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Articleshttps://doi.org/10.1038/s41589-020-0528-7
Discovery of a NAPE-PLD inhibitor that modulates
emotional behavior in mice
Elliot D. Mock 1, Mohammed Mustafa2, Ozge Gunduz-Cinar3, Resat Cinar 4, Gavin N. Petrie5,
Vasudev Kantae1,6, Xinyu Di6, Daisuke Ogasawara 7, Zoltan V. Varga8, Janos Paloczi8,
Cristina Miliano9, Giulia Donvito2, Annelot C. M. van Esbroeck 1, Anouk M. F. van der Gracht1,
Ioli Kotsogianni1, Joshua K. Park4, Andrea Martella1, Tom van der Wel 1,10, Marjolein Soethoudt1,
Ming Jiang1,10, Tiemen J. Wendel1, Antonius P. A. Janssen 1,10, Alexander T. Bakker1,
Colleen M. Donovan3, Laura I. Castillo3, Bogdan I. Florea11, Jesse Wat12, Helma van den Hurk12,
Matthias Wittwer13, Uwe Grether13, Andrew Holmes3, Constant A. A. van Boeckel1,12,
Thomas Hankemeier6, Benjamin F. Cravatt 7, Matthew W. Buczynski9, Matthew N. Hill5,
Pal Pacher 8, Aron H. Lichtman2,14 and Mario van der Stelt 1,10 ✉
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 inhibi-
tor. 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.
I
n recent years, lipids have come to the foreground as signaling systems are, however, poorly studied, partly because of a lack of
mediators in the central nervous system (CNS)1,2. While classi- pharmacological tools to modulate their biosynthetic enzymes8.
cal neurotransmitters are stored in synaptic vesicles and released NAPE-PLD is generally considered a principal NAE bio-
on fusion with the plasma membrane of neurons, due to their lipo- synthetic enzyme9,10. Biochemical and structural studies have
philic nature, lipids readily diffuse through membranes and are not demonstrated that NAPE-PLD is a membrane-associated, consti-
stored in vesicles. It is, therefore, generally accepted that signaling tutively active zinc hydrolase with a metallo-β-lactamase fold11.
lipids are produced ‘on demand’ and are rapidly metabolized to The enzyme generates a broad range of NAEs by hydrolysis of the
terminate their biological action3. In particular, NAEs, including phosphodiester bond between the phosphoglyceride and the NAE
N-palmitoylethanolamine (PEA), N-oleoylethanolamine (OEA) and in N-acylphosphatidylethanolamines (NAPEs)12. Knockout (KO)
the endocannabinoid anandamide (N-arachidonoylethanolamine, studies have shown that the Ca2+-dependent conversion of NAPE
AEA) have emerged as key lipid signaling molecules. Genetic dele- to NAEs bearing both saturated and polyunsaturated fatty acyl
tion or pharmacological inhibition of the main NAE hydrolytic groups are fivefold reduced in brain lysates from mice that geneti-
enzyme, fatty acid amide hydrolase (FAAH), revealed elevated cally lack Napepld13. In accordance, reduced levels of saturated and
anandamide, PEA and OEA levels in brain and implicated these mono-unsaturated NAEs were observed in the brains of NAPE-PLD
molecules in the modulation of various physiological processes KO mice13–15. Anandamide levels were not reduced in the trans-
such as pain, stress, anxiety, appetite, cardiovascular function and genic model reported by Leung et al., which suggested the presence
inflammation4–7. The physiological effects resulting from pertur- of compensatory mechanisms13. Indeed, multiple alternative bio-
bation of the production of anandamide and other NAEs in living synthetic pathways for anandamide have been discovered since10.
1
Department of Molecular Physiology, Leiden Institute of Chemistry, Leiden University, Leiden, the Netherlands. 2Department of Pharmacology and
Toxicology, Virginia Commonwealth University, Richmond, VA, USA. 3Laboratory of Behavioral and Genomic Neuroscience, National Institute on
Alcoholism and Alcohol Abuse (NIAAA), National Institute of Health (NIH), Bethesda, MD, USA. 4Laboratory of Physiologic Studies, NIAAA, NIH,
Bethesda, MD, USA. 5Hotchkiss Brain Institute, University of Calgary, Calgary, Alberta, Canada. 6Analytical Biosciences and Metabolomics, Division of
Systems Biomedicine and Pharmacology, Leiden Academic Centre for Drug Research, Leiden University, Leiden, the Netherlands. 7Department of Chemical
Physiology, The Scripps Research Institute, La Jolla, CA, USA. 8Laboratory of Cardiovascular Physiology and Tissue Injury, NIAAA, NIH, Bethesda, MD,
USA. 9School of Neuroscience, Virginia Polytechnic Institute and State University, Blacksburg, VA, USA. 10Oncode Institute, Leiden, the Netherlands.
11
Bio-organic Synthesis, Leiden Institute of Chemistry, Leiden University, Leiden, the Netherlands. 12Pivot Park Screening Centre B.V., Oss, the Netherlands.
13
Roche Innovation Center Basel, F. Hoffman-La Roche Ltd, Basel, Switzerland. 14Department of Medicinal Chemistry, Virginia Commonwealth University,
Richmond, VA, USA. ✉e-mail: m.van.der.stelt@chem.leidenuniv.nl
Nature Chemical Biology | VOL 16 | June 2020 | 667–675 | www.nature.com/naturechemicalbiology 667
,Articles NATUrE ChEmIcAL BIOLOgy
Of note, two other NAPE-PLD KO strains have been generated, which of 396 Da, calculated partition coefficient c-logP = 3.84) (Fig. 1d
did show a decreased brain anandamide content14,15. Constitutive and Supplementary Table 2). On resynthesis, chemical analysis and
genetic mouse models are, however, poorly suited to study the rapid retesting, the identity and submicromolar activity (Ki = 0.30 μM,
and dynamic formation of NAEs and their biological functions. 95% CI = 0.22–0.38 μM) of the hit was confirmed (Fig. 2 and
Potent CNS-active inhibitors of NAPE-PLD are currently not avail- Supplementary Notes). To improve the inhibitory activity of com-
able. Previously described NAPE-PLD inhibitors lack the potency, pound 2, a medicinal chemistry program was initiated in which 112
selectivity and/or physicochemical properties to study the role of this derivatives were synthesized. A full report of the medicinal chemistry
enzyme in in vivo systems in an acute setting16–18. Thus, a need exists program will be described elsewhere; however, in brief, we discov-
for pharmacological tools that inhibit NAE biosynthesis in the brain. ered that replacing the morpholine for a (S)-3-hydroxypyrrolidine
Here, we report the discovery of LEI-401 (1) as a brain active (7) improved activity (Ki = 0.086 μM, 95% CI, 0.058–0.14 μM),
NAPE-PLD inhibitor. High-throughput screening (HTS) and a while simultaneously decreasing lipophilicity (c-logP = 3.46)
medicinal chemistry program led to the identification of LEI-401 (Fig. 2a,b). The absolute configuration of the hydroxyl was not
as a potent and selective NAPE-PLD inhibitor. LEI-401 reduced a essential for binding, as the (R)-enantiomer 8 showed similar
broad range of NAEs including anandamide in neuronal cells in a potency (Ki = 0.11 μM, 95% CI, 0.096–0.13 μM). Conformational
NAPE-PLD-dependent manner. Anandamide and other NAE levels restriction of the phenethylamine moiety to a (S)-3-phenylpiperidine
were reduced in the brain of wild-type (WT) mice treated with LEI- (1) further increased the potency (Ki = 0.027 μM, 95% CI, 0.021–
401, but not in NAPE-PLD KO mice. The compound activated the 0.033 μM), whereas its (R)-enantiomer 9 was three times less active
hypothalamus–pituitary–adrenal (HPA) axis and impaired extinc- (Ki = 0.094 μM, 95% CI, 0.071–0.12 μM) (Fig. 2b). In view of the bio-
tion of an aversive memory in mice, thereby mimicking the effects logical activity profile, we decided to further characterize compound
of cannabinoid CB1 receptor antagonism. These behavioral effects 1 (termed LEI-401).
were prevented by a FAAH inhibitor. Taken together, LEI-401 is To profile NAPE-PLD inhibitors in mouse models, we assessed
a first-in-class brain active NAPE-PLD inhibitor that blocks NAE whether LEI-401 showed any species difference using recombi-
biosynthesis in the brain of freely moving mice, thereby revealing a nant mouse NAPE-PLD expressed in HEK293T cells. LEI-401
possible endogenous tone of this lipid family in emotional behavior. retained activity on mouse NAPE-PLD in the PED6 assay, albeit at a
reduced potency (Ki = 0.18 μM, 95% CI, 0.15–0.21 μM). Next, we
Results investigated the activity of LEI-401 on NAPE-PLD in a secondary
New NAPE-PLD inhibitor chemotypes are identified by HTS. To in vitro assay to confirm an on-target effect of the inhibitor. To this
identify new inhibitors for NAPE-PLD, we initiated a HTS campaign end, we measured the NAPE-PLD enzyme activity in brain lysates of
in collaboration with the European Lead Factory (Supplementary WT and NAPE-PLD KO mice by quantifying the formation of NAE
Table 1)19. A previously reported fluorescent NAPE-PLD activity (C17:0) from a synthetic C17:0-NAPE using an LC–MS-based assay21.
assay20 using the surrogate substrate N-((6-(2,4-dinitrophenyl) LEI-401 reduced C17:0-NAE formation using brain homogenates of
amino)hexanoyl)-2-(4,4-difluoro-5,7-dimethyl-4-bora-3a,4a- WT mice, but not from KO mice (Supplementary Fig. 3), thereby con-
diaza-s-indacene-3-pentanoyl)-1-hexadecanoyl-sn-glycero-3-p firming that NAPE hydrolysis and its inhibition by LEI-401 in ex vivo
hosphoethanol-amine triethylammonium salt (PED6) was converted preparations is dependent on NAPE-PLD in mouse brain. Following
to a HTS-compatible 1,536-well format (Fig. 1a). As an enzyme this, we assessed the activity profile of LEI-401 for the receptors and
source, we used membrane fractions of human embryonic kidney metabolic enzymes of the endocannabinoid system. No inhibitory
293T (HEK293T) cells that were transiently transfected with human activities were measured at 10 μM for the cannabinoid receptors type
NAPE-PLD (Supplementary Fig. 1). This resulted in a robust assay 1 and type 2 (Supplementary Table 3) as well as for the other enzymes
with a Z’ = 0.87, a signal-to-background ratio of 6.7 and intraplate involved in anandamide biosynthesis and degradation, such as phos-
variability (coefficient of variability) of <3.3%. Approximately 350,000 pholipase A2 group 4E (PLA2G4E) and FAAH (Supplementary
compounds were screened at a single concentration (10 μM) in 294 Table 4), respectively. In addition, LEI-401 did not inhibit enzymes
plates over 3 d, affording 8,321 actives (using the cut-off value of Z involved in the biosynthesis and degradation of the other endocan-
score ≤ −7) (Fig. 1b,c). Hit validation at the same concentration yielded nabinoid 2-arachidonoylglycerol (2-AG), including diacylglycerol
3,885 confirmed actives. To reduce the number of compounds, the lipases (DAGLα or DAGLβ), monoacylglycerol lipase (MAGL) and
percentage effect on the enzyme activity (≥40%) was used to obtain α,β-hydrolase domain 6 (ABHD6) (Supplementary Table 4).
a list of 1,120 actives. Next, dose-response curves were generated in
the presence and absence of ZnSO4 (100 μM) to remove promiscuous Photoaffinity labeling shows NAPE-PLD target engagement. To
Zn2+-ion chelators, which yielded 352 hits. Visual inspection of the assess target engagement and selectivity of LEI-401 in cellular sys-
352 compounds revealed the presence of potential assay interfering tems, we turned to a photoaffinity labeling approach, which was pre-
compounds that absorbed light within the visible wavelength; there- viously successfully applied for other metallo-enzymes22. Based on
fore, a second deselection assay was developed. The substrate PED6 the structure-activity relationships of LEI-401, a photoactivatable
was incubated with lysate to obtain the maximum fluorescent signal, alkyne probe (10) was synthesized in which the 3-phenyl-piperidine
followed by incubation with different concentrations of the confirmed was replaced with a trifluoromethyl-diazirine-benzyl-piperazine as
actives to determine the ability of the compounds to quench the a photoreactive group (Fig. 3a and Supplementary Notes). In addi-
fluorescent signal under the primary assay conditions. Compounds tion, the methylcyclopropyl group was substituted for a propargyl
demonstrating no effect (half-maximal inhibitory concentration with group to serve as a ligation handle to introduce reporter groups
negative log (pIC50) < 5) in the second deselection assay were selected by copper(I)-catalyzed azide-alkyne cycloaddition (CuAAC,
(Supplementary Table 2). The compounds were checked for purity or ‘click’) chemistry to study target engagement. First, we con-
(>85%) and correct molecular weight by liquid chromatography– firmed that compound 10 inhibited NAPE-PLD with a Ki of
mass spectrometry (LC–MS) analysis. This afforded a qualified hit list 0.18 μM (95% CI, 0.11–0.27 μM) in the biochemical PED6-assay
of five compounds (2–6) with four different chemotypes (Fig. 1d). (Supplementary Fig. 4). To assess whether compound 10 was able
to visualize NAPE-PLD, HEK293T cells were transfected with
Hit optimization provides nanomolar inhibitor LEI-401. hNAPE-PLD-FLAG or control (mock) plasmid for 48 h, followed
Compound 2 (N-(cyclopropylmethyl)-2-(methyl(phenethyl)amino)- by in situ treatment with compound 10 (2 μM) for 30 min and then
6-morpholinopyrimidine-4-carbox-amide) displayed the most opti- irradiated at 350 nm for 10 min. After lysis and clicking of a Cy5-N3
mal biological and physicochemical characteristics (molecular weight fluorophore, the samples were resolved by SDS-polyacrylamide gel
668 Nature Chemical Biology | VOL 16 | June 2020 | 667–675 | www.nature.com/naturechemicalbiology
, NATUrE ChEmIcAL BIOLOgy Articles
a b
F Primary activity screen (10 µM)
N B F O 40
N
O –
O NO2
O H
C15H31 O O O N
P N 20
H
O O
NO2
PED6
quenched 0
z score
–7
H2O –20
NAPE-PLD
F
N B F O –40
N
O –
O NO2
O H
C15H31 O O OH + HO N
P N –60
H
O O
NO2 0 100,000 200,000 300,000
Fluorescent
Compound no.
c d
No. compounds
N HO O O
OH
349,525 Screening library H O
N N O N N
H
N
N N O OH
8,321 Primary actives O N
O O
2 O 3 4
3,885 Confirmed actives K i = 0.26 µM K i = 0.51 µM K i = 1.3 µM
c-logP = 3.84 c-logP = 4.63 c-logP = 3.42
1,120 ≥40% effect MW = 396 MW = 449 MW = 398
O CN
352 First deselection
O O O
5 Second deselection
O O
Qualified hit list O O
N N
O O
5 6
Ki = 1.2 µM Ki = 2.3 µM
c-logP = 6.23 c-logP = 5.75
MW = 464 MW = 431
Fig. 1 | HTS of a NAPE-PLD activity assay provided potent new inhibitors 2–6. a, Cleavage of PED6 generated a fluorescent read-out of NAPE-PLD
activity. b, Primary activity screen. c, Hit triage of the HTS campaign. d, Structures, potency data and physicochemical parameters of hit compounds
(2–6). Data represent mean values ± s.e.m. for two independent experiments with two biological replicates (n = 4). Dose-response curves are depicted in
Supplementary Fig. 2. Complete HTS data are listed in Supplementary Tables 1 and 2.
electrophoresis (SDS–PAGE) and protein labeling was visualized HEK293T cells (Fig. 3f and Supplementary Fig. 8). In summary,
by in-gel fluorescence scanning (Supplementary Fig. 5–7). An addi- the development of photoaffinity probe 10 enabled the detection
tional band at the expected molecular weight of ~46 kDa was appar- of NAPE-PLD in living cells and visualization of target engagement
ent in the hNAPE-PLD-transfected cells but not in mock-treated by LEI-401 with NAPE-PLD.
cells, at the same height as the signal of an anti-FLAG antibody
on western blot (Fig. 3b). The intensity of the fluorescent band LEI-401 lowers NAEs in WT but not in NAPE-PLD KO cells.
at 46 kDa was reduced on coincubation by the inhibitor LEI-401 Having established that LEI-401 is a cell-permeable inhibitor and
(Fig. 3b), indicating that LEI-401 bound to NAPE-PLD in living engages with NAPE-PLD, we then investigated whether LEI-401
cells. Concentration-response experiments revealed that LEI-401 inhibited endogenous NAPE-PLD in living cells. To this end, the
dose-dependently reduced the labeling of NAPE-PLD with an IC50 mouse Neuro-2a neuroblastoma cell line was selected, since it
of 0.86 μM (95% CI, 0.60–1.2 μM) (Fig. 3c,d). To establish the selec- expressed endogenous NAPE-PLD as determined by quantita-
tivity of LEI-401, a label-free chemical proteomics experiment was tive PCR (qPCR; Supplementary Table 5) and western blot using
performed23. Cell lysates prepared from hNAPE-PLD-transfected NAPE-PLD antibodies (Supplementary Fig. 9). Targeted lipidomics
HEK293T cells incubated with photoprobe 10 were clicked with on lipid extracts of Neuro-2a cells allowed the quantification of
biotin-N3, which allowed for enrichment of probe-labeled pro- eight different NAEs by LC–MS (Fig. 4a). As an appropriate nega-
teins using avidin agarose beads, followed by trypsin digestion and tive control, two NAPE-PLD KO cell lines were generated using
protein identification by mass spectrometry. Proteomic analysis CRISPR/Cas9. The cell line KO-2 showed more efficient ablation
allowed identification of 136 proteins (Fig. 3e). NAPE-PLD was of NAPE-PLD protein expression as demonstrated by western blot
enriched by probe 10 in an ultraviolet (UV)-dependent manner analysis compared to KO-1 and was therefore chosen for further anal-
and its labeling could be prevented by coincubation with LEI-401 ysis (Supplementary Fig. 9). Next, we incubated both WT and KO
(Fig. 3f). Furthermore, LEI-401 did not compete for other pro- cells with LEI-401 (10 μM) for 2 h. A significant twofold reduction
tein targets of probe 10 in this analysis or in mock-transfected of anandamide was apparent in WT cells but not in NAPE-PLD KO
Nature Chemical Biology | VOL 16 | June 2020 | 667–675 | www.nature.com/naturechemicalbiology 669
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