1-, 2- and 3-AG as substrates of the endocannabinoid enzymes and endogenous ligands of the cannabinoid receptor 1.

2-Arachidonoylglycerol (2-AG) is the most potent and abundant endocannabinoid that acts as a full agonist at the cannabinoid 1 (CB1) and 2 (CB2) receptors. It serves as a substrate for several serine hydrolases, including monoacylglycerol lipase (MGL), α/β hydrolase domain 6 (ABHD6) and fatty acid amide hydrolase (FAAH). However, 2-AG's rapid conversion to 1-AG (the S stereoisomer) and 3-AG (the R stereoisomer) complicates in vivo signaling. Here, we present the interaction profiles of 2-AG and its isomerization products, 1- and 3-AG, with the endocannabinoid MGL, ABHD6 and FAAH enzymes as well as the CB1 receptor. The 1- and 3-AG enantiomers are less prone to isomerization, and their affinities to endocannabinoid enzymes and potencies at CB1 receptor are quite different compared to 2-AG. Although MGL is the principal hydrolytic enzyme of 2-AG, 3-AG (the R isomer) appears to be the best substrate for hMGL. Contrarily, 1-AG (the S isomer) demonstrates the worst substrate profile, indicating that the stereochemistry of 1(3)-monoacylglycerols is very important for MGL enzyme. On the other hand, both 1- and 3-AG (the sn1 monoacylglycerols) are efficiently hydrolyzed by hABHD6 without preference, while 2-AG (the sn2 monoacylglycerol) has the lowest rate of hydrolysis. FAAH, the principal hydrolytic enzyme for arachidonoylethanolamide (anandamide, AEA), catalyzes the hydrolysis of all three isomers with similar efficiencies. In a functional cAMP assay at CB1 receptor, all three isomers behaved as agonists, with 2-AG being the most potent, followed by 3-AG then 1-AG. The presented data provides stereochemical insights to design chemically stable AG analogs with preferential stability against enzymes of interest.