Mechanistic Modeling of Monoglyceride Lipase Covalent Modification Elucidates the Role of Leaving Group Expulsion and Discriminates Inhibitors with High and Low Potency

Inhibition of monoglyceride lipase (MGL), also known as monoacylglycerol lipase (MAGL), has emerged as a promising approach for treating neurological diseases. To gain useful insights in the design of agents with balanced potency and reactivity, we investigated the mechanism of MGL carbamoylation by the reference triazole urea SAR629 (IC50 = 0.2 nM) and two recently described inhibitors featuring a pyrazole (IC50 = 1800 nM) or a 4-cyanopyrazole (IC50 = 8 nM) leaving group (LG), using a hybrid quantum mechanics/molecular mechanics (QM/MM) approach. Opposite to what was found for substrate 2-arachidonoyl-sn-glycerol (2-AG), covalent modification of MGL by azole ureas is controlled by LG expulsion. Simulations indicated that changes in the electronic structure of the LG greatly affect reaction energetics with triazole and 4-cyanopyrazole inhibitors following a more accessible carbamoylation path compared to the unsubstituted pyrazole derivative. The computational protocol provided reaction barriers able to discriminate between MGL inhibitors with different potencies. These results highlight how QM/MM simulations can contribute to elucidating structure–activity relationships and provide insights for the design of covalent inhibitors.


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Potential of mean force for MGL acylation by 1. Figure S3. Evolution of the free-energy profiles at different simulation times for MGL acylation by 1. PMF convergence is achieved after 400 ps of umbrella sampling (US) simulations, for each window.

Superposition of MGL acylated by 1 and two X-ray structures of MGL and a glycerol
molecule. Figure S4. Superposition of MGL acylated by 1 (grey and yellow carbon atoms, respectively), representing the products of the reaction, and two X-ray structures of MGL in complex with a glycerol molecule: 6AX1 (A, cyan carbon atoms) and 3HJU (B, purple carbon atoms).

Gas-phase energy calculations on (E) and (Z)-configurations for piperazine azole urea fragments taken from compound 4-6.
Table S1. Gas phase energies (kcal•mol -1 ) are computed at DFTB3 or M06-2X-D3/cc-PVDZ level.  1.4 ± 0.6 -1.9 ± 0.14 6 5.4 ± 0.82 -0.77 ± 0.14 S10 Steered MD simulation for MGL carbamoylation by 4. Figure S6. Work profile from a SMD simulation modeling the carbamoylation of MGL by 4. Work profile is reported over the reaction coordinate accounting for the nucleophilic attack of Ser122 to the carbonyl carbon of the reactive urea group of 4, and for the expulsion of the triazole ring as

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Potential of mean force for MGL carbamoylation by 4. Figure S7. Evolution of the free-energy profiles at different simulation times for MGL carbamoylation by 4. PMF convergence is achieved after 400 ps of US simulation, for each window. Figure S8. Superposition of MGL carbamoylated by 4 (grey and light green carbon atoms, respectively), representing the product of the reaction, and the X-ray structure of 4 (dark green carbon atoms) covalently bound to Ser122.

Steered MD simulation for MGL carbamoylation by 5 and 6.
Figure S10. Work profiles from SMD simulations modeling the carbamoylation of MGL by 5 and 6. Work profiles are reported over the reaction coordinate accounting for the nucleophilic attack of Ser122 to the carbonyl carbon of the reactive urea group of 5 and 6, and for the expulsion of the azole ring as LG.

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Potential of mean force for MGL carbamoylation by 5. Figure S11. Evolution of the free-energy profiles at different simulation times for MGL carbamoylation by 5. PMF convergence is achieved after 400 ps of US simulation for each window.

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Potential of mean force for MGL carbamoylation by 6. Figure S12. Evolution of the free-energy profiles at different simulation times for MGL carbamoylation by 6. PMF convergence is achieved after 400 ps of US simulation for each window.

Polar interaction between 6 and Arg57 during US simulations.
Figure S13. Representation of a snapshot from an US simulation of the transition state (TS) configuration of MGL carbamoylation by 6 (cyan carbon atoms), which forms a polar interaction with Arg57 side chain (green dashes).

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Docking model of 7 within MGL binding site. Figure S14. Binding mode of 7 (teal carbon atoms) within MGL active site (grey carbon atoms).

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Steered MD simulation for MGL carbamoylation by 7. Figure S15. Work profile from a SMD simulation modeling the carbamoylation of MGL by 7.
Work profile is reported over the reaction coordinate accounting for the nucleophilic attack of Ser122 to the carbonyl carbon of the reactive urea group of 7, and for the expulsion of the pyrazole-4-carboxamide ring as LG.

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Potential of mean force for MGL carbamoylation by 7. Figure S16. Evolution of the free-energy profiles at different simulation times for MGL carbamoylation by 7. PMF convergence is achieved after 500 ps of US simulation for each window.

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List of restrained residues.
In order to maintain the structure of monoglyceride lipase (MGL) close to the X-ray coordinates, and to avoid long-range conformational variations, a harmonic restraint of 5 kcal•mol -1 Å -2 was applied on the alpha carbon atoms of residues situated 5 Å from the active site region. This restrained region comprises all alpha carbon atoms of all the residues, excluding Gly50 to Asp53, Gly120 to Ile127, Ile145 to Leu148, Leu150 to Asn152, Phe209 to Val217, Leu241, Cys242, His269, Val270.

Definition of the QM region for the Michaelis complexes under study.
Figure S17. 2D and 3D representation of the atoms and of the link atoms included in the QM region for the Michaelis complex of MGL and 1 (A), 4 (B), 5 (C), 6 (D) and 7 (E).