©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Tepoxalin, a Novel Dual Inhibitor of the Prostaglandin-H Synthase Cyclooxygenase and Peroxidase Activities (*)

Susanna S. C. Tam , Daniel H. S. Lee , Elizabeth Y. Wang , Donald G. Munroe , Catherine Y. Lau (§)

From the (1) From Discovery Research, The R. W. Johnson Pharmaceutical Research Institute, Don Mills, Ontario, M3C 1L9 Canada

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Prostaglandin-H synthase-1, the rate-limiting enzyme in prostaglandin synthesis, has both cyclooxygenase (CO) and peroxidase (PO) activities. While most nonsteroidal anti-inflammatory drugs (NSAIDs) inhibit only the CO activity, we describe an inhibitor, tepoxalin, that inhibits both the CO (IC = 0.1 µM) and the PO (IC = 4 µM) activities. Unlike many NSAIDs which are competitive inhibitors of CO, tepoxalin is a noncompetitive inhibitor of CO and its inhibitory effect on PO but not CO is reversed by excess heme. Moreover, inhibition of the PO activity by tepoxalin is not dependent on the enzymatic turnover of the CO activity. The hydroxamic acid of tepoxalin is responsible for the PO inhibition since a carboxylic acid derivative of tepoxalin retains full CO but not PO inhibition. We postulated that the hydroxamic group might confer the ability to inhibit PO on conventional CO inhibitors. This idea was supported by the observation that naproxen hydroxamic acid, but not naproxen showed PO inhibition. Furthermore, tepoxalin's carboxylic acid analogue and naproxen each competitively relieved PO inhibition by their respective hydroxamic acids. The intracellular activity of PO as monitored by the release of reactive oxygen species was also inhibited by both tepoxalin and naproxen hydroxamic acid. These observations suggest a strategy for design of novel compounds to inhibit prostaglandin synthase PO. The therapeutic implications of these novel PO inhibitors are discussed.


INTRODUCTION

The heme containing enzyme prostaglandin-H synthase-1 (PGHS1, EC 1.14.99.1)() catalyzes the first committed step in the biosynthesis of prostaglandins, thromboxanes, and prostacyclins (Smith and Marnett, 1991; DeWitt, 1991; Smith et al., 1991). It exhibits two distinct enzymatic activities: cyclooxygenase (CO) activity which converts arachidonic acid (AA) to the hydroperoxide, prostaglandin G (PGG), and a peroxidase (PO) activity which reduces PGG to the alcohol PGH through a heme-mediated, two-electron transfer process (Marnett and Maddipati, 1991; Lambeir et al., 1985). The relationship between these two activities of the synthase is of considerable interest because of the importance of the synthase in the overall control of eicosanoid biosynthesis. It is now known that the two activities occur at distinct, albeit interacting, sites on the membrane-bound PGHS1 molecule (Kulmacz and Lands, 1983; Shimokawa and Smith, 1991). X-ray crystallography studies with the ovine enzyme suggest that the CO active site consists of a long narrow, hydrophobic channel extending from the protein-membrane interface through the protein interior approaching the heme prosthetic group at its apex (Picot et al., 1994). On the other hand, the PO site is found on the exterior of the protein. Despite the new insight provided by the x-ray crystallography data, the symbiotic relationship between the two catalytic functions remains puzzling.

In order for the CO to remove the 13-pro (S) hydrogen from the AA, the enzyme must be activated by a process that requires peroxide (Lands et al., 1976; Hemler et al., 1979; Hemler and Lands, 1980). However, CO is also readily inactivated by high levels of peroxides (Hemler and Lands, 1980). This leads to the well known self-inactivation process that PGHS1 undergoes since the product of CO, PGG, is a peroxide. Reducing cosubstrates that activate PO are also excellent protectors of CO activity (Markey et al., 1987; Kulmacz et al., 1994). Recent spectral and computer analyses suggest that the PO of PGHS1 is a heme-dependent peroxidase similar to horseradish peroxidase (Kulmacz et al., 1994). In this scheme, the resting form of the synthase reacts with the hydroperoxide to form the two higher oxidation states, namely Compound I and Compound II (Ple and Marnett, 1989; Dietz et al., 1988), with a subsequent two-electron transfer to the resting state. The CO activity is proposed to occur by an intramolecular step governed by the conversion of Compound I to a catalytic intermediate. This intermediate, generated during the PO reaction cycle, produces a tyrosyl radical (Dietz et al., 1988) which interacts as an oxidant with AA to remove a hydrogen atom from C-13 of the fatty acid to initiate the CO reaction. The enzyme bound fatty acyl radical in turn interacts with molecular oxygen and rearranges to form the enzyme bound PGG which then dissociates from the CO site of the enzyme. This model accounts for the stimulation of PGHS1 CO activity by exogenous hydroperoxides and also the inhibition by hydroperoxide scavengers.

The use of selective inhibitors for one or both of the catalytic activities of PGHS1 also provides intriguing insight into the relationship between the two enzymatic activities. Most NSAIDs such as aspirin, indomethacin, and ibuprofen compete with AA for binding to the enzyme (Mizuno et al., 1982). These NSAIDs do not affect the PO activity (Mizuno et al., 1982; Kulmacz et al., 1985). Antioxidants on the other hand can either activate or inhibit PO depending on the dose used. At low doses, the NSAID BW755C is readily oxidized by the PO of PGHS1 and thus activates CO in a dose-related manner. However, at higher doses it inhibits CO activities (Marnett et al., 1982). The ability to stimulate CO at low concentrations and inhibit at high concentrations is typical of compounds which are reducing cofactors for the PO component of PGHS1. Compounds for which this has been documented include MK447 (Kuehl et al., 1977), phenol (Hemler and Lands, 1980), guaiacol (Egan et al., 1980), sulfides (Ple and Marnett, 1989), and acetaminophen (Robak et al., 1978). However, because of the biphasic activities and the low binding affinity of these compounds, they cannot be used effectively to delineate the relationship between the two catalytic activities. Compounds that are potent, dual inhibitors of both CO and PO activities are likely to provide novel insight and further understanding into the relationship between CO and PO of PGHS1. In this report, we describe the mode of action of tepoxalin, previously identified as a dual CO/5-lipoxygenase inhibitor (Argentieri et al., 1994), to be a potent inhibitor of both the CO and PO activities of the sheep PGHS1. This novel compound allows us to probe into both the symbiotic and interdependent relationships between CO and PO. The important therapeutic application of compounds that block one or both of the enzyme activities is discussed.


MATERIALS AND METHODS

Reagents

PGHS1 was purchased from Oxford Biomedical Research Inc., Oxford, MI. The purity of the enzyme was about 95% as demonstrated by Western blot assay carried out in-house. Tris, tryptophan, hematin, AA sodium salt, N,N,N`,N`-tetramethyl-p-phenylenediamine dihydrochloride (TMPD), phenol, hydrogen peroxide, aspirin, naproxen sodium, indomethacin, diclofenac sodium salt, piroxicam, pyrrolidinedithiocarbamate (PDTC), deferoxamine mesylate, phenylbutazone, and EDTA were obtained from Sigma. Tepoxalin and its analogues were synthesized as described previously (Wachter and Ferro, 1992). 2`,7`-Dichlorofluorescein diacetate was obtained from Molecular Probes (Eugene, OR).

PGHS1 CO Assay

CO activity was obtained by measuring the oxidation of TMPD at 611 nm. The assay was carried out at 37 °C according to the method of Raz and Needleman(1990) with a slight modification. An absorption coefficient of 13,500 M cm at 611 nm was used to calculate the rate of TMPD oxidation. A stoichiometric ratio of 2 mol of TMPD oxidized/mol of hydroperoxide reduced was used to calculate specific activity (Kulmacz, 1987) assuming 1 mol of AA would be consumed for each mole of hydroperoxide reduced in a TMPD coupled reaction. Each assay mixture contained 80 µM AA, 1 µM hematin, 200 µM TMPD, 5 mM tryptophan, and 1.25 µg of ovine PGHS1 in a final volume of 1 ml of 0.1 M Tris-HCl, pH 8.0. The reaction was initiated by the addition of AA. For inhibition studies, drugs were dissolved in ethanol and added simultaneously with AA except for aspirin where the enzyme was pretreated with aspirin for 30 min at room temperature in 0.1 M Tris-HCl, pH 8.0, before the assay. The effect of compounds on spontaneous TMPD oxidation was measured in the absence of the enzyme and subtracted as blank for each measurement. Enzyme velocity was calculated according to the formula: OD at 611 nm/min/0.027 enzyme (µg/µl) volume of the enzyme. Oxygen consumption was performed according to the method described by DeWitt et al.(1981) using a YSI model 5300 biological oxygen monitor. In some studies, the PGE level was measured by radioimmunoassay (DuPont NEN Research Products, Boston, MA).

PGHS1 Hydroperoxidase Assay

The spectrophotometric assay for the hydroperoxidase activity was performed as described for CO activity except that AA was replaced by freshly prepared 100 µM HO and the amount of enzyme used was reduced to 0.5 µg. For inhibition studies, drugs dissolved in ethanol were incubated with the enzyme for 1 min at 37 °C before the addition of hematin, except for aspirin where the enzyme was pretreated with aspirin for 30 min at room temperature before the assay.

Other Peroxidases

The Myeloperoxidase Assay

This was a modification of the published method of Suzuki et al.(1983). Briefly, dorsal skin homogenate was prepared from mice injected intradermally with lipopolysaccharide 6 h earlier. The homogenate was centrifuged at 4 °C at 12,000 g for 20 min. The supernatant was mixed with 3,3`,5,5`-tetramethylbenzidine in phosphate buffer to a final concentration of 1.6 mM. HO (150 µl of 3%) was added to initiate the reaction and the mixture was incubated at 37 °C for 2 min whereupon the reaction was stopped by the addition of 200 mM sodium phosphate buffer. The absorbance was measured spectrophotometrically at 655 nm using a Beckman DU 640 spectrophotometer.

The Horseradish Peroxidase Assay

This was performed according to the method of Harlow and Lane(1988).

Measurement of ROS in Jurkat Cells

Jurkat cells were pretreated with various drugs for 1 h at 37 °C and were washed twice. The cells were resuspended in phosphate-buffered saline supplemented with 2 mM glucose and incubated with 5 µM 2`,7`-dichlorofluorescein diacetate (DCFH-DA) and 2 µg/ml propidium iodide for 20 min at 37 °C. The cells were then stimulated with 100 µMt-butyl hydroperoxide for 45 min at 37 °C. Intracellular oxidative burst activities in the t-butyl hydroperoxide stimulated and unstimulated cells were measured by the oxidation of non-fluorescent DCFH-DA to the highly fluorescent 2`,7`-dichlorofluorescein as detected by flow cytometry using FACScan (Becton Dickinson, CA). Dead cells stained by propidium iodide were excluded from the analysis by electronic gating and 10,000 events were analyzed for each sample. For COS7 cells, ROS released was measured using a microfluorometric assay according to the method described by Wan et al.(1993).


RESULTS

Tepoxalin Inhibits Both CO and PO Activities in Ovine PGHS1

In order to compare the CO and PO activities of PGHS1 under similar assay conditions, their activities were measured by monitoring the oxidation of TMPD using AA or HO as substrates, respectively. However, to ensure that the CO activity measured under these conditions was comparable to published results, initial studies were conducted with an O consumption assay to validate the AA/TMPD assay. The two enzyme assays were comparable with the O consumption assay yielding an activity of 37.5 nmol of O/min/µg of protein (18.75 nmol of AA/min/µg of protein) as compared to 15 nmol of AA/min/µg of protein by the spectrophotometric assay with TMPD as the reducing cosubstrate and the color indicator. The validity of this assay was further confirmed by demonstrating that the IC of known NSAIDs () obtained in this assay were similar to previously published results (O'Neill et al., 1994; Meade et al., 1993). Fig. 1compares the inhibitory effects of tepoxalin with piroxicam (a known CO inhibitor with an IC similar to tepoxalin) on CO and PO activities; compares tepoxalin's IC on CO and PO activities with several known NSAIDs. Unlike many NSAIDs, tepoxalin inhibited both CO and PO activities with an IC of 0.1 and 4 µM, respectively (Fig. 1, ). This was not due to nonspecific reduction of TMPD by tepoxalin since, in the absence of PGHS1, tepoxalin did not affect oxidation of TMPD by hematin. Tepoxalin's inhibitory effect on PGHS1 also was not altered in the presence of different concentrations of cosubstrates (tryptophan 1-5 mM or TMPD 100-400 µM), suggesting that tepoxalin is not competing with cosubstrates in the peroxidase reaction. Moreover, as previously reported (Kazmi et al., 1995), tepoxalin is only a poor antioxidant compared to other known inhibitors of PGHS1 (). Although tepoxalin's potency for PO inhibition is about 40 times less than for CO inhibition, this is not surprising since the PO activity has a turnover rate that is 10-15 times higher than CO activity (Markey et al., 1987). The specificity of tepoxalin on PGHS1 PO activity was further confirmed by the observation that tepoxalin tested at 50 µM had no inhibitory effect on related peroxidases such as horseradish peroxidase or myeloperoxidase (legend to ).


Figure 1: Effect of tepoxalin and piroxicam on PGHS1 CO (a) and PGHS1 PO (b). The CO and PO assays in the presence of increasing concentrations of tepoxalin or piroxicam were conducted as described under ``Experimental Procedures.'' The % control response is the ratio of tepoxalin- or piroxicam-treated samples to vehicle-treated samples. The enzyme activity of CO and PO in the presence of vehicle alone is 22.56 nmol of AA/min/µg of protein and 10.65 nmol of HO/min/µg of protein, respectively. Effects of tepoxalin on CO and PO were tested three to four times with similar results.



As can be seen in , known non-antioxidant type NSAIDs inhibit only the CO activity and even at concentrations 50-100-fold over their IC for CO activity, no inhibition of PO activity was observed. The PO activity, however, was inhibited by strong antioxidants such as PDTC, phenylbutazone, or iron chelating agents for heme such as deferoxamine. EDTA, interestingly, had no effect on PO or CO activity even at 10 mM. The antioxidant PDTC and the chelating agent deferoxamine inhibited only the PO but not the CO activity, which seemingly contradicts an earlier suggestion that the PO activity is absolutely required for the CO activity (Ohki et al., 1979). However, recent findings from site-directed mutagenesis indicate that it is possible to obtain a mutant (histidine 386 replaced by alanine) that retains 40% of CO activity with undetectable levels of PO activity (Shimokawa and Smith, 1991).

Unique Inhibitory Activity of Tepoxalin on PGHS1

Tepoxalin Is a Noncompetitive Inhibitor of CO Activity

The mode of inhibition of tepoxalin on CO activity was compared to the well known CO inhibitor naproxen. As reported previously (Van der Ouderaa et al., 1980; Mizuno et al., 1982; O'Neill et al., 1994), naproxen competitively inhibited CO, since increasing concentrations of AA relieved its inhibitory effect (Fig. 2a). Tepoxalin, on the other hand, exhibited a typical noncompetitive mode of inhibition with reduced V and unaltered K in the presence of increasing concentrations of the substrate. Lineweaver-Burk double-reciprocal plots confirmed the competitive and noncompetitive nature of naproxen and tepoxalin, respectively (Fig. 2b). It is unlikely, however, that tepoxalin acts like aspirin, another NSAID which noncompetitively inhibits CO by forming a covalent linkage with PGHS1, because extensive dialysis of the enzyme after drug treatments can reverse the inhibitory effect of tepoxalin but not that of aspirin (data not shown).


Figure 2: Effect of increasing concentrations of AA on the inhibition of CO by naproxen and tepoxalin. a, vehicle, 0.5 µM naproxen, or 0.1 µM tepoxalin was incubated with increasing concentrations of AA (20-400 µM) in a standard CO reaction mixture as described under ``Experimental Procedures.'' Velocity (V) was expressed as the change in OD reading at 611 nm/min and can be converted to AA oxidized/min/µg protein as described under ``Experimental Procedures.'' b, Lineweaver-Burk double-reciprocal plot of initial velocity against substrate concentrations. The experiment was repeated three times with reproducible results.



CO Inactivation Does Not Prevent Inhibition of PO Activity by Tepoxalin

CO and PO activities can be measured sequentially by monitoring the oxidation of TMPD, using first AA and then HO as the substrates. This type of spectrophotometric assay allows both combined CO + PO activities and residual PO activity alone to be measured in the same enzyme preparation (Raz and Needleman, 1990). As shown in Fig. 3a, upon addition of AA to PGHS1, a rapid change in optical density occurred which reached a plateau around 120 s. Addition of more AA at this time did not produce another burst of activity suggesting inactivation of CO or combined CO + PO activities (data not shown). However, addition of HO triggered a second wave of oxidation presumably due to the decomposition of HO by the residual PO activity. This suicidal type of inactivation of CO by AA while leaving most PO intact has been previously reported (Smith and Lands, 1972; Hemler et al., 1979; Marshall et al., 1987; Kulmacz and Lands, 1983). Addition of naproxen inhibited the CO phase but not the PO activity which is consistent with earlier data (). In contrast, the addition of tepoxalin inhibited not only the CO phase but also the second phase made up of only the PO activity (Fig. 3a). To test whether inactivation of CO activity precludes the inhibition of PO activity by tepoxalin, the drug was added after the cessation of CO activities triggered by AA as indicated in Fig. 3b. One minute after addition of tepoxalin, HO was added to activate residual PO. As can be seen, tepoxalin was still quite capable of inhibiting PO after CO activities had been inactivated. This result suggests that CO inactivation does not prevent specific binding of tepoxalin or inhibition of PGHS1 PO by tepoxalin.


Figure 3: Spectrophotometric measurement of the effect of tepoxalin and naproxen on the PO and combined CO + PO activities. Ovine PGHS1 was preincubated with 1 µM hematin as described under ``Experimental Procedures.'' a, at the zero time point, AA and TMPD were added with tepoxalin, naproxen, or vehicle and TMPD oxidation was measured anywhere from 0 to 150 s. HO was then added and oxidation of TMPD was measured for another 150 s. The calculated activities of CO in the presence of vehicle, 10 µM tepoxalin, and 1 µM naproxen are 15, 0.13, and 0.93 nmol of AA/min/µg of protein, respectively, the corresponding PO activities are 9.55, 4.21, and 9.12 nmol of HO/min/µg of protein. b, tepoxalin was added around 150 s after addition of AA followed 1 min later by HO. The calculated activities of PO in the presence of vehicle or tepoxalin are 7.85 and 4.0 nmol of HO/min/µg of protein, respectively. The experiment was carried out twice with reproducible results.



Heme Concentration Dependence of PO but Not CO Inhibition by Tepoxalin

As reported previously, both the CO and the PO activities of PGHS1 display an absolute requirement for heme (Ohki et al., 1979; Miyamoto et al., 1976). Addition of 1 µM hematin to the enzyme mixture resulted in nearly maximal activity for both CO and PO. As can be seen in , tepoxalin's inhibitory effect on CO activity could not be reversed by increasing concentrations of hematin; however, its inhibitory effect on PO activity was dependent on the concentration of hematin in the reaction mixture as the presence of excess heme reduced the suppressive effect of tepoxalin on PO activity. To test whether tepoxalin is a nonspecific iron chelator, its effect on the iron-dependent decomposition of HO was monitored. At 30 µM, tepoxalin had no effect on HO decomposition. In contrast, deferoxamine which contains four hydroxamic acid groups blocked Fe-dependent breakdown of HO by 50% at 200 µM (data not shown). Deferoxamine is relatively ineffective in blocking the PO activity (IC = 400 µM, see ).

The Hydroxamic Acid in Tepoxalin Is Critical for PO Inhibition

Since increasing concentrations of hematin reduced tepoxalin's inhibitory effect on PO but not CO and since the chemical structure of tepoxalin consists of a modified pyrazole attached to hydroxamic acid (I), it is possible that the hydroxamic acid which chelates iron plays a key role in the inhibition of PO. To test this possibility, a tepoxalin carboxylic derivative lacking the hydroxamic acid (RWJ 20142) was tested on both CO and PO functions. RWJ 20142 exhibited no effect on PO activity while maintaining a potency similar to tepoxalin in inhibiting CO activity (IC = 0.1 µM) (I). This difference suggests that the hydroxamic acid of the tepoxalin molecule plays an essential role in PO but not CO inhibition. Interestingly, naproxen hydroxamic acid, in which naproxen was modified by the addition of hydroxamic acid, inhibited PO with an IC of 25 µM. Since naproxen was not active on the PO function even when tested at 50 µM, addition of hydroxamic acid on a pure CO inhibitor can convert it into a dual CO/PO inhibitor as demonstrated by both tepoxalin and naproxen.

PO Inhibition by Tepoxalin Can be Relieved by a Structural Analogue Lacking Hydroxamic Acid

The importance of the hydroxamic acid of tepoxalin for PO inhibition was further tested by using structurally related analogues. As mentioned earlier, RWJ 20142, a tepoxalin analogue which has a carboxylic acid substitution for the hydroxamic acid exhibited only CO inhibition. This compound relieved tepoxalin's inhibitory effect on PO in a dose-related manner (Fig. 4a). Naproxen or indomethacin, on the other hand, had no effect on tepoxalin's inhibitory effects (Fig. 4a). These data suggest that PO inhibition by tepoxalin depends on binding to a site on PGHS1 which can be occupied by a tepoxalin analogue but not by structurally unrelated naproxen or indomethacin. On the other hand, naproxen effectively relieved the PO inhibitory effect of naproxen hydroxamic acid, its structurally similar analogue (Fig. 4b). RWJ 20142, however, had no effect on the PO inhibitory effect of naproxen hydroxamic acid. Studies with these two classes of structurally dissimilar compounds demonstrate that a pure CO inhibitor can be converted into an inhibitor for both CO and PO by the appropriate substitution. The dual inhibitors thus formed bind to the same site as their analogues which are pure CO inhibitors. Tepoxalin Inhibits PO Activities in Intact Cells-To determine whether the ability of tepoxalin to inhibit PO activity could be observed in intact cells, Jurkat cells were stimulated with t-butyl hydroperoxide, a chemical shown previously to activate the AA metabolism through PGHS1 (Robison and Forman, 1990). The combined CO + PO activity of the intact cells was measured by quantitating the amount of PGE released into the supernatant. The PO activity was measured by monitoring the intracellular ROS production with the intracellular probe DCFH-DA which is converted to the fluorescent 2`,7`-dichlorofluorescein (DCF) upon interaction with oxygen metabolites or a variety of peroxides (Cathcart et al., 1983; Bass et al., 1983; Boissy et al., 1989). Within 15-30 min of t-butyl hydroperoxide treatment, a dramatic increase in oxidation of DCFH-DA to DCF was observed which paralleled the increase in PGE production in the supernatant of t-butyl hydroperoxide-stimulated Jurkat cells (Fig. 5a). Dose response studies with t-butyl hydroperoxide also showed a correlation between ROS production and PGE synthesis. The ROS release peaked around 30 min and started to decline probably due to the action of intracellular oxidant scavengers such as glutathione peroxidase or catalase. On the other hand, the PGE level continued to increase for up to 1 h. All four compounds tested, tepoxalin, naproxen, RWJ 20142, and naproxen hydroxamic acid blocked PGE synthesis (Fig. 5b) but only tepoxalin and naproxen hydroxamic acid, the two PO inhibitors blocked ROS production in Jurkat cells (Fig. 5c). The fact that the release of ROS in Jurkat cells stimulated with t-butyl hydroperoxide can be largely attributed to PGHS1 PO activation is further confirmed by two observations. 1) Both tepoxalin and naproxen hydroxamic acid had no effect on the endogenous increase in ROS in unstimulated Jurkat cells as measured by an increase in fluorescence in DCFH-DA loaded, unstimulated cells as a function of time. 2) Failure of t-butyl hydroperoxide to activate ROS release in COS7, a cell line previously reported to be PGHS1 negative (Hla and Neilson, 1992). DCFH-DA loaded COS7 cells were stimulated with buffer or 100 µMt-butyl hydroperoxide for 30 min. The fluorescence emitted was measured in a microplate fluorometer and results for stimulated or unstimulated samples were 1572 ± 260 and 1490 ± 400 fluorescence units, respectively.


Figure 4: Displacement of tepoxalin or naproxen hydroxamic acid by their analogues. a, the effect of 2.5 µM tepoxalin on PO was measured in the presence of increasing concentrations of RWJ 20142 (tepoxalin analogue), naproxen, or indomethacin. Naproxen had no effect even when tested at 50 µM (data not shown). b, the effect of 25 µM naproxen hydroxamic acid on PO was measured in the presence of increasing concentrations of naproxen or the tepoxalin analogue RWJ 20142 (the highest concentration attainable was 150 µM). The uninhibited PO activity is 9.045 nmol of HO/µg of protein. The experiments were carried out twice with similar results.




Figure 5: Tepoxalin and naproxen hydroxamic acid inhibit PO activities in intact cells. a, t-butyl hydroperoxide stimulated production of PGE and also ROS in Jurkat cells. Jurkat cells were stimulated with different concentrations of t-butyl hydroperoxide for 30 min and the level of PGE in the supernatant was measured by the radioimmunoassay as described under ``Experimental Procedures.'' ROS production was measured by monitoring the conversion of DCFH-DA to DCF using FACScan (Hockenbery et al., 1993) and the mean fluorescence of stimulated cells-nonstimulated cells was shown as a function of t-butyl hydroperoxide concentrations. b, suppression of PGE production by various compounds as measured by radioimmunoassay. c, histograms illustrate the suppression of ROS release by tepoxalin and naproxen hydroxamic acid in Jurkat cells treated for 30 min with 100 µMt-butyl hydroperoxide.




DISCUSSION

The bifunctional catalytic activities of PGHS1 have been well recognized for years and recent x-ray data further highlight the intriguing structural relationships between the two enzyme activities (Picot et al., 1994). The availability of a potent dual inhibitor for both CO and PO will provide insight into the symbiotic relationship between the two functions and also supply novel information on the biological significance of PO both in vitro and in vivo. This study using tepoxalin, a putative inhibitor for both CO and PO, has fulfilled this mission by: 1) providing additional data to delineate the interdependence between CO and PO; 2) a novel method to design dual inhibitors; and 3) the possible biological role of PO in intact cells.

Studies with an inhibitor of both CO and PO activities provide insight into the symbiotic and functional relationship between the PGHS1 CO and PO functions. PGHS1 is an unusual enzyme with two catalytic activities. The CO function which is rate-limiting and substrate specific, bears little homology to other proteins. The PO function, on the other hand, bears striking similarity to other peroxidases (Picot et al., 1994). In this report, by using different inhibitors, we have demonstrated that CO and PO can function quite independently. Tepoxalin was still effective in inhibiting the PO after the CO was inactivated by the addition of the substrate AA, and several antioxidants including PDTC and deferoxamine can inhibit PO with little effect on CO. Clearly PO is required for converting the product of CO namely PGG to PGH. However, the turnover rate of PO is so much higher than CO that, even when PO was inhibited to an almost undetectable level, there was still sufficient PO to convert the product of CO into the final more stable form. Similar observations were made when the PO function was inactivated by site-directed mutagenesis (Shimokawa and Smith, 1991). Co-crystallization studies of tepoxalin or other inhibitors with PGHS1 would undoubtedly provide further insight into the intriguing relationship between PO and CO functions and suggest novel approaches to the design of more potent inhibitors.

The work carried out in this report with tepoxalin and naproxen hydroxamic acid also suggests rational approaches for design of PO inhibitors. Since PGHS1 PO, like many peroxidases, is heme dependent, one strategy would be to design PO inhibitors that complex hemic iron to prevent redox cycling. However, simple hydrophilic chelating agents such as EDTA or deferoxamine were not active at nontoxic concentrations. We propose that, for a chelator to selectively inhibit PO, it must be brought to the vicinity of the metal group. This molecular specificity could be achieved by incorporating a chelating group (hydroxamic acid) into the structure of conventional CO inhibitors (NSAIDs) that are known to bind to PGHS1 selectively and with high affinity.

In this study, we provided two examples of such inhibitors: 1) tepoxalin, a bis-substituted pyrazole structure which is a potent noncompetitive inhibitor of CO activity with an IC of 0.1 µM. Extensive structure-activity relationship studies of tepoxalin and its analogues revealed that the CO inhibition activity of tepoxalin is governed mainly by the pyrazole group (data not shown). Its hydroxamic acid, an iron chelator, appears to be absolutely required for PO inhibition. The importance of the hydroxamic acid in the inhibition of the PO was further supported by the observations that (a) increasing concentrations of hematin reversed tepoxalin's suppressive effect on PO but not on CO activity and (b) substitution of the hydroxamic acid with carboxylic acid eliminated completely the PO inhibition but retained full CO suppression.

2) Attaching hydroxamic acid to another known CO inhibitor, naproxen, resulted in its conversion to an active PO inhibitor. This observation supports the proposition that adding the hydroxamic acid functionality to a high affinity ligand for PGHS1 can result in PO inhibition. However, while tepoxalin potently inhibited CO with IC similar to its analogue RWJ 20142 lacking the hydroxamic group, attaching the hydroxamic acid to naproxen decreased the CO inhibitory activity of the compound (IC increased from 1 to 10 µM). Thus, similar modifications of a CO inhibitor may produce compounds with various degrees of inhibitions for CO and PO.

Our data also suggest that attaching hydroxamic acid to a CO inhibitor to convert it to a combined CO + PO inhibitor could be a general formula that can be applied to CO inhibitors. Tepoxalin and naproxen are structurally unrelated compounds and their mode of inhibition on CO is different with naproxen being competitive and tepoxalin being noncompetitive. Their binding sites on PGHS1 are likely to be different since naproxen cannot relieve tepoxalin's inhibitory effect on PO while the tepoxalin's analogue, RWJ 20142, could not relieve the inhibitory effect of naproxen hydroxamic acid. However, despite the differences in structure and binding profile, the addition of hydroxamic acid resulted in the acquisition of PO inhibitory function by structurally diverse CO inhibitor.

The binding site of tepoxalin on PGHS1 is currently unknown. However, being a non-competitive inhibitor, it is unlikely that it binds to the site occupied by AA in the hydrophobic channel of PGHS1. Tepoxalin may in fact be binding close to and altering the conformation around tyrosine 384, the tyrosyl radical known to be crucial to the hydrolysis of AA. Being close to tyrosine 385 also places tepoxalin in the vicinity of the peroxidase, allowing the hydroxamic acid of tepoxalin to effectively chelate the heme group and inactivate the peroxidase.

The biological importance of PGHS1 PO inhibitors is further illustrated in intact cell studies. Upon t-butyl hydroperoxide stimulation, a dramatic increase in intracellular levels of ROS was observed. Although t-butyl hydroperoxide is a poor substrate of PGHS1, it has been demonstrated previously that t-butyl hydroperoxide can rapidly and selectively stimulate AA metabolism through the PGHS1 pathway in alveolar macrophages (Robison and Forman, 1990), probably through the activation of phospholipase A (Charkraborti et al., 1993). Since the dose response of ROS release to t-butyl hydroperoxide stimulation in Jurkat cells paralleled that of PGE production (Fig. 5a), it is reasonable to postulate that the ROS release was due to emission of free radicals during the reduction of PGG to PGH. Dual CO/PO inhibitors, tepoxalin and naproxen hydroxamic acid, but not pure CO inhibitors such as RWJ 20142 or naproxen, suppressed the production of free radicals upon t-butyl hydroperoxide stimulation, although all four compounds suppressed PGE production. The basal level of ROS release in Jurkat cells and the ROS production in t-butyl hydroperoxide stimulated COS7, a PGHS1 negative cell line, however, were not affected by tepoxalin or naproxen hydroxamic acid suggesting that the ROS release in t-butyl hydroperoxide stimulated cells is related to PGHS1. One can speculate from these results that dual CO/PO inhibitors, besides arresting prostaglandin synthesis, can also dampen the production of ROS which are causative for oxidative damages (Dargel, 1992). ROS and lipid peroxidation, the chain reaction that ROS initiates are implicated in the pathogenesis of atherosclerosis, cancer, inflammatory processes, hyperoxic injury, and ischemic-reperfusion injury (Holmsen and Robkin, 1977; Burk et al., 1978; Akerboom et al., 1982). Thus the therapeutic application of CO + PO inhibitors such as tepoxalin probably goes beyond treatment of prostaglandin-mediated inflammatory processes.

In summary we have illustrated, in this report, that the PO of PGHS1 can be inhibited by attaching hydroxamic acid to a binder of the enzyme. This strategy worked for PGHS1 with tepoxalin and naproxen hydroxamic acid, the examples described above. Recently, a similar strategy has been employed to design inhibitors for the metallo-protease responsible for tumor necrosis factor processing (Gearing et al., 1994; McGeehan et al., 1994; Mohler et al., 1994). Hydroxamic acid attached to a peptide mimetic-like structure created several potent inhibitors for the protease. It is likely that attaching a metal chelating function to a competitive or noncompetitive structure that binds to the enzyme with high affinity will become a useful way of designing inhibitors for metallo-enzymes.

  
Table: 0p4in With the myeloid peroxidase assay, 50 µM tepoxalin and vehicle showed 0.856 ± 0.06 and 0.890 ± 0.07 unit of OD, respectively. With the horseradish peroxidase assay performed in ELISA format, readings for 50 µM tepoxalin and vehicle were 0.773 ± 0.07 and 0.787 ± 0.08, respectively.(119)

  
Table: Effects of hematin concentration on CO and PO inhibition by tepoxalin


  
Table: Effect of tepoxalin and naproxen derivatives on CO and PO activities



FOOTNOTES

*
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: Discovery Research, The R. W. Johnson Pharmaceutical Research Institute, 19 Green Belt Dr., Don Mills, Ontario, M3C 1L9 Canada. Tel.: 416-442-2500 (ext. 2427); Fax: 416-442-2502.

The abbreviations used are: PGHS, prostaglandin-H synthase; CO, cyclooxygenase; PO, peroxidase; NSAIDs, nonsteroidal anti-inflammatory drugs; AA, arachidonic acid; PGG, prostaglandin G; TMPD, N,N,N`,N`-tetramethyl-p-phenylenediamine dihydrochloride; PDTC, pyrrolidinedithiocarbamate; ROS, reactive oxygen species; DCFH-DA, 2`,7`-dichlorofluorescein diacetate; DCF, 2`,7`-dichlorofluorescein.


ACKNOWLEDGEMENTS

We thank Dr. Syed M. I. Kazmi for helpful discussion, and Dr. J. Booth, Anna Bahnesli, and Linda Traeger for their assistance in the manuscript preparation.


REFERENCES
  1. Akerboom, T. P. M., Bilzer, M., and Sies, H.(1982) J. Biol. Chem. 257, 4248-4252 [Abstract/Free Full Text]
  2. Argentieri, D. C., Ritchie, D. M., Ferro, M. P., Kirchner, T., Wachter, M. P., Anderson, D. W., Rosenthale, M. E., and Capetola, R. J.(1994) J. Pharmacol. Exp. Ther. 271, 1399-1408 [Abstract]
  3. Bass, D. A., Parce, J. W., Dechatelet, L. R., Szejda, P., Seeds, M. C., and Thomas, M.(1983) J. Immunol. 130, 1910-1917 [Abstract/Free Full Text]
  4. Boissy, R. E., Trinkle, L. S., and Nordlund, J. J.(1989) Cytometry 10, 779-787 [Medline] [Order article via Infotrieve]
  5. Burk, R. F., Nishiki, K., Lawrence, R. A., and Chance, B.(1978) J. Biol. Chem. 253, 43-46 [Medline] [Order article via Infotrieve]
  6. Cathcart, R., Schwiers, E., and Ames, B. N.(1983). Anal. Biochem. 134, 111-116 [Medline] [Order article via Infotrieve]
  7. Charkraborti, S., Michael, J. R., Gurtner, S. S., Dutta, G., Merker A. (1993) Biochem. J. 292. 585-589
  8. Dargel, R.(1992) Exp. Toxic Pathol. 44, 169-181 [Medline] [Order article via Infotrieve]
  9. DeWitt, D. L.(1991) Biochim. Biophys. Acta 1083, 121-134 [Medline] [Order article via Infotrieve]
  10. DeWitt, D. L., Rollins, T. E., Day, J. S., Ganger, J. A., and Smith, W. L.(1981) J. Biol. Chem. 256, 10375-10382 [Free Full Text]
  11. Dietz, R., Nastainczyk, W., and Ruf, H. H.(1988) Eur. J. Biochem. 171, 321-328 [Abstract]
  12. Egan, R. W., Gale, P. H., Beveridge, G. C., Marnett, L. J., and Kuehl, F. A., Jr.(1980) Advances in Prostaglandin and Thromboxane Research (Samuelsson, B., Ramwell, P. W., and Paoletti, R., eds) Vol. 6, pp. 153-155, Raven Press, New York [Medline] [Order article via Infotrieve]
  13. Gearing, A. J. H., Beckett, P., Christodoulou, M., Churchill, M., Clements, J., Davidson, A. H., Drummond, A. H., Galloway, W. A., Gilbert, R., Gordon, J. L., Leber, T. M., Mangan, M., Miller, K., Nayee, P., Owen, K., Patel, S., Thomas, W., Wells, G., Wood, L. M., and Woolley, K.(1994) Nature 370, 555-557 [CrossRef][Medline] [Order article via Infotrieve]
  14. Harlow, E., and Lane, D.(1988) Antibodies: A Laboratory Manual, pp. 553-612, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY
  15. Hemler, M. E., and Lands, W. E. M.(1980) J. Biol. Chem. 255, 6253-6261 [Abstract/Free Full Text]
  16. Hemler, M. E., Cook, H. W., and Lands, W. E. M.(1979) Arch. Biochem. Biophys. 193, 340-345 [Medline] [Order article via Infotrieve]
  17. Hla, T., and Neilson, K.(1992) Proc. Natl. Acad. Sci. U.S.A. 89, 7384-7388 [Abstract]
  18. Hockenbery, D. M., Oltvai, Z. N., Yin, X.-M., Milliman, C. L., and Korsmeyer, S. J.(1993) Cell 75, 241-251 [Medline] [Order article via Infotrieve]
  19. Holmsen, H., and Robkin, L.(1977) J. Biol. Chem. 252, 1752-1757 [Abstract]
  20. Kazmi, S. M. I., Plante, R. K., Visconti, V., Taylor, G. R., Zhou, L., and Lau, C. Y.(1995) J. Cell. Biochem. 57, 299-310 [Medline] [Order article via Infotrieve]
  21. Kuehl, F. A., Jr., Humes, J. L., Egan, R. W., Ham, E. A., Beveridge, G. C., and Van Arman, C. G.(1977) Nature 265, 170-173 [Medline] [Order article via Infotrieve]
  22. Kulmacz R. J.(1987) Prostaglandins 34, 225-240 [CrossRef][Medline] [Order article via Infotrieve]
  23. Kulmacz, R. J., and Lands, W. E. M.(1983) Prostaglandins 25, 531-540 [CrossRef][Medline] [Order article via Infotrieve]
  24. Kulmacz, R. J., Miller, J. F., Jr., and Lands, W. E. M.(1985) Biochem. Biophys Res. Commun. 130, 918-923 [Medline] [Order article via Infotrieve]
  25. Kulmacz, R. J., Pendleton, R. B., and Lands, W. E. M.(1994) J. Biol. Chem. 269, 5527-5536 [Abstract/Free Full Text]
  26. Lambeir, A. M., Markey, C. M., Dunford, H. B., and Marnett, L. J. (1985) J. Biol. Chem. 260, 14894-14896 [Abstract/Free Full Text]
  27. Lands, W. E. M., Cook, H. W., and Rome, L. H.(1976) in Advances in Prostaglandin and Thromboxane Research (Samuelsson, B., and Paoletti, R., eds) Vol. I, pp. 7-17, Raven Press, New York
  28. Markey, C. M., Alward, A., Weller, P.E., and Marnett, L. J.(1987) J. Biol. Chem. 262, 6266-6279 [Abstract/Free Full Text]
  29. Marnett, L. J., and Maddipati, K. R.(1991) in Peroxidase in Chemistry and Biology (Everse, J., Everse, K. E., and Grisham, M. B., eds) Vol. I, pp. 293-334, CRC Press, Boca Raton, FL
  30. Marnett, L. J., Siedlik, P. H., and Fung, L. W. M.(1982) J. Biol. Chem. 257, 6957-6964 [Abstract/Free Full Text]
  31. Marshall, P. J., Kulmacz, R. J., and Lands, W. E. M.(1987) J. Biol. Chem. 262, 3510-3517 [Abstract/Free Full Text]
  32. McGeehan, G. M., Becherer, J. D., Bast, R. C., Jr., Boyer, C. M., Champion, B., Connolly, K. M., Conway, J. G., Furdon, P., Karp, S., Kidao, S., McElroy, A. B., Nichols, J., Pryzwansky, K. M., Schoenen, F., Sekut, L., Truesdale, A., Verghese, M., Warner, J., and Ways, J. P.(1994) Nature 370, 558-561 [CrossRef][Medline] [Order article via Infotrieve]
  33. Meade, E. A., Smith, W. L., and DeWitt, D. L.(1993) J. Biol. Chem. 268, 6610-6614 [Abstract/Free Full Text]
  34. Miyamoto, T., Ogino, N., Yamamoto, S., and Hayaishi, O.(1976) J. Biol. Chem. 251, 2629-2636 [Abstract]
  35. Mizuno, K., Yamamoto, S., and Lands, W. E. M.(1982) Prostaglandins 23, 743-757 [CrossRef][Medline] [Order article via Infotrieve]
  36. Mohler, K. M., Sleath, P. R., Fitzner, J. N., Cerretti, D. P., Alderson, M., Kerwar, S. S., Torrance, D. S., Otten-Evans, C., Greenstreet, T., Weerawarna, K., Kronheim, S. R., Petersen, M., Gerhart, M., Kozlosky, C. J., March, C. J., and Black, R. A.(1994) Nature 370, 218-220 [CrossRef][Medline] [Order article via Infotrieve]
  37. O'Neill, G. P., Mancini, J. A., Kargman, S., Yergey, J., Kwan, M. Y., Falgueyret, J.-P., Abramovitz, M., Kennedy, B. P., Ouellet, M., Cromlish, W., Culp, S., Evans, J. F., Ford-Hutchinson, A. W., and Vickers, P. J.(1994) Mol. Pharmacol. 45, 245-254 [Abstract]
  38. Ohki, S., Ogino, N., Yamamoto, S., and Hayaishi, O.(1979) J. Biol. Chem. 254, 829-836 [Abstract]
  39. Picot, D., Loll, P. J., and Garavito, R. M.(1994) Nature 367, 243-249 [CrossRef][Medline] [Order article via Infotrieve]
  40. Ple, P., and Marnett, L. J.(1989) J. Biol. Chem. 264, 13983-13993 [Abstract/Free Full Text]
  41. Raz, A., and Needleman, P.(1990) Biochem. J. 269, 603-607 [Medline] [Order article via Infotrieve]
  42. Robak, J., Wieckowski, A., and Gryglewski, R.(1978) Biochem. Pharmacol. 27, 393-396 [Medline] [Order article via Infotrieve]
  43. Robison, T. W., and Forman, H. J.(1990) Prostaglandins 40, 13-28 [Medline] [Order article via Infotrieve]
  44. Shimokawa, T., and Smith, W. L.(1991) J. Biol. Chem. 266, 6168-6173 [Abstract/Free Full Text]
  45. Smith, W. L., and Lands, W. E. M.(1972) Biochemistry 11, 3276-3285 [Medline] [Order article via Infotrieve]
  46. Smith, W. L., and Marnett, L. J.(1991) Biochim. Biophys Acta 1083, 1-17 [Medline] [Order article via Infotrieve]
  47. Smith, R. C., Reeves, J., McKee, M. L., and Daron, H.(1987) Free Rad. Biol. Med. 3, 251-257 [Medline] [Order article via Infotrieve]
  48. Smith, W. L., Marnett, L. J., and DeWitt, D.L.(1991) Pharmacol. Ther. 49, 153-179 [CrossRef][Medline] [Order article via Infotrieve]
  49. Suzuki, K., Ota, S., Sakatani, T., Sasagawa, S., and Fujikawa, T. (1983) Anal. Biochem. 132, 345-352 [Medline] [Order article via Infotrieve]
  50. Van der Ouderaa, F. J., Buytenhek, M., Nugteren, D. H., and Van Dorp, D. A.(1980) Eur. J. Biochem. 109, 1-8 [Abstract]
  51. Wachter, M. P., and Ferro, M. P.(1992) U. S. Patent 5,164,381
  52. Wan, C. P., Myung, E., and Lau, B. H. S.(1993) J. Immunol. Methods 159, 131-138 [CrossRef][Medline] [Order article via Infotrieve]

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