URB602 inhibits monoacylglycerol lipase and selectively blocks 2-arachidonoylglycerol degradation in intact brain slices

Abstract

The N-aryl carbamate URB602 (biphenyl-3-ylcarbamic acid cyclohexyl ester) is an inhibitor of monoacylglycerol lipase (MGL), a serine hydrolase involved in the biological deactivation of the endocannabinoid 2-arachidonoyl-sn-glycerol (2-AG). Here, we investigated the mechanism by which URB602 inhibits purified recombinant rat MGL by using a combination of biochemical and structure-activity relationship (SAR) approaches. We found that URB602 weakly inhibits recombinant MGL (IC(50) = 223 +/- 63 microM) through a rapid and noncompetitive mechanism. Dialysis experiments and SAR analyses suggest that URB602 acts through a partially reversible mechanism rather than by irreversible carbamoylation of MGL. Finally, URB602 (100 microM) elevates 2-AG levels in hippocampal slice cultures without affecting levels of other endocannabinoid-related substances. Thus, URB602 may provide a useful tool by which to investigate the physiological roles of 2-AG and explore the potential interest of MGL as a therapeutic target.

Figure 1. Expression of Recombinant MGL

(A) Coomassie staining (left) and Western blot (right) analyses of purified recombinant MGL expressed in E.coli. (B) Time-course of oleic acid (OA) production from 2-oleoyl-sn-glycerol (2-OG) in the presence of purified MGL (10 ng). Results are the average of two experiments, each performed in duplicate.

Figure 2. Inhibition of MGL Activity by Various Agents

(A, B) Effects of URB602 (circles, n=4), MAFP (triangles, n=3), or NAM (squares, n=3 on either purified MGL expressed in E.coli (A) or MGL expressed in HeLa cells (B). Results are expressed as mean ± SEM, each assay performed in duplicate. (C) Inhibition of MGL activity in cerebellar membranes by URB602, MAFP, or NAM. Results are expressed as mean ± SEM (n=3), each assay performed in triplicate. *p<0.05, **p<0.01, ANOVA, followed Dunnett's test; ^#p<0.05, unpaired Student's t-test.

Figure 3. Characterization of the Mechanism of MGL Inhibition by URB602

(A) Michaelis-Menten analysis of the MGL reaction in the presence of vehicle (DMSO, 1%) (circles, n=4), 0.3 mM URB602 (triangles, n=3), or 1 mM URB602 (squares, n=3). Results are expressed as mean ± SEM. (B) Time-course of the MGL reaction in the presence of 1% DMSO (circles) or 0.3 mM URB602 (triangles). Results are expressed as mean ± SEM (n=5). (C) Changes in MGL inhibition over time were replotted as percent of maximal inhibition (closed squares); also shown are changes in URB602 concentration over time, plotted as percent of initial concentration (open triangles). Results are expressed as mean ± SEM (n=3). (D) Effects of dialysis (24 h, 0-4°C) on MGL activity by URB602. Results are expressed as mean ± SEM (n=6). **p<0.01, ***p<0.001, unpaired Student's t-test.

Figure 4

Structure of MGL inhibitor SPB 01403

Figure 5. Effects of URB602 on Endocannabinoid Levels in Rat Brain Slices

Effects of URB602 (100 μM) or vehicle (0.1% DMSO in DMEM) on (A) 2-AG levels; (B) anandamide levels, and (C) PEA levels in rat organotypic hippocampal slices in culture. Results are expressed as mean ± SEM (n=4-5). **p<0.01, ns = not significant, unpaired Student's t-test.