Inhibitors of CoA-Independent Transacylase Block the Movement of Arachidonate into 1-Ether-Linked Phospholipids of Human Neutrophils

The enzyme CoA-independent transacylase (CoA-IT) has been proposed to mediate the movement of arachidonate between phospholipid subclasses and influence the formation of arachidonic acid metabolites and platelet-activating factor. To substantiate the critical role of CoA-IT, we have developed two structurally diverse inhibitors of CoA-IT activity, SK&F 98625 [diethyl 7-(3,4,5-triphenyl- 2-oxo-2,3-dihydro-imidazole-1-yl)heptane phosphonate] and SK&F 45905 [2-[2-(3-4-chloro-3-(trifluoromethyl)phenyl)ureido]-4-(trifluoromethyl)phenoxy]-4,5-dichlorobenzenesulfonic acid]. These compounds were tested for their capacity to block microsomal CoA-IT activity using two assay systems, the transacylation of l-alkyl-2-lyso-5n-glycero-3-phosphocholine (GPC) and the transfer of [14C]arachidonate from 1 -acyl-2-[¹⁴C]arachidonoy 1-GPC to lyso-PE. Both SK&F 98625 and SK&F 45905 inhibited CoA-IT activity (IC50s 6-19 pM) in these two assays. In contrast, SK&F 98625 or SK&F 45905 had little or no effect on other lipid-modifying activities, including CoA-dependent acyltransferase or acetyltransferase. Kinetic analysis revealed that both SK&F 98625 and SK&F 45905 interact directly with the enzyme and prevented the acylation of lysophospholipids in a competitive manner. In intact human neutrophils, both SK&F 98625 and SK&F 45905 completely blocked the movement of [3H]arachidonate from 1-acyl-linked phospholipids into l-alkyl-2-arachidonoyl-GPC and l-alk-r-enyl-2-arachidonoyl-GPE. In contrast, these compounds did not inhibit the incorporation of free arachidonic acid into cellular lipids indicating that they did not alter CoA-dependent acyl transferase activities in the intact cell. This is the first report to utilize an inhibitor to address the importance of CoA-IT in arachidonate-phospholipid remodeling. These results provide further evidence that CoA-IT mediates the movement of arachidonate into the large pools of 1 -ether-linked phospholipids in human neutrophils and suggest that it may be possible to regulate AA levels in cellular phospholipids with CoA-IT inhibitors.