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Comparative Study
. 2007 Jan;150(2):186-91.
doi: 10.1038/sj.bjp.0706971. Epub 2006 Dec 4.

Lack of selectivity of URB602 for 2-oleoylglycerol compared to anandamide hydrolysis in vitro

S Vandevoorde  1 , K-O Jonsson, G Labar, E Persson, D M Lambert, C J Fowler
Affiliations
Comparative Study

Lack of selectivity of URB602 for 2-oleoylglycerol compared to anandamide hydrolysis in vitro

S Vandevoorde et al. Br J Pharmacol. 2007 Jan.

Abstract

Background and purpose: Two compounds, URB602 and URB754, have been reported in the literature to be selective inhibitors of monoacylglycerol lipase, although a recent study has questioned their ability to prevent 2-arachidonoyl hydrolysis by brain homogenates and cerebellar membranes. In the present study, the ability of these compounds to inhibit monoacylglycerol lipase and fatty acid amide hydrolase has been reinvestigated.

Experimental approach: Homogenates and cell lines were incubated with test compounds and, thereafter, with either [(3)H]-2-oleoylglycerol or [(3)H]-anandamide. Labelled reaction products were separated from substrate using chloroform: methanol extraction.

Key results: In cytosolic fractions from rat brain, URB602 and URB754 inhibited the hydrolysis of 2-oleoylglycerol with IC(50) values of 25 and 48 microM, respectively. Anandamide hydrolysis by brain membranes was not sensitive to URB754, but was inhibited by URB602 (IC(50) value 17 microM). Hydrolysis of 2-oleoylglycerol by human recombinant monoacylglycerol lipase was sensitive to URB602, but not URB754. The lack of selectivity of URB602 for 2-oleoylglycerol compared to anandamide hydrolysis was also observed for intact RBL2H3 basophilic leukaemia cells. C6 glioma expressed mRNA for monoacylglycerol lipase, and hydrolyzed 2-oleoylglycerol in a manner sensitive to inhibition by methyl arachidonoyl fluorophosphonate but not URB754 or URB597. MC3T3-E1 mouse osteoblastic cells, which did not express mRNA for monoacylglycerol lipase, hydrolyzed 2-oleoylglycerol in the presence of URB597, but the hydrolysis was less sensitive to methyl arachidonoyl fluorophosphonate than for C6 cells.

Conclusions and implications: The data demonstrate that the compounds URB602 and URB754 do not behave as selective and/or potent inhibitors of monoacylglycerol lipase.

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Figures

Figure 1
Figure 1
(a and b) Inhibition of the hydrolysis of [3H]AEA (membrane preparations), [3H]2-OG (cytosolic [‘cy'] and URB597-treated membrane [‘m'] preparations) by (a) URB 602 and (b) URB 754. Shown are means and s.e.m. (vertical lines), n=3–6, of the activity as percentage of the corresponding vehicle controls. The experiments were performed in Belgium, and the insets show the corresponding data from experiments performed in Sweden. The substrate concentrations were 2 μM and incubation times 10 min. (c) Representative PCR analyses for MAGL from C6 (upper) and RBL2H3 (lower) cells. Lane 1: 100 bp ladder, lane 2: rat MAGL mRNA (30 PCR cycles), expected PCR fragment size was 327 bp. The identity of the PCR fragment as MAGL was confirmed by sequencing, as described in Methods section. (d) Inhibition of 0.25 μM [3H]2-OG hydrolysis in intact C6 cells by URB597, URB754, URB602 and MAFP. Compounds (10 μl in ethanol or dimethyl sulphoxide (DMSO), as appropriate) were preincubated for 10 min before the addition of substrate and incubation for a further 5 min at 37°C. Corresponding data for URB597 and 0.25 μM [3H]AEA hydrolysis are also shown in the figure. (e) Semi-quantitative PCR analysis of MAGL in MC3T3-E1 cells and mouse brain. The PCR reactions were normalized with glyceraldehyde-3-phosphate dehydrogenase, and the numbers in the figure indicate the number of PCR cycles. (f) Time course of 0.25 μM [3H]2-OG hydrolysis in the absence and presence of 1 μM URB597 by the MC3T3-E1 cells. Shown are means and s.e.m., n=3–4, of the hydrolysis for wells seeded with 2.5 × 105 cells after subtraction of the observed hydrolysis for the wells alone. (g) Effects of URB754 and MAFP on the hydrolysis of 0.25 μM [3H]2-OG. Compounds were preincubated for 10 min before the addition of [3H]2-OG and incubation for a further 20 min. URB597 (1 μM) was present during the preincubation phase (means and s.e.mean are shown, n=4–8 of the activities expressed as percentage of the corresponding vehicle controls).
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