Platelet-activating factor acetylhydrolase: selective inhibition by potent
Gary B. Quistad, Karl J. Fisher, Sarah C. Owen, Rebecka Klintenberg, John E. Casida*
Environmental Chemistry and Toxicology Laboratory, Department of Environmental Science, Policy and Management, University of California,
Berkeley, CA 94720-3112, USA
Received 25 August 2004; accepted 28 September 2004
Available online 11 November 2004
Platelet-activating factor (PAF) is a potent endogenous phospholipid modulator of diverse biological activities, including inflammation
and shock. PAF levels are primarily regulated by PAF acetylhydrolases (PAF-AHs). These enzymes are candidate secondary targets of
organophosphorus (OP) pesticides and related toxicants. Previously known OP inhibitors of other serine hydrolases were tested with PAF-AH
from mouse brain and testes of established functional importance compared with the structurally different human plasma enzyme. Several
key OP pesticides and their oxon metabolites were very poor inhibitors of mouse brain and human plasma PAF-AH in vitro but moderately
active for mouse brain and blood PAF-AH in vivo (e.g., tribufos defoliant and profenofos insecticide, presumably following oxidative
bioactivation). OP compounds were then designed for maximum in vitro potency and selectivity for mouse brain PAF-AH vs.
acetylcholinesterase (AChE). Lead compounds were found in a series of benzodioxaphosphorin 2-oxides. Ultrahigh potency and selectivity
were achieved with n-alkyl methylphosphonofluoridates (long-chain sarin analogs): mouse brain and testes IC50 V 5 nM for C8–C18analogs
and 0.1–0.6 nM for C13and C14compounds; human plasma IC50 V 2 nM for C13–C18analogs. AChE inhibitory potency decreased as chain
length increased with maximum brain PAF-AH/AChE selectivity (N3000-fold) for C13–C18compounds. The toxicity of i.p.-administered
PAF (LD50 ca. 0.5 mg/kg) was increased less than 2-fold by pretreatment with tribufos or the C13n-alkyl methylphosphonofluoridate. These
studies with a mouse model indicate that PAF-AH is not a major secondary target of OP pesticide poisoning. The optimized PAF-AH
inhibitors may facilitate investigations on other aspects of PAF metabolism and action.
D 2004 Elsevier Inc. All rights reserved.
Keywords: Acetylhydrolase; Methylphosphonofluoridate; Organophosphorus inhibitor; Pesticide; Platelet-activating factor; Tribufos
Platelet-activating factors (PAFs) are 1-O-alkyl-2-acetyl-
sn-glycero-3-phosphocholine phospholipids with potent,
diverse physiological actions. They are strong mediators
of inflammation (Karasawa et al., 2003; Prescott et al.,
2000). Increased platelet activation triggers acute vascular
syndromes, including ischemic stroke and myocardial
infarction, and antiplatelet drugs are now a routine part of
treatment to prevent additional occurrences (Zhao et al.,
2001). PAF is highly toxic to mice, inducing severe shock
and death at 40 Ag/kg i.v. (Fukuda et al., 2000; Tanniere-
Zeller et al., 1989).
PAF-acetylhydrolase (AH) plays an important role in
human health by hydrolyzing the sn-2 ester bond and
0041-008X/$ - see front matter D 2004 Elsevier Inc. All rights reserved.
Abbreviations: AChE, acetylcholinesterase; AH, acetylhydrolase;
BDPO, benzodioxaphosphorin 2-oxide; CPO, chlorpyrifos oxon; DFP,
diisopropyl fluorophosphate; ED50, dose inhibiting 50% of enzyme
activity; IC50, concentration inhibiting 50% of enzyme activity; DMSO,
dimethyl sulfoxide; 2-N-methylcarbamyl PAF, 1-O-palmityl-2-(N-
methylcarbamyl)-sn-glycero-3-phosphocholine; OP, organophosphorus;
PAF, platelet-activating factor; phenyl-BDPO, phenyl-benzodioxaphos-
phorin 2-oxide; PMSF, phenylmethanesulfonyl fluoride; SAR, structure–
activity relationship; TMPF, n-tridecyl methylphosphonofluoridate.
* Corresponding author. Environmental Chemistry and Toxicology
Laboratory, Department of Environmental Science, Policy and Manage-
ment, University of California, 115 Wellman Hall, Berkeley, CA 94720-
3112. Fax: +1 510 642 6497.
E-mail address: email@example.com (J.E. Casida).
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