The ceramides are a family of bioactive lipid-derived messengers involved in the control of cellular senescence, inflammation, and apoptosis. Ceramide hydrolysis by acid ceramidase (AC) stops the biological activity of these substances and influences survival and function of normal and neoplastic cells. Because of its central role in the ceramide metabolism, AC may offer a novel molecular target in disorders with dysfunctional ceramide-mediated signaling. Here, a class of benzoxazolone carboxamides is identified as the first potent and systemically active inhibitors of AC. Prototype members of this class inhibit AC with low nanomolar potency by covalent binding to the catalytic cysteine. Their metabolic stability and high in vivo efficacy suggest that these compounds may be used as probes to investigate the roles of ceramide in health and disease, and that this scaffold may represent a promising starting point for the development of novel therapeutic agents.

The ceramides are a family of bioactive lipid-derived messengers involved in the control of cellular senescence, inflammation, and apoptosis. Ceramide hydrolysis by acid ceramidase (AC) stops the biological activity of these substances and influences survival and function of normal and neoplastic cells. Because of its central role in the ceramide metabolism, AC may offer a novel molecular target in disorders with dysfunctional ceramide-mediated signaling. Here, a class of benzoxazolone carboxamides is identified as the first potent and systemically active inhibitors of AC. Prototype members of this class inhibit AC with low nanomolar potency by covalent binding to the catalytic cysteine. Their metabolic stability and high in vivo efficacy suggest that these compounds may be used as probes to investigate the roles of ceramide in health and disease, and that this scaffold may represent a promising starting point for the development of novel therapeutic agents.

Background and purpose:  Monoacylglycerol lipase (MGL) is a presynaptic serine hydrolase that inactivates the endocannabinoid neurotransmitter, 2-arachidonoyl-sn-glycerol. Recent studies suggest that cysteine residues proximal to the enzyme active site are important for MGL function. In the present study, we characterize the role of cysteines in MGL function and identify a series of cysteine-reactive agents that inhibit MGL activity with nanomolar potencies by interacting with cysteine residue 208.

Experimental approach:  A series of cysteine traps were screened for the ability to inhibit MGL in vitro. Rapid dilution assays were performed to determine reversibility of inhibition. Molecular modelling and site-directed mutagenesis were utilized to identify cysteine residues targeted by the inhibitors.

Key results:  The screening revealed that 2-octyl-4-isothiazolin-3-one (octhilinone) inhibited purified rat recombinant MGL (IC₅₀= 88 ± 12 nM) through a partially reversible mechanism. Initial structure–activity relationship studies showed that substitution of the n-octyl group of octhilinone with a more lipophilic oleoyl group increased inhibitor potency (IC₅₀= 43 ± 8 nM), while substitution with a methyl group produced the opposite effect (IC₅₀= 239 ± 68 nM). The inhibitory potency of octhilinone was selectively decreased by mutating cysteine 208 in MGL to glycine (IC₅₀; wild-type, 151 ± 17 nM; C208G, 722 ± 74 nM), but not by mutation of other cysteine residues (C32, C55, C201, C208 and C242).

Conclusions and implications:  The results indicated that cysteine 208 plays an important role in MGL function and identified a novel class of isothiazolinone-based MGL inhibitors with nanomolar potency in vitro.

The fatty acid ethanolamides are a class of signaling lipids that include agonists at cannabinoid and α type peroxisome proliferator-activated receptors (PPARα). In the brain, these compounds are primarily hydrolyzed by the intracellular serine enzyme fatty acid amide hydrolase (FAAH). O-aryl carbamate FAAH inhibitors such as URB597 are being evaluated clinically for the treatment of pain and anxiety, but interactions with carboxylesterases in liver might limit their usefulness. Here we explore two strategies aimed at overcoming this limitation. Lipophilic N-terminal substitutions, which enhance FAAH recognition, yield potent inhibitors but render such compounds susceptible to attack by broad-spectrum hydrolases and inactive in vivo. By contrast, polar electron-donating O-aryl substituents, which decrease carbamate reactivity, yield compounds, such as URB694, that are highly potent FAAH inhibitors in vivo and less reactive with off-target carboxylesterases. The results suggest that an approach balancing inhibitor reactivity with target recognition produces FAAH inhibitors that display significantly improved drug-likeness.