| The phospholipase A2 (PLA2) superfamily consists of many Groups of enzymes defined by their ability to catalyze the hydrolysis of the middle (sn-2) ester bond of substrate phospholipids. One of the members of the superfamily, the cytosolic Group IVA phospholipase A2 (GIVAPLA2) is the principal provider of free lysophospholipid and polyunsaturated fatty acids (e.g. arachidonic acid), the precursors of platelet activating factor and eicosanoids, respectively. Physiological increases in intracellular Ca2+ lead to enhanced membrane affinity and translocation of GIVAPLA2 from the cytosol to its substrate membranes. Ca2+-dependent GIVAPLA2 translocation and subsequent activity are mediated by its Ca2+-dependent phospholipid binding C2 domain. We have shown that phosphatidylinositol 4,5-bisphosphate (PI-4,5-P 2) also binds with high affinity and specificity to GIVAPLA2 in a 1:1 stoichiometry, facilitating membrane binding and activity. We have demonstrated that GIVAPLA2 possesses full activity in the absence of Ca2+ when PI-4,5-P2 or PI-3,4,5-P3 are present. When GIVAPLA2 was expressed without its Ca 2+-binding C2 domain (ΔC2), this construct was still able to bind to phosphoinositides (PIPn's), but there was no interfacial activity. This demonstrated that the C2 domain plays a novel, critical role in GIVAPLA2 interfacial activity. The catalytic domain residues, K488, K541, K543, and K544 were confirmed to be essential for PI-4,5-P 2-dependent activity increases. Through its downstream products, including platelet activating factor and eicosanoids, GIVAPLA2 is a key drug target to prevent inflammation, pain, and associated pathophysiological conditions. A novel class of potent human GIVAPLA2 inhibitors was developed. These inhibitors were designed to contain the 2-oxoamide functionality and a free carboxy group. Using cellular models of inflammation, we had previously shown that the production of inflammatory eicosanoids depends on GIVAPLA 2. We now show that the most potent 2-oxoamide inhibitors of GIVAPLA 2 block the production of prostaglandin E2 induced by lipopolysaccharide (LPS) or LPS/PAF in these cellular models. We have also further tested these inhibitors in rat models of inflammation and pain, and have shown that these inhibitors are anti-inflammatory and analgesic. |
