MAGLi 432 blocks MAGL in human and mouse brain lysates in a selective and potent manner.

(A) Chemical structure of MAGLi 432 with an IC50 = 4.2 nM in human MAGL as determined by enzymatic assays. (B) X-ray co-crystal structure of MAGLi 432 (cyan) with human MAGL (blue). View of the active site of human MAGL (blue) reveals a non-covalent binding of the inhibitor. Interactions of MAGLi 432 with the catalytic Ser122 are mediated through the amide carbonyl group via a water molecule located in the oxyanion hole. Hydrogen bonds are depicted as yellow dashed lines. Selective MAGL inhibition by MAGLi 432 in human (C-E) and mouse (F-H) brain lysates was determined by competitive activity based protein profiling (ABPP). Brain lysates were incubated with DMSO, 10 μM MAGLi 432 or 10 μM JZL 184 and then with either a broad serine hydrolase activity-based probe (TAMRA-FP, green) or the MAGL-specific probe (red) before samples were loaded on a gel. Green fluorescent bands reveal serine hydrolases not inhibited by MAGLi 432. Red fluorescent bands reveal active MAGL not blocked by MAGLi 432. The normalized signal intensity was quantified as the average signal intensity for each MAGL fluorescent band (green or red) divided by the total protein signal intensity (Coomassie Brilliant Blue staining, S1 Fig) in human (D, E) and mouse (G, H) brain lysates (n = 2). (I-L) Assessment of MAGL potency was measured by competitive ABPP after incubation with ascending doses of MAGLi 432 in human (I) and mouse brain lysates (J) followed by incubation with the MAGL-specific probe. (K, L) The average signal intensities of active MAGL and total MAGL protein in human (K) and mouse (L) brain lysates were quantified as the total detectable active MAGL signal (ABPP) divided by the total the MAGL protein/β-actin signal (n = 2).