Vol. 298, Issue 1, 376-385, July 2001
BMS-229724 Is a Tight-Binding Inhibitor of Cytosolic
Phospholipase A2 That Acts at the Lipid/Water Interface and
Possesses Anti-Inflammatory Activity in Skin Inflammation Models
James R.
Burke,
Lynda B.
Davern,
Paul L.
Stanley,
Kurt R.
Gregor,
Jacques
Banville,
Roger
Remillard,
John W.
Russell,
Patrick J.
Brassil,
Mark R.
Witmer,
Graham
Johnson,
Jeffrey A.
Tredup and
Kenneth M.
Tramposch
Dermatology Drug Discovery, Bristol-Myers Squibb Pharmaceutical
Research Institute, Buffalo, New York (L.B.D., P.L.S., K.M.T.); and
Candiac, Quebec, Canada (J.B., R.R.), Wallingford, Connecticut (P.J.B.,
G.J., J.W.R.), Princeton, New Jersey (J.R.B., K.R.G., M.R.W., J.A.T.)
 |
Abstract |
Cytosolic phospholipase A2 (cPLA2) catalyzes
the selective release of arachidonic acid from the sn-2
position of phospholipids and is believed to play a key cellular role
in the generation of arachidonic acid. BMS-229724
(4-[4-[2-[2-[bis(4-chlorophenyl)methoxy]ethyl-sulfonyl]ethoxy]phenyl]-1,1,1-trifluoro-2-butanone) was found to be a selective inhibitor of cPLA2
(IC50 = 2.8 µM) in that it did not inhibit secreted
phospholipase A2 in vitro, nor phospholipase
C and phospholipase D in cells. The compound was active in inhibiting
arachidonate and eicosanoid production in U937 cells, neutrophils,
platelets, monocytes, and mast cells. With a synthetic covesicle
substrate system, the dose-dependent inhibition could be defined by
kinetic equations describing competitive inhibition at the lipid/water
interface. The apparent equilibrium dissociation constant for the
inhibitor bound to the enzyme at the interface
(KI*app) was determined to be
1 · 10
5 mol% versus an apparent dissociation
constant for the arachidonate-containing phospholipid of 0.35 mol%.
The unit of concentration in the interface is mole fraction (or mol%),
which is related to the surface concentration of substrate, rather than
bulk concentration that has units of molarity. Thus, BMS-229724
represents a novel inhibitor of cPLA2, which partitions
into the phospholipid bilayer and competes with phospholipid substrate
for the active site. This potent inhibition of the enzyme translated
into anti-inflammatory activity when applied topically (5%, w/v) to a
phorbol ester-induced chronic inflammation model in mouse ears,
inhibiting edema and neutrophil infiltration, as well as prostaglandin
and leukotriene levels in the skin. In hairless guinea pigs, BMS-229724
was active orally (10 mg/kg) in a UVB-induced skin erythema model in
hairless guinea pigs.
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Introduction |
Leukotrienes and prostaglandins
are derived from arachidonic acid and are potent lipid mediators of
inflammation and pain. The hydrolysis of arachidonoyl-containing
phospholipids to produce arachidonate is catalyzed by phospholipase
A2 (PLA2). The 85-kDa group
IV cytosolic phospholipase A2
(cPLA2) appears to be the most likely candidate
to catalyze this hydrolysis, since the enzyme is highly selective for
arachidonoyl-containing phospholipids and is tightly regulated by
receptor-mediated stimulation and intracellular calcium levels (Clark
et al., 1995
). Because of its putative role in the generation of
pro-inflammatory eicosanoids, the enzyme has received considerable
medicinal interest.
In addition to the cell biological studies that have been performed to
investigate the role of cPLA2 in eicosanoid
production, the use of
cPLA2/ "knockout" mice
has provided compelling evidence for the role of
cPLA2 in inflammatory disorders. When compared
with wild-type mice, the
cPLA2/ mice showed markedly
reduced allergen-induced anaphylactic responses and near complete
blockade of leukotriene production and neutrophil infiltration when
challenged with LPS/zymosan to the lungs (Uozumi et al., 1997; Nagase
et al., 2000). Interestingly, postischemic brain injury was also
reduced in these cPLA2/
mice, indicating a role of cPLA2 in neuronal
injury (Bonventre et al., 1997).
In unstimulated cells, the enzyme is normally located in the cytosol,
but translocates to the membrane in response to submicromolar concentrations of calcium (Clark et al., 1995). The catalytic mechanism
of cPLA2 is thought to be much like that of
serine esterases and proteases. The enzyme forms an acyl enzyme
intermediate between arachidonate of the phospholipid substrate and an
active site serine residue. Site-directed mutagenesis has been used to
provide evidence that serine-228 is this putative active site
nucleophile (Sharp et al., 1994). Since the phospholipid substrate on
which the enzyme acts is in the form of an aggregate rather than
water-soluble monomers, it has been difficult to identify inhibitors of
the enzyme that have suitable pharmaceutical properties for in vivo measurements of anti-inflammatory activity.
The identification of inhibitors of cPLA2 is
further complicated since the enzyme must first bind to the surface of
the lipid/water interface before abstracting a single phospholipid into
its active site. Therefore, four classes of inhibitors can be
envisioned: 1) compounds that act to promote the desorption of the
interface-bound enzyme by altering the physical nature of the interface
(i.e., a "detergent" effect); 2) compounds that bind to the
interfacial recognition site of the enzyme in the aqueous phase and
inhibit the adsorption to the interface; 3) compounds that bind to the active site of the enzyme in the aqueous phase; and 4) compounds that
bind to the active site when the enzyme is bound to the lipid/water interface and, therefore, are competitive with respect to individual phospholipid molecules (i.e., competitive inhibition at the interface).
In this report, we investigate the mechanism of action of BMS-229724, a
novel inhibitor of cPLA2, and show that it is a
competitive inhibitor at the interface. We also demonstrate that it has
potent anti-inflammatory activity when administered both topically and orally in skin inflammation models in mice and guinea pigs.
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Experimental Procedures |
Materials.
All nonradiolabeled phospholipids were obtained
from Avanti Polar Lipids (Birmingham, AL) except for DMPM, which was
from Calbiochem (San Diego, CA). The radiolabeled
[14C]PAPC was from PerkinElmer Life Science
Products (Boston, MA) (55 mCi/mmol). The human, premonocytic U937 cell
line, was obtained from the American Type Culture Collection (Manassas,
VA). [3H]Arachidonate-labeled U937 membranes
were prepared from U937 cells that had been prelabeled for 16 h
with [3H]arachidonate (100 Ci/mmol) as
previously described (Burke et al., 1997c). BMS-229724 was synthesized
as previously described (Banville et al., 1999).
The human, recombinant cPLA2 was expressed in sf9
insect cells as previously described (Burke et al., 1995b). The human
nonpancreatic sPLA2 (group IIA), which had been
purified from human platelets, was obtained from Dr. R. C. Franson
(Virginia Commonwealth University), along with the
[1-14C]oleic acid-labeled Escherichia
coli membrane substrate (3000 dpm/nmol phospholipid).
Enzyme Assays.
Sonicated phospholipid covesicles, comprised
of DMPM and containing [14C]PAPC or other
phospholipids, were prepared using the general methods described
previously (Jain et al., 1989; Jain and Gelb, 1991; Diez et al., 1992).
With this synthetic substrate, enzymatic assays were performed using
the general procedure of Burke et al. (1995b), in which
cPLA2 (210-390 ng/ml) was added to solutions of
the radiolabeled covesicles (150-270 µM bulk phospholipid) in 25 mM
BisTris propane containing 7 mM CaCl2, 0.4 mg/ml
albumin (bovine serum albumin, essentially fatty acid free), and 4 M
glycerol at pH 8. All components except enzyme were incubated at 37°C
for 5 min before the addition of enzyme. At various times, 100-µl aliquots were removed and quenched by addition into 1.9 ml of tetrahydrofuran. The hydrolyzed, radiolabeled fatty acid was then isolated using aminopropyl solid-phase extraction columns as described previously (Tramposch et al., 1992). The rate of hydrolysis was calculated after an initial burst of product formation (Burke et al.,
1999a). Under these assay conditions, the high calcium concentration
catalyzes vesicle fusion with continuous exchange of phospholipids
(Jain et al., 1986; Burke et al., 1995b).
Assays of cPLA2 activity using the
[3H]arachidonate-labeled U937 membranes as
substrate typically used 0.6 µg/ml enzyme and membrane substrate (22 µM) in 20 mM HEPES buffer, pH 8, containing 6 mM
CaCl2, 0.9 mg/ml human serum albumin, and
0 to 4 M glycerol (Burke et al., 1997c). Enzyme assays were allowed to
proceed for 20 min at 37°C before quenching with tetrahydrofuran. The
radiolabeled fatty acid product was isolated as described above. Assays
of sPLA2 using radiolabeled E. coli
membrane as substrate were performed as described previously (Tramposch
et al., 1992).
Differential Scanning Calorimetry.
Differential scanning
calorimetry was performed with a MCS DSC instrument as previously
described (Burke et al., 1999b).
Cell Assays.
For measurements of the effects of
cPLA2 inhibitors on cellular arachidonate
production, dibutyryl-cAMP-differentiated U937 cells were used
according to the previously published procedure (Burke et al., 1997a).
In short, cells were preincubated with the inhibitors for 5 min at
37°C prior to stimulating with the chemotactic peptide, fMLP. After 5 min of stimulation, the reactions were terminated in an ice bath and
the mass of arachidonate determined by gas chromatography/electron
capture of the derivatized product (Burke et al., 1997a).
For the effect on the products of phospholipase C (PLC), the
fMLP-induced production of inositol trisphosphates in these same cells
was carried out as previously described. (Burke et al., 1997a).
The activation of PLD in differentiated U937 cells was followed by
measuring the amount of phosphatidylethanol produced in the presence of
ethanol as previously described (Anthes et al., 1991). The radiolabeled
production of phosphatidylethanol was monitored by thin-layer chromatography.
The LPS-induced production of eicosanoids and cytokines with peripheral
blood monocytes was carried out using the procedure of Liebler et al.
(1994). All stimulations were for 4 h except when measuring
IL-1
, which required 18 h of stimulation. IL-6, IL-8, TNF
,
and IL-1 in the media were measured using specific enzyme
immunoassays from Endogen Corporation (Woburn, MA).
LTB4 and PGE2 were measured
using radioimmunoassays from Perceptive Biosystems (Framingham, MA).
For measurements of arachidonate and eicosanoids in platelets,
neutrophils, mast cells, and keratinocytes, the procedures of Bartoli
et al. (1994), Burke et al. (1997b), Nakamura et al. (1991), and McCord
et al. (1994), respectively, were used. The production of PAF in human
neutrophils was measured by the procedure of Tramposch et al. (1994).
Chronic Skin Inflammation Model in Mice.
A chronic
persistent skin inflammation in the ears of CD-1 mice was induced by
the repeated treatment of phorbol ester using the procedure of Stanley
et al. (1991). Briefly, TPA (10 µl, 0.01% in acetone/water at 99:1)
was applied to ears on days 0, 2, 4, 7, and 9. BMS-229724 in the same
vehicle was applied twice daily on days 7 to 9 and once (morning) on
day 10. Six hours after the last application, ear punch biopsies were
removed, weighed for assessment of edema, then snap-frozen for
later analysis of myeloperoxidase content, an enzyme marker of
neutrophil infiltration, and for eicosanoid levels measured by
high-performance liquid chromatography/radioimmunoassay. For
PGE2 levels, biopsies were taken on day 9.
UVB-Induced Erythema in Hairless Guinea Pigs.
Hairless
guinea pigs (female, 350-400 g, Charles River Laboratories,
Wilmington, MA) were used so depilation methods would not interfere
with the quality of the skin. Animals were exposed on the masked flanks
to UVB radiation (350 mJ) emitted from a bank of six fluorescent tubes.
BMS-229724 was administered orally in peanut oil by gavage tube 90 min
prior to UVB exposure, and animals were graded for intensity of
erythema 6 h after irradiation as follows: grade 0, no erythema;
grade 1, very slight erythema; grade 2, mild erythema with uniform
redness and well-defined edges; grade 3, moderate erythema (bright even
redness); and grade 4, severe erythema (intense red with slight edema).
Grading was performed in a blinded fashion. After the guinea pigs were
evaluated for erythema, tissue biopsies (
inch) of the
inflamed sites were taken for PGE2 and
LTB4 measurements using the procedure of
Tramposch et al. (1994).
Pharmacokinetics in Rats.
A solution of BMS-229724 in
water/cremophor/ethanol (75:12.5:12.5) at 2.0 mg/ml was administered to
one group (n = 3) of male Sprague-Dawley-1 rats
(cannulated; Hilltop, Scottdale, PA) as a zero order intravenous
infusion of 10 min duration or at 1.0 mg/ml to a second group as a zero
order intraportal infusion of 20 min duration to provide a dose of 4 mg/kg to each group. A third group of rats (n = 3) was
administered a solution of BMS-229724 (4.0 mg/ml in
water/cremophor/ethanol, 75:12.5:12.5) by oral gavage to provide a dose
of 20 mg/kg. Serial blood samples were collected up to 24 h after
beginning either infusion or administration of the oral dose. Protein
in separated plasma was precipitated with acetonitrile, and the
resulting supernatants were analyzed for BMS-229724 by a reverse-phase
chromatography (high-performance liquid chromatography system)
interfaced to a tandem mass spectrometer operated in the negative ion
electrospray, selected reaction monitoring mode.
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Results |
U937 cells treated with [3H]arachidonate
incorporate this radiolabeled fatty acid into phospholipid pools. An
assay using membranes isolated from these radiolabeled U937 cells as
substrate was used to measure the activity of recombinant, human
cPLA2. When assayed in this manner, BMS-229724
was identified as an inhibitor of cPLA2. As shown
in Fig. 1, the dose-dependent inhibition
gave an IC50 value of 2.8 µM. Against the group
IIA sPLA2 from human platelets using membranes
from E. coli as substrate (sPLA2 does
not effectively hydrolyze mammalian membranes), BMS-229724 showed less
than 30% inhibition at concentrations as high as 600 µM, which is
the solubility limit of the compound in this assay. Additional
selectivity measurements were taken in cells (vide
infra).
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Fig. 1.
Dose-dependent inhibition of cPLA2 and
sPLA2 by BMS-229724.  , activity of cPLA2
assayed using membranes from [3H]arachidonate-labeled
U937 cells as substrate;  , activity of sPLA2. Data are
represented as the percentage of control without inhibitor and are the
average of triplicate measurements. See Experimental
Procedures for details.
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Mechanism of Inhibition.
Because the enzyme acts at the
lipid/water interface, kinetic analysis of the enzyme (and the
inhibition mechanism) are quite different from solution-phase
enzymology. Indeed, the equilibrium binding of
cPLA2 to the substrate involves a dependence on
two processes as defined in Scheme 2
(Burke et al., 1995b) where E is defined as the free enzyme, A is the
phospholipid vesicle, EA* is the enzyme bound to the lipid/water
interface, S is the phospholipid substrate within the vesicle, and EAS*
is the interface-bound enzyme containing an active site-bound
phospholipid substrate. These include the intrinsic equilibrium binding
to the interface (defined by a dissociation constant,
KS) and the equilibrium binding of
phospholipid substrate to the active site at the interface (defined by
a dissociation constant, KM*).
To characterize the mechanism of inhibition by BMS-229724, a covesicle
substrate system comprised of PAPC dispersed within DMPM (which is not
hydrolyzed by the enzyme) was used. This substrate system greatly
simplifies the kinetic analysis of the enzyme at the interface, since
there is only one substrate (PAPC) within the bilayer, and its
concentration (in units of mole fraction of the bilayer) at the
interface can be easily controlled. Indeed, this covesicle substrate
allows for the determination of the equilibrium dissociation constants
of phospholipids and inhibitors from the active site at the interface
(Burke et al., 1995b), and the benefits of using this covesicle
substrate system when performing kinetic analyses of inhibitors has
recently been demonstrated (Burke et al., 1999a). Using these
covesicles, the concentration of BMS-229724 was varied while measuring
the cPLA2-catalyzed rate of hydrolysis of the
covesicles containing different mole percentages of
[14C]PAPC. As long as the bulk phospholipid
concentration is large enough to ensure that essentially all of the
enzyme is at the interface (e.g., at 270 µM phospholipid over 98% of
the enzyme is interface-bound; Burke et al., 1995b), the following
equation describing competitive inhibition of
cPLA2 at the interface is valid (Burke et al.,
1995b, 1997d).1
|
(1)
|
where
(v0)o/(v0)I
is the ratio of initial rates in the absence to that in the presence of
a competitive inhibitor;
KM*app and
KI*app are
defined as the apparent dissociation constants for the substrate and
inhibitor,
respectively,2
XI is the concentration of inhibitor
(in units of mole fraction), and
XSo is the mole
fraction of radiolabeled substrate ([14C]PAPC)
in the absence of inhibitor. The unit of concentration in the interface
is a mole fraction that is related to the surface concentration of
substrate, rather than bulk concentration, which has units of molarity.
With BMS-229724, the XI value was
determined by assuming that all of the inhibitor was partitioned within
the phospholipid bilayer (later shown not to be true).
Using these covesicles as substrate for cPLA2,
the dose-dependent inhibition by BMS-229724 was plotted in Fig.
2 as
(v0)o/(v0)I
versus XI/(1 
XI) at each
XSo
value.3 As
predicted by eq. 1, linear correlations were obtained. Nonlinear regression analysis of the data as fit to eq. 1 yielded
KM*app and
KI*app values of
0.35 and 0.005 mol%, respectively. The value of
KM*app
determined here is in agreement with the value of 0.3 ± 0.1 mol% determined previously (Burke et al., 1995b, 1997d). Only an inhibitor that acts competitively at the interface would show this relationship.
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Fig. 2.
Correlation of the inhibition of hydrolysis of
[14C]PAPC/DMPM covesicles with the concentration of
BMS-229724 (XI). Enzyme was assayed in a
solution containing radiolabeled covesicles as substrate. These
[14C]PAPC/DMPM covesicles contained 0 to 6.9 mol%
BMS-229724 and had XSo values
(in terms of [14C]PAPC concentrations) of 6 mol% (),
8 mol% (), 10 mol% ( ). Data were fit to eq. 1, which describes
competitive inhibition at the interface. [Enzyme] = 390 ng/ml,
[phospholipid] = 268 µM. The rate was measured at equilibrium,
after burst and time-dependent inhibition (see Fig. 4). Data represent
the average of duplicate measurements. To determine the
XI values, the inhibitor was assumed to be
completely partitioned into the bilayer.
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Verification that BMS-229724 partitions into the bilayer was provided
by the effect of the compound on the thermal transition of DMPM
vesicles. Differential scanning calorimetry showed that the addition of
BMS-229724 at 10 and 100 µM resulted in a dose-dependent lowering and
broadening of the phase transition temperature of these vesicles,
consistent with a partitioning of the inhibitor into the bilayer
(results not shown).
Additional evidence that BMS-229724 acts as a competitive, reversible
inhibitor at the lipid/water interface comes from the observation that
BMS-229724 was 10 times less potent against cPLA2 with covesicle substrate containing high amounts of cholesterol (cholesterol/phospholipid ratio of 0.6:1, results not shown). The
presence of cholesterol in a phospholipid bilayer at this concentration
has been shown to greatly reduce the partition coefficient of drugs
into phospholipid bilayer vesicles (Herbette et al., 1991). This
results, presumably, from an increase in the packing density of the
bilayer. This effect of cholesterol on the inhibition by BMS-229724
provides further support to the conclusion that BMS-229724 acts by
partitioning into the phospholipid bilayer and competing with
phospholipid monomers for the active site of cPLA2.
The assumption that the inhibitor was completely partitioned into the
phospholipid bilayer of covesicle substrate (without cholesterol) was
tested by determining the effect of reaction volume on the inhibition.
If the inhibitor is completely partitioned into the vesicle, the degree
of inhibition from a constant mole amount of inhibitor will be
independent of the reaction volume, as long as all of the enzyme is
bound to the interface since the mole fraction of inhibitor within the
bilayer will remain constant (Lin and Gelb, 1993). However, if the
inhibitor is only partially partitioned into the vesicle, the
inhibition will decrease with increasing volume as more of the
inhibitor partitions into the aqueous phase. Indeed, Fig.
3 shows that the inhibition of the linear
rate was dependent on the reaction volume. The relationship between the
degree of inhibition and the reaction volume can be defined by the
following (rearranged equation from Burke et al., 1995a):
|
(2)
|
where L is the volume of the reaction mixture,
P and IT are the mole
amounts of phospholipid and inhibitor, respectively, C is
the partition
coefficient,4
M is a
constant,5
U is the volume that the bilayer occupies (equal to
1.21 · 106 ml/nmol; see Bassolino-Klimas
et al., 1993); and
(v0)o/(v0)I
is as defined earlier.
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Fig. 3.
Effect of the reaction volume on the inhibition of
cPLA2-catalyzed hydrolysis of [14C]PAPC/DMPM
covesicles by BMS-22974. cPLA2 was assayed in the presence
of 536 nmol of phospholipid (covesicles of 92:8
DMPM/[14C]PAPC) with and without 1.43 nmol of BMS-229724
while varying the reaction volume. The solid line represents a fit of
the data to eq. 2 to yield a partition coefficient of 22. See text for
details.
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A partition coefficient of 22 was determined using a nonlinear
regression analysis to fit the data. This corresponds to partial partitioning of less than 1% BMS-229724 into the bilayer under the
conditions of Fig. 2. After correcting for this partial partitioning, the true Ki* value was determined to
be 1 · 105 mol%, which is 4 orders of
magnitude smaller than the dissociation constant of the phospholipid
substrate (KM* = 0.35 mol%).
Part of the reason that BMS-229724 partitions to such a small extent
into phospholipid bilayers in vitro is that it forms aggregates with
itself in solution. This was evidenced by laser light-scattering
experiments, which showed that aggregate formation of BMS-229724
occurred when the compound was added in dimethyl sulfoxide to aqueous
solutions (results not shown). Further evidence of this aggregation was
provided by the observation that less than 5% of a 100 µM solution
of BMS-229724 was recovered in the filtrate after passing the solution
through a 0.10-µm filter (results not shown). In contrast, no
light-scattering was observed with a 100 µM solution BMS-229724 in
methanol, and complete recovery of compound was obtained with this
methanol solution after filtration. The propensity to form aggregates
at lower concentrations of BMS-229724 was not measured. The aggregation
of BMS-229724 in aqueous solutions shifts the phospholipid-to-water
distribution equilibrium toward the aqueous phase. Therefore, most of
the enzyme and cell experiments detailed in this section will vastly
underestimate the potency of BMS-229724, since the compound is not
effectively partitioned into the phospholipid bilayer. Indeed, as will
be shown later, BMS-229724 is considerably more potent in vivo, which
may reflect a higher degree of partitioning.
Hemiketal Formation with an Active Site Serine.
There is NMR
evidence that the arachidonyl trifluoromethyl ketone
(AACOCF3) forms a hemiketal with a serine residue
in the active site of cPLA2 (Trimble et al.,
1993). Characteristic of this hemiketal formation is the slow,
tight-binding inhibition observed with AACOCF3.
The slow-binding inhibition with trifluoromethyl ketone inhibitors of
esterases and proteases is thought to result from one of two possible
mechanisms: 1) the active site serine can only interact with the
nonhydrated trifluoromethylketone, the concentration of which may be so
small (BMS-229724 in aqueous solvents is >99% hydrated at the ketone
as determined by NMR) that the effect on rate is correspondingly
diminished; or 2) a conformational change in the enzyme must occur
(e.g., release of a water molecule from the active site) to accommodate
the hydrated ketone in the active site (Stein et al., 1987). To
determine whether BMS-229724 may also be forming a hemiketal with the
active site serine residue, an investigation of the time-dependent
(i.e., slow-binding) inhibition with this inhibitor was performed.
As shown in Fig. 4, the
cPLA2-catalyzed hydrolysis of synthetic substrate
(DMPM/PAPC) was inhibited by the presence of BL-763, a
cPLA2 inhibitor that lacks a trifluoromethyl
ketone (Burke et al., 1999b). Consistent with the fact that BL-763 is a
competitive, readily reversible inhibitor is the observation that there
is no time dependence to the inhibition. That is, the inhibition was
immediate and constant (an initial burst of product formation was
observed under these conditions).
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Fig. 4.
Time-dependent inhibition of cPLA2.
cPLA2-catalyzed hydrolysis of [14C]PAPC/DMPM
covesicles by cPLA2 inhibitors. Enzyme was assayed in a
solution containing 270 µM radiolabeled covesicles as substrate (8:92
ratio of [14C]PAPC to DMPM). , no inhibitor; , 10 µM BL-763; , 0.5 µM BMS-229724.
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In contrast, the inhibition of the enzymatic hydrolysis by BMS-229724
was time-dependent. In this case, the inhibition was not apparent at
the early time points, but was pronounced at later time points. This
demonstrates that BMS-229724, like AACOCF3, is a
slow-binding inhibitor presumably due to formation of a hemiketal with
an active site serine (Scheme 3).
Evidence for tight-binding inhibition of cPLA2 by
BMS-229724 was obtained by preincubating the enzyme with BMS-229724 in
the presence of phosphatidylmethanol vesicles for 10 min. Dilution of
this preincubated enzyme into the synthetic substrate assay (to dilute
out the inhibitor) yielded a rate of hydrolysis that was inhibited for
several minutes before enzyme activity was eventually regenerated (Fig.
5).
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Fig. 5.
Slow regeneration of enzymatic activity after
preincubation with BMS-229724. Nonradiolabeled (270 µM) PAPC/DMPM (8 mol% PAPC) vesicles were preincubated with BMS-229724 for 10 min, then
diluted 14-fold into an assay with 270 µM
[14C]PAPC/DMPM covesicles (8 mol%
[14C]PAPC) as substrate to dilute out the inhibitor. ,
no inhibitor in preincubation; , 1 µM BMS-229724 in
preincubation.
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Thus, BMS-229724 is a slow-binding, tight-binding inhibitor while
acting competitively at the lipid/water interface. Greatly reduced
inhibition measured with methyl ketones and trifluoromethyl alcohols
provides additional evidence that BMS-229724 forms a hemiketal with an
active site serine (results not shown).
Activity in Cells.
It has been shown that stimulation of
differentiated U937 cells with the chemotactic peptide fMLP results in
a robust release of arachidonate. Moreover, this release is the result
of the action of cPLA2 (Burke et al., 1997a).
fMLP stimulation of these cells also leads to the activation of PLC and
PLD, which appear to be upstream of cPLA2
activation (Burke et al., 1997a). Consistent with the role of
cPLA2 in agonist-induced production of
arachidonate (and eicosanoids derived from arachidonate), BMS-229724
dose dependently inhibited fMLP-stimulated arachidonate and
PGE2 production in differentiated U937 cells with
identical IC50 values of 2 µM (Fig. 6). The selectivity for
cPLA2 was further evidenced in these cells by the
lack of activity against PLC and PLD, as measured by the production of
inositol 1,4,5-trisphosphate and phosphatidylethanol, respectively, in
U937 cells.
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Fig. 6.
Inhibition of fMLP-induced production of
arachidonate, PGE2, inositol 1,4,5-trisphosphate, or
phosphatidylethanol in differentiated U937 cells. , mass of
arachidonate produced; , amount of PGE2 produced;  ,
amount of PLC-dependent [3H]inositol 1,4,5-trisphosphate
produced;  , amount of PLD-dependent phosphatidylethanol produced
(stimulated in the presence of ethanol). Data are represented as the
percentage of control without inhibitor after subtracting unstimulated
values and are the average of triplicate measurements. See
Experimental Procedures for details.
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Interestingly, the activity of BMS-229724 against agonist-induced
arachidonate (and eicosanoid) production was observed in a wide range
of inflammatory cells, including platelets, mast cells, keratinocytes,
and monocytes (Table 1). The activity
against eicosanoids cannot be explained by inhibition of cyclooxygenase or 5-lipoxygenase, since BMS-229724 did not inhibit either of these
enzymes in this concentration range (results not shown). The compound
also inhibited the production of platelet-activating factor, a
pro-inflammatory lipid mediator resulting from the lysophospholipid product of cPLA2-catalyzed hydrolysis. These
results point to the important role of cPLA2 in
generating inflammatory mediators.
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TABLE 1
Inhibition of arachidonate and eicosanoids by BMS-229724 in
inflammatory cells
See Experimental Procedures for details.
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Recent reports have implicated a role for arachidonate in the
activation of NF-
B (Camandola et al., 1996; Thommesen et al., 1998).
Accordingly, we tested BMS-229724 as an inhibitor of LPS-stimulated production of TNF, IL-6, IL-1, and IL-8 in human monocytes. As
shown in Fig. 7, BMS-229724 inhibited
production of these cytokines with IC50 values in
the range of 4 to 7 µM. This tracked with the
IC50 value against PGE2 of
6 µM in these cells (arachidonate release could not be directly
measured in this experiment due to the small number of cells).
Consistent with this effect being regulated on the level of
arachidonate rather than the eicosanoid metabolites, neither
indomethacin nor MK-886 (inhibitors of the cyclooxygenase and
5-lipoxygenase pathways, respectively) inhibited the production of
these cytokines at concentrations where complete inhibition of
PGE2 and LTB4 production,
respectively, was observed (results not shown). BMS-229724 was also
shown to inhibit LPS-induced cytokine and nitric oxide production in
primary cultured microglia (R. Pasmantur, unpublished observation).
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Fig. 7.
Inhibition by BMS-229724 of LPS-stimulated cytokine
production in human monocytes. , IL-6; , TNF; , IL-8;  ,
IL-1. Data are represented as the percentage of control without
inhibitor and are the average of triplicate measurements. All LPS
stimulations were for 4 h, except measurements of IL-1 where
the stimulation was allowed to proceed for 18 h to achieve
measurable levels.
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Pharmacology and Pharmacokinetics.
As a first measure of the
anti-inflammatory activity of BMS-229724, the compound was administered
topically in a mouse model of chronic skin inflammation induced by
repeated exposure to phorbol ester. The skin inflammation in this model
is persistent and has been useful in assessing whether topically
applied compounds are able to resolve an existing inflammatory lesion
(Stanley et al., 1991). A 5% (w/v) solution of BMS-229724
significantly reduced the edema (Fig. 8A)
and cell infiltration (Fig. 8B) in this model. Consistent with its
mechanism of action, BMS-229724 reduced the levels of prostaglandin and
leukotriene biosynthesis in the inflamed skin with reductions of 96%
and 74%, respectively, measured at the 5% dose (Fig.
9). Since systemic anti-inflammatory
agents, including glucocorticoids, are not active in this model,
another model was used to assess the systemic anti-inflammatory
activity of BMS-229724 (see below).
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Fig. 8.
Effect of topical application of BMS-229724 on ear
weight (A) and myeloperoxidase activity (B) in the phorbol
ester-induced chronic model of skin inflammation in mice. Mice were
treated with topical phorbol ester over 11 days (see
Experimental Procedures for details). Beginning on day
7, BMS-229724 was applied topically in acetone/water (99:1) to inflamed
skin twice daily. Ears were excised on day 10 and weighed to measure
edema. Tissue myeloperoxidase content was determined as a measure of
neutrophil influx. N = 10 animals per group, with
the error bars representing standard deviations. Drug concentrations
are in w/v percentage. *p < 0.05, compared with
TPA control.
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Fig. 9.
Effect of topical application of BMS-229724 on
PGE2 (A) and LTB4 (B) in inflamed skin from the
phorbol ester-induced chronic model of skin inflammation in mice. Mice
were treated with topical phorbol ester over 11 days (see
Experimental Procedures for details). Beginning on day
7, BMS-229724 was applied topically in acetone/water (99:1) to inflamed
skin twice daily. Ears were excised on either day 9 for
PGE2 or day 10 for LTB4 determinations.
N = 10 animals per group, with the error bars
representing standard deviations. Drug concentrations are in w/v
percentage. *p < 0.05, **p < 0.01, compared with TPA control.
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To assess the pharmakinetic profile of BMS-229724, intra-arterial- or
intraportal-cannulated rats were used. After i.a. administration, the
plasma concentrations of BMS-229724 were observed to decline with a
mean elimination half-life estimated to be 2.5 ± 0.5 h. The
total clearance and steady-state volume of distribution
(Vdss) were estimated to be 46.1 ± 15.1 ml/min · kg and 1.2 ± 0.5 l/kg, respectively.
Based on a comparison of the dose-normalized mean AUC after intraportal
(i.p.t.) and oral administration with the mean AUC after i.a.
administration, the estimated i.p.t. and oral bioavailabilities of
BMS-229724 in the rat were 91% and 12%, respectively. The high i.p.t.
bioavailability indicates that the relatively low oral bioavailability
is not due to first-pass hepatic metabolism. Nearly identical oral
exposure was obtained when BMS-229724 was administered in peanut oil. A
summary of the mean pharmacokinetic parameters for BMS-229724 in the
rat is shown in Table 2.
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TABLE 2
Summary of mean pharmacokinetic parameters for BMS-229724
BMS-229724 was administered by oral (20 mg/kg), intraportal (4 mg/kg),
or intra-arterial (4 mg/kg) routes to the rat in
cremophor/ethanol/water vehicle except as
noted.a
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To evaluate its systemic activity against skin inflammation, BMS-229724
was tested in hairless guinea pigs in which skin erythema was induced
by UVB irradiation. In this model, increased synthesis of
PGE2 is characteristic of the inflammatory
response after acute exposure to UVB light (Hruza and Pentland, 1993),
and these increases parallel the development of the erythema (Snyder,
1976). The role of PGE2 in mediating UV-induced
erythema was demonstrated by the ability of cyclooxygenase inhibitors
such as indomethacin to suppress erythema up to 24 h after
irradiation (Snyder, 1976). Recent results have shown that
cPLA2 synthesis occurs in skin when exposed to doses of UV sufficient to cause erythema, suggesting that
cPLA2 participates in UVB-induced inflammation
(Gresham et al., 1996).
When dosed orally in this model, BMS-229724 exhibited a dose-response
effect on erythema (Fig. 10A). At 10 mg/kg, the erythema was significantly reduced and the response was
similar to the positive control, ibuprofen. BMS-229724 also produced a
corresponding dose-responsive reduction of PGE2
(Fig. 10B). These data are consistent with recent work suggesting that
cPLA2 plays an important role in the
prostaglandin-dependent erythema in skin induced by UVB irradiation.
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Fig. 10.
Effects of BMS 229724 on erythema (A) and
PGE2 (B) levels in the guinea pig UVB model of
inflammation. BMS-229724 was administered by oral gavage in peanut oil
to hairless guinea pigs. Erythema was graded 6 h after UVB
irradiation and skin biopsies taken for PGE2 level
determinations. N = 7 animals per group with the
error bars representing standard deviations. *p < 0.05, **p < 0.01, compared with UVB-exposed,
vehicle control.
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Discussion |
We have shown that BMS-229724 inhibits
cPLA2 at the interface in a manner that is
competitive with respect to phospholipid molecules for the active site.
Inhibitors of this type may be pharmacologically advantageous in vivo,
compared with inhibitors that act on the enzyme in the aqueous phase
since the degree of inhibition for the latter will depend on the
fraction of enzyme bound to the interface.
After taking into account the partial partitioning of BMS-229724 into
the bilayer in these in vitro kinetic analyses, the inhibitor was shown
to have a dissociation constant 30,000 times lower than that of the
arachidonoyl-containing phospholipid substrate. This corresponds to
needing only one molecule of BMS-229724 in a sea of over 10,000 arachidonoyl-containing phospholipid molecules to show inhibition. This
potency appears to result primarily from the reversible formation of a
covalent bond between a serine residue and the carbonyl carbon of the
inhibitor (i.e., formation of a hemiketal) as evidenced by the
slow-binding, tight-binding inhibition observed with BMS-229724.
Consistent with the role of cPLA2 in the
production of arachidonate used for eicosanoid production by cells,
BMS-229724 was active in a wide range of cell types in reducing a
number of inflammatory mediators, including PGE2,
LTB4, and PAF. An inhibitor of
cPLA2, therefore, would be expected to have
anti-inflammatory efficacy greater than that of cyclooxygenase
inhibitors and leukotriene D4/E4 antagonists that
individually affect only a subset of these lipid mediators.
Of additional interest is the observation that BMS-229724 inhibited the
NF-B-dependent expression of pro-inflammatory cytokines, such as
TNF, IL-1, IL-8, and IL-6 in human monocytes. Although the exact
mechanism is unclear, it appears that cPLA2
regulates NF-B or AP-1 activation (Camandola et al., 1996; Woo et
al., 2000). The results shown here indicate that the role of
cPLA2 may be mediated through arachidonate
directly rather than the cyclooxygenase- or 5-lipoxygenase-derived
metabolites (although arachidonate metabolites derived from routes
other these oxygenases cannot be ruled out). Indeed, it has been shown
that dual inhibition of the cyclooxygenase and 5-lipoxygenase pathways
by tebufelone actually enhanced LPS-stimulated TNF and IL-1
production in human monocytes (Sirko et al., 1991), presumably due to
the build up of arachidonate in these cells in the presence of
tebufelone. Inhibition of cytokine production by BMS-229724 suggests
that a cPLA2 inhibitor may have a more
efficacious and broader anti-inflammatory activity in vivo than the
classical nonsteroidal anti-inflammatory drugs, which do not directly
affect cytokine production.
The chronic skin inflammation model in mouse ears provided a good
opportunity to test for in vivo anti-inflammatory activity with
BMS-229724. First, there is evidence that this inflammation is
mediated by prostaglandins and leukotrienes (Tramposch et
al., 1994). Second, the model allows for compounds to be evaluated for
activity against an existing inflammatory lesion since the inflammation
is established prior to drug administration. The observation that
BMS-229724 displayed topical anti-inflammatory activity in this animal
model is particularly relevant, since the model can be considered to be
clinically relevant and suitable for selecting useful drug candidates
for the treatment of chronic skin diseases, such as psoriasis and
atopic dermatitis.
The compound was also quite effective when dosed orally to hairless
guinea pigs in a UVB-induced erythema model. Although this erythema
does not strictly model any chronic inflammatory skin disease, it is
prostaglandin-mediated and would, therefore, be predictive of activity
in inflammatory skin disorders, such as psoriasis and atopic
dermatitis, where eicosanoids derived from the
cPLA2-mediated production of arachidonate may
play a role in pathogenesis (Duell et al., 1988; Fogh et al., 1989).
These results together demonstrate that the potent
cPLA2 inhibitor BMS-229724 is orally bioavailable
and possesses anti-inflammatory activity both topically and orally in
skin inflammation models. BMS-229724 is the first demonstration of a
cPLA2 inhibitor with anti-inflammatory activity
in vivo. We will be performing additional studies to determine
whether BMS-229724 is active in other models of inflammatory disorders,
such as asthma and arthritis.
| |
Acknowledgments |
We thank Professor F. Chilton (Wake Forest University) for help
in measuring arachidonate production in antigen-stimulated mouse bone
marrow-derived mast cells.
| |
Footnotes |
Accepted for publication March 22, 2001.
Received for publication February 2, 2001.
1 This equation is valid, since the
active site dissociation constant for DMPM has been shown to be more
than 330 times greater than the value for PAPC (Burke et al., 1997d).
2 KM*app
and KI*app are related to the
intrinsic dissociation constants (KM* and
KI*) by the equations:
KM*app = KM* (1 + 1/KL*)
and KI*app = KI* (1 + 1/KL*),
where KL* is the active site dissociation constant for DMPM at the interface (Burke et al., 1997d).
3 When determining the equilibrium
dissociation constants from the active site, the mole amount of
substrate ([14C]PAPC in this case) and the mole amount of
DMPM were held constant, while varying the mole amount of inhibitor
(Burke et al., 1995b, 1997d). This has the effect of actually
decreasing the mole fraction of both substrate and DMPM as the
inhibitor concentration is increased. Thus,
XSO in eq. 1 equals the mole
fraction of substrate phospholipid without inhibitor. The mole fraction
of substrate in the presence of the inhibitor is correspondingly less.
4 The partition coefficient,
C, is defined as the concentration (molarity) of
inhibitor in the phospholipid bilayer divided by the concentration in
the aqueous phase.
5
In eq. 2, M is defined as:
where IT equals the
total mole amount of inhibitor present. The definitions of all other
variables are defined in the text.
Address correspondence to: Dr. James R. Burke,
Bristol-Myers Squibb, P.O. Box 4000, Princeton, NJ 08543. E-mail:
james.burke{at}bms.com
| |
Abbreviations |
PLA2, phospholipase A2;
cPLA2, cytosolic PLA2;
sPLA2, secreted PLA2;
LPS, lipopolysaccharide;
DMPM, 1,2-dimyristoyl-sn-glycero-3-phosphomethanol;
PAPC, 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine;
[14C]PAPC, 1-palmitoyl-2-arachidonoyl-[arachidonoyl-1-14C]-sn-glycero-3-phosphocholine;
fMLP, N-formyl-methionyl-leucyl-phenylalanine;
PLC, phospholipase C;
PLD, phospholipase D;
IL, interleukin;
TNF, tumor
necrosis factor-;
LTB4, leukotriene B4;
PGE2, prostaglandin E2;
PAF, platelet-activating factor;
TPA, 12-O-tetradecanoylphorbol-13-acetate;
BisTris, 2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diol;
AACOCF3, arachidonyl trifluoromethyl ketone;
NF-B, nuclear factor-B;
AP-1, activating protein-1;
AUC, area under the
curve;
i.p.t., intraportal;
Vdss, steady-state volume of distribution.
| |
References |
-
Anthes JC,
Krasovsky J,
Egan RW,
Siegel ME and
Billan MM
(1991)
Sequential degradation of choline phosphoglycerides by phospholipase D and phosphatidate phosphohydrolase in dibutyryl cAMP-differentiated U937 cells.
Arch Biochem Biophys
287:
53-59[Medline].
-
Banville J,
Gai Y,
Johnson G,
Zusi FC and
Burke JR
(1999)
inventors, Bristol-Myers Squibb Company, assignee. Selective cPLA2 inhibitors. International Patent Application WO 99/15129. 1999 Apr 1.
-
Bartoli F,
Lin HK,
Ghomashchi F,
Gelb MH,
Jain MK and
Apitz-Castro R
(1994)
Tight binding inhibitors of 85-kDa phospholipase A2 but not 14-kD phospholipase A2 inhibit release of free arachidonate in thrombin-stimulated human platelets.
J Biol Chem
269:
15625-15630[Abstract/Free Full Text].
-
Bassolino-Klimas D,
Alper HE and
Stouch TR
(1993)
Solute diffusion in lipid bilayer membranes: an atomic level study by molecular dynamics simulation.
Biochemistry
32:
12624-12637[Medline].
-
Bonventre JV,
Huang Z,
Taheri MR,
O'Leary E,
Li E,
Moskowitz MA and
Sapirstein A
(1997)
Reduced fertility and postischaemic brain injury in mice deficient in cytosolic phospholipase A2.
Nature (Lond)
390:
622-625[Medline].
-
Burke JR,
Davern LB,
Gregor KR,
Todderud G,
Alford JG and
Tramposch KM
(1997a)
Phosphorylation and calcium influx are not sufficient for the activation of cytosolic phospholipase A2 in U937 cells: requirement for a Gi-type G-protein.
Biochim Biophys Acta
1341:
223-237[Medline].
-
Burke JR,
Davern LB,
Gregor KR and
Tramposch KM
(1997b)
Leukotriene B4 stimulates the release of arachidonate in human neutrophils via the action of cytosolic phospholipase A2.
Biochim Biophys Acta
1359:
80-88[Medline].
-
Burke JR,
Gregor KR and
Tramposch KM
(1995a)
Mechanism of inhibition of human nonpancreatic phospholipase A2 by the anti-inflammatory agent BMS-181162.
J Biol Chem
270:
274-280[Abstract/Free Full Text].
-
Burke JR,
Guenther MG,
Witmer MR,
Tredup J,
Hail ME,
Micanovic R and
Villafranca JJ
(1997c)
Presence of glycerol masks the effects of phosphorylation on the catalytic efficiency of cytosolic phospholipase A2.
Arch Biochem Biophys
341:
177-185[Medline].
-
Burke JR,
Witmer MR and
Tredup JA
(1999a)
The size and curvature of anionic covesicle substrate affects the catalytic action of cytosolic phospholipase A2.
Arch Biochem Biophys
365:
239-247[Medline].
-
Burke JR,
Witmer MR,
Tredup J,
Micanovic R,
Gregor KR,
Lahiri J,
Tramposch KM and
Villafranca JJ
(1995b)
Cooperativity and binding in the mechanism of cytosolic phospholipase A2.
Biochemistry
34:
15165-15174[Medline].
-
Burke JR,
Witmer MR,
Tredup J,
Micanovic R,
Gregor KR,
Lahiri J,
Tramposch KM and
Villafranca JJ
(1997d)
Cooperativity and binding in the mechanism of cytosolic phospholipase A2.
Biochemistry
36:
6854.
-
Burke JR,
Witmer MR,
Zusi FC,
Gregor KR,
Davern LB,
Padmanabha R,
Swann RT,
Smith D,
Tredup JA,
Micanovic R,
Manly S,
Villafranca JJ and
Tramposch KM
(1999b)
Competitive, reversible inhibition of cytosolic phospholipase A2 at the lipid-water interface by choline derivatives that partially partition into the phospholipid bilayer.
J Biol Chem
274:
18864-18871[Abstract/Free Full Text].
-
Camandola S,
Leonarduzzi TM,
Varesio L,
Carini R,
Scavazza A,
Chiarpotto E,
Baeuerle PA and
Poli G
(1996)
Nuclear factor B in activated by arachidonic acid but not by eicosapentaenoic acid.
Biochem Biophys Res Commun
229:
643-647[Medline].
-
Clark JD,
Schievella AR,
Nalefski EA and
Lin L-L
(1995)
Cytsolic phospholipase A2.
J Lipid Mediat Cell Signal
12:
83-117[Medline].
-
Diez E,
Louis-Flamberg P,
Hall RH and
Mayer RJ
(1992)
Substrate specificities and properties of human phospholipases A2 in a mixed vesicle model.
J Biol Chem
267:
18342-18348[Abstract/Free Full Text].
-
Duell EA,
Ellis CN and
Voorhees JJ
(1988)
Determination of 5,12, and 15-lipoxygenase products in keratomed biopsies of normal and psoriatic skin.
J Invest Dermatol
91:
446-450[Medline].
-
Fogh K,
Herlin T and
Kragballe K
(1989)
Eicosanoids in skin of patients with atopic dermatitis: prostaglandin E2 and leukotriene B4 are present in biologically active concentrations.
J Allergy Clin Immunol
83:
450-455[Medline].
-
Gresham A,
Masferrer J,
Chen X,
Leal-Khouri S and
Pentland AP
(1996)
Increased synthesis of high molecular weight cPLA2 mediates early UV-induced PGE2 in human skin.
Am J Physiol
270:
C1037-C1050[Abstract/Free Full Text].
-
Herbette LG,
Rhodes DG and
Mason RP
(1991)
New approaches to drug design and delivery based on drug-membrane interactions.
Drug Des Deliv
7:
75-118[Medline].
-
Hruza LL and
Pentland AP
(1993)
Mechanisms of UV-induced inflammation.
J Invest Dermatol
100:
355-415.
-
Jain MK and
Gelb MH
(1991)
Phospholipase A2-catalyzed hydrolysis of vesicles: uses of interfacial catalysis in the scooting mode.
Methods Enzymol
197:
112-125[Medline].
-
Jain MK,
Rogers J,
Jahagirdar DV,
Marecek JF and
Ramirez F
(1986)
Kinetics of interfacial catalysis by phospholipase A2 in intravesicle scooting mode, and heterofusion of anionic and zwitterionic vesicles.
Biochem Biophys Acta
860:
435-447[Medline].
-
Jain MK,
Yuan W and
Gelb MH
(1989)
Competitive inhibition of phospholipase A2 in vesicles.
Biochemistry
28:
4135-4139[Medline].
-
Liebler JM,
Kunkel SL,
Burdick MD,
Standiford TJ,
Rolfe MW and
Strieter RM
(1994)
Production of IL-8 and monocyte chemotactic peptide-1 by peripheral blood monocytes. Disparate responses to phytohemagglutinin and lipopolysaccharide.
J Immunol
152:
241-249[Abstract].
-
Lin HK and
Gelb MH
(1993)
Competitive inhibition of interfacial catalysis by phospholipase A2: differential interaction of inhibitors with the vesicle interface as a controlling factor of inhibitor potency.
J Am Chem Soc
115:
3932-3942.
-
McCord M,
Chabot-Fletcher M,
Breton J and
Marshall LA
(1994)
Human keratinocytes possess an sn-2 acylhydrolase that is biochemically similar to the U937-derived 85-kDa phospholipase A2.
J Invest Dermatol
102:
980-986[Medline].
-
Nagase T,
Uozumi N,
Ishii S,
Kume K,
Izumi T,
Ouchi Y and
Shimizu T
(2000)
Acute lung injury by sepsis and acid aspiration: a key role for cytosolic phospholipase A2.
Nat Immunol
1:
42-46[Medline].
-
Nakamura T,
Fonteh AN,
Hubbard WC,
Triggiani M,
Inagaki N,
Ishizaka T and
Chilton FH
(1991)
Arachidonic acid metabolism during antigen and ionophore activation of the mouse bone marrow derived mast cell.
Biochim Biophys Acta
274:
1338-1347.
-
Sharp JD,
Pickard RT,
Chiou XG,
Manetta JV,
Kovacevic S,
Miller JR,
Varshavsky AD,
Roberts EF,
Strifler BA and
Brems DN
(1994)
Serine 228 is essential for catalytic activities of 85-kDa cytosolic phospholipase A2.
J Biol Chem
269:
23250-23254[Abstract/Free Full Text].
-
Sirko SP,
Schindler R,
Doyle MJ,
Weisman SM and
Dinarello CA
(1991)
Transcription, translation and secretion of interleukin 1 and tumor necrosis factor: effects of tebufelone, a dual cyclooxygenase/5-lipoxygenase inhibitor.
Eur J Immunol
21:
243-250[Medline].
-
Snyder DS
(1976)
Effect of topical indomethacin on UVR-induced redness and PGE2 levels in sunburned guinea pig skin.
Prostaglandins
11:
631-643[Medline].
-
Stanley PL,
Steiner S,
Havens M and
Tramposch KM
(1991)
Mouse skin inflammation induced by multiple topical applications of 12-O-tetracanoylphorbol-13-acetate.
Skin Pharmacol
4:
262-271[Medline].
-
Stein RL,
Strimpler AM,
Edwards PD,
Lewis JJ,
Mauger RC,
Schwartz JA,
Stein MM,
Trainor DA,
Wildonger RA and
Zottola MA
(1987)
Mechanism of slow-binding inhibition of human leukocyte elastase by trifluoromethyl ketones.
Biochemistry
26:
2682-2689[Medline].
-
Thommesen L,
Sjursen W,
Gasvik K,
Hanssen W,
Brekke O-L,
Skattebol L,
Holmeide AK,
Espevik T,
Johansen B and
Laegreid A
(1998)
Selective inhibitor of cytosolic or secretory phospholipase A2 block TNF-induced activation of transcription factor nuclear factor-B and expression of ICAM-1.
J Immunol
161:
3421-3430[Abstract/Free Full Text].
-
Tramposch KM,
Chilton FH,
Stanley PL,
Franson RL,
Havens MB,
Nettleton DO,
Davern LB,
Darling IM and
Bonney RJ
(1994)
Inhibitor of phospholipase A2 blocks eicosanoid and platelet activating factor biosynthesis and has topical anti-inflammatory activity.
J Pharmacol Exp Ther
271:
852-859[Abstract/Free Full Text].
-
Tramposch KM,
Steiner SA,
Stanley PL,
Nettleton DO,
Franson RD,
Lewin AH and
Carroll FI
(1992)
Novel inhibitor of phospholipase A2 with topical anti-inflammatory activity.
Biochem Biophys Res Commun
189:
272-279[Medline].
-
Trimble LA,
Street IP,
Perrier H,
Tremblay NM,
Weech PK and
Bernstein MA
(1993)
NMR structural studies of the tight complex between a trifluoromethyl ketone inhibitor and the 85-kDa human phospholipase A2.
Biochemistry
32:
12560-12565[Medline].
-
Uozumi N,
Kume K,
Nagase T,
Nakatani N,
Ishii S,
Tashiro F,
Konagata Y,
Maki K,
Ikuta K,
Ouchi Y,
Miyazaki J and
Shimizu T
(1997)
Role of cytosolic phospholipase in allergic response and parturition.
Nature (Lond)
390:
618-622[Medline].
-
Woo C-H,
Kim B-C,
Kim K-W,
Yoo M-H,
Eom Y-W,
Choi E-J,
Na DS and
Kim J-H
(2000)
Role of cytosolic phospholipase A2 as a downstream mediator of rac in the signaling pathway to JNK stimulation.
Biochem Biophys Res Commun
268:
231-236[Medline].
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Abstract
Introduction
