Investigating DAGLβ expression and activity in human monocytes via proteomic and lipidomic profiling

Abstract

Abstract Lipid-metabolizing enzymes are important targets for small-molecule inhibitor discovery as the regulation of inflammation responses have been linked to lipid metabolism cascades controlled by these enzymes. Non-steroidal anti-inflammatory drugs inhibit enzymes in the COX pathway preventing conversion of arachidonic acid (AA) into inflammation-inducing prostaglandins. An upstream precursor to AA is the endocannabinoid 2-arachidonoyl-glycerol (2-AG), which is biosynthesized from the cleavage of arachidonate-containing diacylglycerols (DAGs) by diacylglycerol lipase beta (DAGLβ). Previous work to identify DAGLβ’s role in inflammatory function led to the creation of KT109, a DAGLβ-specific 1,2,3-triazole urea inhibitor. Primary mouse macrophages treated with KT109 showed reductions in AA and 2-AG and an increase in arachidonate-containing DAGs, demonstrating that DAGLβ is key in proinflammatory lipid regulation and signaling. These studies focused on the effects of inhibiting mouse DAGLβ, but information regarding KT109 inhibition of human DAGLβ is still lacking. Herein, we applied activity-based protein profiling (ABPP) and quantitative proteomic and lipidomic methods to determine the effectivity of KT109 inhibition in human monocytic cell line THP1. Potency (IC 50values) for DAGLβ inhibition by KT109 in human THP1 cells were comparable to the magnitude of inhibition in mouse cells. Lipidomic analysis of KT109-treated monocytic and macrophage-differentiated THP1s did not demonstrate an accumulation of arachidonate-containing DAGs. These findings show a potential difference in the expression and activity of human DAGLβ when compared to mouse DAGLβ and additional targets of KT109 in human cells. Funding and disclosures: This work was supported by the National Institute on Drug Abuse grant no. R01 DA043571.