Activity-Based Probe for N-Acylethanolamine Acid Amidase

Abstract

N-Acylethanolamine acid amidase (NAAA) is a lysosomal cysteine hydrolase involved in the degradation of saturated and monounsaturated fatty acid ethanolamides (FAEs), a family of endogenous lipid signaling molecules that includes oleoylethanolamide (OEA) and palmitoylethanolamide (PEA). Among the reported NAAA inhibitors, α–amino–β–lactone (3–aminooxetan–2–one) derivatives have been shown to prevent FAE hydrolysis in innate-immune and neural cells and to reduce reactions to inflammatory stimuli. Recently, we disclosed two potent and selective NAAA inhibitors, the compounds ARN077 (5-phenylpentyl N-[(2S,3R)-2-methyl-4-oxo-oxetan-3-yl]carbamate) and ARN726 (4-cyclohexylbutyl-N-[(S)-2-oxoazetidin-3-yl]carbamate). The former is active in vivo by topical administration in rodent models of hyperalgesia and allodynia, while the latter exerts systemic anti-inflammatory effects in mouse models of lung inflammation. In the present study, we designed and validated a derivative of ARN726 as the first activity-based protein profiling (ABPP) probe for the in vivo detection of NAAA. The newly synthesized molecule 1 is an effective in vitro and in vivo click-chemistry activity based probe (ABP), which is able to capture the catalytically active form of NAAA in Human Embryonic Kidney 293 (HEK293) cells overexpressing human NAAA as well as in rat lung tissue. Competitive ABPP with 1 confirmed that ARN726 and ARN077 inhibit NAAA in vitro and in vivo. Compound 1 is a useful new tool to identify activated NAAA both in vitro and in vivo, and to investigate the physiological and pathological roles of this enzyme.

Second‐Generation Non‐Covalent NAAA Inhibitors are Protective in a Model of Multiple Sclerosis

“…Third, we investigated whether 8 prevents the binding of the covalent activity-based probe 5 to NAAA. [9] As previously shown, [9] 5 labeled hNAAA in cell extracts,a nd this effect was blocked by incubation with 4 ( Figure 3C). In contrast, incubation with 8 only partially antagonized hNAAA labeling by 5 and at short incubation times ( Figure 3C).…”

“…Structures of published NAAA inhibitors ( 3 , [6] 4 , [5b] 6 [8] ), activity-based NAAA probe ( 5 ) [9] (top), and compounds reported here (bottom).…”

“…NAAA hydrolyzes saturated and monounsaturated fatty acid ethanolamides (for example, PEA (1)) into fatty acid (for example, palmitic acid (2)) and ethanolamine. (3, [6] 4, [5b] 6 [8] ), activity-based NAAA probe (5) [9] (top), and compounds reported herein (bottom). member of this class,i sp otent, selective for NAAA, and orally available.Moreover, 8 crosses the blood-brain barrier, elevates PEA and OEA levels in the CNS,a nd is strongly active in am ouse model of MS.…”

“…To improve potency, we launched as tructure-activity relationship (SAR) study that started with the benzamide portion of 7.R emoval of the o-methyl group (9), or replacement of such ag roup with ah alogen (10,11), caused ac omplete loss of activity.B yc ontrast, substitutions with am ethoxy (12), methylsulfonyl (13), or ethylsulfonyl (14) group yielded compounds of progressively greater potency. Since moving the ethylsulfonyl substituent to the meta-o r para-position of the phenyl ring strongly reduced activity (15,16), we focused our exploration on o-sulfonyl derivatives containing linear, branched, or cyclic alkyl groups ( Table 2).…”

Second‐Generation Non‐Covalent NAAA Inhibitors are Protective in a Model of Multiple Sclerosis

“… Structures of published NAAA inhibitors ( 3 , 4 , 6 ), activity‐based NAAA probe ( 5 ) (top), and compounds reported herein (bottom).…”