The molecular mechanism of the inhibition by licofelone of the biosynthesis of 5-lipoxygenase products

Abstract

Background and purpose: Licofelone is a dual inhibitor of the cyclooxygenase and 5-lipoxygenase (5-LO) pathway, and has been developed for the treatment of inflammatory diseases. Here, we investigated the molecular mechanisms underlying the inhibition by licofelone of the formation of 5-LO products.

Experimental approach: The efficacy of licofelone to inhibit the formation of 5-LO products was analysed in human isolated polymorphonuclear leukocytes (PMNL) or transfected HeLa cells, as well as in cell-free assays using respective cell homogenates or purified recombinant 5-LO. Moreover, the effects of licofelone on the subcellular redistribution of 5-LO were studied.

Key results: Licofelone potently blocked synthesis of 5-LO products in Ca(2+)-ionophore-activated PMNL (IC(50)=1.7 microM) but was a weak inhibitor of 5-LO activity in cell-free assays (IC(50)>>10 microM). The structures of licofelone and MK-886, an inhibitor of the 5-LO-activating protein (FLAP), were superimposable. The potencies of both licofelone and MK-886 in ionophore-activated PMNL were impaired upon increasing the concentration of arachidonic acid, or under conditions where 5-LO product formation was evoked by genotoxic, oxidative or hyperosmotic stress. Furthermore, licofelone prevented nuclear redistribution of 5-LO in ionophore-activated PMNL, as had been observed for FLAP inhibitors. Finally, licofelone as well as MK-886 caused only moderate inhibition of the synthesis of 5-LO products in HeLa cells, unless FLAP was co-transfected.

Conclusions and implications: Our data suggest that the potent inhibition of the biosynthesis of 5-LO products by licofelone requires an intact cellular environment and appears to be due to interference with FLAP.

Figure 1

Inhibition of 5-LO product synthesis by licofelone and MK-886 in intact PMNL. Freshly isolated PMNL (7.5 × 10⁶ ml⁻¹ PGC buffer) were preincubated with licofelone (a) or MK-886 (b) as indicated at 37°C. After 15 min, 2.5 μM ionophore plus the indicated concentrations of AA were added and after 10 min, the formation of 5-LO products was determined as described. Values are given as mean+s.e., n=3–4. 5-LO product synthesis in the absence of test compounds (100%, control) was 37.4±3.8 (ionophore), 57.9±5.7 (ionophore+3 μM AA), 105.1±8.4 (ionophore+10 μM AA), 176.6±31.1 (ionophore+30 μM AA), 145.1±24.4 (ionophore+60 μM AA) and 105.8±30.5 (ionophore+90 μM AA) ng per 10⁶ PMNL. AA, arachidonic acid; 5-LO, 5-lipoxygenase; PGC buffer, PBS containing 1 mg ml⁻¹ glucose and 1 mM CaCl₂; PMNL, polymorphonuclear leukocytes.

Figure 2

Comparison of the structures of licofelone and MK-886. (a) Chemical structures of licofelone and MK-886. (b) Flexible alignment of licofelone and MK-886 showing mutual PPPs. Green shapes represent lipophilic features, red ones show potential hydrogen-bond acceptors, and the polar group (carboxy function) is displayed in magenta. PPP, potential pharmacophore point.

Figure 3

Inhibition of 5-LO activity by licofelone in cell-free assays; influence of DTT. (a) Inhibition of the activity of 5-LO in intact PMNL, in homogenates of PMNL, and of purified 5-LO. Intact PMNL were preincubated with the test compounds (or DMSO as vehicle, w/o) for 15 min before addition of 2.5 μM ionophore plus 40 μM AA. For determination of the inhibition of the activity of 5-LO in homogenates or of purified human recombinant 5-LO, test compounds were added to the incubations 5 min before addition of 1 mM Ca²⁺ plus 40 μM AA. Values are given as mean+s.e., n=4; data were analysed by ANOVA followed by Tukey-HSD post hoc test: ^***P<0.001 vs control (vehicle). (b) Influence of DTT on the efficacy of licofelone for 5-LO inhibition in a cell-free assay. Homogenates of 7.5 × 10⁶ PMNL in PG buffer containing 1 mM EDTA were prepared by sonication. ATP (1 mM) and licofelone together with or without 1 mM DTT was added and samples were kept on ice for 5–10 min. Samples were preincubated at 37°C for 30 s before addition of 2, 10 or 40 μM AA plus 2 mM CaCl₂, each. After 10 min, 5-LO products formed were determined. Values are given as mean+s.e., n=4; data were analysed by ANOVA followed by Tukey-HSD post hoc test. P>0.05. AA, arachidonic acid; DMSO, dimethylsulphoxide; DTT, dithiothreitol; 5-LO, 5-lipoxygenase; PG buffer, PBS containing 1 mg ml⁻¹ glucose; PMNL, polymorphonuclear leukocytes.

Figure 4

The potency of licofelone depends on the stimulus to activate 5-LO. PMNL (7.5 × 10⁶ in 1 ml PGC buffer, final volume) were preincubated with licofelone or MK-886 (or DMSO as vehicle, w/o) as indicated at 37°C. After 12 min, cells were pretreated with 10 μM SA, 500 μM diamide or 300 mM NaCl or left untreated. After 3 min untreated and diamide-treated cells were stimulated with 2.5 μM ionophore plus 40 μM AA, and SA- or NaCl-treated cells were supplemented with 40 μM AA. After another 10 min, the formation of 5-LO products was determined. 5-LO product synthesis in the absence of test compounds (100%, control) was 177.1 ± 20.2 (ionophore+AA), 145.0±56.0 (ionophore+diamide+AA), 55.4±7.6 (SA+AA), and 61.7±11.8 (NaCl+AA) ng per 10⁶ PMNL. Values are given as mean+s.e., n=4–5; data were analysed by ANOVA followed by Tukey-HSD post hoc test: ^**P<0.01, ^***P<0.001 vs vehicle control (w/o). AA, arachidonic acid; DMSO, dimethylsulphoxide; 5-LO, 5-lipoxygenase; PGC buffer, PBS containing 1 mg ml⁻¹ glucose and 1 mM CaCl₂; PMNL, polymorphonuclear leukocytes; SA, sodium arsenite.

Figure 5

Licofelone inhibits translocation of 5-LO to the nuclear membrane. (A) Freshly isolated PMNL (3 × 10⁷ ml-1 PGC buffer) were preincubated with licofelone as indicated at 37°C. After 15 min, 2.5 μM ionophore was added and after 10 min, the samples were chilled on ice. After detergent (0.1% NP-40) lysis and subcellular fractionation, 5-LO was determined in nuclear and non-nuclear fractions by western blotting. The results shown are representative of at least three independent experiments. (B) Indirect immunofluorescence microscopy. PMNL were preincubated with licofelone (10 μM) or MK-886 (300 nM) or vehicle (DMSO) as indicated, centrifuged onto poly-L-lysine-coated glass coverslips and activated by ionophore A23187 (2.5 μM) as indicated. After 3 min, cells were fixed, permeabilized and incubated with anti-5-LO serum (1551, AK-7). After addition of Alexa Fluor 488 goat anti-rabbit IgG the fluorescence was analysed. Left panel: staining for 5-LO; middle panel: staining for nuclei (DAPI); right panel: overlay of 5-LO and nuclear staining. (a) Vehicle, (b) vehicle+A23187, (c) licofelone+A23187, (d) MK-886+A23187. The photographs shown are representative of four similar samples. DAPI, diamidino-2-phenylindole; DMSO, dimethylsulphoxide; G; 5-LO, 5-lipoxygenase; PGC buffer, PBS containing 1 mg ml⁻¹ glucose and 1 mM CaCl₂; PMNL, polymorphonuclear leukocytes.

Figure 6

Licofelone is a potent inhibitor of FLAP-mediated 5-LO product formation. HeLa cells were transiently transformed with plasmids pcDNA3.1–5LO, pSG5-FLAP and pSG5 empty vector (10 μg, each) as indicated. (a) Expression of 5-LO and FLAP. HeLa cells (2 × 10⁶) were re-suspended in 100 μl PGC buffer, mixed with the same volume of SDS-b and heated for 6 min at 95°C. Samples were analysed by western blotting for 5-LO and FLAP proteins. (b) HeLa cells (2 × 10⁶) transfected with 5-LO alone were re-suspended in 1 ml PGC buffer and incubated with licofelone or MK-886 (or DMSO as vehicle, w/o) as indicated. After 15 min at 37°C, cells were stimulated with 5 μM ionophore plus 2, 10 or 40 μM AA for another 10 min. 5-LO products were extracted and determined by HPLC. (c) HeLa cells (2 × 10⁶) transfected with 5-LO plus pSG5 empty vector (5-LO/- (3)) or with 5-LO plus FLAP (5-LO/FLAP (2)) were re-suspended in 1 ml PGC buffer and incubated with 3 μM licofelone or 1 μM MK-886 (or DMSO as vehicle, w/o) as indicated. After 15 min at 37°C, cells were stimulated with 5 μM ionophore plus 2, 10 or 40 μM AA for another 10 min. 5-LO products were extracted and determined by HPLC. Results are given as mean+s.e., n=3–4; data were analysed by ANOVA followed by Tukey-HSD post hoc test: ^*P<0.05, ^**P<0.01, ^***P<0.001 vs vehicle control (w/o). AA, arachidonic acid; DMSO, dimethylsulphoxide; FLAP, 5-lipoxygenase-activating protein; 5-LO, 5-lipoxygenase; PGC buffer, PBS containing 1 mg ml⁻¹ glucose and 1 mM CaCl₂; SDS-b, 2 × sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS–PAGE) sample loading buffer.