Novel reversible, irreversible and fluorescent inhibitors of platelet-activating factor acetylhydrolase as mechanistic probes.
Phosphatidylcholines (1-O-alcoxy-2-amino-2-desoxy-phosphocholines and 1-pyrene-labeled analogs) were synthesized and used to examine interactions with recombinant human PAF-acetylhydrolase (PAF-AH), an enzyme purified from plasma, and with macrophage-like U937 cells. Novel phosphatidylcholines containing a sn-2-carbamoylester group such as 1-O-hexadecyl-2-desoxy-2-amino-methylcarbamoyl-2-methyl-rac-glycer o-3-phosphocholine 11 were found to act as site-specific irreversible enzyme inhibitors with Ki-values up to 83 (K(irev)) and 177 (Ki(inact)) microm. The compounds exhibit only marginal inhibition of Ca2+-dependent phospholipases. Kinetic data show that phosphocholines carrying a terminal sn-1-pyrene moiety inhibit PAF-AH activity with an effectivity similar to analogs with an aliphatic chain. 1-O-Decyloxy-[10-(4-pyrenyl)-butoxy]-2-desoxy-2-amino-carbamoyl-me thyl-rac(-glycero-3-phosphocholine 13 could be used for enzyme labeling and to demonstrate an inhibitor-enzyme stoichiometry of 0.7:1. At 8 degrees C, the compound accumulated in the membranes of U937 cells, at 37 degrees C it was internalized into intracellular compartments. Structure activity studies in a mixed micelle assay indicated that the inhibition power of reversible and irreversible inhibitors increases along with the (sn)-1-chain length similar to the structure-dependent binding of ether phospholipids to the PAF-receptor. Unlike the situation at the (sn)-1-position, increasing chain length at the sn-2-position, or an alkyl branching of the glycerol backbone significantly reduced the inhibitory potency.
Deigner
H P
HP
Pharmazeutisch-Chemisches Institut, Universität Heidelberg, Germany. hans-peter.deigner@urz.uni-heidelberg.de
Kinscherf
R
R
Claus
R
R
Fyrnys
B
B
Blencowe
C
C
Hermetter
A
A
D043203:1-Alkyl-2-acetylglycerophosphocholine Esterase; D001665:Binding Sites; D015415:Biomarkers / Q000032:analysis; D002478:Cells, Cultured / Q000737:chemistry / Q000378:metabolism; D004347:Drug Interactions; D005456:Fluorescent Dyes / Q000032:analysis* / Q000138:chemical synthesis*; D006801:Humans; D008264:Macrophages / Q000378:metabolism*; D010741:Phospholipases A / Q000037:antagonists & inhibitors*; D010972:Platelet Activating Factor / Q000037:antagonists & inhibitors*; D012680:Sensitivity and Specificity; D013329:Structure-Activity Relationship
D015415:Biomarkers; D005456:Fluorescent Dyes; D010972:Platelet Activating Factor; D010741:Phospholipases A; D043203:1-Alkyl-2-acetylglycerophosphocholine Esterase