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

Abstract

Palmitoylethanolamide (PEA) and oleoylethanolamide (OEA) are endogenous lipid mediators that suppress inflammation. Their actions are terminated by the intracellular cysteine amidase, N-acylethanolamine acid amidase (NAAA). Even though NAAA may offer a new target for anti-inflammatory therapy, the lipid-like structures and reactive warheads of current NAAA inhibitors limit the use of these agents as oral drugs. A series of novel benzothiazole-piperazine derivatives that inhibit NAAA in a potent and selective manner by a non-covalent mechanism are described. A prototype member of this class (8) displays high oral bioavailability, access to the central nervous system (CNS), and strong activity in a mouse model of multiple sclerosis (MS). This compound exemplifies a second generation of non-covalent NAAA inhibitors that may be useful in the treatment of MS and other chronic CNS disorders.

Keywords: N-acylethanolamine acid amidase; cysteine hydrolase; fatty acylethanolamides; multiple sclerosis; neuroinflammation.

Figure 1

NAAA hydrolyzes saturated and monounsaturated fatty acid ethanolamides (e.g., PEA, 1) into fatty acid (e.g., palmitic acid, 2) and ethanolamine.

Figure 2

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

Figure 3

Compound 8 inhibits NAAA via a non-covalent mechanism. (A) LC-MS tracings showing (top) that the covalent inhibitor 4 forms an adduct with a peptide containing NAAA's catalytic C126 (C126TSIVAQDSR), as illustrated in the inset, whereas (bottom) 8 or its vehicle (DMSO) have no such effect. (B) Covalent inhibitor 4 (top) or 8 (bottom) was incubated with NAAA (filled bars) or buffer alone (open bars) and quantified in supernatant after protein precipitation. Bars: mean ± SEM, n =3. (C) Lysosomal extracts of hNAAA-overexpressing HEK293 cells were incubated with vehicle (2% DMSO), 4 or 8 for 2 h before addition of probe 5. A rhodamine fluorophore was inserted by click chemistry. The arrowhead indicates NAAA. Top, fluorescence; bottom, Coomassie blue staining (loading control).

Figure 4

(A) Michaelis–Menten analysis of the reaction of hNAAA in the presence of vehicle (DMSO, black circle) or 8 (50 nM, filled triangle; 200 nM, filled square). (B) Lineweaver–Burk plot indicates uncompetitive inhibition (inset: magnification of the plot close to its origin).

Figure 5

PK and pharmacodynamic profiles of 8 in mice. (A) Levels of 8 in plasma (black circles) or brain (gray triangles) after oral administration (3 mg/kg). (B) Time course of the effects of 8 (30 mg/kg) on PEA, OEA and anandamide (AEA) levels in brain. Results are expressed as mean ± SEM, n = 3. * P< 0.05; **, P < 0.01; ***, P < 0.001, one-way ANOVA.

Figure 6

Time-course of the effects of 8 or vehicle on (A) clinical score and (B) body weight in EAE mice and sham-immunized controls. 8 was administered at 30 mg/kg twice daily. MOG: myelin oligodendrocyte glycoprotein 35–55, antigen used to induce EAE. Results are expressed as mean ± SEM, n = 30. * P< 0.05; **, P < 0.01; ***, P < 0.001, two-way ANOVA followed by Bonferroni post hoc test.

Scheme 1

a) (2S)-2-methylpiperazine, NaHCO₃, EtOH/ H₂O, reflux, 15 h, quant.; b) EtI, 2M NaOH, EtOH, rt, 15 h, quant.; c) Oxone, H₂O, 40°C, 15 h, 96%; d) HATU, Et₃N, CH₃CN, rt, 15 h, 43%.