Competitive, reversible inhibition of cytosolic phospholipase A(2) at the lipid-water interface by choline derivatives that partially partition into the phospholipid bilayer

Cytosolic phospholipase A(2) (cPL(2)) catalyzes the selective release of ar achidonic acid from the sn-2 position of phospholipids and is believed to p lay a key cellular role in the generation of arachidonic acid. When assayin g the human recombinant cPLA(2) using membranes isolated from [H-3] arachid onate-labeled U937 cells as substrate, 2-(2'-benzyl-4-chlorophenoxy)ethyl-d imethyl-n-octadecyl-ammonium chloride (compound 1) was found to inhibit the enzyme in a dose-dependent manner (IC50 = 5 mu M). It was over 70 times mo re selective for the cPLA(2) as compared with the human nonpancreatic se cr eted phospholipase A(2), and it did not inhibit other phospholipases, Addit ionally, it inhibited arachidonate production in N-formyl-methionyl-leucyl- phenylalanine-stimulated U937 cells. To further characterize the mechanism of inhibition, an assay in which the enzyme is bound to vesicles of 1,2-dim yristoyl-sn-glycero-3-phosphomethanol containing 6-10 mol % of 1-palmitoyl- 2-[1-C-14]arachidonoyl-sn-glycero-3-phosphocholine was employed. With this substrate system, the dose-dependent inhibition could be defined by kinetic equations describing competitive inhibition at the lipid-water interface. The apparent equilibrium dissociation constant for the inhibitor bound to t he enzyme at the interface (K-I(*app)) was determined to be 0.097 +/- 0.032 mol % versus an apparent dissociation constant for the arachidonate-contai ning phospholipid of 0.3 +/- 0.1 mol %. Thus, compound 1 represents a novel structural class of inhibitor of cPLA(2) that partitions into the phosphol ipid bilayer and competes with the phospholipid substrate for the active si te. Shorter n-alkyl-chained (C-4, C-6, C-8) derivatives of compound 1 were shown to have even smaller K-I(*app) values. However, these short-chained a nalogs were less potent in terms of bulk inhibitor concentration needed for inhibition when using the [H-3]arachidonate-labaled U937 membranes as subs trate. This discrepancy was reconciled by showing that these shorter-chaine d analogs did not partition into the [H-3]arachidonate-labeled U937 membran es as effectively as compound 1. The implications for in vivo efficacy that result from these findings are discussed.