Benzisothiazolinone Derivatives as Potent Allosteric Monoacylglycerol Lipase Inhibitors That Functionally Mimic Sulfenylation of Regulatory Cysteines

Abstract

We describe a set of benzisothiazolinone (BTZ) derivatives that are potent inhibitors of monoacylglycerol lipase (MGL), the primary degrading enzyme for the endocannabinoid 2-arachidonoyl-sn-glycerol (2-AG). Structure–activity relationship studies evaluated various substitutions on the nitrogen atom and the benzene ring of the BTZ nucleus. Optimized derivatives with nanomolar potency allowed us to investigate the mechanism of MGL inhibition. Site-directed mutagenesis and mass spectrometry experiments showed that BTZs interact in a covalent reversible manner with regulatory cysteines, Cys201 and Cys208, causing a reversible sulfenylation known to modulate MGL activity. Metadynamics simulations revealed that BTZ adducts favor a closed conformation of MGL that occludes substrate recruitment. The BTZ derivative 13 protected neuronal cells from oxidative stimuli and increased 2-AG levels in the mouse brain. The results identify Cys201 and Cys208 as key regulators of MGL function and point to the BTZ scaffold as a useful starting point for the discovery of allosteric MGL inhibitors.

Structure-Guided Discovery of the Novel Covalent Allosteric Site and Covalent Inhibitors of Fructose-1,6-Bisphosphate Aldolase to Overcome the Azole Resistance of Candidiasis

“…As illustrated in Figure , compound 1 exhibited the highest CaFBA inhibition (IC 50 = 17.7 μM) compared with other compounds, suggesting that compound 1 could easily conjugate to a thiol group of CaFBA . Therefore, we considered 1 as a suitable probe for the discovery of novel potent covalent inhibitors of CaFBA.…”

“…As illustrated in Figure 1, compound 1 exhibited the highest CaFBA inhibition (IC 50 = 17.7 μM) compared with other compounds, suggesting that compound 1 could easily conjugate to a thiol group of CaFBA. 55 Therefore, we considered 1 as a suitable probe for the discovery of novel potent covalent inhibitors of CaFBA. To further identify the functional cysteine site that regulates the catalytic activity of CaFBA, all three cysteines in CaFBA were mutated to serine (C157S, C111S, and C292S), and kinetic parameter determination and an inhibitory assay against wild-type (WT) and mutant CaFBA were performed (Table 1) using compound 1 as a probe.…”

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

“…These findings have promoted MGL as a promising drug target, with carbamate-based inhibitors being currently evaluated in clinical phase . From a biochemical standpoint, MGL has been largely investigated over the years, often with a focus on allosteric regulation and oxidative stress. , Only recently, systematic investigations on the role of active site residues of MGL during the catalytic cleavage of 1 have appeared in the literature . Despite these recent advances, an atomistic understanding of the catalytic mechanism employed by MGL is still lacking.…”

“…In recent years, the search for potent inhibitors of MGL has led to the identification of different classes of compounds, 10 , 11 ranging from allosteric cysteine-targeting modulators 12 to orthosteric inhibitors, comprising both covalent agents (i.e., carbamates 13 and tertiary ureas 14 ) and noncovalent inhibitors. 15 Covalent inhibitors of MGL include JZL184 (compound 2 , Figure 1 ) 16 reported as the first agent able to carbamoylate Ser122, and the first-in-class inhibitor Lu AG06466/ABX-1431 (compound 3 , Figure 1 ), 17 which recently completed a phase 2 clinical trial for the treatment of Tourette’s syndrome 18 and is currently being evaluated for multiple sclerosis.…”

Pyrrolidin‐2‐one linked benzofused heterocycles as novel small molecule monoacylglycerol lipase inhibitors and antinociceptive agents

“…A benzoylpiperidine derivative (compound 23, IC 50 = 80 n m ) is reported as potent and selective reversible MAGL inhibitor (Granchi et al., 2019). A benzisothiazolinone (BTZ) derivative (compound 13; Figure 1) has been revealed recently as MAGL inhibitor (IC 50 = 34.1 n m ), targeting allosteric regulatory cysteine residues (Cys201 and Cys208; Castelli et al., 2020). Our group have identified two novel human MAGL inhibitors, ZINC24092691 (IC 50 = 10 n m ) and ZINC12863377 (IC 50 = 39 n m ), by docking‐based virtual screening of ZINC database.…”