(Received for publication, July 29, 1994; and in revised form, October 26, 1994)
From the
Many important mediators of inflammation result from the
liberation of free arachidonic acid from phospholipid pools which is
thought to result from the action of phospholipase A (PLA
). It is believed, therefore, that the inhibition
of PLA
would be an important treatment in many inflammatory
disease states. The anti-inflammatory agent BMS-181162
(4-(3`-carboxyphenyl)-3,7-dimethyl-9-(2",6",6"-trimethyl-1"-cyclohexenyl)-2Z,4E,6E,8E-nonatetraenoic
acid) selectively inhibits PLA
and has been shown to block
arachidonic acid release in whole cells. The mechanism of inhibition of
human nonpancreatic-secreted PLA
by BMS-181162 is
investigated in this paper. A scooting mode assay in which the enzyme
is irreversibly bound to vesicles of
1,2-dimyristoyl-sn-glycero-3-phosphomethanol containing 5 mol
% of
1-palmitoyl-2-[1-
C]arachidonoyl-sn-glycero-3-phosphocholine,
was used to characterize the inhibition. With this assay system,
BMS-181162 inhibited the enzyme in a dose-dependent manner. Compounds
which inhibit in the scooting mode have been shown to be competitive
inhibitors in the interface (Gelb, M. H., Berg, O., and Jain, M. K.
(1991) Curr. Op. Struct. Biol. 1, 836-843). This was
verified by demonstrating that the inhibition was not due to the
desorption of the enzyme from the lipid-water interface. Additionally,
the compound did not measurably affect the rate of association onto the
vesicles. Therefore, the inhibition was not the result of a modulation
of the bilayer morphology nor an interaction with the interfacial
binding site on the enzyme. The degree of inhibition was dependent on
the reaction volume which indicates that the inhibitor is only
partially partitioned into the bilayer. After compensating for this
partitioning, the dose-dependent inhibition could be defined by kinetic
equations describing competitive inhibition at the interface. The
equilibrium dissociation constant for the inhibitor bound to the enzyme
at the interface (K
) was
determined to be 0.013 mol fraction, thus demonstrating that BMS-181162
represents a novel structural class of tight-binding competitive
inhibitors of human nonpancreatic secreted PLA
. Using Escherichia coli membranes as substrate, to which the enzyme
binds to the interface reversibly, the inhibition showed a
nonclassical kinetic pattern which is also consistent with a partial
partitioning of the inhibitor into the bilayer. This was verified by a
direct measurement of the amount of inhibitor remaining in solution.
The implications for in vivo efficacy which result from this
mechanism are discussed.
Phospholipase A catalyzes the hydrolysis of the sn-2 ester of phospholipids to release fatty acids and
lysophospholipids. Because of its putative involvement in generating
arachidonic acid, the precursor for the biosynthesis of leukotrienes
and prostaglandins, as well as lysophospholipids which are converted
into platelet activating factor, the role of the nonpancreatic secreted
phospholipase A
in the formation of these three important
classes of inflammatory mediators has received considerable medicinal
interest. Moreover, it is believed that inhibition of this enzyme will
be important in a wide variety of inflammatory disease states such as
psoriasis, rheumatoid arthritis, septic shock, and
ischemia(1, 2, 3) .
The human form of
nonpancreatic secreted phospholipase A (hnps-PLA
) (
)has been isolated from both
rheumatoid synovial fluid (4, 5) and human platelets (6) . Since the same gene encodes the hnps-PLA
from
both sources, they are believed to be identical(6) . The gene
for the enzyme has been cloned (7) and the enzyme's
crystal structure solved(8, 9) . Recently, the
hnps-PLA
has also been found in the the
spleen(10) , placenta(11, 12) , and adult
cartilage(13) .
Since the phospholipid substrate on which the enzyme acts is in the form of an aggregate rather than water-soluble monomers, the enzyme must first bind to the surface of the lipid/water interface before abstracting a single phospholipid molecule into its active site(14, 16) . Substrate binding is assisted by an active-site calcium which also acts as a Lewis acid to facilitate the hydrolysis of the sn-2 ester(15) . Because of the reversible interfacial binding step, this catalytic mechanism has been termed the ``hopping mode'' since the enzyme is continually hopping on and off the interface.
Because of the interfacial binding step, the kinetics are not as straightforward as solution catalysis. The analysis of enzymatic catalysis at the interface has received considerable investigation, however, and can be quantitated with the use of anionic phospholipid vesicles where the enzyme is tightly and irreversibly bound to the interface (i.e. the ``scooting mode''; (16) ). In such an assay, each enzyme-containing vesicle behaves identically in time under conditions where there is at most one enzyme per vesicle, the components are mixed at the interface, there is no exchange of any component between vesicles, and the vesicles are of uniform size and composition(17) . The kinetics can then be described using the classical Michaelis-Menten kinetic theory applied to interfacial catalysis(18) .
BMS-181162 is a specific
inhibitor of phospholipase A in that it does not inhibit
phospholipase A
, phospholipase C, or phospholipase D in
vitro. It has been shown to block arachidonic acid release as well
as leukotriene B
and platelet activating factor
biosynthesis in human polymorphonuclear leukocytes(38) . In a
phorbol ester-induced mouse skin inflammation, topically applied
BMS-181162 effectively blocked the inflammation (19) while
dose-dependently inhibiting leukocyte infiltration. Moreover, this
agent also lowered tissue levels of leukotriene B
and
prostaglandin E
(38) .
In this paper we provide
a detailed kinetic characterization of the inhibition of hnps-PLA by BMS-181162 using anionic phospholipid vesicles, on which the
enzyme acts in the scooting mode, and E. coli membrane
vesicles onto which the enzyme binds reversibly (i.e. the
hopping mode). It is shown that inhibition does not result from the
modulation of the amount of enzyme bound to the interface. This may
have been the case if the compound altered the physical nature of the
interface to cause desorption of the enzyme or if it interacted with
the interfacial recognition site on the enzyme. Additional data are
presented which demonstrate that the inhibitor partially partitions
into the bilayer and binds tightly and reversibly to the active site of
hnps-PLA
in a manner which is competitive with respect to
substrate monomers at the interface.
The effect of various concentrations of
BMS-181162 on the action of hnps-PLA (73 ng/ml) on
covesicles of DMPM and [
C]PAPC (21:1) was
measured under the conditions described above. The concentration of
radiolabeled vesicles was 375 µM and the inhibitor
concentrations used were 0, 15, 40, 50, 65, and 90 µM.
Assays of hnps-PLA utilizing radiolabeled E. coli membrane as substrate were performed as described by Tramposch et al. (19) except that the reaction time was 5 min
instead of 30 min.
With the covesicles of
21:1 DMPM and [C]PAPC used here, the formation
of radiolabeled arachidonic acid is followed. As shown in Fig. 1, the same type of reaction progress curve is observed.
However, the curve shows a small initial burst through the first few
minutes (see inset) before a linear zero-order portion from 4 to 20 min
is observed.
Figure 1:
Reaction progress curve for the action
of hnps-PLA on covesicles of DMPM and
[
C]PAPC (21:1). Enzyme (73 ng/mL) in a solution
containing 375 µM radiolabeled vesicles. The solid
line is curve fitted to the data according to . Inset: expansion of the initial time points with the solid
line being a linear regression through the points from 4 to 20
min.
The initial burst in this system arises from the
differences in the rate of hydrolysis of DMPM and
[C]PAPC by hnps-PLA
. That is, DMPM
is hydrolyzed approximately 10 times faster than
[
C]PAPC (23) and, during the first few
minutes, most of the DMPM is hydrolyzed. Afterwards, the enzyme
encounters both [
C]PAPC substrate (
5 mol %)
and DMPM hydrolysis products (
95 mol %). Since the dissociation
constant for the DMPM products is much smaller than the DMPM substrate
(0.12 mol fraction and >1 mol fraction, respectively(25) ),
the DMPM hydrolysis products act to greatly inhibit the hydrolysis of
[
C]PAPC. Because of this, the initial zero-order
rate, from 4 to 20 min, and the first-order portion of the progress
curve are both measured in the presence of DMPM products.
The Michaelis-Menten equation and its integrated form adapted to catalysis in the scooting mode are shown below (derived according to (18) ).
Here K and K
are the dissociation constants for
the PAPC substrate and products (1:1 mixture of fatty acid and
lysophospholipid), respectively, in the interface, while K
is the dissociation constant for the
DMPM products. The unit of concentration in the interface is mole
fraction, which is related to the surface concentration of substrate,
rather than bulk concentration which has units of molarity. The term X
is the mole fraction of radiolabeled
PAPC substrate (0.045), X
is the mole
fraction of DMPM products (0.955), N
is the number
of phospholipid molecules in the outer layer of the vesicle, and k
is the first-order relaxation constant obtained
by curve fitting the reaction progress curve to the following equation
which relates the amount of product formed at time t (P
) to the total product at the end of the
reaction (P
).
The value of Nk
in is the turnover number under first-order conditions and
will equal the apparent second-order rate constant in the presence of
product inhibition(18, 24) .
Figure 2:
Representative effects of BMS-181162 on
the action of hnps-PLA on radiolabeled covesicles.
Conditions are identical to those described in Fig. 1: open
circles, no inhibitor; solid circles, 40 µM BMS-181162; open triangles, 90 µM BMS-181162. The data represents the average of two experiments.
The solid lines are computer fits to yielding P
values of 0.80, 0.80, and 0.79
µM, respectively. See ``Experimental
Procedures'' for details.
The effect of BMS-181162 on the
trapping of enzyme onto anionic vesicles was also investigated. As
shown in Fig. 3, enzyme added to vesicles containing 100% DMPM
was no longer accessible to the vesicles which contained 5 mol %
[
C]PAPC added 1 min later (solid
triangles). This indicates that the enzyme had been immediately
trapped on the nonradiolabeled vesicles (the small degree of hydrolysis
observed may be due to a minor amount of nonprocessivity which has been
observed with the human form of this enzyme; (25) ). Under the
same conditions, the presence of 70 µM BMS-181162 (open circles) did not affect this trapping. As a control,
enzyme added to a mixture of radiolabeled and nonradiolabeled vesicles
resulted in the formation of radiolabeled product (closed
circles) since the radiolabeled vesicles were accessible to the
enzyme.
Figure 3:
Trapping of hnps-PLA onto
vesicles of DMPM. Enzyme (73 ng/ml) in a solution containing vesicles
of 100% DMPM (500 µM) and radiolabeled covesicles of 21:1
DMPM/[
C]PAPC (340 µM). Order of
addition: solid triangles, radiolabeled vesicles added last, 1
min after the addition of enzyme; open circles, same, but in
the presence of 70 µM BMS-181162; solid circles,
enzyme added last.
In order to fit the dose-dependent inhibition data to
kinetic equations, the mole fraction of inhibitor in the bilayer (X) must be known. Since the enzyme is
irreversibly bound to the vesicle, the reaction progress curve is not
affected by the volume of the reaction(16, 22) . 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 (26) . If, however, 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, Table 1shows that the inhibition
of both the initial velocity and the N
k
value decreased upon
increasing the volume. The following equation can be used to determine
the amount of partitioning (derived from ):
where (V)
/(V
)
is the ratio of initial rate (from 4 to 20 min) in the absence to
that in the presence of a competitive inhibitor, L is the
volume of the reaction mixture, P is the mole amount of
phospholipid, B is a constant, (
)C is the
partition coefficient, (
)and U is the volume which
the bilayer occupies (equal to 1.21
10
ml/nmol; (27) ).
Using the data from Table 1, a plot of (V)
/(V
)
versusL(1 - (V
)
/(V
)
)
is linear (see Fig. 4) and the slope was used to calculate a
partition coefficient, C, of 2900. A value of 2700 was
obtained when the ratio (N
k
)
/(N
k
)
was used instead. Using this partition coefficient, the mole
fraction of inhibitor, defined as X
, at constant
phospholipid concentration can be calculated for the dose-dependent
inhibition described above and represented in Fig. 2. At the
phospholipid concentration in that experiment, 55% of the inhibitor
would be partitioned into the bilayer and 45% would be in the aqueous
phase.
Figure 4:
Effect of the reaction volume on the
inhibition of hnps-PLA hydrolysis of radiolabeled vesicles
by BMS-181162. The data from Table 1was plotted according to . The slopes are, therefore, related to the partition
coefficient. See text for details.
This partition coefficient was determined assuming that the inhibitor partitions into both the outer and inner leaflets of the vesicle. Consistent with this assumption is the fact that the inhibition by BMS-181162 is the same whether the inhibitor was added to preformed vesicles or if the vesicles were prepared in the presence of inhibitor (results not shown). If the inhibitor had only partitioned into the outer leaflet when added to previously formed vesicles, the inhibition would have been greater than when the vesicles were prepared in the presence of inhibitor where the inhibitor is assumed to incorporate into both leaflets.
The equations describing competitive inhibition in the scooting mode are given by and . The validity of this type of analysis has been demonstrated previously(18, 22, 28, 29) .
Here, K is the dissociation
constant for the inhibitor in the interface. With BMS-181162, the
dose-dependent inhibition data (as represented by Fig. 2) was
plotted in Fig. 5as (V
)
/(V
)
versusX
/(1-X
)
and a linear plot with a y intercept of 1 was obtained. The
value of X
, defined as the amount
of inhibitor in the interface necessary for 50% inhibition of the
initial rate, was calculated from the slope to be 0.12 mol fraction.
Figure 5:
Correlation of the dose-dependent
inhibition to the mole fraction of BMS-181162 in the bilayer. The data
represented by Fig. 2was fit to which describes
competitive inhibition in the scooting mode. A partition coefficient of
2700 was used to determine the X values.
An analogous plot of (Nk
)
/(N
k
)
versusX
/(1-X
)
is also linear (plot not shown) and yields an n
value of 0.10 mol fraction, which
is the amount of inhibitor in the interface needed to inhibit the value
of N
k
by 50%.
The K in has been
demonstrated for phosphocholine-containing lipids to be greater than 1
mol fraction under similar conditions while K
equals 0.12 mol
fraction(25) . Using these values in and , the n
and X
values can be used to estimate a K
of 0.013 mol fraction for BMS-181162
and a K
of 0.023 for PAPC.
Figure 6:
Hanes
analysis of the inhibition of hnps-PLA by BMS-181162 using radiolabeled
E. coli membranes as substrate. Substrate concentrations are
expressed as the bulk concentration of phospholipid. Fixed bulk
concentrations of BMS-181162 are as follows: open circles, no
inhibitor; solid circles, 10 µM inhibitor; open triangles, 25 µM inhibitor; solid
triangles, 45 µM inhibitor; open squares, 70
µM inhibitor. Rates are expressed as
dpmml
min
. Each point
represents the average of at least two experiments. The solid lines are a computer fit to , which describes competitive
inhibition at the interface in the hopping mode, with a partial
partitioning of the inhibitor into the bilayer as defined by a
partition coefficient of 4400. See text for
details.
Here, X and K
have their previously defined meanings with X
calculated using a partition coefficient of 4400 (see below);
[S] is the bulk concentration of phospholipid; X
is the concentration of
[1-
C]oleic acid-labeled phospholipid substrate
in terms of mole fraction of the interface; K
is the apparent equilibrium
dissociation constant for the substrate at the interface; and K
is the equilibrium constant for dissociation of
the enzyme from the interface of E. coli membranes. It should
be noted, however, that the kinetic constants in cannot be
obtained from the computer fit since there are to many unknown
constants in the equation.
As a direct measure of the partitioning of BMS-181162 into E. coli membranes, the concentration of membranes was varied while keeping the bulk concentration of inhibitor constant. As shown is Fig. 7, the concentration of inhibitor in the aqueous phase decreased as the phospholipid concentration increased. A computer-generated fit of the data to partial partitioning (solid line) yielded a partition coefficient of 4300. An analogous experiment where the concentration of inhibitor was varied (data not shown) was also consistent with partial partitioning and yielded a partition coefficient of 4500.
Figure 7:
Direct measurement of the partitioning of
BMS-181162 into E. coli membranes. A fixed bulk concentration
of BMS-181162 (50 µM) was incubated with varying
concentrations of membranes. Conditions were identical to those for the
assay of hnps-PLA shown in Fig. 6. The samples were
then centrifuged to remove the membranes and the concentration of
inhibitor in the aqueous phase was determined by measuring the
absorbance at 345 nm (
= 31.5
mM
cm
). Membrane
concentrations are expressed as the bulk concentration of phospholipid.
The solid line is a computer fit to a partial partitioning
assuming that the amount of inhibitor in the bilayer is the difference
between the total amount of inhibitor present and the amount of
inhibitor remaining in solution. Each value was run in
duplicate.
The principal objective of the present research was to
determine the mechanism of inhibition of hnps-PLA by
BMS-181162. For reversible inhibitors, four classes of inhibitors can
be envisioned: (a) compounds which act to promote the
desorption of the interface-bound enzyme by altering the physical
nature of the interface, (b) compounds which bind to the
interfacial recognition site of the enzyme in the aqueous phase and
inhibit the adsorption to the interface, (c) compounds which
bind to the active site of the enzyme in the aqueous phase, and (d) compounds which bind to the active site when the enzyme is
bound to the interface and, therefore, are competitive with respect to
individual phospholipid molecules.
Since inhibitors of type a do not interact directly with the enzyme, they are nonspecific and have been shown not to inhibit the enzyme in the scooting mode due, most probably, to the extremely strong association of the enzyme with anionic vesicles(28, 31, 32) . Since BMS-181162 did act to inhibit the enzyme in the scooting mode, this indicates that the inhibitor is not of type a. Moreover, the progress curves shown in Fig. 2proceeded to the same end point. This demonstrates that the inhibition is not a result of desorption of the enzyme from the vesicle. Otherwise the progress curve in the presence of inhibitor would have proceeded beyond this end point as the enzyme ``hops'' to other vesicles after desorption(33) .
Since the enzyme is bound irreversibly to anionic vesicles, inhibitors of type c would, most probably, also show no inhibition in the scooting mode(33) . It could be envisioned, however, that a type c inhibitor may show inhibition in a scooting mode assay if it binds to the active site before the enzyme binds to the interface and is unable to dissociate from the active site once at the interface. Inhibition of this sort, however, would result in scooting mode progress curves that proceed to a lower end point than without inhibitor since active site-occupied enzyme would be inactive.
Compounds of type b may show inhibition in the scooting mode if the rate of association onto vesicles in the presence of inhibitor is on the order of minutes rather than milliseconds when no inhibitor is present. The inhibition observed would result from the fact that less enzyme is initially bound to the vesicle. The trapping experiment represented by Fig. 3demonstrates that the adsorption onto vesicles is not affected by the presence of BMS-181162.
Since inhibition does not
result from class a, b, or c inhibition, only class d remains as a
possible mechanism. Indeed, all inhibitors reported to date which
inhibit PLA in the scooting mode have been shown to be
competitive inhibitors in the interface(32) . Moreover, the
present results show that the dose-dependent inhibition of
hnps-PLA
by BMS-181162 was defined by and . From and , the following
equation was shown to be a further kinetic proof of competitive
inhibition(18, 33) .
From the n and X
values, the ratio from was calculated to be 1.2 while the ratio (N
k
)
/(V
)
obtained from the progress curve without inhibitor was also 1.2.
The inhibition pattern observed with E. coli membranes as
substrate is also consistent with competitive inhibition at the
interface. This substrate contains a high amount of zwitterionic
phospholipids such as phosphatidylethanolamine and phosphatidylcholine.
Since sPLA has a low affinity for zwitterionic membranes (24, 33) , the enzyme is thought to catalyze
hydrolysis of these membranes in the hopping mode in which the enzyme
binds to the interface reversibly. Indicative of a hopping mode
mechanism is the fact that the rate of hydrolysis is dependent on the
bulk phospholipid concentration with an apparent K
of 90 µM (see Fig. 6, no inhibitor).
Consequently, in this assay the interfacial binding step becomes
relevant in the kinetic analysis and the apparent K
, as defined by standard Michaelis-Menten
equations, is defined by the combination of two dissociation
constants(34) : the equilibrium constant for dissociation of
the enzyme from the interface (K
in )
and the equilibrium constant for dissociation of the substrate molecule
from the enzyme active site in the interface (K
in ).
The Hanes plot of the inhibition (Fig. 6) showed nonlinear relationships but approached competitive inhibition (parallel lines) for the limiting case of high bulk substrate concentrations. For an inhibitor which only partially partitions into the bilayer, a Hanes analysis would not be expected to show linearity at fixed bulk inhibitor concentrations. This is because the concentration of inhibitor in the bilayer (in units of mole fraction) would change as the bulk phospholipid concentration is increased. Only at high concentrations of substrate, where most of the inhibitor is partitioned into the bilayer, would linear competitive inhibition be observed with a Hanes plot. Indeed, the data in Fig. 6can be fit to , which defines the kinetics for competitive inhibition at the interface for the enzyme in the hopping mode, with a partial partitioning of the inhibitor into the bilayer.
The direct measurement of the partition coefficient with E. coli membranes (Fig. 7) yielded a value somewhat greater than
that determined indirectly with covesicles of DMPM containing 5
mol % PAPC (4400 versus 2800). This is not surprising since
the anionic inhibitor would be expected to have a more favorable
interaction with zwitterionic phospholipids such as phosphatidylcholine
and phosphatidylethanolamine as compared to the anionic
phosphatidylmethanol. The high amount of phosphatidylethanolamine in E. coli membranes may, therefore, explain the enhanced
partitioning of BMS-181162 into these membranes.
While BMS-181162
did not protect the hnps-PLA from inactivation from the
active site-directed alkylating agent PNBr, it did protect the porcine
pancreatic sPLA
from inactivation (K
= 14 µM). Moreover, the monomeric
phospholipids 1-octanoyl-2-hydroxy-sn-glycero-3-phosphocholine
and 1,2-dicaproyl-sn-glycero-3-phosphocholine also did not
protect the hnps-PLA
from inactivation (results not shown).
However, these two phospholipids are able to protect the porcine
pancreatic sPLA
from alkylation(39) .
The lack
of protection of the hnps-PLA with these agents is most
probably due to the fact that the dissociation constants of substrates
and inhibitors (in units of mole fraction of the bilayer) from the
hnps-PLA
have been shown to be 1-2 orders of
magnitude larger with hnps-PLA
as compared to porcine
pancreatic PLA
(25) . Therefore, it would be
expected that much lower concentrations of inhibitor or phospholipid
(in units of molarity) would be required to protect the porcine
pancreatic sPLA
. However, the fact that BMS-181162 protects
the porcine pancreatic sPLA
from alkylation provides
further evidence that it is an active site-directed inhibitor of
sPLA
.
Affinity for the active site and implications for in vivo efficacy. For BMS-181162, the value of K, the equilibrium constant for
dissociation of the inhibitor from the enzyme active site in the
interface, was determined to be 0.013 mol fraction using covesicles of
21:1 DMPM/[
C]PAPC. This value can be compared to K
values of 0.03, 0.003, and 0.003 mol
fraction for the PLA
inhibitors MG14, Ro 23-9358, and all-cis-5,8-tetradecadienamide, respectively (25, 35, 37) . (
)To the best of
our knowledge, these molecules are the only classes of effective
inhibitors that have been demonstrated, using a scooting mode assay, to
be active site-directed. However, MG-14 and all-cis-5,8-tetradecadienamide have not been shown to have
anti-inflammatory activity in vivo. It is interesting to note
that BMS-181162 and Ro 23-9358 are diacids which may bind to the active
site Ca
atom.
It should be noted that molecules
which have comparable dissociation constants do not necessarily have
comparable affinities for the active site. That is, the value of a
dissociation constant at the interface is a measure of the relative
affinity of the molecule for the active site as compared to the
affinity of the molecule for the bilayer(36) . For example, two
molecules which interact identically with the active site may have
different dissociation constants if one interacts more strongly with
the bilayer(26) . In the case of BMS-181162, the inhibitor does
not interact as strongly with the bilayer as compared to phospholipids.
This is evidenced by the fact that BMS-181162 only partially partitions
into the bilayer. It is, therefore, easier for the enzyme to
``abstract'' BMS-181162 from the bilayer. Thus, the decreased
interaction with the bilayer, as compared to phospholipids, contributes
to the low K value for BMS-181162.
In conclusion, BMS-181162 has been shown to inhibit hnps-PLA at the interface in a manner which is competitive with respect to
phospholipid molecules for the active site. Inhibitors which act at the
interface will always show inhibition since the enzyme must be at the
interface in order to hydrolyze the phospholipid. Inhibitors of this
type may be pharmacologically advantageous in vivo as compared
to inhibitors which 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. Indeed, BMS-181162 represents the first
example of an active site-directed PLA
inhibitor which
blocks arachidonate release in whole cells as well as having in vivo anti-inflammatory activity. Another type of inhibition
can be envisioned in which an inhibitor in the aqueous phase can bind
to the active site when the enzyme is bound to the interface. However,
it is difficult to imagine that the active site of interface-bound
enzyme would be accessible to inhibitors in the aqueous
phase(33) .
BMS-181162 represents an important step in the
development of potent in vivo inhibitors of hnps-PLA since, in addition to inhibiting the enzyme at the interface, it
has weak interactions with the phospholipid bilayer. Therefore, when
designing further generations of inhibitors, it may be advantageous to
focus on decreasing interactions between the inhibitor and the bilayer
as well as increasing the interactions between the inhibitor and the
active site. In this regard, inhibitors of type d which have a
strong affinity for the active site, but partition very poorly into the
bilayer, may seem to be poor inhibitors in terms of bulk inhibitor
concentration in an in vitro assay. This is due to the fact
that a small fraction of the inhibitor would be present in the bilayer
since the ratio of the volumes of the bilayer to the aqueous phase is
low (
10
). However, as reviewed by Gelb et
al.(17) , when looking at the inhibition in vivo,
where a much larger fraction of the inhibitor would be present in the
bilayer since the ratio of the lipid to aqueous volumes is about 0.1,
such an inhibitor may prove to be very potent.
Where I equals the mole amount of inhibitor in the bilayer and I is the mole amount of inhibitor in solution. The value of all other variables are defined in the text. In a similar way, can be used to derive a relationship analogous to :
where A is defined as the following.