidonic acid (AANO2), a novel anti-inflammatory signaling mediator. Herein, the interaction of AANO2 with PGHS was analyzed. AANO2 inhibited oxygenase activity of PGHS-1 but not PGHS-2. AANO2 exhibited time- and concentration-dependent inhibition of peroxidase activity in both PGHS-1 and -2. The plot of kobs versus AANO2 concentrations showed a hyperbolic function with kinact = 0.045 s−1 and Ki*app = 0.019 μm for PGHS-1 and kinact = 0.057 s−1 and Ki*app = 0.020 μm for PGHS-2. Kinetic analysis suggests that inactivation of PGHS by AANO2 involves two sequential steps: an initial reversible binding event (described by Ki) followed by a practically irreversible event (Ki*app) leading to an inactivated enzyme. Inactivation was associated with irreversible disruption of heme binding to the protein. The inhibitory effects of AANO2 were selective because other nitro-fatty acids tested, such as nitrooleic acid and nitrolinoleic acid, were unable to inhibit enzyme activity. In activated human platelets, AANO2 significantly decreased PGHS-1-dependent thromboxane B2 formation in parallel with a decrease in platelet aggregation, thus confirming the biological relevance of this novel inhibitory pathway.

Nitrite and nitrate-dependent generation of anti-inflammatory fatty acid nitroalkenes

“…Consequently, synthetic homologs of endogenous nitroalkenes induce human coronary artery endothelium to alter the expression of over 400 genes related to metabolism, tissue repair and inflammatory responses [12]. Nitroalkylation also directly inhibits the catalytic activity of enzymes including xanthine oxidoreductase [33], cyclooxygenase [37] and soluble epoxide hydrolase (Cys521) [34], reactions that result in significant beneficial physiological responses. These alkylation reactions are reversible, with the exception of xanthine oxidoreductase, and can be modulated by competing tissue nucleophiles such as cysteine, glutathione (GSH) and hydrogen sulfide [11, 38].…”

Characterization and quantification of endogenous fatty acid nitroalkene metabolites in human urine

“…Potential mechanisms underlying these actions include a ) inhibition of neutrophil function and platelet activation ( 43,44 ), b ) serving as partial agonists for PPAR ␥ ( 14,15,(45)(46)(47), c ) inhibition of cytokine expression via inhibition of DNA binding by the p65 unit of NF-B ( 48 ), and d ) upregulation of phase 2 gene expression via Keap1/Nrf2-dependent ( 5,49,50 ) and -independent mechanisms ( 51 ). Critical pro-infl ammatory enzymatic activities are also inhibited by fatty acid nitroalkenes, including xanthine oxidoreductase and cyclooxygenase-2 ( 52,53 ). These actions result in antiinfl ammatory responses in diverse animal models of disease including limiting restenosis after vessel injury ( 54 ), attenuation of weight gain and loss of insulin sensitivity in murine models of metabolic syndrome ( 6, 55 ), inhibition of sepsis-induced renal failure ( 56 ), prevention of ischemia-reperfusion injury ( 31,32,57 ), reduction of plaque formation in a murine ApoE Ϫ / Ϫ atherosclerosis model, and the reduction of chemically-induced infl ammatory bowel disease ( 47 ).…”

Modulation of Nitro-fatty Acid Signaling

“…Consistent with this notion, nitroalkenes alkylate multiple functionally significant cysteines in the transcriptional regulatory protein Keap1, the p65 subunit of NFB, and the ligand binding domain of PPAR␥, leading to the regulation of Ͼ300 genes critical for metabolic, antioxidant defense, and tissue repair responses (12,15,52). Although electrophilic reactivity and signaling actions are often similar for NO 2 -OA, NO 2 -LA, NO 2 -CLA, and NO 2 -arachidonic acid, there are also unique and functionally significant reactions of NO 2 -OA with xanthine oxidoreductase and NO 2 -arachidonic acid with cyclooxygenase (53,54).…”