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Vol. 53, Issue 2, 322-329, February 1998
-Lactams SB 212047 and SB 216754 Are Irreversible,
Time-Dependent Inhibitors of Coenzyme A-Independent
Transacylase
Departments of Immunopharmacology (J.D.W., C.-M.S., M.C.-F., D.E.G., L.A.M., R.J.M.) and Medicinal Chemistry (W.B.), SmithKline Beecham Pharmaceuticals, King of Prussia, Pennsylvania 19406, and Section on Pulmonary and Critical Care Medicine and Department of Biochemistry (F.H.C.), Bowman Gray School of Medicine, Winston-Salem, North Carolina 27103
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Summary |
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Summary
Introduction
Procedures
Results
Discussion
References
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The enzyme coenzyme A-independent transacylase (CoA-IT) has been
demonstrated to be the key mediator of arachidonate remodeling, a
process that moves arachidonate into 1-ether-containing phospholipids. Blockade of CoA-IT by reversible inhibitors has been shown to block the
release of arachidonate in stimulated neutrophils and inhibit the
production of eicosanoids and platelet-activating factor. We describe
novel inhibitors of CoA-IT activity that contain a 
-lactam nucleus.
-Lactams were investigated as potential mechanism-based inhibitors
of CoA-IT on the basis of the expected formation of an acyl-enzyme
intermediate complex. Two -lactams, SB 212047 and SB 216754, were
shown to be specific, time-dependent inhibitors of CoA-IT activity
(IC50 = 6 and 20 µM, respectively, with a
10-min pretreatment time). Extensive washing and dilution could not
remove the inhibition, suggesting it was irreversible. In stimulated human monocytes, SB 216754 decreased the production of eicosanoids in a
time-dependent manner. In an in vivo model of phorbol
ester-induced ear inflammation, SB 216754 was able to inhibit indices
of both edema and cell infiltration. Taken together, the results
support two hypotheses: 1) CoA-IT activity is important for the
production of inflammatory lipid mediators in stimulated cells and
in vivo and 2) the mechanism by which CoA-IT acts to
transfer arachidonate is through an acyl-enzyme intermediate.
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Introduction |
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Summary
Introduction
Procedures
Results
Discussion
References
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CoA-IT
is an enzyme responsible for the remodeling of arachidonate between
different phospholipids (Snyder et al., 1992
; Winkler and
Chilton, 1995). Specifically, CoA-IT seems to selectively remodel
arachidonate and other long-chained, unsaturated fatty acids (Chilton
et al., 1983; Kramer and Deykin, 1983; Sugiura et
al., 1987; Winkler et al., 1991). Additional studies
showed that it is responsible for the remodeling of arachidonate from 1-acyl-containing phospholipids into 1-alkyl- and 1-alkenyl-containing phospholipids (Chilton and Murphy, 1986; Sugiura et al.,
1987). To explore the role of CoA-IT in inflammatory cells, we reported previously the characterization of tool compounds that block CoA-IT activity and arachidonate remodeling (Chilton et al., 1995).
Using these tools, we demonstrated that this CoA-IT-mediated movement of arachidonate is important for several functions of stimulated inflammatory cells, including the production of PAF, release of free
AA, and production of prostanoids (Winkler et al., 1995).
Because of the importance of CoA-IT to inflammatory cell function, it
is meaningful to gain insight to its mechanism of action. We
hypothesized previously that the enzyme CoA-IT is part of a family of
transacylases, typified by lecithin cholesterol acyl-transferase (Winkler and Chilton, 1993). This hypothesis details a catalytic mechanism in which an active site nucleophile in CoA-IT attacks the
acyl carbonyl of the sn-2 arachidonate of phospholipids,
forming a covalent interaction between arachidonate and CoA-IT and
liberating lyso phospholipid. The covalently attached arachidonate then
can be donated to a suitable lyso phospholipid acceptor. Based on this
mechanism, the same CoA-IT nucleophile could attack the ketone within a
-lactam ring, opening the ring and forming a covalent interaction
between CoA-IT and the -lactam-containing compound. Further reaction
by decomposition of -lactams, such as penicillins and
cephalosporins, resulting in stable enzyme/product coupling, is well
documented for a number of enzymes (Brenner and Knowles, 1981; Chabin
et al., 1993; Vilain et al., 1993; Green et
al., 1995). We examined a number of -lactams for their ability
to inhibit CoA-IT activity and identified some compounds that seem to
undergo a mechanism-based reaction as described.
We report here the characterization of two -lactam inhibitors
of CoA-IT activity: SB 212047 [4-methoxybenzyl(3S, 4R)-6-bromo-6-[(1-methyl-1,2,3-triazol-4-yl)-hydroxymethyl] penicillanate]
and SB 216754 [(3S,4R)-4-[(isobutenyloxy)-3-triphenylmethylamino)]azetidin-2-one]. The data demonstrate that these compounds are time-dependent, selective
inhibitors of CoA-IT activity. They seem to interact with CoA-IT in an
irreversible manner, supporting the proposed mechanism of action of
CoA-IT. In addition, inhibition of CoA-IT by a -lactam compound
reduced the production of PAF and eicosanoids by isolated, stimulated
inflammatory cells and blocked indices of inflammation in
vivo, supporting the notion that CoA-IT activity is critical for
inflammatory cell function.
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Experimental Procedures |
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Summary
Introduction
Procedures
Results
Discussion
References
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Materials. [3H]Acetic acid, sodium salt (50-100 Ci/mmol), [3H]arachidonate-labeled Escherichia coli (10 mCi/mmol, 4-8 nmol Pi/10 µl), and 1-[3H]alkyl-2-lyso-GPC (30-60 Ci/mmol) were purchased from New England Nuclear Research Products (Boston, MA). Histopaque-1077, Percol, and common laboratory chemicals were purchased from Sigma Chemical (St. Louis, MO). 1-Alkyl-2-lyso-GPC was purchased from BIOMOL Research Laboratories (Plymouth Meeting, PA). Silica gel G plates were from Analtech (Newark, DE). Essentially fatty acid-free BSA was obtained from Calbiochem (San Diego, CA). Silica gel columns were from Baker (Phillipsburg, NJ). All fatty acids were from Nu-Chek Prep (Elysian, MN).
Preparation of cells.
Neutrophils were prepared from
heparinized venous blood collected from healthy donors and isolated
according to the procedure of Boyum (1968) using the Histopaque-1077
technique as described previously (Winkler et al., 1993).
The final leukocyte preparation was suspended in Dulbecco's PBS (137 mM NaCl, 8.8 mM
Na2HPO4, 1.5 mM
KH2PO4, and 2.7 mM KCl; GIBCO, Grand Island, NY) and was >95% viable and
pure, as determined by trypan blue exclusion and histological
examination. Neutrophils were resuspended at a concentration of
107 cells/ml and stimulated with A23187 as
described in the figure legends.
CoA-IT activity.
CoA-IT activity in U937 microsomes was
measured as described previously (Winkler et al., 1991) in a
total volume of 100 µl. Microsomes (5-20 µg of protein) from a
centrifugation at 100,000 × g for 60 min (Winkler
et al., 1991) were diluted in PBS with 1 mM EGTA
to the desired protein concentration. The reaction was initiated by the
addition of 1-[3H]alkyl-2-lyso-GPC (0.1 µCi/tube) and 1 µM final cold 1-alkyl-2-lyso-GPC in
assay buffer with 0.25 mg/ml fatty acid-poor BSA. The reaction was run
for 2-10 min at 37°, times chosen to be on the linear portion of the
product production curve. The reaction was stopped, the lipids were
extracted (Bligh and Dyer, 1959), and materials from an aliquot of the
chloroform extract were separated by TLC in chloroform/methanol/acetic
acid/water (50:25:8:4, v/v/v/v; System I, Analtech, Newark, DE) and
visualized by radioscanning (Bioscan, Washington, D.C.). The product,
1-[3H]alkyl-2-acyl-GPC, was scraped and
quantified by liquid scintillation spectroscopy. With this TLC system,
the RF values for synthetic standards
of 1-alkyl-2-lyso-GPC and 1-alkyl-2-acyl-GPC were ~0.24 and ~0.64,
respectively.
Enzyme assays.
PLA2 activities were
determined using [3H]arachidonate-labeled
Escherichia coli membranes by measuring the liberation of
free [3H]AA (Marshall and McCarte-Roshak,
1992). Human type II, 14-kDa PLA2 was purified
from a clone expressed in Chinese hamster ovary cells (Stadel et
al., 1992). Cytosolic 85-kDa PLA2 (Kramer
et al., 1991) was obtained from a clone expressed in
baculovirus-infected insect cells (Amegazie et al., 1993).
5LO activity was measured by monitoring O2
consumption in a reaction mixture of 10 µM AA, 5 µM sonicated dioleoyl phosphatidylcholine, 150 mM NaCl, 5% ethylene glycol, and 0.2 mM
CaCl2 (Marshall et al., 1991). 5LO
activity was obtained from the cytosolic fraction (100,000 × g, 60 min) of RBL-1 cells. Cyclooxygenase activity was
measured using the enzyme from ram seminal vesicles, incubated with 50 µM AA (Rabinovici et al., 1993). The reaction
was terminated by the addition of 10% trichloroacetic acid, and
products were quantified by absorbance at 532 nm. CoA-dependent
acylation activity was determined using U937 microsomes that were
diluted in Tris buffer (100 mM, pH 7.4) and incubated with
12-15 µM acyl-CoA and 0.015 µCi
1-[14C]acyl-2-lyso-GPC with 1 µM cold 1-acyl-2-lyso-GPC. After 10 min at 37°, the
samples were extracted and developed on TLC plates (System I), and the
amount of 1,2-diacyl-GPC product was determined. PLA2 activities were determined using E. coli membranes by measuring the liberation of free
[3H]AA (Marshall and McCarte-Roshak, 1992).
Lyso PAF:acetyltransferase activity was determined as described
previously (Winkler et al., 1993), by measuring the
production of [3H]PAF from 1-alkyl-2-lyso-GPC
and [3H]acetyl-CoA in broken neutrophils.
PAF production by intact neutrophils.
The incorporation of
[3H]acetate into 1-radyl-2-lyso-GPC during cell
activation was used to quantify PAF biosynthesis (Mueller et
al., 1983; Winkler et al., 1993). Cell suspensions
(10 × 106 cells/ml) in PBS containing 1 mM Ca2+ and 1.1 mM
Mg2+ were incubated at 37° in a volume of 950 µl and exposed to drugs or vehicle for the indicated times. Then, 50 µl of a solution containing [3H]acetic acid
(30 µCi) with A23187 (1 or 2 µM final) in PBS with Ca2+, Mg2+, and 1 mg/ml BSA
was added to the cell suspensions. After 10 min at 37°, the reactions
were terminated by the addition of 1 volume of chloroform/methanol
(1:2, v/v), and the lipids were extracted (Winkler et al.,
1991). After extraction of total lipids, individual phospholipids were
separated by TLC on silica gel G plates developed in
chloroform/methanol/glacial acetic acid/water (50:25:8:4, v/v/v/v) and
localized by radioscanning (Bioscan). The area corresponding to PAF was
then scraped and quantified by liquid scintillation counting. In human
neutrophils, ~90% of the
1-radyl-2-[3H]acetyl-GPC produced under these
conditions is 1-alkyl-2-acetyl-GPC (Triggiani et al., 1991).
Eicosanoid assays. After stimulation of inflammatory cells, the cells were pelleted by brief centrifugation, and the supernatant fluids were collected. The mole quantities of three different eicosanoids (LTB4, LTC4, PGE2) in the supernatants were determined with enzyme immunoassay kits (Cayman, Ann Arbor, MI) used according to the manufacturer's directions.
Mouse ear inflammation.
The inner and outer surfaces of the
left ears of BALB/c male mice (six per group) were treated with vehicle
or phorbol ester (4 µg/ear, phorbol-12-myristate-13-acetate; Sigma).
SB 216754 was applied topically immediately after the challenge, and
its effects were compared with those of the dimethylsulfoxide vehicle. After 4 hr, ear thickness was measured as an index of the edematous response, and the myeloperoxidase activity of the inflamed ears was
determined on tissue homogenates as an index of cellular infiltration (Griswold et al., 1991).
Protein analysis.
Protein concentrations were determined
according to the method of Bradford (1976) with reagents purchased from
BioRad (Hercules, CA).
Data analysis. Each displayed experiment was performed in triplicate and is representative of two to four experiments performed on different donor animals. The results are mean ± standard error, with statistical analysis performed on original data with Student's t test or analysis of variance with Scheffé post hoc tests.
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Results |
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Top
Summary
Introduction
Procedures
Results
Discussion
References
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Selectivity profiles of SB 212047 and SB 216754. The ability of SB 212047 and SB 216754 to inhibit CoA-IT activity in microsomes of U937 cells in a concentration-dependent manner was determined, using a 10-min preincubation time, as shown in Fig. 1. As initial assessment of time-dependent inhibition, the compounds were incubated with U937 microsomes for 80 min before the activity measurement. As can be observed in Fig. 1, a longer preincubation time decreases the apparent IC50 value.
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Kinetics of CoA-IT inhibition. Several series of experiments were performed to characterize further the mechanism by which these compounds inhibited CoA-IT activity; these included studies on the kinetics of CoA-IT inhibition, ability of substrate to protect against inhibition, and reversibility of the inhibition.
Microsomes from U937 cells containing CoA-IT activity were treated with different concentrations of-lactam SB 212047, and the loss of
activity was measured over time (Fig.
2A). The data show that the inhibition of
CoA-IT was progressive over time and essentially complete by 80 min. In
a similar fashion, SB 216754 and two compounds of related structures
(SB 216610 and SB 219204; 10-min IC50 = 20 and 75 µM, respectively) caused inhibition of CoA-IT activity
that was dependent on pretreatment time (Fig. 2B).
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-lactam inhibitors, experiments were performed in which 1 µM lyso-PAF substrate was added at the same time as the
inhibitor and CoA-IT activity was measured in a 10-min assay. Under
such experimental conditions, both SB 212047 (20 µM) and SB 216754 (20 µM) do not inhibit CoA-IT activity (data
not shown), suggesting substrate protection is effective.
Experiments were performed to attempt to recover CoA-IT activity after
inhibition by -lactams. Microsomes containing CoA-IT activity were
incubated with SB 212047 (40 µM) for 80 min, such that
>90% of the activity was inhibited. These microsomes then were washed
with PBS and centrifuged (100,000 × g, 60 min) three separate times. After each centrifugation, CoA-IT activity was measured
and still found to be inhibited by >90% (data not shown). In
addition, microsomes of U937 cells containing CoA-IT activity were
incubated with -lactam SB 216754 for 0 or 30 min and then diluted
20-fold directly into assay conditions; CoA-IT activity was measured.
As shown in Fig. 3, minimal inhibition of
CoA-IT activity is observed at the final concentration of inhibitor if the microsomes are diluted immediately after the addition of the compound. In contrast, when the compound was incubated with the microsomes for 30 min before dilution, inhibition of activity is
observed, indicating that inhibition is a time-dependent process. Similar results were obtained with SB 212047 (data not shown).
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-lactam
inhibition was measured over longer time periods. U937 microsomes were
treated with SB 216754 (300 µM) and diluted to decrease
the effective concentration of the compound to 15 µM, and
then CoA-IT activity was measured over a 6-hr period. The results (Fig.
4) show no indication of recovery of
CoA-IT activity over the 6-hr period.
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Influence of SB 212047 and SB 216754 on lipid mediator
production.
We have shown previously that inhibition of CoA-IT
activity in inflammatory cells, using competitive inhibitors, resulted in decreases in the ability of those cells to produce inflammatory mediators (Winkler et al., 1995). We extended this study to
examine the cellular pharmacology of the irreversible -lactam
compounds. Treatment of human neutrophils with up to 30 µM SB 212047 had no effect on the ability of those cells
to produce PAF (20% inhibition compared with A23187 control response;
three experiments; p = N.S.). One possible explanation
of this lack of cellular effect of this compound is a lack of cellular
stability. This hypothesis was tested in vitro by the
addition of glutathione to mimic the reducing environment within the
cell. Glutathione (100 µM) reduced the ability of 30 µM SB 212047 to inhibit CoA-IT in microsomes [CoA-IT
activity (mean ± standard error) for control, 16,287 ± 477 dpm; glutathione, 16,153 ± 418 dpm (99%); SB 212047, 677 ± 205 dpm (4%); and glutathione plus SB 212047, 15,466 ± 330 dpm (95%)]. This suggests a possible mechanism by which the compound may
lack stability within the cell and thus have no inhibitory effect on
PAF production
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Effect of inhibition of CoA-IT on inflammation in
vivo.
The profile of SB 216754 as an inhibitor of CoA-IT
is that of a compound causing a broad reduction in the production of a variety of lipid mediators in isolated inflammatory cells. To test
whether this profile would translate into anti-inflammatory effects in
an in vivo setting, we selected phorbol ester-induced inflammation in the mouse ear, a model of inflammation in which a
variety of lipid mediators are produced. In this model, inflammation has been attributed to the production of a variety of lipid
inflammatory mediators, including LTs, PGs, and PAF (Griswold et
al., 1991; Merlos et al., 1991). We extensively
characterized this model, and we and others have shown previously that
inflammation can be blocked by inhibitors of PLA2
and by dexamethasone (Tramposch et al., 1992; Miyake
et al., 1993; Marshall et al., 1994, 1995). In
addition, we have shown that topical application of the CoA-IT inhibitor SK&F 98625 or SK&F 45905 to the ear prevented both edema and
inflammatory cell influx induced by the inflammatory stimulus (Winkler
et al., 1995). In a similar fashion, the current study shows
that SB 216754 inhibited both indices of the inflammatory response in
the mouse ear (Table 2).
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73%) and inhibition of CoA-IT activity (62%) (control, 34 ± 2; phorbol ester, 42 ± 6; phorbol ester plus SB 216754, 37 ± 1 pmol/min/mg). An additional experiment was performed to
examine whether this correspondence continued during a chronic
inflammatory response. Mice were treated with phorbol ester or vehicle
and then with SB 216754 (0.75 mg/ear twice daily for 3 days), after
which the tissue content of CoA-IT activity was determined. Phorbol
ester treatment increased CoA-IT activity, whereas SB 216754 inhibited
this increase by 70% (control, 38 ± 13; phorbol ester, 58 ± 6; phorbol ester plus SB 216754, 45 ± 8 pmol/min/mg). In the
same mice, SB 216754 demonstrated statistically significant decreases
in both edema (26%) and myeloperoxidase (47%). These results
strengthen the hypothesis that the anti-inflammatory effects observed
with SB 216754 could be ascribed to inhibition of CoA-IT activity.
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Discussion |
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Summary
Introduction
Procedures
Results
Discussion
References
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Although much is known about the activity of CoA-IT, key
information on its structure and mechanism of action remains to be uncovered. As a working model, we proposed that the mechanism of action
of CoA-IT may be similar to that determined for lecithin cholesterol
acyl-transferase (Jauhiainen and Dolphin, 1986; Winkler and Chilton,
1993). This proposal is based on inhibitor studies showing that the
activity of CoA-IT is inhibited by the serine esterase inhibitor
N-tosyl-L-phenylalanine chloromethyl ketone, the
histidine modifier diethyl pyrocarbonate, and agents that modify
cysteine residues, such as N-ethylmaleimide (Kramer and Deykin, 1983; Winkler et al., 1991). However, this proposed
mechanism of action of CoA-IT remains difficult to prove in the absence of purified enzyme with sequence details. One attempt to further our
understanding of this enzyme involves the investigation of -lactams
as mechanism-based inhibitors.
A large number of -lactam-containing compounds were screened for
inhibition of CoA-IT, but only a few were found to be inhibitors. The
experiments described herein demonstrate that specific -lactam compounds can inhibit CoA-IT activity with many characteristics used to
define mechanism-based inhibition: concentration dependence, time
dependence, irreversibility, selectivity, and substrate protection. Furthermore, SB 216754 was shown to block the production of
inflammatory lipid mediators in human neutrophils and monocytes and to
inhibit indices of the inflammatory response in vivo. Two
major conclusions can be drawn from these results. The first is that
these studies provide further support for the critical role that CoA-IT
plays in the inflammatory response. The second is that these results show that specific -lactams can interact with CoA-IT to irreversibly inactivate the enzyme.
Concerning the mechanism by which -lactams inhibit CoA-IT, we
hypothesize the first step to be the opening of the -lactam by an
active site nucleophile, normally involved in the hydrolysis of the
sn-2 fatty ester of the donor substrate. The resulting acyl-enzyme intermediate then could be a stable, in effect
irreversible, complex, or it may undergo rearrangement and further
reactions, such as that classically proposed for clavulanic acid
(Brenner and Knowles, 1981). The irreversible nature of the complex, if an acyl-intermediate, suggests that water is excluded rigorously from
the active site. This would not be surprising for a membrane-bound, lipid-metabolizing enzyme.
Concerning the role of CoA-IT in inflammation, previous studies have
shown a link between CoA-IT activity and PAF biosynthesis, both in
broken-cell preparations (Uemura et al., 1991; Venable et al., 1991; Winkler et al., 1992) and in whole
cells (Sugiura et al., 1990). Moreover, CoA-IT activity
seems to be linked to the transfer, and the rate of transfer, of
arachidonate between specific subcellular pools (Fonteh and Chilton,
1992; Winkler et al., 1994). Two previously described
inhibitors of CoA-IT, SK&F 98625 and SK&F 45905, were able to inhibit
arachidonate movement as well as PAF, LT, and PG production (Winkler
et al., 1995). These effects in isolated cell systems
translated into anti-inflammatory effects in a specific in
vivo setting of inflammation. The current results with the
-lactam CoA-IT inhibitor SB 216754 support these previous findings.
The data showing SB 216754 reduced lipid mediator production in a
time-dependent fashion is particularly strong support for the role of
CoA-IT in this response.
As with any tool compound, selectivity always is a concern. SB 216754 was found to inhibit 5LO and acetyltransferase at higher concentrations but not in a time-dependent manner. In addition, the effects of this compound on other enzymes, such as those involved in de novo phospholipid biosynthesis, are unknown. Thus, cellular and in vivo results should be viewed with proper caution. It is encouraging, however, that three structurally diverse compounds that are inhibitors of CoA-IT (SB 216754, SK&F 45905, and SK&F 98625) have the same pharmacological effects in cells and in vivo.
In summary, specific -lactam compounds can inhibit CoA-IT activity
in a concentration- and time-dependent manner. Treatment of
inflammatory cells with a -lactam CoA-IT inhibitor resulted in a
time-dependent reduction in the ability of those cells to produce
inflammatory mediators. The characteristics of inhibition of CoA-IT by
the -lactam compounds support a mechanism of action involving a
covalent interaction between the inhibitors and CoA-IT enzyme.
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Footnotes |
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Received June 11, 1997; Accepted October 31, 1997
This work was supported in part by National Institute of Health Grants AI24985 and AI26771.
Send reprint requests to: Dr. James D. Winkler, Department of Immunopharmacology, UW-2532, SmithKline Beecham Pharmaceuticals, 709 Swedeland Road, P.O. Box 1539, King of Prussia, PA 19406. E-mail: james_d_winkler{at}sbphrd.com
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Abbreviations |
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CoA-IT, coenzyme A-independent
transacylase;
AA, arachidonic acid;
BSA, bovine serum albumin;
GPC, sn-glycero-3-phosphocholine;
5LO, 5-lipoxygenase;
PLA2, phospholipase A2;
PBS, phosphate-buffered
saline;
PAF, platelet-activating factor;
TLC, thin layer
chromatography;
HBSS, Hanks' balanced salt solution;
EGTA, ethylene
glycol bis(-aminoethyl
ether)-N,N,N
,N-tetraacetic
acid;
HEPES, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid;
LT, leukotriene;
PG, prostaglandin.
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References |
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Summary
Introduction
Procedures
Results
Discussion
References
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-lactams.
Biochemistry
32:
8970-8980[Medline]. 
mediates endotoxin-induced lung injury in platelet activating factor-primed rats.
J Pharmacol Exp Ther
267:
1550-1557.
Biochim Biophys Acta
1215:
133-140[Medline]. This article has been cited by other articles:
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  E. Boilard and M. E. Surette Anti-CD3 and Concanavalin A-induced Human T Cell Proliferation Is Associated with an Increased Rate of Arachidonate-Phospholipid Remodeling. LACK OF INVOLVEMENT OF GROUP IV AND GROUP VI PHOSPHOLIPASE A2 IN REMODELING AND INCREASED SUSCEPTIBILITY OF PROLIFERATING T CELLS TO CoA-INDEPENDENT TRANSACYLASE INHIBITOR-INDUCED APOPTOSIS J. Biol. Chem., May 11, 2001; 276(20): 17568 - 17575. [Abstract] [Full Text] [PDF]
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