Novel 2-Oxoamide Inhibitors of Human Group IVA Phospholipase A₂

Abstract

A novel class of potent human cytosolic phospholipase A(2) (GIVA PLA(2)) inhibitors was developed. These inhibitors were designed to contain the 2-oxoamide functionality and a free carboxyl group. Among the compounds tested, a long-chain 2-oxoamide containing L-gamma-norleucine was the most potent inhibitor, causing a 50% decrease in GIVA PLA(2) activity at 0.009 mole fraction.

In Vivo Phospholipase Activity of the Pseudomonas aeruginosa Cytotoxin ExoU and Protection of Mammalian Cells with Phospholipase A2 Inhibitors

“…ExoU was syringe-loaded into these labeled cells, and after a 20-min incubation, medium from the cells was collected. The medium was extracted using a modified Dole protocol to isolate free fatty acids, and the radioactivity in the extract was quantified (25). Using this in vivo phospholipase assay, we find that syringe loading ExoU results in the production of large amounts of free fatty acids, consistent with high levels of phospholipase activity in ExoU (Fig.…”

“…Samples were vortexed and incubated at 40°C for 60 min. Products of this and following radioassays were quenched and analyzed using a modified Dole protocol (25). Mixed micelles were made in the following way: 190 l of 100 mM KCl, 20 mM HEPES, pH 7.7, were added to phospholipids and vortexed followed by addition of 10 l of 150 mM TX-100 and vortexing.…”

“…Approximately 10 6 cells were syringe-loaded as described above, except that cells were incubated in the syringe-loading solution for 20 min and then centrifuged at 720 ϫ g for 1 min. The supernatant was used for LDH quantification and extracted using the modified Dole protocol (25). Radioactivity in the sample extracted by the Dole protocol was quantified by scintillation counting.…”

Essential Ca2+-independent Role of the Group IVA Cytosolic Phospholipase A2 C2 Domain for Interfacial Activity

“…The mixed micelle substrate was prepared as described previously (23,37). The micelles were initially made up in 20 mM HEPES and 100 mM KCl (190 l/assay) to form a cloudy white solution of multilamellar vesicles upon vortexing.…”

“…This brought the final volume to 500 l and the desired final concentrations as noted above. After a 60-min incubation, the reaction was quenched, and the fatty acids were extracted using a modified Dole protocol (38) as previously described (23,37). The final radioactive cpm were doubled in the calculation of specific activity because only 1 of 2 ml of heptane were counted.…”