Volume 20, Issue 3, Pages 379-390 (March 2013) Piperazine and Piperidine Triazole Ureas as Ultrapotent and Highly Selective Inhibitors of Monoacylglycerol Lipase  Niina Aaltonen, Juha R. Savinainen, Casandra Riera Ribas, Jani Rönkkö, Anne Kuusisto, Jani Korhonen, Dina Navia-Paldanius, Jukka Häyrinen, Piia Takabe, Heikki Käsnänen, Tatu Pantsar, Tuomo Laitinen, Marko Lehtonen, Sanna Pasonen-Seppänen, Antti Poso, Tapio Nevalainen, Jarmo T. Laitinen  Chemistry & Biology  Volume 20, Issue 3, Pages 379-390 (March 2013) DOI: 10.1016/j.chembiol.2013.01.012 Copyright © 2013 Elsevier Ltd Terms and Conditions

Figure 1 Comparative In Vitro Evaluation of the 2-AG Hydrolase Inhibitors MAFP, JZL184, and NAM (A) Dose-response curves for MAFP, NAM, and JZL184 to inhibit 2-AG hydrolysis in rat cerebellar (Rcm) and mouse whole brain (Mbm) membranes. Membranes were pretreated for 30 min with DMSO (control) or with the indicated concentrations of the inhibitors. Glycerol liberated from 2-AG hydrolysis was determined as described in Experimental Procedures. The data points as well as the logIC50 values shown in the box are means ± SEM from three independent experiments. IC50 values (mean) are shown in parenthesis. Note that, in contrast to MAFP, which comprehensively blocks 2-AG hydrolysis, ∼20% residual activity remains after maximally effective concentrations of the MAGL-selective inhibitors NAM and JZL184 (dashed horizontal line). (B) Functional autoradiography visualizing endocannabinoid-dependent, CB1R-mediated Gi/o protein activity. Basal represents G protein activity in the absence of added agonist. Pretreatment with MAFP results in endogenous 2-AG accumulation and subsequent CB1R activity in brain regions, such as the caudate putamen (CPu), the cerebral cortex (Cx), the hippocampus (Hip), and the molecular layer of the cerebellum (Cbm). For comparative purposes, CB1R response to the synthetic cannabinoid agonist CP55,940 is shown. Note that, in contrast to MAFP, the MAGL-selective inhibitors NAM and JZL184 do not generate the CB1R signal. Similarly, the broad spectrum lipase inhibitor THL (orlistat) or the ABHD6-selective inhibitor WWL70 do not evoke the MAFP-mimicking response. Scale bar, 2 mm. The data are representative images from at least three independent experiments. Three images in (B) (Basal, MAFP 10−6 M and CP55,940 5 × 10−6 M) were previously presented in a review article illustrating CB1R activity evoked by endogenous and exogenous cannabinoid agonists (Savinainen et al., 2012). Chemistry & Biology 2013 20, 379-390DOI: (10.1016/j.chembiol.2013.01.012) Copyright © 2013 Elsevier Ltd Terms and Conditions

Figure 2 The MAGL Inhibitors JJKK-046 and JJKK-048 Activate CB1Rs Indirectly through Elevating Brain 2-AG Levels (A) The inhibitors evoke MAFP-mimicking and AM251-sensitive, [35S]GTPγS-binding responses in functional autoradiography. CB1R-dependent G protein activity is evident in various brain regions, including the CPu, the Cx, the Hip, and the molecular layer of the Cbm. For comparative purposes, response to the synthetic cannabinoid agonist CP55,940 is also shown. Scale bar, 2 mm. The experiment was repeated twice with similar outcome. (B) JJKK-048 selectively increases 2-AG levels in rat brain sections. Sections were treated for 1 hr with the indicated concentrations of the inhibitors, washed, and incubated thereafter for 90 min in assay buffer. The buffer was removed and tissue endocannabinoid content determined with LC-MS/MS, as detailed in Experimental Procedures. For reference purposes, the global serine hydrolase inhibitor, MAFP, and the FAAH-selective inhibitor, PF-750, were included. Values are mean + SEM from triplicate slides with two horizontal brain sections in each. Statistical comparison was performed using one-way Anova followed by Tukey’s multiple comparisons. Significance level ∗∗∗p < 0.001. See also Figures S2 and S4, and Table S1. Chemistry & Biology 2013 20, 379-390DOI: (10.1016/j.chembiol.2013.01.012) Copyright © 2013 Elsevier Ltd Terms and Conditions

Figure 3 JJKK-048 Potently Inhibits MAGL in Intact Human Cells (A) Treatment for 1 hr with JJKK-048 inhibits MAGL activity in intact HEK293 cells overexpressing hMAGL. For comparative purposes, the response to JZL184 (10−6 M) is shown. (B) ABPP of serine hydrolases in membranes and lysates of human melanoma cell lines using the TAMRA-FP probe reveals highest MAGL expression in C8161 cells, evident both in membranes and lysates (squares). Treatment for 1 hr with JJKK-048 (10−7 M) selectively blocks TAMRA-FP labeling of both the ∼33 and ∼35 kDa forms of MAGL, with no additional targets evident in any of the cell lines. To facilitate localization of MAGL, mouse whole brain membranes (MBM) were run in the same gel. (C) Treatment of intact C8161 melanoma cells for 1 hr with JJKK-048 or JZL184 at the indicated doses significantly inhibited 2-AG hydrolase activity, as assessed from lysates of inhibitor-treated cells. Values are means ± SD of duplicate determinations from one experiment that was repeated twice with similar outcome. See also Figure S6 and Supplemental Discussion. Chemistry & Biology 2013 20, 379-390DOI: (10.1016/j.chembiol.2013.01.012) Copyright © 2013 Elsevier Ltd Terms and Conditions