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-C-14]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-I*) 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.
