Design, Synthesis, and Evaluation of Phospholipid Analogues as Inhibitors of the Bacterial Phospholipase C from Bacillus cereus

Article abstract of DOI:10.1021/jo00096a024

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 (S_p)-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 as follows: 7 ca. 11 ca. 10 < 4 ca. 8 < 5 ca. 6.