(Received for publication, December 11, 1996, and in revised form, January 23, 1997)
From the Departments of Biochemistry and Molecular
Biology and § Medicinal Chemistry, Merck Frosst,
Kirkland, Quebec, H9H 3L1 Canada
Modeling of the active site of prostaglandin G/H
synthase-2 (PGHS-2) onto PGHS-1 utilizing the known crystal structure
of PGHS-1 shows that the only residues impinging directly on the active
site that were not conserved in the two enzymes are
His513 and Ile523 of PGHS-1
(Arg499 and Val509 of PGHS-2). These residues
of human PGHS-1 were each mutated to the corresponding PGHS-2 residues
(His513 Arg and Ile523
Val) and a
double mutant (His513
Arg,Ile523
Val)
containing both residues was also constructed. The mutant enzyme forms
were expressed in COS-7 cells, and their properties were compared with
those of the normal isoforms using microsomal membranes. The mutated
enzyme forms all had apparent Km values within
1.4-fold that of the wild type enzyme, and the specific activity of the
mutants were within 2-fold of that of PGHS-1. DuP697, NS-398, DFU, and
SC-58125 are selective PGHS-2 inhibitors that act as
time-dependent inhibitors of PGHS-2 and rapidly reversible competitive inhibitors of PGHS-1. The single Ile523
Val
mutation increased the sensitivity to each of these selective inhibitors with most of the effect detected using instantaneous inhibition assays, except for DuP697, whose potency was further increased by preincubation with the enzyme. The double PGHS-1 His513
Arg,Ile523
Val mutant became
more sensitive to inhibition by NS-398 and DFU than the single IV
mutant, and time-dependent inhibition was observed. In
contrast, the single HR mutation did not increase the sensitivity to
inhibition by the selective PGHS-2 inhibitors. The potency of a
selective PGHS-1 inhibitor, L-745,296, was decreased 5- and 13-fold in
the HR and HR-IV mutants, respectively. All the results indicate that
mutations of His513 and Ile523 residues of
PGHS-1 can strongly increase sensitivity to selective PGHS-2 inhibition
and restore time-dependent inhibition. They also suggest
that the corresponding Arg499 and Val509
residues of PGHS-2 are essential determinants in differentiating between the interaction of nonselective NSAIDs and selective PGHS-2 inhibitors and their mechanism of action.
Prostaglandins are derived from arachidonic acid and act as mediators of pain, fever, and other inflammatory responses (1). Prostaglandin G/H synthase (PGHS)1 converts arachidonic acid into prostaglandin G2 by the addition of molecular oxygen (a cyclooxygenase step) and then catalyzes the conversion of prostaglandin G2 to prostaglandin H2 by a peroxidase reaction (2, 3). Prostaglandin H2 is the precursor to the formation of all prostaglandins, thromboxane, and prostacyclin. Nonsteroidal anti-inflammatory drugs (NSAIDs), such as aspirin and indomethacin, abrogate prostaglandin synthesis through inhibition of the cyclooxygenase reaction of PGHS (4).
A second isoform of PGHS has been discovered (PGHS-2) that is induced in inflammatory situations in response to cytokines or growth factors (5-10). This has lead to the development of selective PGHS-2 inhibitors, which have demonstrated that inhibition of PGHS-2 alone is sufficient to obtain an anti-inflammatory effect while eliminating the gastric ulceration seen in animal models with NSAIDs, which inhibit both PGHS-1 and -2 without a large degree of selectivity (9, 11, 12). The observation with selective PGHS-2 inhibitors and the difference in the regulation of the expression of the two isoforms has led to the suggestion that PGHS-1 is responsible for normal physiological PG synthesis, whereas PGHS-2 is the main isoform responsible for elevated PG production during inflammatory responses (9). Recent examples of selective PGHS-2 inhibitors are DuP697 (13), NS-398 (14), SC-58125 (9), and DFU (5,5-dimethyl-3-(3-fluorophenyl)-4-(4-methylsulfonyl)phenyl-2(5H)-furanone).2 These compounds have similar mechanisms of action, and they inhibit PGHS-2 by a time-dependent mechanism, whereas inhibition of PGHS-1 is through a time-independent and competitive mechanism (13, 16). The molecular basis for this time-dependent inhibition has not been elucidated.
The cDNA and corresponding amino acid sequences of both human PGHS isoforms have been published, and the two enzyme forms have 63% sequence identity (17). The x-ray crystal structure of sheep seminal vesicle PGHS-1 has demonstrated that the cyclooxygenase active site is comprised of a long hydrophobic channel that is also the site for binding of NSAIDs (18). Several distinctive features of the active site are: 1) an Arg120 residue at the mouth of the channel that has been demonstrated to be important for binding of arachidonic acid and NSAIDs containing a carboxylic acid residue (19, 20); 2) a Tyr385 residue at the upper portion of the active site that is involved in the formation of a radical at the C-13 position of arachidonic acid (21); and 3) a Ser530 residue (just below Tyr385) is the residue that is the site of acetylation by aspirin (22). The aspirin acetylated PGHS-2 results in an enzyme form that oxidizes arachidonic acid to 15-HETE (23) with altered sensitivity to inhibition by certain NSAIDs (24). Molecular modeling of the active site of PGHS-2 using the coordinates of PGHS-1 and site-directed mutagenesis have identified Val509 of PGHS-2 as essential for inhibition by PGHS-2 selective inhibitors (25, 26). We have utilized molecular modeling of the active site of PGHS-2 to determine residues of PGHS-1 that could be mutated to recover inhibition of PGHS-2 selective inhibitors on a modified form of PGHS-1.
Diclofenac was obtained from Sigma and indomethacin and arachidonic acid were purchased from Cayman Chemical Co. Sulindac sulfide, DFU, DuP697, NS-398, SC-58125, and L-745,296 (compound 23) (27) were synthesized by the Medicinal Chemistry Department of the Merck Frosst Center for Therapeutic Research. Hematin and glutathione were purchased from Sigma; phenol was obtained from Life Technologies, Inc.
Molecular ModelingHuman PGHS-2 was homology built based upon an amino acid sequence alignment with residues 33-586 of sheep PGHS-1 using GCG and the published crystal structure of sheep PGHS-1. For amino acid changes between the two isoforms, the sheep PGHS-1 side chains were replaced with those corresponding to human PGHS-2. Following manual correction of the worst steric clashes of the introduced side chains, an all atom model was built and progressively minimized using AMBER 4.1 (28, 29). Restraints to the sheep PGHS-1 peptide backbone were maintained at all times. The protein was heated to 300 K over 10 ps and equilibrated over 45 ps in vacuo using a distance-dependent dielectric constant. Snapshots of the trajectory at 0, 15, 30, and 45 ps were cooled and minimized; comparison of the resulting structures showed little structural variation. In the active site where flurbiprofen was shown to bind in sheep PGHS-1, there was almost no structural variation between the human PGHS-2 minimized structures. A solvent accessible surface area was generated (water probe radius, 1.4 Å) around the area corresponding to the flurbiprofen binding site in sheep PGHS-1.
Mutagenesis of PGHS-1The three mutants of PGHS-1
(His513 Arg; Ile523
Val; and
His513
Arg,Ile523
Val) were constructed
by using the Sculptor in vitro mutagenesis system (Amersham
Corp.). The coding region of PGHS-1 was subcloned into the multiple
cloning site of Bluescript SK (Stratagene), and single stranded DNA was
obtained using M13 K07 helper phage (Life Technologies, Inc.) as
described by the manufacturer's instructions. Oligonucleotides were
obtained from Research Genetics (Huntsville, AL) with a one- or
two-nucleotide mismatch. The oligonucleotides were hybridized to the
single stranded DNA, and mutants were generated according to the
manufacturer's instructions. The mutants were confirmed by sequencing
using a Prism DyeDeoxy Terminator Kit (Applied Biosystems) and an ABI
373 DNA sequencer. The correct mutants were completely sequenced on
both cDNA strands to confirm no misincorporation of nucleotides
during the mutagenesis procedure. The cDNA of interest was
subcloned into the pcDNA3 vector (Invitrogen) for transient
expression in COS-7 (ATCC) cells.
PGHS-1 cDNA constructs were transfected into COS-7 cells using a calcium phosphate transfection kit (Life Technologies, Inc.). The cells were washed with fresh medium 24 h after transfection and harvested 72 h post-transfection. The harvested cells were washed with phosphate-buffered saline (Life Technologies, Inc.) and resuspended in 100 mM Tris-HCl, pH 7.4, 10 mM EDTA, 2 µg/ml aprotinin, 2 µg/ml leupeptin, 2 µg/ml soybean trypsin inhibitor, 0.5 mM phenylmethylsulfonyl fluoride. The cells were disrupted with a Kontes Micro Ultrasonic Cell Disruptor for 3 × 10 s. The crude homogenate was centrifuged at 1000 × g for 10 min at 4 °C. The supernatant fraction was then subjected to a 100,000 × g spin for 1 h at 4 °C. The resulting microsomal pellet fraction for each mutant and the wild type PGHS-1 was resuspended in 100 mM Tris-HCl, pH 7.4, 10 mM EDTA. The protein concentration was quantitated by using the Pierce Coomassie protein assay as described by the manufacturer. Protein expression was quantitated by immunoblot analysis. Protein samples were subjected to electrophoresis on 10% SDS-polyacrylamide gels and transferred electrophoretically to nitrocellulose membranes. The PGHS-1 protein was detected with a PGHS-1 polyclonal antiserum (30), and a secondary antibody developed with enhanced chemiluminesce (DuPont NEN). The immunoblot was exposed to Kodak Biomax MR film. The developed film was analyzed for expression levels by laser densitometric scanning using a Molecular Dynamics Laser Densitometer.
Enzyme AssaysPGHS activity was determined based on the conversion of arachidonic acid to PGE2 by radioimmunoassay. Microsomal membrane protein preparations (final concentration, 15 µg/ml) of PGHS-1 were incubated in the absence or the presence of inhibitor for various times in 100 mM Tris-HCl, pH 7.4, 10 mM EDTA, 1 mM glutathione, 0.5 mM phenol, 100 µM hematin. The reaction was initiated with either 2 or 10 µM arachidonic acid and was terminated at 1 or 3 min by acidification with 0.1 N HCl (final concentration). This mixture was neutralized with an equivalent amount of NaOH and analyzed for PGE2 formation using the methyl oximated PGE2 RIA kit (Amersham Corp.).
A time course of PGE2 formation for all of the microsomal preparations was obtained and found to result in a rapid product formation that was linear for the first minute and reached a maximum after 3-5 min. The Km was determined using a reaction time of 1 min over a broad range of arachidonic acid concentrations (0.1-10 µM) and was calculated using a hyperbolic regression program created by Dr. J. S. Easterby (University of Liverpool, UK). The IC50 calculations of the inhibitors tested were determined ± standard error from at least four values. Microsomal PGHS-2 was prepared from COS-7 cells infected with a vaccinia virus PGHS-2 construct as described previously (12).
The
development of selective PGHS-2 inhibitors has led to attempts to
identify residues of PGHS-2 involved in the binding of these
inhibitors. Molecular modeling of the three-dimensional structures of
the human isoforms was performed utilizing the published crystal
structure of sheep PGHS-1 (18) to identify residues of PGHS-1 that can
be converted to PGHS-2 to mimic the inhibitor binding site of PGHS-2.
The predicted structure of PGHS-2 demonstrates that the majority of the
changes between the two isoforms occur at the N-terminal region forming
the amphipathic helices and at the C-terminal tail. The cyclooxygenase
active site of PGHS-1 and 2 are very similar. The important features of
the active site are depicted in Fig. 1 with
Tyr385 (PGHS-1 residues, Fig. 1A) representing
the upper part of the active site located just below the heme moiety
and presumably responsible for the abstraction of the hydrogen atom at
the C-13 position of arachidonic acid. The Ser530 residue
is the site of aspirin acetylation and is equivalent to
Ser516 in PGHS-2 (Fig. 1B). Arg120
(Arg106 in PGHS-2) is the proposed interaction site of the
acid moiety of several NSAIDs and of the carboxylic acid group of
arachidonic acid. His513 of PGHS-1 (Arg499 of
PGHS-2) and Ile523 of PGHS-1 (Val509 of PGHS-2)
are two residues that impinge on the active site and that are not
conserved between the two isoforms. The solvent accessible space in the
active site of these isoforms (depicted as blue dots in Fig.
1) shows that the His513 and Ile523 of PGHS-1
allow for only a small available space in the vicinity of these
residues for inhibitor interaction. Specifically, Ile523
appears to severely restrict access to this space. In contrast, the
Val509 and Arg499 of PGHS-2 result in the
creation of a larger active site of PGHS-2 accessible to substrates and
inhibitors. We have replaced the residues His513 and
Ile523 of PGHS-1 with the corresponding residues of PGHS-2
to mimic the inhibitor binding site of PGHS-2.
Expression and Activity of Mutants of PGHS-1
The double
mutant of PGHS-1 (PGHS-1 His513 Arg,Ile523
Val (HR-IV)) was constructed to completely mimic the lower end of
the active site of PGHS-2 and the single mutants (PGHS-1
His513
Arg (HR) and PGHS-1 Ile523
Val
(IV)) were constructed to establish the contribution of the single
residue on the inhibitor selectivity. Levels of PGHS protein expression
of the various mutants in microsomal membranes from transfected COS-7
cells were quantitated by immunoblot analysis using a specific
anti-PGHS-1 antiserum (Fig. 2). Laser densitometric scanning indicates that the level of expression of each of the various
proteins was very similar with relative levels of 0.9-, 0.6-, and
0.9-fold that of PGHS-1 for HR, IV, and HR-IV mutants, respectively.
The PGHS activity of the various mutants, as assessed by conversion of
arachidonic acid to PGE2 ranged from 0.6 to 2-fold that of
the wild type PGHS-1 (Table I). Membranes from mock
transfected COS-7 cells contained less than 3% of the PGHS activity
measured for any of the recombinant PGHS proteins. The time course of
PGE2 production was similar for all recombinant PGHS
preparations with a rapid substrate conversion over the first minute of
the reaction and a plateau of product formation after a 3-5-min
reaction as previously reported (19). The apparent
Km values for arachidonic acid were determined and
found to be very similar for PGHS-2, PGHS-1, and the mutants of PGHS-1
(0.5-0.9 µM) (Table I).
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The effect of the various mutations introduced into PGHS-1 on inhibitor sensitivity was evaluated using selective PGHS-2 inhibitors (DuP697, NS-398, SC-58125, and DFU), nonselective NSAIDs (sulindac sulfide, indomethacin, and diclofenac), and a selective PGHS-1 inhibitor (L-745,296). The results are summarized in Table II, and titration curves for the selective PGHS-2 inhibitors are shown in Fig. 3. The HR mutant resulted in an enzyme form that was inhibited by NSAIDs with similar potencies as observed for wild type PGHS-1, and there was no significant change in sensitivity to PGHS-2 selective inhibitors. The most significant change for the HR mutant was a 5-fold increase in the IC50 for the selective PGHS-1 inhibitor, L-745,296. In contrast to the modest effect of the HR mutation on inhibitor sensitivity, the IV mutant was found to have an increased sensitivity to all of the PGHS-2 inhibitors. For example, DuP697 was about 300-fold and SC-58125 at least 15-fold more potent at inhibiting the IV mutant than the wild type PGHS-1. The IV mutant was also more sensitive to inhibition by NS-398 or DFU, and the IC50 for these selective inhibitors was at least 4-fold lower than that observed for PGHS-1. The double mutant HR-IV was more sensitive to inhibition by the selective PGHS-2 compounds, NS-398, SC-58125, and DFU, than either of the single point mutants. DFU and SC-58125, which both have IC50s of >50 µM for the inhibition of PGHS-1, inhibited HR-IV with IC50 values of 1.6 and 1.8 µM, respectively. The HR-IV mutant remained sensitive to inhibition by the PGHS-1 selective inhibitor L-745,296, although the potency of this inhibitor was decreased by more than 10-fold as compared with native PGHS-1.
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Mechanism of Inhibition of Mutant Forms of PGHS-1
The
inhibition of PGHS-2 by selective compounds such as DuP697 is
time-dependent for PGHS-2 and rapidly reversible for PGHS-1 (13, 16). This difference is readily observed with microsomal preparations where only the inhibition of PGHS-2 was found to depend on
the time of preincubation with enzyme as shown in Fig. 4
(A and B). We examined this time dependence for
inhibition of the IV mutant, and as shown in Fig. 4C,
inhibition of this mutant by DuP697 follows a similar pattern as that
seen for inhibition of PGHS-2. DuP697 was also found to be a
time-dependent inhibitor of HR-IV (data not shown).
SC-58125 was a time-independent inhibitor of both the IV and HR-IV
mutant (data not shown).
Both instantaneous inhibition assays in which the inhibitor was added at the time of initiation of the reaction and time-dependent inhibition have been used to evaluate the effect of NSAIDs and PGHS-2 inhibitors on enzyme activity (31). IC50 values were determined for both the IV and HR-IV mutants using assays with either no preincubation or a 15-min preincubation of the inhibitor prior to initiation of the enzyme reaction. These results are summarized in Table III for DuP697, DFU, and NS-398. The single IV mutation caused only a slight increase in inhibitor potency as determined for instantaneous inhibition, which was not further modified by increasing the preincubation time in the case of DFU and NS-398. In contrast, DuP697, for which an IC50 value of 0.1 µM for PGHS-1 is observed independently of the time of preincubation, was a more potent inhibitor of the IV mutant after a 15-min preincubation (IC50 = 0.3 nM), consistent with the time-dependent inhibition observed in Fig. 4C. The introduction of the second HR mutation did not further change the parameters for the inhibition by DuP697. For DFU and NS-398, the second HR mutation caused a further decrease in IC50 values for instantaneous inhibition to 5.2 and 2.6 µM, respectively. In addition, the IC50 values for these two inhibitors were about 3-fold lower using the 15-min preincubation assay. All of these selective PGHS-2 inhibitors are essentially inactive when tested for instantaneous inhibition versus PGHS-2 (IC50 > 50 µM). Altogether, the results of Table III and Fig. 4 demonstrate that the IV mutant is inhibitable in a time-dependent fashion by DuP697, and the HR-IV mutant is inhibitable in a time-dependent fashion by DuP697, NS-398, and DFU.
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The discovery of a second isoform of PGHS several years ago has led to the identification of several selective inhibitors of PGHS-2 that have anti-inflammatory properties and a significantly decreased capacity for gastric ulceration. The mechanism of action of these selective PGHS-2 inhibitors is through a two-step mechanism. The first step involves the formation of an enzyme inhibitor complex (EI) followed by the slower formation of a tightly bound EI* complex as shown in the equation below.
![]() |
(Eq. 1) |
The single IV mutation in PGHS-1 results in an enzyme form that is more sensitive to inhibition by all four of the PGHS-2 selective inhibitors, although NS-398 and DFU are much weaker inhibitors of this mutant compared with either the double HR-IV mutant or PGHS-2. The single IV mutant was also more sensitive to inhibition by nonselective NSAIDs and the selective PGHS-1 inhibitor L-745,296, although the effect was most pronounced with the PGHS-2 selective inhibitors. The increase in potency for the IV mutant as compared with the wild type PGHS-1 is observable at instantaneous inhibition for DFU, SC-58125, and NS-398 with a further increase in the potency of DuP697 by preincubation of the inhibitor with enzyme before the measurement of activity. These results suggest that the IV mutation has increased the affinity of PGHS-1 for all selective PGHS-2 inhibitors but without a change in the mechanism of inhibition (rapidly reversible) for DFU, SC-58125, and NS-398. However, this single mutation was sufficient to change the rapidly reversible mechanism of inhibition of PGHS-1 by DuP697 to the time-dependent mechanism characteristic of PGHS-2 inhibition. Reconstitution of time-dependent inhibition would be consistent with a two-step mechanism as shown above for PGHS-2, although alternate mechanisms cannot be ruled out. The introduction of the second HR mutation to generate the HR-IV mutant resulted in an enzyme form very sensitive to inhibition by all four of the PGHS-2 selective inhibitors with DFU and NS-398 now showing time-dependent inhibition in addition to DuP697. It is of interest to note that the changes of the HR mutation on inhibitor potency were observed on the IV mutant of PGHS-1 rather than on PGHS-1, demonstrating the advantage of combining mutations to show the importance of the various residues. Another difference in the active site of PGHS-2 as compared with PGHS-1 is the increased capacity of PGHS-2 to synthesize 15-R-HETE from arachidonic acid when acetylated by aspirin (23, 24). Both the single IV and the double mutant HR-IV did not synthesize any appreciable 15-R-HETE as compared with PGHS-2 when treated with 100 µM aspirin (data not shown). This suggests that the major determinants for the binding of selective PGHS-2 inhibitors by the PGHS-1 mutants are not sufficient to restore all of the properties of the active site of PGHS-2.
Two recent reports have shown that mutation of Val509 of PGHS-2 to the corresponding Ile523 in PGHS-1 results in the loss of sensitivity to PGHS-2 inhibition (25, 26). Our results demonstrate that the converse in PGHS-1 is sufficient to gain partial sensitivity to PGHS-2 inhibitors, but the double HR-IV mutation results in an enzyme form that is more sensitive to a greater number of PGHS-2 selective inhibitors from three diverse structural classes, with most having a similar mechanism of inhibition. These data suggest that the molecular modeling of PGHS-2 has accurately predicted residues that are important for PGHS-2 inhibitor selectivity. During preparation of this manuscript, the crystal structure of PGHS-2 was published and confirms the major points depicted by the molecular modeling (15). The crystal structure data demonstrate the importance of Val509 as the residue that provides access to a larger active site for PGHS-2 as compared with PGHS-1. In conclusion, we have demonstrated that the combination of molecular modeling, mutagenesis, and inhibitor characterization have delineated both residues Arg499 and Val509 to be essential determinants in the differentiation between interaction of nonselective NSAIDs and selective PGHS-2 inhibitors and their mechanism of inhibition.