A β-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-labelled U937 cells as substrate, 3.3-Dimsthyl-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 μM). This β-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 (K(I)(*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.