Assay and Inhibition of the Purified Catalytic Domain of Diacylglycerol Lipase Beta.

The diacylglycerol lipases (DAGLα and DAGLß) hydrolyze DAG to generate 2-arachidonoylglycerol (2-AG), the principal endocannabinoid and main precursor of arachidonic acid (AA). The DAGLs make distinct tissue specific contributions toward 2-AG and AA levels, and therefore, selective modulators for these enzymes could play crucial roles toward harnessing their therapeutic potential. Relatively high-throughput assays have recently been reported for DAGLα and have proven useful toward the characterization of inhibitors of this enzyme. Similar assays are also warranted for DAGLß which was the aim of this study. We first adapted previously reported DAGLα membrane assays (using PNPB and DiFMUO as substrates) to measure recombinant DAGLß activity in membranes. In contrast to results with DAGLα, both substrates provided a relatively limited signal window for measuring DAGLß activity, however, an improved window was obtained when employing a third commercially available substrate, EnzChek. In order to further improve on the assay parameters, we successfully purified the glutathione S-transferase (GST) tagged catalytic domain of DAGLß. Activity of the enzyme was confirmed using EnzChek as well as two DAGL inhibitors (THL and OMDM-188). The purified DAGLß catalytic domain assay described here provides the basis for a relatively clean and convenient assay with the potential to be adapted for high-throughput drug discovery efforts.

2-Linoleoylglycerol Is a Partial Agonist of the Human Cannabinoid Type 1 Receptor that Can Suppress 2-Arachidonolyglycerol and Anandamide Activity.

Introduction: The cannabinoid type 1 (CB1) receptor and cannabinoid type 2 (CB2) receptor are widely expressed in the body and anandamide (AEA) and 2-arachidonoylglycerol (2-AG) are their best characterized endogenous ligands. The diacylglycerol lipases (diacylglycerol lipase alpha and diacylglycerol lipase beta) not only synthesize essentially all the 2-AG in the body but also generate other monoacylglycerols, including 2-linoleoylglycerol (2-LG). This lipid has been proposed to modulate endocannabinoid (eCB) signaling by protecting 2-AG from hydrolysis. However, more recently, 2-LG has been reported to be a CB1 antagonist. Methods: The effect of 2-LG on the human CB1 receptor activity was evaluated in vitro using a cell-based reporter assay that couples CB1 receptor activation to the expression of the ß-lactamase enzyme. Receptor activity can then be measured by a ß-lactamase enzymatic assay. Results: When benchmarked against 2-AG, AEA, and arachidonoyl-2'-chloroethylamide (a synthetic CB1 agonist), 2-LG functions as a partial agonist at the CB1 receptor. The 2-LG response was potentiated by JZL195, a drug that inhibits the hydrolysis of monoacylglycerols. The 2-LG response was also fully inhibited by the synthetic CB1 antagonist AM251 and by the natural plant derived antagonist cannabidiol. 2-LG did not potentiate, and only blunted, the activity of 2-AG and AEA. Conclusions: These results support the hypothesis that 2-LG is a partial agonist at the human CB1 receptor and capable of modulating the activity of the established eCBs.