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-pyr ene-labeled analogs) were synthesized and used to examine interactions with recombinant human PAF-acetylhydrolase (PAF-AH), an enzyme purified from pl asma, and with macrophage-like U937 cells. Novel phosphatidylcholines conta ining a sn-2-carbamoylester group such as 1-O-hexadecyl-2-desoxy-2-amino-me thylcarbamoyl-2-methyl-rac-glycero-3-phosphocholine 11 were found to act as site-specific irreversible enzyme inhibitors with K-i-values up to 83 (K-i rev) and 177 (K-i(inact)) mu m. The compounds exhibit only marginal inhibit ion of Ca2+-dependent phospholipases. Kinetic data show that phosphocholine s carrying a terminal sn-1-pyrene moiety inhibit PAF-AH activity with an ef fectivity similar to analogs with an aliphatic chain. 1-O-Decyloxy-[10-(4-p yrenyl)-butoxy]-2-desoxy-2-amino-carbamoyl-methyl-rac-glycero-3-phosphochol ine 13 could be used for enzyme labeling and to demonstrate an inhibitor-en zyme stoichiometry of 0.7:1. At 8 degrees C, the compound accumulated in th e membranes of U937 cells, at 37 degrees C it was internalized into intrace llular compartments. Structure-activity studies in a mixed micelle assay in dicated that the inhibition power of reversible and irreversible inhibitors increases along with the (sn)-1-chain length similar to the structure-depe ndent binding of ether phospholipids to the PAF-receptor. Unlike the situat ion at the (sn)-1-position, increasing chain length at the sn-2-position, o r an alkyl branching of the glycerol backbone significantly reduced the inh ibitory potency. (C) 1999 Elsevier Science Ireland Ltd. All rights reserved .