Symmetrically substituted dichlorophenes inhibit N-acyl-phosphatidylethanolamine phospholipase D

Abstract

N-Acyl-phosphatidylethanolamine phospholipase D (NAPE-PLD) (EC 3.1.4.4) catalyzes the final step in the biosynthesis of N-acyl-ethanolamides. 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 N-acyl-ethanolamide biosynthesis in these processes. The endogenous bile acid lithocholic acid (LCA) inhibits NAPE-PLD activity (with an IC₅₀ of 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 of 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 that of LCA. We identified 14 potent Nape-pld inhibitors 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 with nontarget 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.

Keywords: N-acyl-ethanolamide; N-acyl-phosphatidylethanolamine; NAPE-PLD; TGR5; bile acid; dichlorophene; endocannabinoid; high-throughput screening (HTS); lipid signaling; metabolic disease; phospholipase D; protein–lipid interaction.