Juha R. Savinainen

The serine hydrolases MAGL, ABHD6 and ABHD12 as guardians of 2-arachidonoylglycerol signalling through cannabinoid receptors

The endocannabinoid 2‐arachidonoylglycerol (2‐AG) is a lipid mediator involved in various physiological processes. In response to neural activity, 2‐AG is synthesized post‐synaptically, then activates pre‐synaptic cannabinoid CB1 receptors (CB1Rs) in a retrograde manner, resulting in transient and long‐lasting reduction of neurotransmitter release. The signalling competence of 2‐AG is tightly regulated by the balanced action between ‘on demand’ biosynthesis and degradation. We review recent research on monoacylglycerol lipase (MAGL), ABHD6 and ABHD12, three serine hydrolases that together account for approx. 99% of brain 2‐AG hydrolase activity. MAGL is responsible for approx. 85% of 2‐AG hydrolysis and colocalizes with CB1R in axon terminals. It is therefore ideally positioned to terminate 2‐AG‐CB1R signalling regardless of the source of this endocannabinoid. Its acute pharmacological inhibition leads to 2‐AG accumulation and CB1R‐mediated behavioural responses. Chronic MAGL inactivation results in 2‐AG overload, desensitization of CB1R signalling and behavioural tolerance. ABHD6 accounts for approx. 4% of brain 2‐AG hydrolase activity but in neurones it rivals MAGL in efficacy. Neuronal ABHD6 resides post‐synaptically, often juxtaposed with CB1Rs, and its acute inhibition leads to activity‐dependent accumulation of 2‐AG. In cortical slices, selective ABHD6 blockade facilitates CB1R‐dependent long‐term synaptic depression. ABHD6 is therefore positioned to guard intracellular pools of 2‐AG at the site of generation. ABHD12 is highly expressed in microglia and accounts for approx. 9% of total brain 2‐AG hydrolysis. Mutations in ABHD12 gene are causally linked to a neurodegenerative disease called PHARC. Whether ABHD12 qualifies as a bona fide member to the endocannabinoid system remains to be established.

Despite substantial degradation, 2‐arachidonoylglycerol is a potent full efficacy agonist mediating CB1 receptor‐dependent G‐protein activation in rat cerebellar membranes

Two endocannabinoids, arachidonoyl ethanolamide (AEA) and 2‐arachidonoylglycerol (2‐AG) bind and activate G‐protein‐coupled cannabinoid receptors, but limited data exist on their relative ability to activate G‐proteins. Here we assess agonist potency and efficacy of various cannabinoids, including 2‐AG, HU‐310 (2‐arachidonoyl glyceryl ether, a third putative endocannabinoid), HU‐313 (another ether analogue of 2‐AG), AEA, R‐methanandamide (an enzymatically stable analogue of AEA), and CP‐55,940 at rat brain CB1 receptors using agonist‐stimulated [35S]‐GTPγS binding to cerebellar membranes and whole brain sections. Degradation of endocannabinoids under experimental conditions was monitored by HPLC. To enhance efficacy differences, agonist dose‐response curves were generated using increasing GDP concentrations. At 10−6 M GDP, all compounds, except HU‐313, produced full agonists responses ∼2.5 fold over basal. The superior efficacy of 2‐AG over all other compounds became evident by increasing GDP (10−5 and 10−4 M). In membrane incubations, 2‐AG was degraded by 85% whereas AEA and HU‐310 were stable. Pretreatment of membranes with phenylmethylsulphonyl fluoride inhibited 2‐AG degradation, resulting in 2 fold increase in agonist potency. Such pretreatment had no effect on AEA potency. Responses in brain sections were otherwise consistent with membrane binding data, but 2‐AG evoked only a weak signal in brain sections, apparently due to more extensive degradation. These data establish that even under conditions of substantial degradation, 2‐AG is a full efficacy agonist, clearly more potent than AEA, in mediating CB1 receptor‐dependent G‐protein activity in native membranes.

An optimized approach to study endocannabinoid signaling: evidence against constitutive activity of rat brain adenosine A1 and cannabinoid CB1 receptors

At nanomolar concentrations, SR141716 and AM251 act as specific and selective antagonists of the cannabinoid CB1 receptor. In the micromolar range, these compounds were shown to inhibit basal G‐protein activity, and this is often interpreted to implicate constitutive activity of the CB1 receptors in native tissue. We show here, using [35S]GTPγS binding techniques, that micromolar concentrations of SR141716 and AM251 inhibit basal G‐protein activity in rat cerebellar membranes, but only in conditions where tonic adenosine A1 receptor signaling is not eliminated. Unlike lipophilic A1 receptor antagonists (potency order DPCPX≫N‐0840 ∼cirsimarin>caffeine), adenosine deaminase (ADA) was not fully capable in eliminating basal A1 receptor‐dependent G‐protein activity. Importantly, all antagonists reduced basal signal to the same extent (20%), and the response evoked by the inverse agonist DPCPX was not reversed by the neutral antagonist N‐0840. These data indicate that rat brain A1 receptors are not constitutively active, but that an ADA‐resistant adenosine pool is responsible for tonic A1 receptor activity in brain membranes. SR141716 and AM251, at concentrations fully effective in reversing CB1‐mediated responses (10−6 M), did not reduce basal G‐protein activity, indicating that CB1 receptors are not constitutively active in these preparations. At higher concentrations (1–2.5 × 10−5 M), both antagonists reduced basal G‐protein activity in control and ADA‐treated membranes, but had no effect when A1 receptor signaling was blocked with DPCPX. Moreover, the CB1 antagonists right‐shifted A1 agonist dose–response curves without affecting maximal responses, suggesting competitive mode of antagonist action. The CB1 antagonists did not affect muscarinic acetylcholine or GABAB receptor signaling. When further optimizing G‐protein activation assay for the labile endocannabinoid 2‐arachidonoylglycerol (2‐AG), we show, by using HPLC, that pretreatment of cerebellar membranes with methyl arachidonoyl fluorophosphonate (MAFP) fully prevented enzymatic degradation of 2‐AG and concomitantly enhanced the potency of 2‐AG. In contrast to previous claims, MAFP exhibited no antagonist activity at the CB1 receptor. The findings establish an optimized method with improved signal‐to‐noise ratio to assess endocannabinoid‐dependent G‐protein activity in brain membranes, under assay conditions where basal adenosinergic tone and enzymatic degradation of 2‐AG are fully eliminated.

Biochemical and pharmacological characterization of human α/β-hydrolase domain containing 6 (ABHD6) and 12 (ABHD12).

In the central nervous system, three enzymes belonging to the serine hydrolase family are thought to regulate the life time of the endocannabinoid 2-arachidonoylglycerol (C20:4) (2-AG). From these, monoacylglycerol lipase (MAGL) is well characterized and, on a quantitative basis, is the main 2-AG hydrolase. The postgenomic proteins α/β-hydrolase domain containing (ABHD)6 and ABHD12 remain poorly characterized. By applying a sensitive fluorescent glycerol assay, we delineate the substrate preferences of human ABHD6 and ABHD12 in comparison with MAGL. We show that the three hydrolases are genuine MAG lipases; medium-chain saturated MAGs were the best substrates for hABHD6 and hMAGL, whereas hABHD12 preferred the 1 (3)- and 2-isomers of arachidonoylglycerol. Site-directed mutagenesis of the amino acid residues forming the postulated catalytic triad (ABHD6: S148-D278-H306, ABHD12: S246-D333-H372) abolished enzymatic activity as well as labeling with the active site serine-directed fluorophosphonate probe TAMRA-FP. However, the role of D278 and H306 as residues of the catalytic core of ABHD6 could not be verified because none of the mutants showed detectable expression. Inhibitor profiling revealed striking potency differences between hABHD6 and hABHD12, a finding that, when combined with the substrate profiling data, should facilitate further efforts toward the design of potent and selective inhibitors, especially those targeting hABHD12, which currently lacks such inhibitors.

Identification of WIN55212-3 as a competitive neutral antagonist of the human cannabinoid CB2 receptor

1 Several G protein‐coupled receptors (GPCRs), including cannabinoid CB1 and CB2 receptors, show constitutive activity under heterologous expression. Such a tonic response is generated in the absence of an activating ligand, and can be inhibited by inverse agonists. Neutral antagonists, however, are silent at such receptors, but can reverse both agonist and inverse agonist responses. To date, no neutral antagonist for the CB2 receptor has been reported. 2 Here, by monitoring receptor‐dependent G protein activation, we demonstrate that WIN55212‐3 acts as a neutral antagonist at the human CB2 (hCB2) receptor. WIN55212‐3 alone, at concentrations 10−4 M, behaved as a silent ligand exhibiting no agonist or inverse agonist activity. However, WIN55212‐3 competitively antagonized cannabinoid agonist CP‐55,940‐stimulated responses (pA2 6.1). Importantly, the inverse agonism evoked by SR144528 in hCB2 was dose‐dependently reversed by WIN55212‐3 (pEC50 5.3±0.2), indicating true neutral antagonist behavior. 3 Furthermore, WIN55212‐3 also antagonized CB1 receptor signaling in a competitive manner (pA2 5.6), but behaved as a partial inverse agonist (pIC50 5.5±0.1) at the constitutively active human CB1. 4 Additionally, WIN55212‐3 antagonized signaling of the human melatonin MT1 receptor, with modest activity at the human muscarinic M4 receptor, but it was inactive towards several other GPCRs. 5 These data identify WIN55212‐3 as a true neutral hCB2 receptor antagonist. WIN55212‐3 offers a valuable tool for further characterization of ligand activities at the CB2 receptor and may serve as a lead compound in further efforts to develop more potent and selective neutral CB2 receptor antagonists.

S-Nitrosothiols modulate G protein-coupled receptor signaling in a reversible and highly receptor-specific manner

BackgroundRecent studies indicate that the G protein-coupled receptor (GPCR) signaling machinery can serve as a direct target of reactive oxygen species, including nitric oxide (NO) and S-nitrosothiols (RSNOs). To gain a broader view into the way that receptor-dependent G protein activation – an early step in signal transduction – might be affected by RSNOs, we have studied several receptors coupling to the Gi family of G proteins in their native cellular environment using the powerful functional approach of [35S]GTPγS autoradiography with brain cryostat sections in combination with classical G protein activation assays.ResultsWe demonstrate that RSNOs, like S-nitrosoglutathione (GSNO) and S-nitrosocysteine (CysNO), can modulate GPCR signaling via reversible, thiol-sensitive mechanisms probably involving S-nitrosylation. RSNOs are capable of very targeted regulation, as they potentiate the signaling of some receptors (exemplified by the M2/M4 muscarinic cholinergic receptors), inhibit others (P2Y12 purinergic, LPA1lysophosphatidic acid, and cannabinoid CB1 receptors), but may only marginally affect signaling of others, such as adenosine A1, μ-opioid, and opiate related receptors. Amplification of M2/M4 muscarinic responses is explained by an accelerated rate of guanine nucleotide exchange, as well as an increased number of high-affinity [35S]GTPγS binding sites available for the agonist-activated receptor. GSNO amplified human M4 receptor signaling also under heterologous expression in CHO cells, but the effect diminished with increasing constitutive receptor activity. RSNOs markedly inhibited P2Y12 receptor signaling in native tissues (rat brain and human platelets), but failed to affect human P2Y12 receptor signaling under heterologous expression in CHO cells, indicating that the native cellular signaling partners, rather than the P2Y12 receptor protein, act as a molecular target for this action.ConclusionThese in vitro studies show for the first time in a broader general context that RSNOs are capable of modulating GPCR signaling in a reversible and highly receptor-specific manner. Given that the enzymatic machinery responsible for endogenous NO production is located in close proximity with the GPCR signaling complex, especially with that for several receptors whose signaling is shown here to be modulated by exogenous RSNOs, our data suggest that GPCR signaling in vivo is likely to be subject to substantial, and highly receptor-specific modulation by NO-derived RSNOs.

Screening of Various Hormone‐Sensitive Lipase Inhibitors as Endocannabinoid‐Hydrolyzing Enzyme Inhibitors

Three classes of chemically diverse hormone-sensitive lipase (HSL) inhibitors, including oxadiazolones, 2H-isoxazol-5-ones and carbamoyl triazoles, were evaluated for their ability to inhibit endocannabinoid-hydrolyzing enzymes, fatty acid amide hydrolase (FAAH) and monoglyceride lipase (MGL, also called monoacylglycerol lipase (MAGL)). All the compounds belonging to these compound classes inhibited both FAAH and MGL with IC50 values varying from the nanomolar to low micromolar range. The most potent FAAH inhibitor was 2H-isoxazol-5-one 10 c with an IC50 value of 0.45 nm, whereas the most promising MGL inhibitor, albeit not selective over FAAH, was 1,3,4-oxadiACHTUNGTRENNUNGazol-2(3H)-one 16 c (IC50 = 78 nm against 2-AG hydrolysis). These results suggest that HSL inhibitors investigated in this paper may provide useful leads for the development of novel FAAH and/or MGL inhibitors. N-arachidonoylethanolamine (anandamide, AEA) and 2arachidonoylglycerol (2-AG) are the two best known and most investigated endocannabinoids that activate cannabinoid CB1 [3] and CB2 [4] receptors and modulate several physiological processes, such as pain sensation and inflammation (for reviews, see Reference [5]). However, endogenous levels of AEA and 2-AG are normally low, as these endocannabinoids are rapidly degraded by the specific enzymes, FAAH (EC 3.5.1.4) and MGL (EC 3.1.1.23), respectively. Inactivation of FAAH and MGL by chemical inhibitors leads to elevated levels of AEA and 2-AG, which have been evidenced to be an attractive and valuable goal in the treatment of a variety of pathological conditions. 8] In recent years, a range of different classes of FAAH inhibitors have been developed, mainly derivatized from the other known serine hydrolase inhibitors (for a review, see Reference [9]). These include various substrate analogues, as well as nonlipid inhibitors such as a-keto heterocycles, carbamates, (thio)hydantoins, piperidine/piperazine ureas and most recently, benzothiazole-based sulfonyl derivatives and boronic acids. Within carbamate-based inhibitors, cyclohexylcarbamic acid biphenyl-3-yl ester (URB597) (1) has been shown to be effica-

Visualization of 2-arachidonoylglycerol accumulation and cannabinoid CB1 receptor activity in rat brain cryosections by functional autoradiography.

In neuronal signalling mediated by the endocannabinoid 2‐arachidonoylglycerol, both synthetic and inactivating enzymes operate within close proximity to the Gi/o‐coupled pre‐synaptic CB1 receptors, thus allowing for rapid onset and transient duration of this lipid modulator. In rat brain, 2‐arachidonoylglycerol is inactivated mainly via hydrolysis by serine hydrolase inhibitor‐sensitive monoacylglycerol lipase activity. We show in this study that comprehensive pharmacological elimination of this activity in brain cryosections by methyl arachidonylfluorophosphonate or hexadecylsulphonyl fluoride results in endocannabinoid‐mediated CB1 receptor activity, which can be visualized by functional autoradiography. URB597, a specific inhibitor of anandamide hydrolysis proved ineffective. TLC indicated that the bioactivity resided in 2‐arachidonoylglycerol‐containing fraction and gas chromatography–mass spectroscopy detected elevated levels of monoacylglycerols, including 2‐arachidonoylglycerol in this fraction. Although two diacylglycerol lipase inhibitors, tetrahydrolipstatin (THL) and RHC80267, blocked the bulk of 2‐arachidonoylglycerol accumulation in methyl arachidonylfluorophosphonate‐treated sections, only THL reversed the endocannabinoid‐dependent CB1 receptor activity. Further studies indicated that at the used concentrations, THL rather specifically antagonized the CB1 receptor. These findings confirm that in brain sections there is preservation of enzymatic pathways regulating the production of endogenous receptor ligands. Furthermore, the presently described methodology may serve as an elegant and intuitive approach to identify novel membrane‐derived lipid modulators operating in the CNS.

Characterization of binding properties of monoglyceride lipase inhibitors by a versatile fluorescence-based technique.

Monoglyceride lipase (MGL) is a serine hydrolase that terminates the signaling of the primary endocannabinoid, 2-arachidonoyl glycerol (2-AG). Versatile high-throughput screening methods allowing the testing of MGL inhibitors are rare, thereby limiting the development and analysis of novel inhibitors. Here we describe an improved fluorescence-based technique that is capable of determining time- and dose-dependent inhibition of MGL with one or multiple binding sites and, at the same time, is capable of revealing the reversibility of inhibitor binding in a simple kinetic assay format. Known reference compounds as well as novel inhibitors, such as JZL184 and CAY10499, were evaluated for their MGL-binding properties and potency.

Chiral 1,3,4-Oxadiazol-2-ones as Highly Selective FAAH Inhibitors

In the present study, identification of chiral 1,3,4-oxadiazol-2-ones as potent and selective FAAH inhibitors has been described. The separated enantiomers showed clear differences in the potency and selectivity toward both FAAH and MAGL. Additionally, the importance of the chirality on the inhibitory activity and selectivity was proven by the simplification approach by removing a methyl group at the 3-position of the 1,3,4-oxadiazol-2-one ring. The most potent compound of the series, the S-enantiomer of 3-(1-(4-isobutylphenyl)ethyl)-5-methoxy-1,3,4-oxadiazol-2(3H)-one (JZP-327A, 51), inhibited human recombinant FAAH (hrFAAH) in the low nanomolar range (IC50 = 11 nM), whereas its corresponding R-enantiomer 52 showed only moderate inhibition toward hrFAAH (IC50 = 0.24 μM). In contrast to hrFAAH, R-enantiomer 52 was more potent in inhibiting the activity of hrMAGL compared to S-enantiomer 51 (IC50 = 4.0 μM and 16% inhibition at 10 μM, respectively). The FAAH selectivity of the compound 51 over the supposed main off-targets, MAGL and COX, was found to be >900-fold. In addition, activity-based protein profiling (ABPP) indicated high selectivity over other serine hydrolases. Finally, the selected S-enantiomers 51, 53, and 55 were shown to be tight binding, slowly reversible inhibitors of the hrFAAH.