Active-Site Inhibitors Modulate the Dynamic Properties of Human Monoacylglycerol Lipase: A Hydrogen Exchange Mass Spectrometry Study

Human monoacylglycerol lipase (hMGL) regulates endocannabinoid signaling primarily by deactivating the lipid messenger 2-arachidonoylglycerol. Agents that carbamylate hMGLs catalytic Ser122 constitute a leading class of therapeutically promising hMGL inhibitors. We have applied peptide-level hydrogen/deuterium exchange mass spectrometry to characterize hMGL’s conformational responses to two potent carbamylating inhibitors, AM6580 (irreversible) and AM6701 (slowly reversible). A dynamic, solvent-exposed lid domain is characteristic of hMGL’s solution conformation. Both hMGL inhibitors restricted backbone enzyme motility in the active-site region and increased substrate binding-pocket solvent exposure. Covalent reaction of AM6580 with hMGL generates a bulkier carbamylated Ser122 residue as compared to the more discrete Ser122 modification by AM6701, a difference reflected in AM6580’s more pronounced effect upon hMGL conformation. We demonstrate that structurally distinct carbamylating hMGL inhibitors generate particular conformational ensembles characterized by regionspecific hMGL dynamics. By demonstrating the distinctive influences of two hMGL inhibitors on enzyme conformation, this study furthers our understanding at the molecular level of the dynamic features of hMGL interaction with small-molecule ligands. The endocannabinoid system is a ubiquitous signaling network involved in numerous (patho)physiological processes including metabolic control, emotional reactivity, pain sensing, and inflammation.1 Cannabinergic signaling in the mammalian central nervous system (CNS) mainly reflects the full-agonist action of the endogenous lipid mediator, 2- arachidonoylglycerol (2-AG), at the cannabinoid 1 G-protein coupled receptor (CB1R).2 As the serine hydrolase mainly responsible for catalytic 2-AG deactivation in vivo, monoacylglycerol lipase (EC 3.1.1.23) (MGL) is a decisive regulator of 2-AG signaling and, consequently, the production of various neurochemicals in the CNS that underlie synaptic efficiency and influence psychobehavioral status.3,4 Increasing interest has focused on MGL inhibition as a therapeutic modality for enhancing indirectly cannabinergic signaling through elevation of tissue 2-AG tone.5–7 Phenotypic benefits associated with genetic or pharmacological disruption of MGL partly recapitulate those of exogenous CB1R agonists while appearing to pose less risk of the undesirable motor and behavioral effects classically associated with systemic CB1Ragonist application.5,7 Indeed, MGL inhibitors may be an attractive means of augmenting, in a site- and event-specific manner, the increased tissue endocannabinoid levels observed under at least some pathological conditions and considered reflective of an intrinsic adaptive response to restore homeostasis.8–10 The role of downstream products from monoacylglycerol hydrolysis in the etiologies of cancer and inflammation provides additional support for the therapeutic potential of MGL inhibitors.11,12 Although tonic potentiation of tissue 2-AG levels may impair 2-AG-mediated antinociception by desensitizing CB1R,13,14 MGL inhibitors have demonstrated therapeutic benefit in acute preclinical models of inflammation, pain, anxiety, and CNS injury without inciting adverse events.15–18 These considerations have promoted the rational design of temporally tuned MGL inhibitors that can potentiate subsets of 2-AG-related signaling circuits to effect a clinically attractive severance of adverse events associated with exogenous CB1R agonists from therapeutic gain. By leveraging the essential role of MGL’s active-site serine nucleophile (Ser122) (Figure 1A) in forming the initial transition state during the catalytic cycle,19 we20–22 and others6,23 have generated several potent, structurally distinct carbamylating inhibitors that inactivate human MGL (hMGL) through covalent enzyme carbamylation at Ser122. Yet optimal translational exploitation of hMGL inhibitors as medicines necessitates a thorough understanding of the inhibitor-interaction profiles that define potent inhibitors with varying hMGL selectivities and durations of action. In this regard, we have utilized two hMGL carbamylating inhibitors, [4-(9H-fluoren-9-yl)piperazin-1-yl][1,2,3]triazolo[4,5-b]-pyridin-1-ylmethanone (AM6580)24 and 5-((biphenyl-4-yl)- methyl)-N,N-dimethyl-2H-tetrazole-2-carboxamide (AM6701) (also known as the 2,5-regioisomer of LY2183240)20,22 (Figure 1B,C) as tool compounds for probing the molecular details of pharmacological hMGL inactivation. Both agents inhibit hMGL in the low nanomolar range through a common mechanism involving rapid carbamylation of Ser122 in the enzyme’s catalytic triad.21,22 Within a 3-h experimental time frame, hMGL inhibition by AM6580 is irreversible, whereas the carbamate group on hMGL Ser122 resulting from enzyme modification byAM6701 begins to be hydrolyzed very slowly such that ≥24 h are required to regenerate catalytically competent hMGL.20,21 This biochemical distinction suggests that the interaction profiles of these two chemically distinct carbamylating inhibitors may impact hMGL conformation in distinctive ways that have contingent implications regarding, for example, the duration and reversibility of hMGL inhibition. Figure 1 Structure of apo-6-His-hMGL and chemical structures of AM6701 and AM6580 and schematic representation of their carbamylation reaction with hMGL. (A) The catalytic triad (Ser129-Asp246-His276, red/white/blue ball-and-stick depiction), lid domain (residues ... We report application of peptide-level, solution-phase hydrogen/deuterium exchange mass spectrometry (H/DXMS) to gain detailed insight into the dynamic transitions in hMGL’s solution structure consequent to AM6580 and AM6701 engagement and inhibitor-induced covalent enzyme modification. H/DX-MS involves mass-spectrometry (MS) measurements of backbone amide hydrogens substituted by the heavier deuterium isotope from deuterated water (D2O) at ambient temperature and physiological pH.25,26 The rate of incorporation of deuterium is modulated by the conformational properties of the protein as well as pH and temperature and thus reports on the combined effects of solvent exposure and the hydrogen-bonding characteristic of the protein backbone. Following acid quenching of the exchange reaction, proteolytic digestion with pepsin is used to localize where along the protein backbone the deuterium had been incorporated. Rapid exchange is characteristic of more disordered, solvent-exposed protein regions, whereas slower and more limited exchange indicates a more compact, solvent-shielded, hydrogen-bonded state. Therefore, the difference between deuterium incorporation into apo versus inhibitor-modified hMGL serves as a proxy for the structural impact of the inhibitor’s interaction with the enzyme. Our results demonstrate that chemically different, small-molecule carbamylating inhibitors generate distinct hMGL conformational ensembles characterized by regionally specific structural responses in the enzyme. To the authors’ best knowledge, these data offer the first direct experimental insight into the molecular dynamics of hMGL in solution upon covalent inhibitor modification. By detailing the hMGL structural excursions characteristic of the hMGL inactive state as induced by two potent, chemically distinct carbamylating inhibitors, the information presented has the potential to aid the design and targeting of new-generation hMGL inhibitors as candidate drugs.