Nitro-oleic Acid, a Novel and Irreversible Inhibitor of Xanthine Oxidoreductase*

Xanthine oxidoreductase (XOR) generates proinflammatory oxidants and secondary nitrating species, with inhibition of XOR proving beneficial in a variety of disorders. Electrophilic nitrated fatty acid derivatives, such as nitro-oleic acid (OA-NO2), display anti-inflammatory effects with pleiotropic properties. Nitro-oleic acid inhibits XOR activity in a concentration-dependent manner with an IC50 of 0.6 μm, limiting both purine oxidation and formation of superoxide \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \((\mathrm{O}_{2}^{{\bar{{\cdot}}}})\) \end{document}. Enzyme inhibition by OA-NO2 is not reversed by thiol reagents, including glutathione, β-mercaptoethanol, and dithiothreitol. Structure-function studies indicate that the carboxylic acid moiety, nitration at the 9 or 10 olefinic carbon, and unsaturation is required for XOR inhibition. Enzyme turnover and competitive reactivation studies reveal inhibition of electron transfer reactions at the molybdenum cofactor accounts for OA-NO2-induced inhibition. Importantly, OA-NO2 more potently inhibits cell-associated XOR-dependent \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(\mathrm{O}_{2}^{{\bar{{\cdot}}}}\) \end{document} production than does allopurinol. Combined, these data establish a novel role for OA-NO2 in the inhibition of XOR-derived oxidant formation.

affirm a key role for XOR in pathophysiology, where inhibition of XOR has proven beneficial in a variety of vascular inflammatory processes (6).
The splanchnic system, the principal site of XOR activity, readily releases XOR into the circulation in response to ischemic or inflammatory insults (7,8). Once released into the vascular compartment, XDH is rapidly converted to XO. Cationic amino acid motifs present on the enzyme confer a high affinity (k d ϭ 6 nM) for negatively charged glycosaminoglycans (GAGs) located on the luminal face of the vascular endothelium (9). This XO-GAG association induces substantial sequestration and thus amplification of local endothelial XO concentration, producing a microenvironment with enhanced O 2 . and H 2 O 2 production. At the same time, GAG association results in resistance to XOR inhibition by oxypurinol, the active metabolite of allopurinol, increasing the K i from 230 nM for soluble XOR to 405 nM for GAG-bound XOR (10,11). Identification of more efficacious inhibitors, especially with regard to endothelium-associated XOR, will facilitate the treatment of vascular inflammatory conditions. The nitration of unsaturated fatty acids represents a convergence of NO, ROS, and lipid-mediated signaling. Current data suggest that oxidative inflammatory conditions that include the generation of NO-derived species induce the nitration of unsaturated fatty acids (12)(13)(14). Two fatty acid nitroalkene derivatives, nitro-oleic acid (OA-NO 2 ; 9-or 10-nitro-9-cis-octadecenoic acids) (26) and nitrolinoleic acid (LNO 2 ; 9-, 10-, 12-, or 13-nitro-octadecadienoic acids) (27) display pleiotropic cell signaling capabilities. For example, these nitroalkenes preferentially activate peroxisome proliferator-activated receptor-␥, inhibit inflammatory cytokine secretion from macrophages, impede platelet activation, and, in aqueous milieu, decay via a Nef-like reaction to yield NO (16 -19).
A central mechanism accounting for nitroalkene-mediated signaling is via post-translational modification of proteins (15). The alkenyl nitro configuration imparts electrophilic reactivity on the ␤-carbon adjacent to the nitro-bonded carbon, facilitating Michael addition reactions with nucleophiles such as cysteine and histidine residues of proteins (15). For example, adduction of OA-NO 2 with the catalytic Cys-149 of glyceraldehyde-3-phosphate dehydrogenase inhibits enzymatic activity and promotes membrane localization due to enhanced hydrophobicity of the nitroalkene-modified enzyme (15). Compared with other biological electrophilic lipids, nitro-fatty acids react with thiols with a high rate constant (20).
Since electrophilic substrates and inhibitors of XOR both interact with the molybdenum cofactor, and long chain alkene modifications augment the effectiveness of XOR inhibitors, the effects of various nitro-fatty acid derivatives on XOR activity were evaluated (21,22). We reveal that OA-NO 2 is a potent and irreversible inhibitor of soluble and GAG-immobilized XOR. These data suggest that nitration of fatty acids to nitroalkene derivatives yields products that can down-regulate XOR-dependent generation of ROS and account for some of the antiinflammatory actions observed for these lipid signaling mediators.
XOR Binding to GAGs-Xanthine oxidase was bound to heparin-Sepharose 6B (HS6B) as previously (11). Briefly, XOR (2 mg/ml) was added to a fixed amount of gel (0.05 g, dry weight), and the mixture was gently stirred in 5 mM KP i , pH 7.4 (2 ml final volume) at 25°C for 30 min. The suspension was centrifuged at 10,000 ϫ g for 5 min and washed, and the pellet was resuspended in 5 mM KP i , pH 7.4.
Anaerobic Experiments-Anaerobic analyses were performed in a table-top glove box (Coy Instruments, Grass Lake, MI) purged with N 2 . Oxygen concentrations were verified by monitoring the atmosphere in the chamber with an O 2 monitor (Maxtec, Salt Lake City, UT). All buffers were equilibrated Ͼ12 h before use. Spectrophotometric determinations were carried out in gas-tight cuvettes, and FAD reduction/oxidation studies were performed by the method of Ichimori (32).
Oxygen Consumption Studies-Real time concentrations of molecular O 2 were determined polarographically using an Apollo 4000 Free Radical Analyzer (World Precision Instruments, Sarasota, FL). Experiments were performed at standard temperature and pressure.
Cellular Studies-Bovine aortic endothelial cells were isolated as previously (11). Primary cell culture, routine passage, and experimental manipulations were all conducted in the absence of proteases. Cells were propagated by subculturing (1:4 ratios) in Medium 199 containing 5% fetal bovine serum and thymidine (10 M). Cells were utilized between passages 4 and 8 and were monitored visually for typical cobblestone morphology indicative of endothelial cells and by staining for von Willebrand factor expression.
EPR Spectrometry-The spin trap DMPO was further purified with activated charcoal, and the concentration was verified spectrophotometrically at 228 nm and ⑀ 228 ϭ 7,800 M Ϫ1 cm Ϫ1 . EPR measurements were performed at 25°C using a Bruker Elexsys E-500 spectrometer equipped with an ER049X microwave bridge and an AquaX cell. Studies were performed with 20 milliwatts microwave power, 100 kHz modulation frequency, 1 G modulation amplitude, 3510 G center field, 100 G sweep width, 164 ms time constant, 168 s sweep time, and 60 dB receiver gain with 1024 data points. Adventitious metals were removed from all buffers and water by treatment with Chelex resin. Buffers were then evaluated for the presence of metals by testing for the appearance of the ascorbyl radical in the EPR upon the addition of 100 M ascorbic acid. The following constituents were added as indicated: DMPO (25 mM), allopurinol (50 -100 M), OA-NO 2 (0 -50 M), SOD (10 units/ml), and xanthine (50 M) to a bovine aortic endothelial cell suspension (3 ϫ 10 6 cells/ml in Chelex-treated PBS, pH 7.4), and the spectrum was recorded after 10 min. Treatment of cells with XO and preparation is as follows. Confluent bovine aortic endothelial cells were exposed to XO (5 milliunits/ml) for 20 min at 25°C, harvested by mechanical dissociation, washed thoroughly (three times with PBS, pH 7.4), resuspended as a singlecell suspension (3 ϫ 10 6 cells/ml), and placed on ice (for less than 1 h) until warmed to 25°C immediately before evaluation in the EPR.
Statistics-Data were analyzed using one-way analysis of variance followed by Tukey's range test for multiple pairwise comparisons. Significance was determined as p Ͻ 0.05.

RESULTS
XOR Inhibition-When purified XOR is exposed to increasing concentrations of either allopurinol or OA-NO 2 , there is a concentration-dependent decrease in the initial rate of uric acid formation (Fig. 1A). OA-NO 2 is significantly more potent than allopurinol in inhibiting XOR activity with IC 50 values of 0.6 versus 2.4 M, respectively. Exposure to 10 M native oleic acid (18:1) or to 125 mM MeOH (vehicle control) did not alter XOR activity (not shown). Similar to its effect on urate production, OA-NO 2 inhibits O 2 . formation in a concentration-dependent manner, with an IC 50 of 0.75 M compared with 2.25 M for allopurinol (Fig. 1B). The addition of up to 2 units/ml SOD does not alter inhibition of uric acid formation, demonstrating that the mechanism of inactivation is not the result of release of NO from nitroalkene (17) and subsequent reaction with O 2 . and ONOO Ϫ formation (13). Furthermore, when the consumption of oxygen was monitored, in the presence of SOD, xanthine-dependent H 2 O 2 production by XOR is inhibited by OA-NO 2 in the same manner as O 2 . formation (not shown). pH dependence of OA-NO 2 inhibition is shown in Fig.  1C. A Dixon plot (Fig. 1D) indicates noncompetitive XOR inhibition by OA-NO 2 .

Influence of Reducing Agents and Other Electrophiles on XOR
Inhibition-Inhibition of XOR by OA-NO 2 was not reversible by the subsequent addition of reducing agents ( Fig. 2A). Treatment of OA-NO 2 -inhibited XOR with GSH (20 mM), ␤-mercaptoethanol (20 mM), or dithiothreitol (20 mM) did not abolish inhibition. However, the addition of these reducing agents to OA-NO 2 prior to exposure of the enzyme abrogated the inhibitory actions of OA-NO 2 . Control experiments in which NAD ϩ (50 -500 M) was added as an electron acceptor did not alter uric acid production, indicating that experimentally induced conversion of XO to XDH does not affect inhibition (not shown).
Nitro-oleic Acid Does Not Affect the FAD Cofactor-In the presence of purine substrate, the addition of DPI prevents FAD-dependent electron transfer, leading to reduction of the enzyme and an inability to reoxidize the molybdenum cofactor, ultimately resulting in inhibition of purine oxidation (Fig. 3A,  line 4). DCPIP can accept electrons from the reduced molybdenum of DPI-inhibited XOR and so restores the capacity to oxidize xanthine to uric acid, (Fig. 3A, line 2). However, when DPI-inhibited XOR is first exposed to OA-NO 2 before the addition of DCPIP and xanthine, uric acid production remained significantly depressed (Fig. 3A, line 3). Control studies revealed no reactions between OA-NO 2 and DPI or DCPIP.
Potential reactions of OA-NO 2 with the FAD cofactor were also addressed (Fig. 3B). Spectrum 1 represents reduced XOR in the absence of OA-NO 2 . When XOR was treated with concentrations of OA-NO 2 , inducing full inhibition under anaerobic conditions, a pronounced absorption (spectrum 2) of the oxidized FAD (450 nm), similar to XOR in the absence of OA-NO 2 (not shown), was observed. A significant loss (spectrum 3) and near abolition (spectrum 4) of absorption was observed upon reduction of the XOR-OA-NO 2 complex with increasing dithionite concentration. Moreover, when a reduced sample (spectrum 4) was exposed to room air for 30 min, a robust absorption appears (spectrum 5), indicating oxidation of the FAD. Analysis of NADH-reduced XOR affirmed this conclusion, since XOR-dependent NADH-mediated O 2 consumption was not affected by OA-NO 2 (Fig. 3C, solid line) There was also no effect of preincubation, in the absence of substrate, of XOR with OA-NO 2 on O 2 consumption (not shown). Nitro-oleic Acid Interaction with the Molybdenum Cofactor of XOR-Xanthine can anaerobically reduce the FAD of XOR following the initial reduction of the molybdenum cofactor (Fig. 4A, line 2). However, when XOR was inhibited by OA-NO 2 , xanthine reduction of the FAD was prevented (line 4). The effect of OA-NO 2 on XOR during enzyme turnover is presented in Fig. 4B (1 and 2). When OA-NO 2 (10 M) was added 15 s after XOR (10 milliunits/ml), turnover was initiated by xanthine addition, and formation of uric acid was inhibited 97%. Under the same conditions in the presence of DCPIP (15 M), the addition of OA-NO 2 yielded 30% inhibition.
The suicide inhibitor allopurinol is oxidized to oxypurinol by XOR at the molybdenum cofactor, where oxypurinol then noncompetitively inhibits enzyme activity. Exposure of XOR (30 milliunits/ml) to allopurinol (100 M) for 5 min (25°C/room air) followed by removal of unbound inhibitor by size exclusion chromatography produced linear rates of uric acid production 12 min after reactivation with xanthine. In contrast, OA-NO 2 (40 M) produced no observable reactivation (Fig.  4C, 1 and 2). However, when XOR was exposed first to allopurinol (5 min) and then OA-NO 2 (5 min), significant reactivation was observed.
Inhibition of Cell-associated XOR by OA-NO 2 -To examine the impact of XOR association with GAGs on the inhibitory actions of OA-NO 2 , XOR was immobilized on a heparin-Sepharose 6B complex (XOR-HS6B) and then exposed to either allopurinol (10 M) or OA-NO 2 (10 M) (Fig. 5A). Although 10 M allopurinol completely inhibited XOR in solution, inhibition of XOR-HS6B was only 50%. In contrast, OA-NO 2 inhibited XOR to similar extents in both free and heparin-bound states. In order to address possible differences in allopurinol and OA-NO 2 inhibition in a model of cell-bound XOR, the impact of OA-NO 2 on cell-associated XOR-dependent O 2 . formation was assessed by EPR spectrometry (Fig. 5B). Control cells, without added XOR, displayed minimal DMPO radical signal in the presence of xanthine (Xan). However, upon cellular GAG association with exogenous XOR, a robust increase in O 2 . generation was observed (XO ϩ Xan). This spectrum contains a combination of both DMPO-OOH and DMPO-OH radical adducts. When XOR-treated cells were exposed to OA-NO 2 , there was a concentration-dependent decrease in radical adduct signal intensity. In contrast, allopurinol treatment only partially inhibited cell-associated O 2 . formation. Cell treatment with oleic acid (18:1) demonstrated minimal effects on radical formation. The addition of SOD (10 units/ml) completely eliminated the radical signal, confirming that O 2 . was the proximal radical detected under these conditions. Controls in which XOR-treated cells (cells that were previously washed to remove free, non-cell-associated-XOR) were spun down, and the supernatant evaluated for radical formation produced no detectable DMPO signal (not shown). In addition, when XORtreated and washed cells were exposed to trypsin (0.25%) for 3 min at 37°C to remove/inactivate cell-associated XOR, no DMPO spin adduct was observed, demonstrating that the source of O 2 . trapped in our experiments was extracellular XOR (not shown).  DECEMBER 26, 2008 • VOLUME 283 • NUMBER 52

DISCUSSION
The pyrazolo-based suicide inhibitor allopurinol has been in clinical use for several decades to treat gout and more recently has been employed to reduce the effect of XOR-derived reactive species in ischemic tissue injury and cardiovascular disease (6).
However, a limited ability of allo-/oxypurinol to inhibit cellassociated XOR has been reported (11). The nature of this limitation, combined with the emerging significance of vessel wallassociated XOR in cardiovascular disease, supports the need for development of new strategies to achieve XOR inhibition. The substrate promiscuity of XOR, combined with the post-translational modification properties of electrophilic nitroalkenes, prompted investigation of OA-NO 2 as a potential inhibitor of XOR.
On an equimolar basis, OA-NO 2 inhibits both uric acid and O 2 . formation more potently than allopurinol, with IC 50 values for OA-NO 2 ϳ4 and ϳ3 times lower, respectively. Inhibition is maximal between pH 7 and 10, suggesting that OA-NO 2 is less effective when the carboxylic acid moiety is protonated (Fig.  1C). Kinetic analysis via Dixon plot reveals nearly parallel slopes for different substrate concentrations, indicating noncompetitive inhibition (Fig. 1D).
The alkenyl nitro configuration of OA-NO 2 confers electrophilic reactivity on the ␤-carbon adjacent to the nitro-bonded carbon, allowing for post-translational modification of proteins via reaction with nucleophilic residues, such as cysteine and histidine (15). In the case of glyceraldehyde-3-phosphate dehydrogenase, this process is reversible by exposure of the OA-NO 2 -inhibited enzyme to reducing agents, such as GSH, that exchange OA-NO 2 from the protein and restore enzymatic activity (15). In contrast to glyceraldehyde-3-phosphate dehydrogenase, high levels (20 mM) of GSH, ␤-mercaptoethanol, and dithiothreitol do not restore activity to OA-NO 2 -inhibited XOR ( Fig. 2A), indicating an irreversible covalent reaction between OA-NO 2 and XOR. However, when these nucleophiles first react with OA-NO 2 before XOR, inhibition capacity is lost.
Inhibition of XOR by OA-NO 2 is specific with respect to other electrophilic lipids, and ethyl pyruvate, a low molecular weight anionic electrophile, does not inhibit XOR activity (Fig.  2B). Furthermore, XOR is not inhibited by several structural variants of OA-NO 2 that differ in the nitroalkenyl and carboxylic acid moieties ( Table 1). The pure preparation of 9-OA-NO 2 regioisomer produced similar inhibition to that observed using the mixture of 9-and 10-OA-NO 2 , indicating nitration of either carbon 9 or 10 is sufficient to inhibit XOR. LNO 2 , a mixture of linoleic acid nitrated at carbon 9, 10, 12, or 13, also inhibited XOR but to a lesser (76%) extent than equimolar concentrations of OA-NO 2 (98%). This difference in inhibition may be proportional to the content of the LNO 2 preparation nitrated at either carbon 9 or 10. Moving the nitro group away from carbon 9 or 10, removal of the double bond between carbons 9 and 10, and/or placing the nitro group in the Z configuration results in loss of inhibitory properties, since exposure of XOR to iso-OA-NO 2 (0 -30 M), nitrostearic acid (0 -30 M), and elaidic acid (30 M), respectively, did not block enzymatic activity. In addition, the presence of a ketone (KODE) or an oxine (OXDE) at carbon 13 resulted in the loss of ability to inhibit XOR. Another requirement for OA-NO 2 inhibition of XOR is an intact carboxylic acid moiety. For example, modification of the carboxylic acid of OA-NO 2 by replacement with an alcohol (OH), a methyl ester, an amide, or a biotin moiety eliminates the inhibitory properties. Together, these data indicate a high level of specificity regarding the structure of OA-NO 2 with respect to its capacity to inhibit XOR, denoting that electrostatic interactions between OA-NO 2 and particular domains on the XOR protein are crucial.
Inhibition of XOR by OA-NO 2 does not occur by abrogation of electron transfer at the FAD cofactor, since OA-NO 2 inhibits uric acid formation from xanthine, even when electrons are transferred to DCPIP, bypassing the FAD (Fig. 3A). Additionally, OA-NO 2 does not materially alter reduction or oxidation spectra of the FAD, as shown in Fig. 3B. These data demonstrate that XOR maintains an electronically responsive FAD when purine oxidation is completely inhibited by OA-NO 2 . Furthermore, OA-NO 2 did not alter the rate of NADH-driven O 2 consumption or O 2 . formation (Fig. 3C). Combined, these data confirm that OA-NO 2 inhibition of XOR is not mediated via reaction with the FAD cofactor. These results prompted investigation of possible effects of OA-NO 2 on the molybdenum cofactor. Anaerobic reduction experiments were performed (as in Fig. 3) using xanthine instead of sodium dithionite to reduce the FAD. This method employs xanthine-derived electrons to sequentially reduce the molybdenum cofactor, the FeS centers, and finally the FAD. When XOR is exposed to OA-NO 2 , the ability of xanthine to reduce the FAD is abolished, suggesting that reaction of OA-NO 2 with XOR affects the molybdenum cofactor (Fig. 4A). Enzyme turnover experiments in which OA-NO 2 (10 M) is added after XOR is primed with xanthine reduce the rate of uric acid formation by 80%, whereas only a 25% decrease in urate formation is observed when DCPIP is present (Fig. 4, B and B1). Since DCPIP associates with and accepts electrons directly from the molybdenum cofactor (33,34), these data indicate that OA-NO 2 competes with DCPIP in close proximity to the molybdenum cofactor. Reactivation studies were designed to further test this theory. Allopurinol is oxidized to oxypurinol at the molybdenum cofactor, where oxypurinol then inhibits enzyme activity. Incubation of XOR with allopurinol for 5 min, followed by size exclusion chromatography to remove free allopurinol, produced reversal of XOR inhibition and full enzyme reactivation within 12 min after the addition of xanthine (Fig. 4, C and C1). Reactivation of XOR could not be observed following treatment with OA-NO 2 . However, when XOR was sequentially treated with allopurinol followed by exposure to OA-NO 2 , a significant (75%) reactivation was observed, indicating that 1) allo-/oxypurinol may have easier access to the molybdenum cofactor and sequential exposure of allo-/oxypurinol followed by exposure to OA-NO 2 results in significant reactivation of XOR, and 2) the molybdenum-allo-/oxypurinol association protects the molybdenum cofactor from inactivation by OA-NO 2 . Together, these data demonstrate that OA-NO 2 inhibition of XOR is mediated via reaction with the molybdenum cofactor. Due to the electrophilic nature of OA-NO 2 , a possible target for adduc-  2), rates of uric acid production before and after the addition of OA-NO 2 in B were calculated for four independent experiments; *, p Ͻ 0.05. C(1), XOR was exposed to either OA-NO 2 or allopurinol (AP) for 5 min, separated from free inhibitor by size exclusion column chromatography (G25 Sephadex), and reactivated by the addition of xanthine (100 M), and uric acid production was determined. Allopurinol and then OA-NO 2 indicates that enzyme was sequentially exposed to allopurinol and then OA-NO 2 . C(2), rates of uric acid production for the three conditions in C(1) were calculated for three independent experiments. Data points represent the mean Ϯ S.E. of at least three independent determinations; *, p Ͻ 0.05.
tion is the dithiolene of the pterin moiety, which serves in the coordination of the molybdenum molecule. Covalent modification of this site could lead to loss of the molybdenum and may explain our inability to reactivate the enzyme. Studies are ongoing to confirm the hypothesis.
Cell association of XOR significantly limits inhibition of XOR by allopurinol or its active metabolite, oxypurinol, even at concentrations far above those observed clinically (11). However, when HS6B-bound XOR was exposed to OA-NO 2 , enzyme activity was inhibited 95% compared with 50% for an equimolar concentration of allopurinol (Fig. 5A). At this concentration (10 M) both inhibitors were equally effective in inhibiting XOR free in solution. To extend this observation to add cell biological context, we exposed confluent bovine aortic endothelial cells to exogenous XOR, using the cells as a platform to immobilize the enzyme on extracellular GAGs, and then evaluated enzyme inhibition by OA-NO 2 and allopurinol. Fig. 5B demonstrates that cell-associated O 2 . generation is more effectively inhibited by OA-NO 2 than by allopurinol, mirroring the results seen when XOR is immobilized by association with HS6B. The spectra in Fig. 5B is composed of a combination of DMPO-OOH and DMPO-OH radical adducts, since the -OOH can rapidly decompose to -OH, especially in a cellular environment where DMPO-OOH can serve as a substrate for glutathione peroxidase or react with adventitious metals (35)(36)(37). Under our experimental conditions, the O 2 . is generated extracellularly, since controls in which extracellular XOR was removed/inactivated by treatment with trypsin produced no detectable DMPO spin adduct. In addition, the absence of contaminating radical species such as ⅐ OH that could serve to augment the spectra in Fig. 5B is confirmed by complete abrogation of the EPR signal in the presence of SOD. The SOD control further demonstrates that the generation of the observed DMPO radical adduct is a result of initial generation of O 2 . .
Nitroalkene derivatives of fatty acids have been detected (low nanomolar range) in human blood plasma, plasma-derived lipoproteins, and activated macrophages (13,38,39). Since fatty acid nitration is induced by oxidative and nitrative inflammatory conditions, these data suggest that fatty acid nitroalkene derivatives represent byproducts that may mediate adaptive anti-inflammatory reactions.
In summary, we have identified electrophilic OA-NO 2 as an XOR inhibitor that is more effective at inhibiting endothelial cell-associated XOR, thus potentially enhancing treatment of vascular inflammation, where XOR-derived ROS alters redoxdependent cell signaling reactions and reduces NO bioavailability. The irreversible inhibition of XOR by OA-NO 2 occurs with a lower IC 50 than allopurinol and encourages further investigation.