Identification and characterization of a new reversible MAGL inhibitor

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Abstract

Monoacylglycerol lipase is a serine hydrolase that play a major role in the degradation of 2-arachidonoylglycerol, an endocannabinoid neurotransmitter implicated in several physiological processes. Recent studies have shown the possible role of MAGL inhibitors as anti-inflammatory, anti-nociceptive and anti-cancer agents. The use of irreversible MAGL inhibitors determined an unwanted chronic MAGL inactivation, which acquires a functional antagonism function of the endocannabinoid system. However, the application of reversible MAGL inhibitors has not yet been explored, mainly due to the scarcity of known compounds possessing efficient reversible inhibitory activities. In this study we reported the first virtual screening analysis for the identification of reversible MAGL inhibitors. Among the screened compounds, the (4-(4-chlorobenzoyl)piperidin-1-yl)(4-methoxyphenyl)methanone (CL6a) is a promising reversible MAGL inhibitor lead (Ki = 8.6 μM), which may be used for the future development of a new class of MAGL inhibitors. Furthermore, the results demonstrate the validity of the methodologies that we followed, encouraging additional screenings of other commercial databases.

Introduction

Monoacylglycerol lipase (MAGL) is a 33 kDa cytosolic serine hydrolase that preferentially cleaves monoacylglycerols into fatty acids and glycerol, with the highest expression in brain, adipose tissue and liver.1 This protein contains two lipase motives (active serine motif GXSXG and the HG dipeptide) with a Ser/His/Asp catalytic triad, and belongs to the ‘α/β hydrolases fold family’, whose conserved three-dimensional structure consists of a central β sheet surrounded by a variable number of α helices. After standing as a little interest enzyme for many years, Piomelli and co-workers found out that it plays a fundamental role in the deactivation of the endocannabinoid 2-arachidonoglycerol (2-AG), a monoglyceride of arachidonic acid esterified at the sn-2 position, which tunes the functionality and plasticity of many synapses.2 2-AG exhibits a full agonist efficacy for both CB1 and CB2 cannabinoid receptors.3 Unlike the classical neurotransmitters, it is produced on demand from membrane lipid precursors and after its action has been effected, it is rapidly taken up by cells and hydrolyzed. Very recently, Nomura et al. showed that in specific tissues such as brain, liver and lung, MAGL acts as a metabolic switch able to connect the endocannabinoid and the eicosanoid lipid signaling systems: through the hydrolysis of 2-AG, MAGL releases a major arachidonic acid precursor pool for the synthesis of pro-inflammatory eicosanoids such as prostaglandins PGE2 and PGD2.4 In agreement with the role of MAGL in modulating the 2-AG-mediated endocannabinoid signaling, in vivo studies showed that the inhibition of MAGL exerts CB1-dependent antinociceptive effects.5 In 2012, Chen et al. demonstrated that the inhibition of MAGL by a covalent inhibitor resulted in significantly diminished amyloid neuropathology, reduced neuroinflammation and degeneration, and improved synaptic and cognitive function in a mouse model of Alzheimer’s disease.6 In aggressive cancer cells and primary tumors, MAGL is upregulated and has a unique role of providing lipolytic source of free fatty acids for the synthesis of oncogenic signaling lipids that promote cancer aggressiveness.7 Other studies showed that MAGL inhibitors impair colorectal cancer tumorigenesis8 and prostate cancer pathogenicity.9 Taken together all these findings strongly suggest that MAGL inhibition could determine significant therapeutic benefits. Over the past five years, great efforts have been made for developing novel MAGL inhibitors;10, 11, 12, 13, 14, 15 however, almost all the reported compounds are characterized by an irreversible MAGL inhibition mechanism and, as reported by Scholsburg et al., the irreversible inhibition of MAGL produced cross-tolerance to CB1 agonists in mice after repeated administration. Chronic MAGL blockade also caused physical dependence, impaired endocannabinoid-dependent synaptic plasticity and desensitized brain CB1 receptors.16 An unexplored issue is represented by the possible utility of reversible MAGL inhibitors that could partially inhibit the enzyme, while leaving the endocannabinoid system intact; however, to date only few examples of MAGL reversible inhibitors are reported in literature.17, 18, 19 With the aim of finding a new MAGL reversible inhibitor lead that can be easily modified from a synthetically point of view, we carried out a virtual screening (VS) study by using a mixed ligand/receptor-based approach (see Fig. 1).

Section snippets

Results and discussion

To identify novel reversible MAGL inhibitors, a structure-based VS approach was developed. The RCSB Protein Data Bank20 was screened and four human MAGL crystal structures were found. Among them, we chose the crystal structure of (2-cyclohexyl-1,3-benzoxazol-6-yl){3-[4-(pyrimidin-2-yl)piperazin-1-yl]azetidin-1-yl}methanone (ZYH) in complex with MAGL (3PE6 PDB code),18 which was the only structure describing the reversible interaction of a ligand with MAGL. As shown in Figure 2, the ZYH

Conclusions

In the present work we reported the first VS study applied to the identification of novel reversible MAGL inhibitors. This study led to the discovery of compound CL6a, as a reversible MAGL inhibitor, which competes with substrate for the enzyme binding site. This compound can be considered as a promising lead, since it shows an inhibition activity which is very similar to that reported for the other known synthetic MAGL reversible inhibitors.19 Moreover, because of its relatively simple

Virtual screening protocol

The Asinex Gold and Platinum database, consisting of 319911 compounds, was subjected to conformational analysis by using OMEGA2.21 Default parameters were used with the following exception: the maxconfs parameter (which sets the maximum number of conformations to be generated) was set to 10,000 (default = 20,000). The resulting database generated by Omega was processed with ROCS software.22 As a reference structure for shape comparison, the

Acknowledgments

Financial support for this project was provided by the Italian Ministero dell’Università e della Ricerca (MIUR), under the National Interest Research Projects framework (PRIN_2010_5YY2HL). Dr. Giorgio Placanica of the University of Pisa is gratefully acknowledged for technical assistance in the analysis of the chemical product.

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