(Received for publication, August 24, 1995)
From the
The therapeutic action of nonsteroidal anti-inflammatory drugs
(NSAIDs) is exerted through the inhibition of prostaglandin G/H
synthase (PGHS), which is expressed as two isoenzymes, termed PGHS-1
and PGHS-2. From the crystal structure of sheep PGHS-1, it has been
proposed that the carboxylic acid group of flurbiprofen is located in a
favorable position for interacting with the arginine 120 residue of
PGHS-1 (Picot, D., Loll, P. J., and Garavito, R. M.(1994) Nature 367, 243-249). Mutation of this Arg
residue to
Glu was performed and expressed in COS-7 cells using a vaccinia virus
expression system. Comparison of microsomal enzyme preparations show
that the mutation results in a 20-fold reduction in the specific
activity of PGHS-1 and in a 100-fold increase in the apparent K
for arachidonic acid. Indomethacin,
flurbiprofen, and ketoprofen, inhibitors of PGHS activity containing a
free carboxylic acid group, do not exhibit any inhibitory effects
against the activity of PGHS-1(Arg
Glu).
Diclofenac and meclofenamic acid, other NSAIDs containing a free
carboxylic acid group, were 50-100-fold less potent inhibitors of
the activity of the mutant as compared with the wild type PGHS. In
contrast, the nonacid PGHS inhibitors,
5-bromo-2-(4-fluorophenyl)-3-(4-methylsulfonyl)thiophene (DuP697) and a
desbromo-sulfonamide analogue of DuP697 (L-746,483), were both more
potent inhibitors of PGHS-1(Arg
Glu) than of the
wild type PGHS-1. Inhibition of PGHS-1(Arg
Glu)
was time-dependent for diclofenac and time-independent for DuP697, as
observed for the wild type enzyme, indicating that the mutation does
not alter the basic mechanism of inhibition. Aspirin is an acid NSAID
that inhibits PGHS-1 through a unique covalent acetylation of the
enzyme and also showed a reduced rate of inactivation of the mutated
enzyme. These data provide biochemical evidence of the importance of
the Arg
residue in PGHS-1 for interaction with
arachidonic acid and NSAIDs containing a free carboxylic acid moiety.
Prostaglandins, which are derived from arachidonic acid, act as
potent mediators of pain, fever, and inflammation(1) . The
enzyme that catalyzes the initial step in their formation is
prostaglandin G/H synthase (PGHS)(2) . ()There are
two isoforms of human PGHS, a constitutively expressed form termed
PGHS-1 and an inducible isoform, termed PGHS-2, which can be induced in
cells and tissues by growth factors and cytokines and also in several
models of
inflammation(3, 4, 5, 6, 7, 8) .
The PGHS-catalyzed reaction consists of the bis-dioxygenation of
arachidonic acid to form prostaglandin G
and the reduction
of this metabolite to prostaglandin H
, which then serves as
the precursor for a variety of biologically active
prostanoids(1, 2, 9) .
Nonsteroidal anti-inflammatory drugs (NSAIDs), which were originally shown to inhibit PGHS-1(10) , have also been demonstrated to inhibit PGHS-2(11, 12, 13, 14) . At least three types of NSAID-mediated inhibition of PGHS isoforms have been reported. In the first case, aspirin has been demonstrated to irreversibly inhibit prostaglandin production of PGHS-1 and stimulate (15R)-hydroxyeicosatetraenoic acid production by PGHS-2 through the acetylation of a specific serine residue in each PGHS isoform(12, 15, 16) . In a second type of NSAID-mediated inhibition, drugs such as indomethacin and flurbiprofen act as time-dependent, irreversible inhibitors of both isoforms without resultant covalent modification(17, 18, 19) . In a third type of PGHS-mediated inhibition, compounds such as N-(2cyclohexyloxy-4-nitrophenyl)methanesulfonamide (NS-398) and 5-bromo-2-(4-fluorophenyl)-3-(4-methylsulfonyl)thiophene (DuP697) act as time-independent, reversible inhibitors of PGHS-1 but as time-dependent, irreversible inhibitors of PGHS-2(17, 18) .
NSAIDs are the most widely
utilized drugs for the treatment of inflammatory disorders such as
arthritis. The major problem in the use of these compounds is their
toxicity profile, which includes ulcerogenicity and renal dysfunction
and has been attributed mainly to inhibition of the PGHS-1 isoform.
Understanding the interaction of NSAIDs with PGHS can provide insight
into the development of NSAIDs with enhanced therapeutic potential and
a decreased capacity for toxicity. The recent determination of the
x-ray crystal structure of sheep PGHS-1 has led to the development of a
model for the topography of the NSAID binding site in
PGHS-1(20, 21, 22) . Docking flurbiprofen in
the long hydrophobic channel of the proposed active site of PGHS shows
the carboxylic acid group of flurbiprofen located in a favorable
position for interacting with the guanidinium group of
Arg. The only other charged residue in the active site
cavity and hydrophobic channel is a Glu
, which is
proposed to form a salt bridge with Arg
.
Various
studies on PGHS have utilized site-directed mutagenesis to demonstrate
a Tyr residue essential for PGHS catalysis(23) ,
essential histidines involved in heme binding of PGHS (24) ,
residues glycosylated on PGHS(25) , and the residue on PGHS
acetylated by aspirin(15, 16) . This study reports on
the mutagenesis of Arg
to glutamate in human PGHS-1 in
order to delineate the interaction between NSAIDs and PGHS. Our
observations show that PGHS-1(Arg
Glu) has a
decreased affinity for arachidonic acid and is no longer sensitive to
inhibition by several NSAIDs containing a carboxylic acid moiety.
Figure 1:
Immunoblot
analysis of PGHS-1 and PGHS-1(Arg-Glu) expressed in COS-7
cells utilizing a vaccinia virus expression system. COS-7 cells were
infected with the indicated viral constructs at a multiplicity of
infection of 1:1, and the cells were harvested after 26 h. Microsomal
proteins from these cells were subjected to SDS-polyacrylamide gel
electrophoresis on 10% precast Tris-glycine acrylamide gels (Novex) and
electrophoretically transferred to nitrocellulose membranes. Immunoblot
detection was performed using a PGHS-1 polyclonal antiserum (29) and
I-protein A as described
previously(28) . Total microsomal protein at 2 and 5 µg
from PGHS-1 expressing cells is shown in lanes 1 and 2, respectively. Total microsomal protein at 2, 5, and 10
µg from hPGHS-1(Arg
Glu) expressing cells is
shown in lanes 3, 4, and 5, respectively.
The immunoblot was exposed to Kodak XAR-2 film at -80 °C for
4 h. The migration position of molecular mass standards is
depicted.
Figure 2:
Dependence of the activity of PGHS-1 and
PGHS-1(Arg
Glu) on arachidonic acid concentration.
Microsomal preparations of COS-7 cells expressing the indicated form of
PGHS-1 were assayed for PGE
production at a concentration
of 15 µg/ml under identical assay conditions. Results are expressed
as a double reciprocal plot with of PGE
production (nmol
PGE
/30 s) as a function of arachidonic acid concentration
for PGHS-1 (A) and PGHS-1(Arg
Glu) (B).
PGHS inhibitors such as
indomethacin, flurbiprofen, and diclofenac inhibit this enzyme by an
essentially irreversible time-dependent
mechanism(17, 18) . The inhibitory potencies of these
NSAIDS were determined using a preincubation time of inhibitor with
enzyme to allow the time-dependent inhibition to develop prior to
initiation of the reaction with 20 µM arachidonic acid.
Flurbiprofen, indomethacin, and ketoprofen are potent inhibitors of
PGHS-1 with IC values of 3.2, 90, and 60 nM,
respectively ( Fig. 3and Table 1). In contrast, no
significant inhibition of PGHS-1(Arg
Glu) at
concentrations up to 24 µM was observed with indomethacin,
flurbiprofen, or ketoprofen. The fenamic acid NSAIDS, diclofenac and
meclofenamic acid, retained some inhibitory effects against PGHS-1 (
Arg-Glu), with IC
values of 1.5 and 5.0
µM for the Arg
Glu mutant,
respectively. Although, these latter NSAIDs inhibited
PGHS-1(Arg
Glu), the potency was decreased
40-100-fold as compared with the inhibition of wild type PGHS-1.
The acid NSAID ibuprofen was also tested and found to inhibit PGHS-1
with an IC
of 100 µM, whereas no detectable
inhibtion of PGHS-1(Arg
Glu) was observed at 240
µM ibuprofen. Clearly, all of the acid NSAIDs tested are
significantly more potent for inhibition of the wild type PGHS-1 as
compared with the mutant PGHS-1(Arg
Glu).
Figure 3:
Inhibition of PGE production
by PGHS-1 and PGHS-1(Arg
Glu). Microsomal
preparations of COS-7 cells expressing PGHS-1 and the mutant were
prepared and assayed for PGE
production as described under
``Materials and Methods.'' The three standard NSAIDs tested
are flurbiprofen (A), indomethacin (B), and
diclofenac (C). These compounds were tested for inhibition of
PGHS-1 activity (
) and PGHS-1(Arg
Glu)
activity (
) at eight different concentrations in duplicate and
are reported as the percent inhibition of the control
reaction.
The
DuP697 compound inhibits PGHS-1 by a competitive reversible inhibitory
mechanism(17, 18) . DuP697 displayed significant
potency for inhibition of both PGHS-1 and PGHS-1(Arg
Glu) with IC
values of 1.2 and 0.1
µM, respectively (Fig. 4A). Also, a
desbromo-sulfonamide analogue of DuP697 (L-746,483) (31) was
found to inhibit both PGHS-1 (IC
= 10
µM) and PGHS-1(Arg
Glu) (IC
= 0.84 µM). The increased potency of
inhibition against the mutant by DuP697 and L-746,483 appears to be
related to the higher K
of the mutant for
arachidonic acid. These compounds are competitive with arachidonic
acid, and because the inhibition was performed at a substrate
concentration (20 µM arachidonic acid) approaching the K
of the PGHS-1(Arg
Glu)
mutant (K
= 60 µM) but higher
than the K
for PGHS-1 (K
= 0.5 µM), the IC
for inhibition
of activity of the mutant is expected to be lower than that of the wild
type. This was confirmed by performing assays with the wild type PGHS-1
at lower arachidonic acid concentration (0.6 µM)
approaching the K
of the microsomal preparation of
PGHS-1 (K
= 0.5 µM). At this
substrate concentration approaching the K
of
PGHS-1, DuP697 results in an IC
of 40 nM, a value
similar to that observed for inhibition of the mutant (data not shown).
In sharp contrast to acid NSAIDs, these results demonstrate that the
nonacid PGHS inhibitors, DuP697 and L-746,483, retain their potency for
inhibition of the mutant PGHS-1(Arg
Glu).
Figure 4:
Inhibition of PGE production
by the nonacid inhibitors DuP697 and L-746,483. Microsomal preparations
were prepared from COS-7 cells expressing PGHS-1 and PGHS-1(Arg
Glu) and were assayed for inhibition of PGE
by
the inhibitors DuP697 (A) and L-746,483 (B) as
described in the legend to Fig. 3. These compounds were tested
for inhibition of PGHS-1 activity (
) and PGHS-1(Arg
Glu) activity (
).
Experiments were performed to confirm the type of inhibition by
diclofenac and DuP697 for PGHS-1(Arg
Glu) by
incubating microsomal preparations for 0-30 min with the
inhibitors prior to initiation of the reaction with arachidonic acid (Fig. 5). The results from these experiments demonstrate that
DuP697 at the given inhibitor concentrations inhibits both PGHS-1 and
PGHS-1(Arg
Glu) in a time-independent manner. In
sharp contrast, diclofenac inhibits both the wild type and mutant
enzyme in a time-dependent mechanism. These results are consistent with
previous reports of the mechanism of inhibition of PGHS-1 by DuP697 and
diclofenac and indicates that the mutation did not alter the mechanism
by which these compounds inhibit the activity.
Figure 5:
Effect of preincubation time of inhibitor
on the inhibition of PGHS by diclofenac and DuP697. Diclofenac or
DuP697 were preincubated for various times with PGHS-1 (A) or
PGHS-1(Arg
Glu) (B) at the indicated
inhibitor concentration prior to the measurement of activity as
described under ``Materials and Methods.'' The results are
reported as a percentage of PGHS activity remaining at the designated
preincubation time points.
Aspirin is an acid
NSAID that is a time-dependent inhibitor of PGHS-1 but whose mechanism
of action is through acetylation of PGHS. Microsomal preparations of
PGHS-1 and PGHS-1(Arg
Glu) were preincubated with
1 mM aspirin for 0-90 min before the addition of
arachidonic acid and measurement of enzymatic activity (Fig. 6).
Aspirin inhibited PGHS-1 in a time-dependent mechanism with a complete
inhibition at 10 min independent of the arachidonic acid concentration
used for the experiment (0.5 or 20 µM). Interestingly,
aspirin at all time points tested only inhibits the mutant
PGHS-1(Arg
Glu) by 40% after a 90-min
preincubation. This is further evidence that an acid NSAID that
inhibits PGHS through a novel covalent modification requires an initial
interaction with the Arg
residue.
Figure 6:
Effect of the Arg
Glu
mutation on the time-dependent inhibition of PGHS activity with
aspirin. Microsomal preparations were preincubated with 1 mM aspirin for 0-90 min before the initiation of the PGHS
reaction with 0.5 or 20 µM arachidonic acid. The results
are reported as the percentage of activity as a function of
preincubation time with aspirin.
The present results demonstrate that the Arg residue of hPGHS-1 is essential for the potent inhibitory effects
of the acid inhibitors flurbiprofen, indomethacin, diclofenac,
ketoprofen, and meclofenamic acid. Also, the less potent PGHS
inhibitors, ibuprofen and aspirin, are acid NSAIDs that also have a
decreased potency for inhibition of PGHS-1(Arg
Glu) as
compared with PGHS-1. Interestingly, Arg
is not essential
for interaction with the nonacid DuP697 and L-746,483 compounds. The
decreased or lack of inhibition of PGHS-1(Arg
Glu)
by acid NSAIDs supports the model proposed by Garavito and his
colleagues in which the Arg
residue of PGHS-1 interacts
with the carboxylic acid group of flurbiprofen(20) . Based on
the x-ray crystal structure of sheep PGHS-1, there are only two charged
residues that are found within the hydrophobic channel leading up to
the cyclo-oxygenase active site, Arg
and
Glu
. In the absence of arachidonic acid or an NSAID,
Arg
and Glu
may form a salt bridge. The
importance of this arginine residue for PGHS activity is demonstrated
by both a large increase in the K
of the mutant
for arachidonic acid and the decrease in both the cyclo-oxygenase and
the peroxidase activities of the enzyme. The conversion of the positive
charged arginine to a negative charged glutamate residue abolishes the
possible interaction between the carboxylic acid group of acid NSAIDs
and arachidonic acid with the guanidinium group of Arg
of
PGHS. This ionic interaction may be essential for the formation of a
tight binding inhibitor complex leading to the time-dependent
inhibition of PGHS by inhibitors such as indomethacin and flurbiprofen.
The presence of the common arginine residue (PGHS-1 and PGHS-2) for the
interaction of the carboxylic acid group from both arachidonic acid and
certain NSAIDs with PGHS is consistent with the competitive binding of
inhibitor preceding the formation of the irreversible enzyme-inhibitor
complex(19) .
The inhibition by DuP697 and L-746,483, which
are nonacid PGHS inhibitors, is not affected by the mutation, implying
that the interaction of DuP697 and PGHS-1 involves amino acids other
than Arg whose spatial arrangement is not affected by the
substitution of Glu for Arg. In addition, the time-independent
inhibition of PGHS-1 by DuP697 is maintained in the mutant
PGHS-1(Arg
Glu), and the time-dependent inhibition
by diclofenac is similar for the mutant as compared with the wild type,
albeit at a higher concentration of inhibitor. The mechanism of
inhibition of the Arg
Glu mutant by DuP697 and
diclofenac shows that some of the essential features for the binding of
inhibitors at the active site are preserved and confirms the
selectivity of the interaction between Arg
and NSAIDs
possessing a free carboxylic acid group. Aspirin is a unique acid NSAID
that inhibits PGHS-1 through a time-dependent covalent acetylation. The
slower time-dependent inhibition of the mutant PGHS-1(Arg
Glu) by aspirin demonstrates the importance of the
interaction of aspirin with Arg
for its ability to
inhibit PGHS.
It has been suggested that PGHS-1 provides prostaglandins for use under normal physiological conditions, whereas PGHS-2 is involved in inflammatory responses(7, 8) . NSAIDs in current therapeutic use inhibit both isoforms of PGHS with no or little selectivity and have associated toxicities (i.e. ulcerogenecity) that have been attributed primarily to their inhibition of PGHS-1(7, 8, 11, 12, 13, 14) . Newly developed PGHS inhibitors, such as NS-398, DuP697, and L-745,337, which display selective inhibition of PGHS-2, retain their anti-inflammatory, analgesic, and anti-pyretic properties but are much more gastric sparing than conventional NSAIDs(7, 8, 32, 33) . Most of the conventional NSAIDs contain a carboxylic acid group. Surprisingly all of the selective inhibitors of PGHS-2 reported to date lack a carboxylic acid group but contain a sulfonyl group. Further understanding of the mode of binding of these NSAIDs with both PGHS-1 and PGHS-2 should provide insight into developing better NSAIDs with a decreased potential for toxicity.