Symmetrically substituted dichlorophenes inhibit N-acyl-phosphatidylethanolamine phospholipase D.

N-acyl-phosphatidylethanolamine phospholipase D (NAPE-PLD) (EC 3.1.4.4) catalyzes the final step in the biosynthesis of N-acyl-ethanolamides (NAEs). Reduced NAPE-PLD expression and activity may contribute to obesity and inflammation, but a lack of effective NAPE-PLD inhibitors has been a major obstacle to elucidating the role of NAPE-PLD and NAE biosynthesis in these processes. The endogenous bile acid lithocholic acid (LCA) inhibits NAPE-PLD activity (with an IC50 68 μM), but LCA is also a highly potent ligand for TGR5 (EC50 0.52 μM). Recently, the first selective small-molecule inhibitor of NAPE-PLD, ARN19874, has been reported (having an IC50 34 µM). To identify more potent inhibitors of NAPE-PLD, here we used a quenched fluorescent NAPE analog, PED-A1, as a substrate for recombinant mouse NAPE-PLD to screen a panel of bile acids and a library of experimental compounds (the "Spectrum Collection"). Muricholic acids and several other bile acids inhibited NAPE-PLD with potency similar to LCA. We identified 14 potent NAPE-PLD inhibitors were identified in the Spectrum Collection, with the two most potent (IC50 ~2 μM) being symmetrically substituted dichlorophenes, i.e. hexachlorophene and bithionol. Structure-activity relationship assays using additional substituted dichlorophenes identified key moieties needed for NAPE-PLD inhibition. Both hexachlorophene and bithionol exhibited significant selectivity for NAPE-PLD compared to non-target lipase activities such as Streptomyces chromofuscus PLD or serum lipase. Both also effectively inhibited NAPE-PLD activity in cultured HEK293 cells. We conclude that symmetrically substituted dichlorophenes potently inhibit NAPE-PLD in cultured cells and have significant selectivity for NAPE-PLD versus other tissue-associated lipases.