title: A beta-lactam inhibitor of cytosolic phospholipase A2 which acts in a competitive, reversible manner at the lipid/water interface.
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, 3,3-Dimethyl-6-(3-lauroylureido)-7-oxo-4-thia-1-azabicyclo[3,2,0] heptane-2-carboxylic acid (1) was found to inhibit the enzyme in a dose-dependent manner (IC50 = 72 microM). This beta-lactam did not inhibit other phospholipases, including the human nonpancreatic secreted phospholipase A2. The inhibition of cPLA2 was found not to be time-dependent. This, along with the observation that the degradation of the inhibitor was not catalyzed by the enzyme, demonstrates that the inhibition does not result from the formation of an acyl-enzyme intermediate with the active site serine residue. Moreover, the ring-opened form of 1 is also able to inhibit cPLA2 with near-equal potency. 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 mole percent of 1-palmitoyl-2-[1-14C]-arachidonoyl-sn-glycero-3-phosphocholine was employed. With this substrate system, the dose-dependent inhibition was defined by kinetic equations describing competitive inhibition at the lipid/water interface. The apparent dissociation constant for the inhibitor bound to the enzyme at the interface (KI*app) was determined to be 0.5 +/- 0.1 mole% versus an apparent dissociation constant for the arachidonate-containing phospholipid of 0.4 +/- 0.1 mole%. Thus, 1 represents a novel structural class of inhibitors of cPLA2 which partitions into the phospholipid bilayer and competes with the phospholipid substrate for the active site.
authors.affiliation: Department of Dermatology Discovery Research, Buffalo, New York, USA.
authors.forename: J R
authors.initials: JR
authors.lastname: Burke
authors.forename: K R
authors.initials: KR
authors.lastname: Gregor
authors.forename: R
authors.initials: R
authors.lastname: Padmanabha
authors.forename: J
authors.initials: J
authors.lastname: Banville
authors.forename: M R
authors.initials: MR
authors.lastname: Witmer
authors.forename: L B
authors.initials: LB
authors.lastname: Davern
authors.forename: S P
authors.initials: SP
authors.lastname: Manly
authors.forename: K M
authors.initials: KM
authors.lastname: Tramposch
mesh_terms: D016718:Arachidonic Acid / Q000378:metabolism; D001667:Binding, Competitive; D002460:Cell Line; D003600:Cytosol / Q000201:enzymology; D004791:Enzyme Inhibitors / Q000138:chemical synthesis / Q000737:chemistry / Q000494:pharmacology*; D006801:Humans; D007527:Isoenzymes / Q000037:antagonists & inhibitors; D007700:Kinetics; D010397:Penicillanic Acid / Q000031:analogs & derivatives* / Q000138:chemical synthesis / Q000737:chemistry / Q000494:pharmacology; D010741:Phospholipases A / Q000037:antagonists & inhibitors*; D054467:Phospholipases A2; D010743:Phospholipids; D011994:Recombinant Proteins / Q000037:antagonists & inhibitors; D014867:Water
chemical_list: C113999:3,3-dimethyl-6-(3-lauroylureido)-7-oxo-4-thia-1-azabicyclo(3,2,0)heptane-2-carboxylic acid; D004791:Enzyme Inhibitors; D007527:Isoenzymes; D010743:Phospholipids; D011994:Recombinant Proteins; D014867:Water; D016718:Arachidonic Acid; D010397:Penicillanic Acid; D010741:Phospholipases A; D054467:Phospholipases A2