Symmetrically substituted dichlorophenes inhibitN-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 major obstacle to elucidating the role of NAPE-PLD and NAE biosynthesis in various physiological processes has been the lack of effective NAPE-PLD inhibitors. The endogenous bile acid lithocholic acid (LCA) inhibits NAPE-PLD activity (IC 50 68 μM) but LCA is also a highly potent ligand for TGR5 (EC 50 0.52 μM). Recently, the first selective small molecule inhibitor of NAPE-PLD, ARN19874, was reported (IC 50 34 μM). To identify more potent inhibitors of NAPE-PLD, we screened compounds using a quenched fluorescent NAPE analog, PED-A1, as a substrate for recombinant mouse NAPE-PLD. Screened compounds included a panel of bile acids as well as a library of experimental compounds (the Spectrum Collection). Muricholic acids and several other bile acids inhibited NAPE-PLD with potency similar to LCA. Fourteen potent NAPE-PLD inhibitors were identified in the Spectrum Collection, with the two most potent (IC 50 ~2 μM) being symmetrically substituted dichlorophenes: hexachlorophene and bithionol. Structure activity relationship assays using additional substituted dichlorophenes identified key moieties needed for NAPE-PLD inhibition. Both hexachlorophene and bithionol showed significant selectivity for NAPE-PLD compared to non-target lipase activities such as S. chromofuscus PLD activity or serum lipase activity. Both also effectively inhibited NAPE-PLD activity in cultured HEK293 cells.