©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Mechanism of Inhibition of Human Nonpancreatic Secreted Phospholipase A by the Anti-inflammatory Agent BMS-181162

(Received for publication, July 29, 1994; and in revised form, October 26, 1994)

James R. Burke (§) Kurt R. Gregor Kenneth M. Tramposch

From the From Dermatology Discovery Research, Bristol-Myers Squibb Pharmaceutical Research Institute, Buffalo, New York 14213

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

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(2) (PLA(2)). It is believed, therefore, that the inhibition of PLA(2) 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(2) and has been shown to block arachidonic acid release in whole cells. The mechanism of inhibition of human nonpancreatic-secreted PLA(2) 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-^14C]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(2). 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.


INTRODUCTION

Phospholipase A(2) 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(2) 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(2) (hnps-PLA(2)) (^1)has been isolated from both rheumatoid synovial fluid (4, 5) and human platelets (6) . Since the same gene encodes the hnps-PLA(2) 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(2) 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(2) in that it does not inhibit phospholipase A(1), phospholipase C, or phospholipase D in vitro. It has been shown to block arachidonic acid release as well as leukotriene B(4) 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(4) and prostaglandin E(2)(38) .

In this paper we provide a detailed kinetic characterization of the inhibition of hnps-PLA(2) 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(2) in a manner which is competitive with respect to substrate monomers at the interface.


EXPERIMENTAL PROCEDURES

Materials

hnps-PLA(2), which had been purified from human platelets according to the procedure of Raghupathi and Franson(20) , 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). The concentration of functional enzyme in these stock solutions of purified enzyme was determined directly using the general procedure of Jain and Gelb(21) . Porcine pancreatic sPLA(2) was obtained from Sigma. The synthesis of 4-(3`-carboxyphenyl)-3,7-dimethyl-9-(2",6",6"-trimethyl-1"-cyclohexenyl)-2Z,4E,6E,8E-nonatetraenoic acid (BMS-181162) has been previously described(19) . DMPM was obtained from Calbiochem and [^14C]PAPC (57 mCi/mmol) purchased from DuPont NEN.

Assays

Small sonicated covesicles of DMPM and [^14C]PAPC of uniform size (10,000 phospholipid molecules on the outer leaflet) were prepared using the general methods described previously(21, 22, 23) . Unless otherwise noted, the desired amount of radiolabeled covesicles in water was added, at 32 °C, to solutions of 25 mM HEPES containing 0.6 mM CaCl(2), 1 mM NaCl, and 1% Me(2)SO at pH 8. Enzyme was then added and 75-µl aliquots were periodically 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(19) .

The effect of various concentrations of BMS-181162 on the action of hnps-PLA(2) (73 ng/ml) on covesicles of DMPM and [^14C]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(2) 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.

Alkylation of sPLA(2) by PNBr

The alkylation of sPLA(2) by the histidine-modifying agent PNBr was carried out essentially as reported(39, 40) . Briefly, sPLA(2), inhibitors or phospholipids (if present), and PNBr were incubated at 25 °C in 22 mM HEPES buffer containing 90 µM CaCl(2) at pH 8. Using the method developed by Scrutton and Utter(41) , the equilibrium constant for dissociation of active site-directed substrates or inhibitors from the enzyme can then be calculated from the degree of protection these agents show against inactivation by PNBr.

Computer Curve Fitting

Data were fit to equations using the curve fit application in the computer program SigmaPlot (Jandel Scientific).


RESULTS

Scooting Mode Assay of Hnps-PLA(2) Using Covesicles of DMPM and [^14C]PAPC

The assay of interfacial catalysis in the scooting mode eliminates the contribution from the interfacial binding step from the kinetic analyses. Using anionic phosphatidylmethanol vesicles, the enzyme never leaves the interface (16, 24) . When small vesicles of DMPM containing 10,000 phospholipid molecules on the outer leaflet are used as substrate in the scooting mode, and the reaction followed by the formation of myristic acid as measured by a pH-stat apparatus, a linear initial rate (zero-order kinetics) is observed for a short time. Then the reaction progress curve takes on a first-order appearance as the substrate becomes depleted and reaction products (fatty acid and lysophospholipid) are formed(18) .

With the covesicles of 21:1 DMPM and [^14C]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(2) on covesicles of DMPM and [^14C]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 [^14C]PAPC by hnps-PLA(2). That is, DMPM is hydrolyzed approximately 10 times faster than [^14C]PAPC (23) and, during the first few minutes, most of the DMPM is hydrolyzed. Afterwards, the enzyme encounters both [^14C]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 [^14C]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(S) and K(P) are the dissociation constants for the PAPC substrate and products (1:1 mixture of fatty acid and lysophospholipid), respectively, in the interface, while K(L) 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(S)^o is the mole fraction of radiolabeled PAPC substrate (0.045), X(L)^o is the mole fraction of DMPM products (0.955), N(S) is the number of phospholipid molecules in the outer layer of the vesicle, and k(i) 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(t)) to the total product at the end of the reaction (P(max)).

The value of N(S)k(i) 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) .

Inhibition of Hnps-PLA(2) by BMS-181162 in the Scooting Mode

The presence of BMS-181162 resulted in a dose-dependent inhibition of the hnps-PLA(2) catalyzed hydrolysis of mixed vesicles of [^14C]PAPC/DMPM as represented in Fig. 2. The inhibition was apparent in both the initial velocity and the first-order portion of the curve. Moreover, the progress curves in the presence of inhibitor proceeded to the same end point as without inhibitor.


Figure 2: Representative effects of BMS-181162 on the action of hnps-PLA(2) 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(max) 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 % [^14C]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(2) 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/[^14C]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(I)) 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(S)k(i) value decreased upon increasing the volume. The following equation can be used to determine the amount of partitioning (derived from ):



where (V(0))^o/(V(0))^I 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, (^2)C is the partition coefficient, (^3)and U is the volume which the bilayer occupies (equal to 1.21bullet10 ml/nmol; (27) ).

Using the data from Table 1, a plot of (V(0))^o/(V(0))^IversusL(1 - (V(0))^o/(V(0))^I) 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(S)k(i))^o/(N^Sk(i))^I was used instead. Using this partition coefficient, the mole fraction of inhibitor, defined as X(I), 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(2) 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(I) 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(0))^o/(V(0))^IversusX(I)/(1-X(I)) and a linear plot with a y intercept of 1 was obtained. The value of X(I), 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 (N(S)k(i))^o/(N(S)k(i))^IversusX(I)/(1-X(I)) is also linear (plot not shown) and yields an n(I) value of 0.10 mol fraction, which is the amount of inhibitor in the interface needed to inhibit the value of N(S)k(i) by 50%.

The K(P) in has been demonstrated for phosphocholine-containing lipids to be greater than 1 mol fraction under similar conditions while K(L) equals 0.12 mol fraction(25) . Using these values in and , the n(I) and X(I) values can be used to estimate a K(I) of 0.013 mol fraction for BMS-181162 and a K(S) of 0.023 for PAPC.

Inhibition of Hnps-PLA(2) by BMS-181162 Using E. coli Membranes as Substrate

Using radiolabeled E.coli membranes as substrate for hnps-PLA(2), BMS-181162 also showed dose-dependent inhibition. A Hanes plot of the inhibition (Fig. 6) yielded curves rather than linear correlations. The data, however, could be fit to which is the kinetic equation for the competitive inhibition of hnps-PLA(2) at the interface in the ``hopping'' mode assuming rapid equilibrium kinetics(30) .


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 dpmbulletmlbulletmin. 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(I) and K(I) have their previously defined meanings with X(I) calculated using a partition coefficient of 4400 (see below); [S] is the bulk concentration of phospholipid; X(S) is the concentration of [1-^14C]oleic acid-labeled phospholipid substrate in terms of mole fraction of the interface; K(m) is the apparent equilibrium dissociation constant for the substrate at the interface; and K(S) 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(2) 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.



Effect of BMS-181162 on the Inactivation of sPLA(2) by PNBr

Using PNBr as an active site-directed alkylating agent, BMS-181162 did not protect the hnps-PLA(2) from inactivation. However, protection was obtained with BMS-181162 against inactivation of porcine pancreatic sPLA(2) yielding a dissociation constant of 14 µM for BMS-181162.


DISCUSSION

The principal objective of the present research was to determine the mechanism of inhibition of hnps-PLA(2) 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(2) 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(2) 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(I) and X(I) values, the ratio from was calculated to be 1.2 while the ratio (N(S)k(i))^o/(V(0))^o 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(2) 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(D) 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(m), 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(S) in ) and the equilibrium constant for dissociation of the substrate molecule from the enzyme active site in the interface (K(m) 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(2) from inactivation from the active site-directed alkylating agent PNBr, it did protect the porcine pancreatic sPLA(2) from inactivation (K(D) = 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(2) from inactivation (results not shown). However, these two phospholipids are able to protect the porcine pancreatic sPLA(2) from alkylation(39) .

The lack of protection of the hnps-PLA(2) 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(2) have been shown to be 1-2 orders of magnitude larger with hnps-PLA(2) as compared to porcine pancreatic PLA(2)(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(2). However, the fact that BMS-181162 protects the porcine pancreatic sPLA(2) from alkylation provides further evidence that it is an active site-directed inhibitor of sPLA(2).

Affinity for the active site and implications for in vivo efficacy. For BMS-181162, the value of K(I), 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/[^14C]PAPC. This value can be compared to K(I) values of 0.03, 0.003, and 0.003 mol fraction for the PLA(2) inhibitors MG14, Ro 23-9358, and all-cis-5,8-tetradecadienamide, respectively (25, 35, 37) . (^4)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(I) value for BMS-181162.

In conclusion, BMS-181162 has been shown to inhibit hnps-PLA(2) 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(2) 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(2) 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.


FOOTNOTES

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§
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(^1)
The abbreviations are: hnps-PLA(2), human nonpancreatic secreted phospholipase A(2); BMS-181162, 4-(3`-carboxyphenyl)-3,7-dimethyl-9-(2",6",6"-trimethyl-1"-cyclohexenyl)-2Z,4E,6E,8E-nonatetraenoic acid; [^14C]PAPC, 1-palmitoyl-2-[1-^14C]-arachidonoyl-sn-glycero-3-phosphocholine; DMPM, 1,2-dimyristoyl-sn-glycero-3-phosphomethanol; PNBr, 2-bromonitroacetophenone; sPLA(2), secreted phospholipase A(2).

(^2)
In , B is equal to:

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.

(^3)
The partition coefficient, C, is defined as the concentration of inhibitor in the phospholipid bilayer divided by the concentration in the aqueous phase.

(^4)
The K values for Ro 23-9358 and all-cis-5,8-tetradecadienamide were determined from the reported X values of 0.003 mol fraction measured using 100% DMPM substrate which has an equilibrium dissociation constant of 4 mol fraction.


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