title: Competitive, reversible inhibition of cytosolic phospholipase A2 at the lipid-water interface by choline derivatives that partially partition into the phospholipid bilayer.
abstract: Cytosolic phospholipase A2 (cPLA2) catalyzes the selective release of arachidonic acid from the sn-2 position of phospholipids and is believed to play a key cellular role in the generation of arachidonic acid. When assaying the human recombinant cPLA2 using membranes isolated from [3H]arachidonate-labeled U937 cells as substrate, 2-(2'-benzyl-4-chlorophenoxy)ethyl-dimethyl-n-octadecyl-ammonium chloride (compound 1) was found to inhibit the enzyme in a dose-dependent manner (IC50 = 5 microM). It was over 70 times more selective for the cPLA2 as compared with the human nonpancreatic secreted phospholipase A2, and it did not inhibit other phospholipases. Additionally, 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-dimyristoyl-sn -glycero-3-phosphomethanol containing 6-10 mol % of 1-palmitoyl-2-[1-14C]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 the enzyme at the interface (KI*app) was determined to be 0.097 +/- 0.032 mol % versus an apparent dissociation constant for the arachidonate-containing phospholipid of 0.3 +/- 0.1 mol %. Thus, compound 1 represents a novel structural class of inhibitor of cPLA2 that partitions into the phospholipid bilayer and competes with the phospholipid substrate for the active site. Shorter n-alkyl-chained (C-4, C-6, C-8) derivatives of compound 1 were shown to have even smaller KI*app values. However, these short-chained analogs were less potent in terms of bulk inhibitor concentration needed for inhibition when using the [3H]arachidonate-labeled U937 membranes as substrate. This discrepancy was reconciled by showing that these shorter-chained analogs did not partition into the [3H]arachidonate-labeled U937 membranes as effectively as compound 1. The implications for in vivo efficacy that result from these findings are discussed.
authors.affiliation: Drug Discovery Research, Bristol-Myers Squibb Pharmaceutical Research Institute, Buffalo, New York 14213, USA. burkej@bms.com
authors.forename: J R
authors.initials: JR
authors.lastname: Burke
authors.forename: M R
authors.initials: MR
authors.lastname: Witmer
authors.forename: F C
authors.initials: FC
authors.lastname: Zusi
authors.forename: K R
authors.initials: KR
authors.lastname: Gregor
authors.forename: L B
authors.initials: LB
authors.lastname: Davern
authors.forename: R
authors.initials: R
authors.lastname: Padmanabha
authors.forename: R T
authors.initials: RT
authors.lastname: Swann
authors.forename: D
authors.initials: D
authors.lastname: Smith
authors.forename: J A
authors.initials: JA
authors.lastname: Tredup
authors.forename: R
authors.initials: R
authors.lastname: Micanovic
authors.forename: S P
authors.initials: SP
authors.lastname: Manly
authors.forename: J J
authors.initials: JJ
authors.lastname: Villafranca
authors.forename: K M
authors.initials: KM
authors.lastname: Tramposch
mesh_terms: D016718:Arachidonic Acid / Q000378:metabolism; D002152:Calorimetry, Differential Scanning; D002784:Cholesterol / Q000378:metabolism; D002794:Choline; D006801:Humans; D015544:Inositol 1,4,5-Trisphosphate / Q000378:metabolism; D007700:Kinetics; D007834:Lasers; D008051:Lipid Bilayers / Q000378:metabolism*; D008055:Lipids; D009240:N-Formylmethionine Leucyl-Phenylalanine / Q000494:pharmacology; D010741:Phospholipases A / Q000037:antagonists & inhibitors*; D054467:Phospholipases A2; D000644:Quaternary Ammonium Compounds / Q000494:pharmacology*; D012542:Scattering, Radiation; D020298:U937 Cells; D014867:Water
chemical_list: C120164:2-(2'-benzyl-4-chlorophenoxy)ethyl dimethyl n-octadecyl ammonium; D008051:Lipid Bilayers; D008055:Lipids; D000644:Quaternary Ammonium Compounds; D014867:Water; D016718:Arachidonic Acid; D009240:N-Formylmethionine Leucyl-Phenylalanine; D015544:Inositol 1,4,5-Trisphosphate; D002784:Cholesterol; D010741:Phospholipases A; D054467:Phospholipases A2; D002794:Choline