title: Screening and characterization of human monoglyceride lipase active site inhibitors using orthogonal binding and functional assays.
abstract: Endocannabinoids such as 2-arachidonylglycerol (2-AG) are ligands for cannabinoid receptors that contribute to the transmission and modulation of pain signals. The antinociceptive effect of exogenous 2-AG suggests that inhibition of monoglyceride lipase (MGLL), the enzyme responsible for degrading 2-AG and arresting signaling, may be a target for pain modulation. Here we describe the characterization of MGLL ligands following a high-throughput screening campaign. Ligands were discovered using ThermoFluor, a label-free affinity-based screening tool that measures ligand binding via modulation of protein thermal stability. A kinetic fluorescent assay using the substrate 4-methylcoumarin butyrate was used to counterscreen confirmed HTS positives. A comparison of results from binding and inhibition assays allowed elucidation of compound mechanism of action. We demonstrate the limit of each technology and the benefits of using orthogonal assay techniques in profiling compounds.
authors.affiliation: GlaxoSmithKline, Oncology Research & Development, Collegeville, PA, USA.
authors.forename: José C
authors.initials: JC
authors.lastname: Clemente
authors.forename: Erica
authors.initials: E
authors.lastname: Nulton
authors.forename: Marina
authors.initials: M
authors.lastname: Nelen
authors.forename: Matthew J
authors.initials: MJ
authors.lastname: Todd
authors.forename: Diane
authors.initials: D
authors.lastname: Maguire
authors.forename: Céline
authors.initials: C
authors.lastname: Schalk-Hihi
authors.forename: Lawrence C
authors.initials: LC
authors.lastname: Kuo
authors.forename: Sui-Po
authors.initials: SP
authors.lastname: Zhang
authors.forename: Christopher M
authors.initials: CM
authors.lastname: Flores
authors.forename: James K
authors.initials: JK
authors.lastname: Kranz
mesh_terms: D001095:Arachidonic Acids / Q000737:chemistry; D020134:Catalytic Domain / Q000187:drug effects*; D063388:Endocannabinoids; D057075:Enzyme Assays / Q000379:methods*; D004791:Enzyme Inhibitors / Q000737:chemistry / Q000494:pharmacology*; D005989:Glycerides / Q000737:chemistry; D057166:High-Throughput Screening Assays; D006801:Humans; D006868:Hydrolysis; D020128:Inhibitory Concentration 50; D007700:Kinetics; D008994:Monoacylglycerol Lipases / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000378:metabolism; D011485:Protein Binding; D012995:Solubility; D013379:Substrate Specificity
chemical_list: D001095:Arachidonic Acids; D063388:Endocannabinoids; D004791:Enzyme Inhibitors; D005989:Glycerides; C094503:glyceryl 2-arachidonate; D008994:Monoacylglycerol Lipases