Two key enzymes implicated in the biosynthesis of 2-AG are diacylglycerol lipase (DAGL)-α and β.² DAGL-α and β are both integral membrane proteins with four predicted transmembrane domains followed by a catalytic domain that conforms to the general sequence requirements for a serine hydrolase (including the presence of the canonical GXSXG active site motif).

Multiple lines of evidence suggest that DAGL-α/β play a role in regulating 2-AG biosynthesis in neurons. For instance, overexpression of DAGL-α in the mouse neuroblastoma cell line Neuro-2a results in a significant increase in basal 2-AG levels.³ Conversely, RNA interference-mediated knockdown of DAGL-α in Neuro-2a cells reduced basal levels of 2-AG and blocked the production of this endocannabinoid stimulated by agonists of group 1 metabotropic glutamate receptors. ³

DAGL-α/β are inhibited by two small-molecule agents, RHC80267 and tetrahydrolipstatin (THL)^2,4 (Figure 1), which have been used to block many of the CB1-dependent forms of neuronal plasticity observed in in vitro preparations.⁵ These findings have invoked 2-AG as the principle endocannabinoid involved in retrograde signaling in the nervous system. However, this conclusion is predicated on the assumption that RHC80267 and THL selectively target DAGL-α/β in the nervous system, a hypothesis that, to date, has gone largely untested. These agents likely inhibit DAGL-α/β by covalent reaction with the enzymes' serine nucleophile (forming carbamoylated and esterified products, respectively), which suggests that the compounds might target additional serine hydrolases in the nervous system via a similar mechanism. This is certainly the case in peripheral tissues, where, for example, THL has found clinical utility as an anti-obesity agent due to blockade of pancreatic lipases in the intestine.⁶

To more globally assess the selectivity of RHC80267 and THL, we analyzed these inhibitors by competitive activity-based protein profiling (ABPP).⁷ ABPP is a chemical proteomic method that utilizes active site-directed small-molecule probes to assess the functional state of numerous enzymes in parallel directly in native biological systems. In competitive ABPP, inhibitors are evaluated for their ability to impair probe labeling of target enzymes.⁸ Because ABPP probes typically label many members from a given enzyme class, competitive profiling experiments provide an excellent assessment of both the potency and selectivity of inhibitors. Inhibitors of DAGL-α/β are most commonly used in nervous system preparations;^3,5 we therefore elected to profile these agents against a mouse brain proteome using fluorophosphonate (FP) ABPP probes, which broadly target enzymes from the serine hydrolase.⁹ For initial comparison, we also analyzed two lipid-based FP inhibitors, O-3841 and MAFP (Figure 1), which have also been shown to inhibit DAGL-α/β.⁴ The mouse brain membrane proteome was treated with each inhibitor across a broad concentration range (0.01- 100 μM) for 30 min, after which reactions were incubated with a rhodamine-tagged FP probe (FP-Rh) for 60 min, separated by SDS-PAGE, and analyzed by in-gel fluorescence scanning.

O-3841 and MAFP were found to inhibit probe labeling of numerous serine hydrolase activities, including fatty acid amide hydrolase (FAAH), KIAA1363, monoacylglycerol lipase (MAGL), ABHD6, and ABHD12 (Figure 2A). Most of these enzymes with the exception of KIAA1363 were more potently inhibited by MAFP than O-3841. In contrast, RHC80267 and THL showed more selective patterns of enzyme inhibition, blocking the labeling of three and two hydrolase activities, respectively (Figure 2B). None of these enzymes correspond to DAGL-α/β, large enzymes (> 70 kDa) that appear to be poorly labeled by FP-probes.¹⁰

To identify the targets of RHC80267 and THL, mouse brain proteome was treated with each inhibitor (50 and 5 μM, respectively) or DMSO (control) for 30 min and then incubated with a biotinylated FP-probe (FP-biotin, 5 μM) for 120 min. Probe-labeled proteins were enriched and characterized by a combination of avidin affinity and liquid chromatography-mass spectrometry steps, following previously described procedures.¹¹ All samples were analyzed in triplicate. This approach, termed ABPP-MudPIT, identified 35 brain serine hydrolases at sufficiently high spectral counts (> 15 average counts in control proteomes) for comparative quantitation between inhibitor- and DMSO-treated proteomes (Figure 3A).

Candidate targets of RHC80267 and THL were defined as serine hydrolases that showed greater than 60% reductions in average spectral counts in inhibitor-treated versus DMSO-treated proteomes. This criterion identified six and three putative targets of RHC80267 and THL, respectively (Figure 3B). These hydrolases included enzymes implicated in endocannabinoid metabolism, such as FAAH¹² and ABHD12,¹³ enzymes that regulate other signaling molecules, such as acetylcholine [acetylcholinesterase (AChE)¹⁴], platelet-activating factor (PLA2g7¹⁵) and lysophosphatidic acid (KIAA1363¹⁶), as well as uncharacterized enzymes (BAT5). Comparison of these results to the gel-based profiles shown in Figure 2 led to the tentative assignment of the 60 kDa hydrolase sensitive to both RHC80267 and THL as BAT5. Additional hydrolases detected by LC-MS analysis (e.g., PLA2g7, AChE) were not visible by gel analysis, underscoring the enhanced sensitivity of the former analytical method. Neither DAGL-α nor β were detected by ABPP-MudPIT, indicating that these hydrolase are either very low abundance in mouse brain and/or weakly labeled by FP probes.¹⁰

We next set out to confirm a subset of the serine hydrolase targets of RHC80267 and THL. HEK293T cells were transfected with cDNAs encoding representative targets of RHC80267 (KIAA1363, FAAH, BAT5, PLA2g7) and THL (ABHD12, BAT5, PLA2g7), and transfected proteomes were then treated with RHC80267 and THL (0.01-100 μM), followed by FP-Rh, which permitted calculation of IC₅₀ values for inhibition of each enzyme (Figure 4). ABHD12, BAT5, and PLA2g7 were inhibited by THL with high potencies (IC₅₀ values < 100 nM) that rivaled the reported potency for THL inhibition of DAGL-α/β (IC₅₀ value of 60 nM⁴). Targets of RHC80267 were inhibited with much lower potency (IC₅₀ values between 10-70 μM), as has also been observed with DAGL-α/β, for which treatment with 30-100 μM of RHC80267 is typically used for inhibition.^2,5