Synthesis and characterization of the first inhibitor of<i>N</i>-acylphosphatidylethanolamine phospholipase D (NAPE-PLD)

Castellani, Beatrice; Diamanti, Eleonora; Pizzirani, Daniela; Tardia, Piero; Maccesi, Martina; Realini, Natalia; Magotti, Paola; Garau, Gianpiero; Bakkum, Thomas; Rivara, Silvia; Mor, Marco; Piomelli, Daniele

doi:10.1039/c7cc07582k

Cited by 34 publications

(29 citation statements)

References 22 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…The IC50 for carbonic anhydrase activity was 107 µM and 178 µM for Inh 1 and Inh 2, respectively ( Figure 5C). Thus, both Inh 1 and Inh HEK293 endogenously express NAPE-PLD (37,43) and were therefore used to assess the ability of these inhibitors to inhibit the NAPE-PLD activity of intact cells. To determine the maximum concentration of Inh 1 and Inh 2 that could be used for cellular inhibition studies without cytotoxicity, HEK293 were treated with 0-100 μM inhibitor and cytotoxicity measured as before.…”

Section: Resultsmentioning

confidence: 99%

“…This assay was performed using the methods of Castellani et al (37). Briefly, 81 μg mL -1 recombinant Nape-pld was pre-incubated with Inh 1 or Inh 2 at 50 µM for 1 h and then samples were diluted 100-fold with 50 mM Tris-HCl (pH=8.0) immediately prior to addition of 4 µM PED-A1 and fluorescence measured at 1-min intervals for 30 min.…”

Section: Rapid Dilution Assaymentioning

confidence: 99%

“…Although its bioavailability and relatively low toxicity allow LCA to be administered in vivo, LCA is also a potent ligand for the bile acid receptor, Tgr5, with an EC50 of 0.53 μM (36) so it cannot be used as a selective inhibitor of NAPE-PLD. More recently, ARN19874 was reported to be a selective NAPE-PLD inhibitor, with an IC50 of 34 µM (37). The absorbance, distribution, metabolism, and excretion (ADME) properties of this compound and whether it has efficacy in vivo have not been reported.…”

Section: Introductionmentioning

confidence: 99%

See 2 more Smart Citations

Symmetrically substituted dichlorophenes inhibit N-acyl-phosphatidylethanolamine phospholipase D

Aggarwal

Zarrow

Mashhadi

et al. 2020

Journal of Biological Chemistry

View full text Add to dashboard Cite

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.

show abstract

Section: Resultsmentioning

confidence: 99%

Section: Rapid Dilution Assaymentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 1 more Smart Citation

Symmetrically substituted dichlorophenes inhibit N-acyl-phosphatidylethanolamine phospholipase D

Aggarwal

Zarrow

Mashhadi

et al. 2020

Journal of Biological Chemistry

View full text Add to dashboard Cite

show abstract

“…All 14 inhibitor compounds (Inh 1-14) were then purchased from commercial sources to validate the putative library compound, analyzed by mass spectrometry to confirm identity and purity, and concentration response curve (CRC) studies performed to establish potency. The IC50 of each of these compounds ranged from 1.6 μM to 19.1 μM, so that each was potentially more potent in vitro than ARN19874 (Table 1) (37).…”

Section: Resultsmentioning

confidence: 99%

“…HEK293 endogenously express NAPE-PLD (37,43) and were therefore used to assess the ability of these inhibitors to inhibit the NAPE-PLD activity of intact cells. To determine the maximum concentration of Inh 1 and Inh 2 that could be used for cellular inhibition studies without cytotoxicity, HEK293 were treated with 0-100 μM inhibitor and cytotoxicity measured as before.…”

Section: Resultsmentioning

confidence: 99%

Symmetrically substituted dichlorophenes inhibitN-acyl-phosphatidylethanolamine phospholipase D

Aggarwal

Zarrow

Mashhadi

et al. 2020

Preprint

View full text Add to dashboard Cite

AbstractN-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 (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, was reported (IC50 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 (IC50 ~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.

show abstract

Recent advances in synthesis of quinazoline‐2,4(1H,3H)‐diones: Versatile building blocks in N‐heterocyclic compounds

Gheidari

Mehrdad

Maleki

2022

Applied Organom Chemis

View full text Add to dashboard Cite

show abstract

Synthesis and characterization of the first inhibitor ofN-acylphosphatidylethanolamine phospholipase D (NAPE-PLD)

Cited by 34 publications

References 22 publications

Symmetrically substituted dichlorophenes inhibit N-acyl-phosphatidylethanolamine phospholipase D

Symmetrically substituted dichlorophenes inhibit N-acyl-phosphatidylethanolamine phospholipase D

Symmetrically substituted dichlorophenes inhibitN-acyl-phosphatidylethanolamine phospholipase D

Recent advances in synthesis of quinazoline‐2,4(1H,3H)‐diones: Versatile building blocks in N‐heterocyclic compounds

Contact Info

Product

Resources

About