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.

Biosynthesis of N-Docosahexanoylethanolamine from Unesterified Docosahexaenoic Acid and Docosahexaenoyl-Lysophosphatidylcholine in Neuronal Cells

“…The cytotoxicity of N- acylphosphatidylethanolamine-phospholipase D (NAPE-PLD) inhibition was assessed in Neuro2A cultures. Previous studies indicated that hexachlorophene and bithionol produced only small decreases in HEK293 cell viability at concentrations of 8 and 10.5 μM, respectively [ 29 ]. On the basis of this information, we tested the effect of these inhibitors on Neuro2A cells at a concentration of 10 μM.…”

“…Effects of the NAPE-PLD inhibitors hexachlorophene and bithionol (Sigma-Aldrich) [ 29 ] on cell viability were tested in the presence of 2 μM FAAH inhibitor URB597. Neuro2A cells were seeded at approximately 40% confluency into a 96-well culture plate in 5% FBS/DMEM medium.…”

A Role of Newly Found Auxiliary Site in Phospholipase A1 from Thai Banded Tiger Wasp (Vespa affinis) in Its Enzymatic Enhancement: In Silico Homology Modeling and Molecular Dynamics Insights

“…Compared with other phospholipase classes, the known PLA1 inhibitor is not commonly reported. The main strategy of PLA1 inhibition is to design a lipid mimetic compound or phospholipid bilayer derivatives [21,22] to compete with a substrate. The idea of the auxiliary site inhibition would be of interest because the site is a solvent-accessible surface.…”

The Liver under the Spotlight: Bile Acids and Oxysterols as Pivotal Actors Controlling Metabolism

“…Although the physiological meaning of this study remains to be assessed in vivo, these results seem promising since BA concentration spans from ~2 to 10 mM in the ileum after meal ingestion and at lower range (µM) in the blood and the liver [ 26 , 161 , 162 ]. More recently, tauroursodeoxycholic acid (TUDCA), taurohyodeoxycholic acid (THDCA) as well as α/β-MCAs and their taurine-conjugated forms have also been identified as NAPE-PLD inhibitors, although slightly less potent than LCA [ 163 ]. These studies paved the way to the design of specific NAPE-PLD modulators [ 164 ].…”