Figure 1.
The strategy of developing NAAA inhibitors.
A–C, the chemical structures of classic NAAA inhibitors including PEA (A), CCP (B), and (S)-OOPP (C); (D) SAR study of 1-Pentadecanyl-carbonyl pyrrolidine; (E) Dose-dependent inhibition of 1-Pentadecanyl-carbonyl pyrrolidine on NAAA activity.
Figure 2.
Compound 16 interacted with NAAA protein.
(A) Computational model illustrated docking of compound 16 at the active site of rat NAAA. (B) Effect of mutant Ala209-NAAA on NAAA activity. Mock, HEK293 cell heterogeneously overexpressing vector control; NAAA, HEK293 cell heterogeneously overexpressing NAAA; Ala209, HEK293 cell heterogeneously overexpressing mutant Ala209-NAAA. ***, P<0.001 vs. NAAA, n = 5.
Figure 3.
Characterization of compound 16 as a reversible and competitive NAAA inhibitor.
(A) Effect of compound 16 (10 µM) on NAAA activity in HEK293 cells heterogeneously overexpressing NAAA. ***, P<0.001 vs. vehicle, n = 4. (B) Concentration-dependent inhibition of NAAA by compound 16 using NAAA recombinant protein derived from HEK293 cell heterogeneously expressing NAAA. (C) Rapid dilution NAAA assay in the presence of vehicle (1% DMSO, open circles) or compound 16 (closed circles). (D) Effect of NAAA activity in the presence of vehicle (open bars) or compound 16 (closed bars) before dialysis (0) and 8 hr after dialysis (8). ***, P<0.001 vs vehicle, n = 4; (E) Michaelis-Menten analysis of the NAAA reaction in the presence of vehicle (open circles) or compound 16 (closed circles). Insert is shown in a Lineweaver-Burk plot.
Figure 4.
Compound 16 reduced LPS-induced inflammation.
(A) Effect of compound 16 (concentrations in µM) or Vehicle on PEA levels (A), mRNA expression levels of iNOS (B) and IL-6 (C) in RAW264.7 treated with vehicle (open bars) or LPS (closed bars). vehicle, 0.1% DMSO; LPS, 0.5 µg/mL. **, P<0.01; ***, P<0.001 vs. vehicle; ##, P<0.01; ###, P<0.001 vs. LPS control, n = 5.