DESIGN, SYNTHESIS, AND EVALUATION OF PHOSPHOLIPID ANALOGS AS INHIBITORS OF THE BACTERIAL PHOSPHOLIPASE-C FROM BACILLUS-CEREUS

Enzymes belonging to the phospholipase C (PLC) family hydrolyze the phosphodiester bond of phospholipids to give a diacylglycerol and a phosphorylated head group. The bacterial phospholipase C from Bacillus cereus (PLC(Bc)) has been studied extensively, and there is a wealth of information regarding those structural features that are important for substrate activity. In contrast, there is virtually no data available regarding structure-activity relationships for inhibitors of this enzyme. To address this shortcoming, a series of optically pure analogues of 1,2-dihexanoyl-sn-glycero-3-phosphocholine (2) containing different replacements of the phosphate group were first synthesized including the phosphoramidates 4 and 8, the phosphonate 5, the (difluoromethylene)phosphonate 6, the thiophosphate 7, the diastereomeric phosphorothioates 9 and 10, and the phosphorodithioate 11. Each of these phosphatidylcholine derivatives was tested for inhibitor or substrate activity with PLC(Bc) using the water-soluble phosphatidylcholine 2 as the monomeric substrate. The measurements were conducted below the critical micellar concentrations of both 2 and the inhibitor. Of the analogues, only 7 and 9 underwent observable enzymatic hydrolysis under the assay conditions used. The k(cat) of the (Sp)-phosphorothioate 9 was approximately one-fifth that of 2, and when compared to 2, 7 was hydrolyzed only very slowly by the enzyme. Kinetic studies indicated that the phospholipid analogues tested were competitive inhibitors with increasing K-i's follows: 7 approximate to 11 approximate to 10 < 4 approximate to 8 < 5 approximate to 6.