Assay and inhibition of diacylglycerol lipase activity

Abstract

A series of N-formyl-α-amino acid esters of β-lactone derivatives structurally related to tetrahydrolipstatin (THL) and O-3841 were synthesized that inhibit human and murine diacylglycerol lipase (DAGL) activities. New ether lipid reporter compounds were developed for an in vitro assay to efficiently screen inhibitors of 1,2-diacyl-sn-glycerol hydrolysis and related lipase activities using fluorescence resonance energy transfer (FRET). A standardized thin layer chromatography (TLC) radioassay of diacylglycerol lipase activity utilizing the labeled endogenous substrate [1″-14C]1-stearoyl-2-arachidonoyl-sn-glycerol with phosphorimaging detection was used to quantify inhibition by following formation of the initial product [1″-14C]2-arachidonoylglycerol and further hydrolysis under the assay conditions to [1-14C]arachidonic acid.

A novel live cell assay to measure diacylglycerol lipase α activity

“…These include low-throughput MS based native substrate assays that employ synthetic DAG as a substrate coupled with the measurement of 2-AG as the product [ 40 , 41 ]. Low-throughput assays that utilize radiolabelled substrates or activity based probes have also been described [ 2 , 13 , 29 – 31 , 41 ]. Previously, relatively higher throughput surrogate substrate (fluorogenic or chromogenic), FRET (using ether lipid reporters) and native substrate (coupled enzyme) assays of particular utility for drug discovery programmes have been reported [ 26 , 28 , 29 ].…”

“…In general though, PNPB is more suitable for measuring DAGLα activity in assays employing overexpressing cell lines as described here where the activity of the recombinant enzyme is several fold higher than the endogenous and/or background activities. Although the use of whole cells (or membranes as opposed to purified protein) preclude K cat calculations, as is the case with other published DAGL assays [ 2 , 13 , 26 , 28 , 29 ], the assay reported here is continuous and employs a chromogenic substrate and so can be used to monitor inhibition kinetics (time course; different inhibitor and substrate concentrations) in a cellular environment [ 26 ]. Improving the signal window of the assay would be beneficial for such studies and would entail three approaches–(1) Increasing DAGLα-V5 activity which could be achieved by generating cell lines expressing higher amounts of DAGLα-V5.…”

Assay of DAGLα/β Activity

“…Since 2-AG biosynthetic enzymes have been identifi ed only recently, little information on methodological approaches for measuring DAGL activities is as yet available. Several efforts have been made to fi ll this gap, culminating in the development of different analytical methodologies that include TLC [ 1 , 7 -9 ], liquid chromatography-mass spectrometry [ 10 ], fl uorescence resonance energy transfer [ 11 ], activitybased protein profi ling [ 12 ], and fl uorescence spectroscopy [ 13 ]. Here, the protocol for assaying DAGL activity by means of the highly sensitive, standardized TLC radioassay that uses labeled 1-oleoyl [1][2][3][4][5][6][7][8][9][10][11][12][13][14] C]-2-arachidonoylglycerol as a substrate for both DAGLα and DAGLβ is presented (Fig.…”

Design and Synthesis of Quenched Activity‐based Probes for Diacylglycerol Lipase and α,β‐Hydrolase Domain Containing Protein 6

“…[8,23] Both ABPs contain at riazole urea as an electrophilict rap, which is ac ommonly used warhead for serine hydrolases (Figure 1a). [12,24] BODIPY-FL [25] and 2,4-DNA [26][27][28][29][30] were chosen as af luorophore-quencher pair for probe 1, [19,31] while Cy5 and cAB40 were selected as af luorophore-quencher pair for qABP 2. [32] Synthesis of probe 1.…”