THE ENDOCANNABINOID ENZYME MONOACYLGLYCEROL LIPASE: DEVELOPMENT OF A NEW FLUORESCENT ASSAY AND NOVEL INHIBITORS

The endocannabinoid system (ECS), comprising CB1/CB2 receptors, endocannabinoids (ECs) and their metabolic enzymes, FAAH (fatty acid amide hydrolase) and MAGL (monoacylglycerol lipase), is responsible for maintaining the homeostatic balance, regulating and modulating the physiological responses to improve general well-being [1]. A dysregulation of ECS is connected to pathological conditions such as pain, inflammation, anxiety, and other disorders [2]. Pharmacological blockade of FAAH and MAGL has emerged as a potentially attractive strategy to augment EC signalling and retain the beneficial effects of cannabinoid receptor activation, while avoiding the undesirable side effects, such as impairments in cognition and motor control, observed with direct cannabinoid receptor agonists [3]. The endogenous serine-hydrolase MAGL is the main enzyme responsible for inactivating endocannabinoid agonist 2-arachidonoylglycerol (2-AG) to arachidonic acid and glycerol. Conventional assays for MAGL activity and inhibitors screening utilize radiolabelled substrates [4] or MAGL-catalysed hydrolysis of p-nitrophenyl alkyl esters allowing the liberation of UV-detectable p-nitrophenol [5]. Furthermore, a fluorescence-based assay has been developed applying 7-hydroxycoumarinyl arachidonate (7-HCA) [6] or HPLC method with fluorescence detection using as fluorogenic probe 1,3-dihydroxypropan-2-yl-4-pyren-1-ylbutanoate [7]. The objective of this PhD project was to: - develop a new sensitive MAGL-activity assay simple, sensitive, and suitable for high-throughput screening (HTS) to test potential inhibitors, both in vitro and in cell cultures; - synthesise more potent and selective inhibitors in order to increase the endogenous level of EC prolonging their action. A first part of this project has been focused on the development of a novel fluorescent method for assaying MAGL. Therefore, we designed and synthesized 7-hydroxyresorufinylarachidonate (7-HRA) which is amenable for high-throughput screening (HTS). MAGL catalyses the hydrolysis of 7-HRA to generate arachidonic acid and a highly red fluorescent resorufin. Our new substrate [8] combines high specific reactivity with the enzyme tested with excellent stability against non-specific degradation if stored in the dark and can be a useful tool in further developments of MAGL inhibitors. Moreover, resorufin’s high red absorption and emission wavelengths should be preferred in cell cultures assays to blue emitting molecules in reason of the lower background absorbance and auto fluorescence of tissues in this spectral region [9]. Three known MAGL inhibitors were used to validate the test assay. A second part of the project was the synthesis of reference molecule URB602 [10] and a library of analogues that were tested against MAGL with our new fluorescent method. Some of them showed encouraging results in terms of inhibitory activity, proving to have an increase in the activity compared to reference URB602. A molecular modelling work was made to understand the possible interactions between each compound and the enzyme and to explain the differences among the different structures. Moreover, the fluorescent method was applied to cancer cell lysates to verify its suitability for in vitro experiments - which was confirmed - and a first screening on the new inhibitors was made on these cell lines, finding that their activity is comparable to that of URB602. References [1] P. Pacher et al., Pharmacol. Rev. 58 (2006) 389-462 [2] J.Z. Long et al., Nat. Chem. Biol. 5 (2009) 37–44 [3] J.Z. Long et al., P. Natl. Acad. Sci. USA 106 (2009) 20270-20275 [4] J. Brengdahl et al., Anal. Biochem. 359 (2006) 40-44 [5] G.G. Muccioli et al., ChemBioChem 9 (2008) 2704-2710 [6] Y. Wang et al., Assay Drug Dev. Techn. 6 (2008) 387-393 [7] A. Holtfrerich et al., Anal. Biochem. 399 (2010) 218-224 [8] S. Lauria et al., Anal. Bioanal. Chem. (2015), 407, 8163-8167 DOI 10.1007/s00216-015-8991-9. [9] M. Fritzsche et al., Anal. Bioanal. Chem. 398 (2010) 181-191 [10] M. Szabo et al., Bioorg. Med. Chem. Lett. 21 (2011) 6782-6787