A monoacylglycerol lipase inhibitor showing therapeutic efficacy in mice without central side effects or dependence

Abstract

Monoacylglycerol lipase (MAGL) regulates endocannabinoid 2-arachidonoylglycerol (2-AG) and eicosanoid signalling. MAGL inhibition provides therapeutic opportunities but clinical potential is limited by central nervous system (CNS)-mediated side effects. Here, we report the discovery of LEI-515, a peripherally restricted, reversible MAGL inhibitor, using high throughput screening and a medicinal chemistry programme. LEI-515 increased 2-AG levels in peripheral organs, but not mouse brain. LEI-515 attenuated liver necrosis, oxidative stress and inflammation in a CCl₄-induced acute liver injury model. LEI-515 suppressed chemotherapy-induced neuropathic nociception in mice without inducing cardinal signs of CB₁ activation. Antinociceptive efficacy of LEI-515 was blocked by CB₂, but not CB₁, antagonists. The CB₁ antagonist rimonabant precipitated signs of physical dependence in mice treated chronically with a global MAGL inhibitor (JZL184), and an orthosteric cannabinoid agonist (WIN55,212-2), but not with LEI-515. Our data support targeting peripheral MAGL as a promising therapeutic strategy for developing safe and effective anti-inflammatory and analgesic agents.

Fig. 1. Identification of reversible MAGL inhibitors in a high-throughput screen guided by activity-based protein profiling.

a MAGL activity assay, employing an enzyme cascade reaction to couple 2-AG hydrolysis to the generation of a fluorescent signal. b HTS triage process. Criteria and cut-offs at each step are indicated. c Results from the primary screen, expressed as z-score per individual compound. Compounds showing ≥50% inhibition (z-score ≤ −4.95) are shown in red. d Hit validation and FAAH selectivity assessment based on orthogonal ABPP assay. e Structure and competitive ABPP labelling profile of compound 1 on mouse brain proteome labelled by FP-TAMRA (n = 1). f Correlation between primary 2-AG hydrolysis activity assay and orthogonal competitive ABPP assay. Corresponding ABPP gels are shown in Supplementary Fig. 1. Complete HTS data are listed in Supplementary Tables 1 and 2.

Fig. 2. Discovery of LEI-515 and its binding pose in human MAGL.

a Hit optimization of compound 1 led to the discovery of LEI-515. Substitution of the ester moiety for CF₂-ketone moiety significantly improved the metabolic stability. b Proposed transition state of LEI-515 bound to MAGL. The catalytic Ser122 of MAGL covalently interacts with the ketone group of LEI-515. c MAGL in vitro activity assay dose-response curves for Hit 1 (pIC₅₀ = 6.4 ± 0.1), 2 (pIC₅ = 8.5 ± 0.1) and LEI-515 (pIC₅₀ = 9.3 ± 0.1). Data represented as mean values (N = 2, n = 2). d and e The X-ray structure of (2S,3S)-isomer of LEI-515 bound to hMAGL. LEI-515 binds to MAGL through a covalently reversible mechanism and the deprotonated hemiketal forms two hydrogen bounds (yellow dotted line) with Ala51 and Met123. Source data are provided as a Source Data file.

Fig. 3. LEI-515 is a reversible MAGL inhibitor and active in cells.

a LEI-515 increases 2-AG concentration in mouse (pIC₅₀ = 6.6) and human brain lysates (pIC₅₀ = 7.0). b Time-dependent recovery of MAGL activity in mouse brain proteome of LEI-515 and ABX-1431 as determined by competitive ABPP using a MAGL selective probe LEI-463-Cy5 (1 µM). Data represented as the fold change in MAGL labelling of the two isoforms as compared to the 5 min chase time. n = 3 biologically independent samples. c Dose-dependent gel-based ABPP profile of LEI-515 in mouse brain membrane proteome using broad spectrum serine hydrolase probes FP-TAMRA (100 nM) and MB064 (250 nM). C = DMSO control, ABX = ABX-1431 (n = 2). d Chemical proteomics-based selectivity profiles of LEI-515 (1 µM, 30 min, 37 °C) on mouse lung proteome using a probe cocktail of MB108 and FP-Biotin (2.5 and 5 μM, respectively, 30 min, 37 °C). Data represented as a volcano plot with cut-off values: unique peptides ≥2; −1 ≤ log₂(fold change) ≥ 1; p < 0.05 using multiple unpaired t-test with FDR 5% (n = 4). e Dose-dependent cellular target engagement of LEI-515 (pEC₅₀ 6.78 ± 0.11) in U-87 MG live cells using LEI-463-Cy5 (10 nM). n = 3 biologically independent samples. f In situ treatment of HS578t cells with LEI-515 (1 μM) time-dependently increased cellular 2-AG levels. n = 5 biologically independent samples. g LEI-515 (1 h) dose-dependently increased cellular 2-AG levels. n = 4 biologically independent samples. h and i In situ treatment of HS578t cells with LEI-515 (1 μM, 1 h) decreased cellular arachidonic acid (AA, p = 0.0011) (h) and anandamide (p = 0.0029) (AEA, i) levels. n = 4 biologically independent samples. Statistical analysis: one-way ANOVA with Dunnett’s post-hoc test (***p < 0.001, **p < 0.01, *p < 0.05 vs. vehicle). All data are represented as mean values ± SEM. Source data are provided as a Source Data file.

Fig. 4. LEI-515 is an orally active, peripherally restricted MAGL inhibitor.

a In vivo pharmacokinetics of LEI-515 in plasma of C57BL/6J mice via intravenous (i.v., 10 mg/kg) or oral (p.o., 10 mg/kg) administration (n = 3). b–d Lipid levels of b 2-AG (colon 100 mg/kg, p = 0.0003), c AA and d anandamide (AEA) in C57BL/6J mice brain and colon after oral administration (30 or 100 mg/kg) of LEI-515. Statistical analysis: Unpaired two-tailed t-test (***p < 0.001, **p < 0.01, *p < 0.05 vs. vehicle; n = 6). Data are presented as mean ± SEM. Source data are provided as a Source Data file.

Fig. 5. LEI-515 reduces acute liver injury.

a Effect of acute CCl₄ treatment with vehicle (n = 16) or LEI-515 (10 and 30 mg/kg i.p., n = 8 and n = 16/group, respectively) on liver transaminases [alanine aminotransferase (ALT, p < 0.0001) and aspartate aminotransferase (AST, p = 0.0084 and <0.0001, respectively)], b and c histopathological liver necrosis and its quantification (n = 15 in CCl₄+veh and n = 14 in CCl₄+LEI 30 mg/kg groups, respectively; p < 0.0001), d and e CD45+ leucocyte infiltration and its quantification (n = 15 in CCl₄+veh and n = 14 in CCl₄+LEI 30 mg/kg groups, respectively; p = 0.0002), f and g lipid peroxidation (4-HNE) 24 h following CCl₄ administration and its quantification (n = 12/group, p = 0.0004). All data are presented as mean ± SEM. Open symbols represent females, whereas closed symbols show male subjects. Statistical analysis: One-way ANOVA and Dunnett post-hoc test (a), unpaired two-tailed t-test (c, e, g) (****p < 0.0001, ***p < 0.001, **p < 0.01 vs. vehicle; n = 8–16/group). The drug had a comparable effect in both male and female mice. Source data are provided as a Source Data file.

Fig. 6. Acute and chronic dosing with peripherally restricted MAGL inhibitor LEI-515, administered p.o. or i.p., suppresses paclitaxel-induced mechanical hypersensitivity in mice of both sexes.

a LEI-515 (0.1, 0.3, 1, 3, 10 and 30 mg/kg p.o.) increases percent maximal antinociceptive effect (%MPE) relative to pre-paclitaxel baseline in male (n = 7) and female (n = 8) mice. ED₅₀ (95% confidence intervals): 1.01 (0.31–3.30) mg/kg and 1.41 (0.30–6.63) mg/kg p.o. in male and female mice, respectively. b Acute LEI-515 (10 mg/kg p.o.) suppressed paclitaxel-induced mechanical hypersensitivity in male and female mice with efficacy lasting over 24 h. ****p < 0.0001 LEI-515 vs. vehicle control groups. c Chronic dosing with LEI-515 (10 mg/kg/day p.o. for 10 days) suppressed paclitaxel-induced hypersensitivity without loss of efficacy in both sexes. ****p < 0.0001 LEI-515 vs. vehicle groups, male LEI-515. d LEI-515 (0.1, 0.3, 1, 3, 10 and 30 mg/kg i.p.) increases %MPE relative to pre-paclitaxel baseline in male and female mice. ED₅₀ (95% confidence intervals): 1.09 (0.26–4.61) and 0.20 (0.02–2.52) mg/kg i.p. in male and female mice, respectively. n = 8 per group. e Acute LEI-515 (10 mg/kg i.p.) suppressed paclitaxel-induced mechanical hypersensitivity in male and female mice with efficacy lasting over 24 h. n = 7–8 per group. f Chronic dosing with LEI-515 (10 mg/kg/day i.p. for 10 days) suppresses paclitaxel-induced hypersensitivity without loss of efficacy in both sexes. ****p < 0.0001 LEI-515 vs. vehicle control (Three-way ANOVA, Tukey post hoc test). LEI-515 trended to increase paw withdrawal thresholds to a greater extent in paclitaxel-treated males than females (p = 0.0569; Tukey post hoc test). No main effect of sex (p = 0.206) or other interactions (Sex × Time: p = 0.2165: Time × Treatment: p = 0.8939; Sex × Treatment: p = 0.3299) were detected. Thresholds were higher in LEI-515-treated males compared to female mice on day 1 of chronic dosing only (+p = 0.0287, Sidak’s post hoc test) (see Supplementary Fig. 7d). Efficacy was preserved at least 4 days following termination of repeated i.p. dosing; ****p < 0.0001 LEI-515 vs. vehicle control. b, c, e, f n = 7–8 per group. All data are represented as mean values ± SEM. Statistical analysis: See Supplementary Tables S8 and S9 for Three-way and Two-way ANOVA results. Source data are provided as a Source Data file.

Fig. 7. The peripherally restricted MAGL inhibitor LEI-515 suppresses mechanical hypersensitivity in male mice through a peripheral CB₂ mechanism.

a LEI-515 (p.o.) suppressed paclitaxel-induced mechanical hypersensitivity in a manner that was blocked equivalently by pretreatment with CNS penetrant (AM630; 5 mg/kg i.p.) and peripherally restricted (SR144528; 2.1 mg/kg i.p.) CB₂ antagonists across the observation interval (p < 0.0001 for all comparisons). Treatment effects were also time dependent ⁺⁺p < 0.01, LEI-515 vs. all other groups; ***p < 0.001, **p < 0.01 vs. vehicle; #p<0.05 vs. SR144528, LEI-515. n = 6 male mice per group. b LEI-515 (p.o.)-induced suppression of paclitaxel-induced mechanical hypersensitivity was not blocked (p = 0.98 vs. AM251, LEI-515; p = 0.2387 vs. AM6545, LEI-515) by pretreatment with either the CNS penetrant (AM251; 5 mg/kg i.p.) or peripherally restricted (AM6545; 10 mg/kg i.p.) CB₁ antagonists throughout the observation interval. Antinociceptive effects of LEI-515 were also time-dependent ****p < 0.0001, ***p < 0.001, **p < 0.01 LEI-515 vs. vehicle. n = 6 male mice per group. c Neither LEI-515 (10 mg/kg p.o.) nor vehicle (i.p.) pretreatment altered mechanical paw withdrawal thresholds in mice receiving cremophor-based vehicles in lieu of paclitaxel (p = 0.4948). Two-way ANOVA with Sidak’s multiple comparison test. n = 6 male mice per group. All data are represented as mean values ± SEM. Statistical analysis: Two-way ANOVA, with Dunnett multiple comparison tests comparing LEI-515 to all other groups. Source data and statistical analyses are provided as a Source Data file.

Fig. 8. The peripherally restricted MAGL inhibitor LEI-515 does not induce CB₁-dependent centrally mediated side effects or physical dependence.

a–c WIN55,212-2 (3 mg/kg i.p.) a increased immobility time in the ring test, b decreased body temperature and c increased tail-flick antinociception whereas LEI-515 (10 mg/kg i.p.) was inactive in all assays (p > 0.8879 vs. vehicle) ⁺⁺p < 0.01 vs. all other groups; **p < 0.01 vs. vehicle; ^##p < 0.01, ^#p < 0.05 vs. LEI-515; ^$p < 0.05 vs. JZL184. JZL184 (n = 7 male mice), all other groups (n = 8 male mice). d–g Rimonabant (10 mg/kg i.p.) precipitated signs of physical dependence including d paw tremors, e head twitches, f withdrawal jumps, and g decreased scratching bouts in mice treated chronically with the global MAGL inhibitor JZL184 (16 mg/kg/day i.p. × 20 days) or the orthosteric cannabinoid agonist WIN55,212-2 (3 mg/kg/day i.p. × 20 days) but not in mice treated chronically with LEI-515 (10 mg/kg/day i.p. × 20 days) (p>0.6554 vs. vehicle for all assays). ⁺⁺p < 0.01 vs. all groups; **p < 0.01, *p < 0.05 vs. vehicle; ^##p < 0.01, ^#p < 0.05 vs. LEI-515. JZL184 (n = 7 male mice), all other groups (n = 8 male mice). All data are represented as mean values ± SEM. Statistical analysis: Two-way ANOVA with Tukey’s multiple comparison test (a–c) or one-way ANOVA with Tukey’s multiple comparison test (d–g). Source data and statistical analyses are provided as a Source Data file.