The major objective of the proposed research is to prepare rationally-designed inhibitors of phospholipase-A2. Phospholipase-A2-catalyzed release of arachidonic acid from the phospholipid pool in cell membranes is the rate-limiting step in the biosynthesis of prostaglandins, leukotrienes, and thromboxanes. Phospholipase-A2 inhibitors are known to inhibit processes such as platelet aggregation and therefore may have uses as antithrombotic pharmaceuticals. Furthermore, selective inhibitors of phospholipase-A2 will be useful experimental tools in the delineation of the major pathways responsible for arachidonic acid release in membranes. Phospholipid analogs in which the ester linkage at the sn-2 position has been replaced by a difluoromethylene ketone will be prepared. Fluoro ketone analogs are known to be potent inhibitors of many hydrolytic enzymes. Fluoro ketones exist predominantly in the hydrated form and it is likely that the hydrated ketone will act as a transition state analog inhibitor of phospholipase-A2. Alternatively, the fluoro ketone might form a stable hemiketal with a nucleophile present at the active site of the enzyme. NMR studies will be conducted in order to establish the mode of binding of the fluoro ketone phospholipid analogs to phospholipase-A2. These studies will help in the understanding of the mechanism of lipolysis catalyzed by phospholipase-A2. The use of non-fluorinated and II-o-alcohol phospholipid analogs as inhibitors of phospholipase-A2 will also be investigated. All of the analogs will be initially tested as inhibitors with the soluble pancreatic enzyme. Eventually the studies will be extended to include phospholipase-A2 derived from platelets and other tissues.
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