Assay and inhibition of diacylglycerol lipase activity

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.

Graphical abstractNew N-formyl-α-amino acid analogs of tetrahydrolipstatin (THL) were synthesized that inhibit human and murine diacylglycerol lipases. New reporter compounds were developed to screen inhibitors of 1,2-diacyl-sn-glycerol hydrolysis activity using fluorescence resonance energy transfer (FRET). Diacylglycerol lipase (DAGL) activities were quantified using [1″-14C]1-stearoyl-2-arachidonoyl-sn-glycerol substrate in a thin layer chromatography (TLC) assay using phosphorimaging analysis.Figure optionsDownload full-size imageDownload as PowerPoint slide