Design, synthesis, and evaluation of water-soluble phospholipid analogues as inhibitors of phospholipase C from Bacillus cereus.
The rate of hydrolysis of natural phospholipids by the phosphatidylcholine-preferring phospholipase C from Bacillus cereus (PLC(Bc)) follows the order phosphatidylcholine > phosphatidylethanolamine >> phosphatidyl-l-serine. To probe the structural basis for this substrate specificity, a series of water-soluble, nonhydrolyzable substrate analogues were needed so their complexes with the enzyme could be studied via X-ray crystallography and isothermal titration calorimetry (ITC). Accordingly the water-soluble dithiophospholipids 2-10 having choline, ethanolamine, and l-serine headgroups were synthesized, and the inhibitory activity of each was determined in an assay using 1,2-dihexanoyl-sn-glycero-3-phosphocholine (C6PC) as the monomeric substrate. The 1,2-dibutanoyl dithiophosphocholine 2 was a weak inhibitor, whereas the related 1,2-dipentanoyl dithiophosphocholine 3 and the ethylene glycol dithiophosphocholines 4 and 5 were moderate inhibitors. The 1,2-omega-hydroxydiacyl dithiophosphocholines 6 and 7 were potent inhibitors, while the related compound 8, which had shorter acyl side chains, was a weak inhibitor. The dithiophosphoethanolamine 9 was a modest inhibitor, whereas the dithiophospho-l-serine 10 was a somewhat weaker inhibitor. Overall, the phospholipid analogues had increasing K(i) values according to the order 2 << 10 < 3 < 4 approximately 5 approximately 8 < 9 << 6 << 7 and increasing solubility according to the sequence 5 approximately 7 < 4 approximately 6 approximately 9 < 3 < 10 < 8 < 2.
Franklin
Christopher L
CL
Department of Chemistry and Biochemistry, The University of Texas at Austin, Austin, Texas 78712, USA. sfmartin@mail.utexas.edu
Li
Hui
H
Martin
Stephen F
SF
D001409:Bacillus cereus / Q000201:enzymology*; D002151:Calorimetry; D018360:Crystallography, X-Ray; D015195:Drug Design; D004791:Enzyme Inhibitors / Q000138:chemical synthesis / Q000494:pharmacology; D007700:Kinetics; D010710:Phosphates / Q000737:chemistry; D010743:Phospholipids / Q000138:chemical synthesis* / Q000494:pharmacology; D011485:Protein Binding; D012995:Solubility; D013329:Structure-Activity Relationship; D013379:Substrate Specificity; D010738:Type C Phospholipases / Q000037:antagonists & inhibitors*
D004791:Enzyme Inhibitors; D010710:Phosphates; D010743:Phospholipids; C083120:phosphorodithioic acid; D010738:Type C Phospholipases