β-Lactones Inhibit N-acylethanolamine Acid Amidase by S-Acylation of the Catalytic N-Terminal Cysteine

Abstract

ABSTRACT:The cysteine amidase N-acylethanolamine acid amidase (NAAA) is a member of the N-terminal nucleophile class of enzymes and a potential target for anti-inflammatory drugs. We investigated the mechanism of inhibition of human NAAA by substituted β-lactones. We characterized pharmacologically a representative member of this class, ARN077, and showed, using high-resolution liquid chromatography−tandem mass spectrometry, that this compound forms a thioester bond with the N-terminal catalytic cysteine in human NAAA. KEYWORDS: NAAA, cysteine amidase, covalent inhibitors, high resolution mass spectrometry, proteomics T he fatty acid ethanolamides (FAEs) are a family of bioactive lipid molecules that have attracted considerable interest due to their potential role in the control of pain, inflammation, and energy metabolism. Two structurally and functionally distinct classes of FAEs have been described. Polyunsaturated FAEs, such as arachidonoylethanolamide (anandamide) and eicosapentaenoylethanolamide, are endogenous agonists of G protein-coupled cannabinoid receptors. 1,2 These compounds are thought to act as retrograde messengers at synapses of the central nervous system and as local mediators in peripheral tissues. In the brain, anandamide-mediated signaling at CB 1 type cannabinoid receptors has been implicated in the regulation of anxiety, 3 depression, 4,5 stress-induced analgesia, 6 and nausea. 7 Outside the brain, anandamide released at sites of tissue injury is thought to control the initiation of emerging pain signals, 8,9 presumably by reducing the local release of proalgesic and proinflammatory factors from nociceptive nerve terminals. 10 The biological actions of anandamide are interrupted by a two-step deactivation process consisting of carriermediated transport into cells 11,12 followed by intracellular hydrolysis, which is catalyzed by the membrane-bound serine hydrolase, fatty acid amide hydrolase (FAAH). Inhibitors of this enzyme protect anandamide from deactivation and selectively magnify its intrinsic actions, producing an array of pharmacological effects that include reduced anxiety, depression, and pain. 13 Saturated and monounsaturated FAEs, which include oleoylethanolamide (OEA) and palmitoylethanolamide (PEA), do not productively interact with cannabinoid receptors. These compounds are, however, potent or moderately potent agonists of nuclear peroxisome proliferator-activated receptor-α (PPAR-α), which is responsible for most of their analgesic and anti-inflammatory properties. 14−16 OEA and PEA are produced in many mammalian tissues, including neurons 17 and innate immune cells, 18 where a selective phospholipase D releases them by cleaving the membrane precursor, N-acyl-phosphatidylethanolamine. 19 They are deactivated either by FAAH, which prefers anandamide and OEA over PEA, or by NAAA, which conversely prefers PEA and OEA over anandamide. 20 Despite its functional similarity with FAAH, NAAA shares no homology with this or other enzymes of the same "amidase signature" family. Rather,...

Therapeutic Potential of Fatty Acid Amide Hydrolase, Monoacylglycerol Lipase, and N-Acylethanolamine Acid Amidase Inhibitors

“…131 Mass spectrometry studies supposed that the nucleophilic thiol group of the Cys131 of rat NAAA interact the carbonyl of β-lactone group to form an acyl-NAAA intermediate. 7,131,132 The β-lactone group has been shown to react readily with nucleophiles, and can be rapidly hydrolyzed in aqueous solution. In fact, most amide-based β-lactone derivatives were rapidly hydrolyzed in aqueous buffer at pH 7.4 with half-lives of less than 20 minutes, whereas the β-substituted methyl group endowed the β-lactone derivatives with improved stability (half-life > 70 min).…”

“…135 Compound 111 rapidly, non-competitively, covalently and reversibly combined with rat NAAA, whereas the inhibition of human NAAA was only partially reversible. 132 The binding mechanism studies indicated that the thiol group of the active cysteine (Cys131 for rat NAAA, Cys126 for human NAAA)…”

A simple and accurate protocol for absolute polar metabolite quantification in cell cultures using quantitative nuclear magnetic resonance

“…Heck293-WT cells, Hek293 cells overexpressing hAC1 (Hek293-hAC1) [19], and Hek293 cells overexpressing human NAAA (Hek293-hNAAA) [20] were plated in 150-mm dishes (5 Â 10 6 cells/ dish) and cultured in complete DMEM containing 10% fetal bovine serum, 2 mM L-glutamine, and antibiotics (100 U/ml penicillin and streptomycin) at 37 C and 5% CO 2 . Cell media were collected 48 h after incubation.…”

Synthesis, Biological Evaluation, and 3D QSAR Study of 2-Methyl-4-oxo-3-oxetanylcarbamic Acid Esters as N-Acylethanolamine Acid Amidase (NAAA) Inhibitors

“…In this regard, it should be pointed out that a hopping exercise aimed at identifying completely novel chemical scaffolds was outside the scope of the present 3D QSAR study. Furthermore, given the covalent nature of the interaction established by these compounds with the enzyme, 36 fair activity predictions based on structure can only be limited to compounds sharing the same mechanism of covalent inhibition. However, each new compound displayed a new structural feature, which incrementally expanded our knowledge of the shape of the pocket, particularly relevant also in light of the lack of a NAAA crystal structure to be utilized in structure-based studies.…”

“…32 Structure−activity relationship (SAR) studies of α-amino-β-lactone derivatives, as carbamic acid esters, investigated the effects on NAAA inhibition of side chain modifications and the stereochemical requirements of the introduction of a β-substitution. 33−35 These works led to the identification of compounds that were highly potent at inhibiting both rat and human NAAA, such as β-lactones 1 (ARN077) 33,34,36 and 2…”