From the
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
The heme containing enzyme prostaglandin-H synthase-1 (PGHS1, EC
1.14.99.1)
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
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.
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
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
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
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
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
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.
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
= 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.
(
)
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.
, 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.
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.
H
O
(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).
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 H
O
triggered a
second wave of oxidation presumably due to the decomposition of
H
O
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, H
O
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 H
O
/min/µg of
protein. b, tepoxalin was added around 150 s after addition of
AA followed 1 min later by H
O
. The calculated
activities of PO in the presence of vehicle or tepoxalin are 7.85 and
4.0 nmol of H
O
/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
H
O
decomposition. In contrast, deferoxamine
which contains four hydroxamic acid groups blocked Fe-dependent
breakdown of H
O
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.
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.
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.
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.
(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.
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
, 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.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.