Fig. 1
From: Endogenous metabolites of vitamin E limit inflammation by targeting 5-lipoxygenase
Molecular insights into 5-LO inhibition by LCMs. a Structures of LCMs. b, c Effect of Triton X-100 (0.01%, c) and of the substrate concentration (AA, c) on 5-LO inhibition by δ-TE-13′-COOH (4h) or α-T (1a). d Reversibility of 5-LO inhibition by δ-TE-13′-COOH (4h) and α-T (1a). Samples were pre-incubated with vehicle or compound for 15 min, 10-fold diluted and incubated for another 5 min before AA was added. Numbers in brackets indicate the diluted compound concentration after pre-incubation. e, f 5-LO pull-down by immobilized δ-TE-13′-COOH (4h; GA-Toyo) or immobilized 4-phenoxy butyric acid (ctrl.), which resembles a truncated form of δ-TE-13′-COOH (4h), in the presence of AA or α-T (1a; 100 µM, each, f). g Scavenging of DPPH radicals by δ-TE-13′-COOH (4h). h Molecular docking simulations of 5-LO showing the interaction of δ-TE-13′-COOH (4h) with Arg101 and Trp102 at the interface of the catalytic and regulatory C2-like domains. i Effect of δ-TE-13′-COOH (4h) on the inhibition of 5-LO product formation by HEK-293 cells that stably express either wild-type 5-LO (wt), triple 5-LO mutant Trp13Ala, Trp75Ala, and Trp102Ala (3 W) or Arg101Asp 5-LO. j Inhibition of purified 5-LO by δ-TE-13′-COOH (4h) in the presence of phosphatidylcholine (PC) or phosphatidylethanolamine (PE; 100 µg/ml, each). Mean ± s.e.m. (b, c, g, i, j) and single data (d–f) from n = 3 (b–e, g, i, j), n = 2 (f) independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001 vs. absence of Triton X-100 (b), 20 µM AA (c), vehicle/control (d–g), wt 5-LO (i) or absence of phospholipids (j); two-tailed paired Student t test (b–e, i, j) or repeated measures one-way ANOVA + Tukey HSD post hoc tests (d, f, g)
