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A critical cysteine residue in monoacylglycerol lipase is targeted by a new class of isothiazolinone-based enzyme inhibitors.

ABSTRACT: Monoacylglycerol lipase (MGL) is a presynaptic serine hydrolase that inactivates the endocannabinoid neurotransmitter, 2-arachidonoyl-sn-glycerol. Recent studies suggest that cysteine residues proximal to the enzyme active site are important for MGL function. In the present study, we characterize the role of cysteines in MGL function and identify a series of cysteine-reactive agents that inhibit MGL activity with nanomolar potencies by interacting with cysteine residue 208.A series of cysteine traps were screened for the ability to inhibit MGL in vitro. Rapid dilution assays were performed to determine reversibility of inhibition. Molecular modelling and site-directed mutagenesis were utilized to identify cysteine residues targeted by the inhibitors.The screening revealed that 2-octyl-4-isothiazolin-3-one (octhilinone) inhibited purified rat recombinant MGL (IC(50)= 88 +/- 12 nM) through a partially reversible mechanism. Initial structure-activity relationship studies showed that substitution of the n-octyl group of octhilinone with a more lipophilic oleoyl group increased inhibitor potency (IC(50)= 43 +/- 8 nM), while substitution with a methyl group produced the opposite effect (IC(50)= 239 +/- 68 nM). The inhibitory potency of octhilinone was selectively decreased by mutating cysteine 208 in MGL to glycine (IC(50); wild-type, 151 +/- 17 nM; C208G, 722 +/- 74 nM), but not by mutation of other cysteine residues (C32, C55, C201, C208 and C242).The results indicated that cysteine 208 plays an important role in MGL function and identified a novel class of isothiazolinone-based MGL inhibitors with nanomolar potency in vitro.

SUBMITTER: King AR

PROVIDER: S-EPMC2737656 | biostudies-literature | 2009 Jul

REPOSITORIES: biostudies-literature

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A critical cysteine residue in monoacylglycerol lipase is targeted by a new class of isothiazolinone-based enzyme inhibitors.

King A R AR Lodola A A Carmi C C Fu J J Mor M M Piomelli D D

British journal of pharmacology 20090526 6

<h4>Background and purpose</h4>Monoacylglycerol lipase (MGL) is a presynaptic serine hydrolase that inactivates the endocannabinoid neurotransmitter, 2-arachidonoyl-sn-glycerol. Recent studies suggest that cysteine residues proximal to the enzyme active site are important for MGL function. In the present study, we characterize the role of cysteines in MGL function and identify a series of cysteine-reactive agents that inhibit MGL activity with nanomolar potencies by interacting with cysteine res ...[more]

PMID: 19486005

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Project description:The endogenous cannabinoid 2-arachidonoylglycerol (2-AG) plays an important role in a variety of physiologic processes, but its rapid breakdown by monoacylglycerol lipase (MAGL) results in short-lived actions. Initial MAGL inhibitors were limited by poor selectivity and low potency. In this study, we tested JZL184 [4-nitrophenyl 4-[bis(2H-1,3-benzodioxol-5-yl)(hydroxy)methyl]piperidine-1-carboxylate] and MJN110 [2,5-dioxopyrrolidin-1-yl 4-(bis(4-chlorophenyl)methyl)piperazine-1-carboxylate], MAGL inhibitors that possess increased selectivity and potency, in mouse behavioral assays of neuropathic pain [chronic constriction injury (CCI) of the sciatic nerve], interoceptive cannabimimetic effects (drug-discrimination paradigm), and locomotor activity in an open field test. MJN110 (1.25 and 2.5 mg/kg) and JZL184 (16 and 40 mg/kg) significantly elevated 2-AG and decreased arachidonic acid but did not affect anandamide in whole brains. Both MAGL inhibitors significantly reduced CCI-induced mechanical allodynia with the following potencies [ED50 (95% confidence limit [CL]) values in mg/kg: MJN110 (0.43 [0.30-0.63]) > JZL184 (17.8 [11.6-27.4])] and also substituted for the potent cannabinoid receptor agonist CP55,940 [2-[(1R,2R,5R)-5-hydroxy-2-(3-hydroxypropyl)cyclohexyl]-5-(2-methyloctan-2-yl)phenol] in the drug-discrimination paradigm [ED50 (95% CL) values in mg/kg: MJN110 (0.84 [0.69-1.02]) > JZL184 (24.9 [14.6-42.5])]; however, these compounds elicited differential effects on locomotor behavior. Similar to cannabinoid 1 (CB1) receptor agonists, JZL184 produced hypomotility, whereas MJN110 increased locomotor behavior and did not produce catalepsy or hypothermia. Although both drugs substituted for CP55,940 in the drug discrimination assay, MJN110 was more potent in reversing allodynia in the CCI model than in producing CP55,940-like effects. Overall, these results suggest that MAGL inhibition may alleviate neuropathic pain, while displaying limited cannabimimetic effects compared with direct CB1 receptor agonists.

Dataset Information

A critical cysteine residue in monoacylglycerol lipase is targeted by a new class of isothiazolinone-based enzyme inhibitors.

Publications

A critical cysteine residue in monoacylglycerol lipase is targeted by a new class of isothiazolinone-based enzyme inhibitors.

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