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Discovery of a NAPE-PLD inhibitor that modulates emotional behavior in mice

Abstract

N-acylethanolamines (NAEs), which include the endocannabinoid anandamide, represent an important family of signaling lipids in the brain. The lack of chemical probes that modulate NAE biosynthesis in living systems hamper the understanding of the biological role of these lipids. Using a high-throughput screen, chemical proteomics and targeted lipidomics, we report here the discovery and characterization of LEI-401 as a CNS-active N-acylphosphatidylethanolamine phospholipase D (NAPE-PLD) inhibitor. LEI-401 reduced NAE levels in neuroblastoma cells and in the brain of freely moving mice, but not in NAPE-PLD KO cells and mice, respectively. LEI-401 activated the hypothalamus–pituitary–adrenal axis and impaired fear extinction, thereby emulating the effect of a cannabinoid CB1 receptor antagonist, which could be reversed by a fatty acid amide hydrolase inhibitor. Our findings highlight the distinctive role of NAPE-PLD in NAE biosynthesis in the brain and suggest the presence of an endogenous NAE tone controlling emotional behavior.

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Fig. 1: HTS of a NAPE-PLD activity assay provided potent new inhibitors 26.
Fig. 2: Hit optimization affords nanomolar potent inhibitor LEI-401.
Fig. 3: Photoprobe 10 visualizes target engagement of LEI-401 with NAPE-PLD in situ.
Fig. 4: LEI-401 reduces NAE levels in Neuro-2a cells, but not in NAPE-PLD KO cells.
Fig. 5: LEI-401 decreases AEA in mouse brain.
Fig. 6: LEI-401 activates HPA axis signaling and diminishes fear extinction in vivo, which can be abolished by inhibition of FAAH.

Data availability

All data generated or analyzed during this study are included in this published article (and its Supplementary Information files) or are available from the corresponding author on reasonable request. The mass spectrometry proteomics data (raw data and ISOQuant output tables for proteins groups and peptides) have been deposited in the ProteomeXchange Consortium (http://proteomecentral.proteomexchange.org) via the PRIDE partner repository with the dataset identifier PXD017586.

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Acknowledgements

The research leading to these results has received support from the Innovative Medicines Initiative Joint Undertaking under grant agreement no. 115489, resources of which are composed of financial contribution from the European Union’s Seventh Framework Programme (no. FP7/2007-2013) and EFPIA companies’ in kind contribution. M.v.d.S. was supported by a VICI-grant from the Netherlands Organization for Scientific Research and funding from Oncode Institute. Leiden University, Faculty of Science ‘Profiling Programme: Endocannabinoids’ is also acknowledged for financial support to E.D.M., V.K., T.H. and M.v.d.S. This research was also supported by National Institutes of Health National Institute on Drug Abuse (grant nos. R01DA039942 and P30DA0339340), Canadian Institutes of Health Research (grant no. FDN-143329 to M.N.H.) as well as start-up funds from the VCU School of Pharmacy to A.H.L. and the Intramural Research Program of NIAAA/NIH (to P.P., O.G.-C., L.I.C., C.M.D. and A.H.). We kindly acknowledge the Pharmaceutical Sciences division of F. Hoffman-La Roche Ltd for their technical assistance with the drug metabolism and pharmacokinetics experiments.

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E.D.M., T.H., M.N.H, P.P., A.H.L. and M.v.d.S. conceived the project and designed the experiments. J.W., H.v.d.H. and C.A.A.v.B. performed the HTS study. E.D.M. and I.K. synthesized the compounds. E.D.M., A.C.M.v.E., A.M.F.v.d.G., A.M., T.v.d.W, M.S., M.J., T.J.W. and A.P.A.J., performed biochemical and cellular experiments. A.T.B. and B.I.F. performed the proteomics measurements. V.K. and X.D. performed the in vitro and cellular lipidomics measurements. E.D.M., M.M., R.C., G.N.P., D.O., Z.V.V., J.P., C.M., G.D., J.K.P., A.P.A.J., B.I.F., M.W., U.G. and M.N.H. performed in vivo pharmacokinetics, lipidomics and behavioral studies. O.G.-C., L.I.C., C.M.D. and A.H. designed, performed and analyzed fear extinction behavior. E.D.M., M.W., U.G., B.F.C., M.W.B., H.v.d.H., C.A.A.v.B., P.P., A.H.L. and M.v.d.S analyzed the data and wrote the manuscript.

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Correspondence to Mario van der Stelt.

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Competing interests

E.M., I.K. C.B. and M.v.d.S. are listed as inventors on patent application WO 2019/229250 A1 filed by Leiden University in which inhibitors of NAPE-PLD are disclosed.

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Supplementary information

Supplemental Information

Supplementary Tables 1–7, Figs. 1–21 and Note.

Reporting Summary

Supplementary Dataset 1

Label-free quantification of mock- and hNAPE-PLD-FLAG transfected HEK293T cells treated with DMSO or LEI-401 (10 µM). Three biological replicates per condition. P values were calculated with a Student’s t-test (two-tailed, unpaired) and Benjamini–Hochberg correction (10% FDR).

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Mock, E.D., Mustafa, M., Gunduz-Cinar, O. et al. Discovery of a NAPE-PLD inhibitor that modulates emotional behavior in mice. Nat Chem Biol 16, 667–675 (2020). https://doi.org/10.1038/s41589-020-0528-7

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