Molecular pharmacological profile of the nonredox-type 5-lipoxygenase inhibitor CJ-13,610

Abstract

5-Lipoxygenase (5-LO) is a crucial enzyme in the synthesis of the bioactive leukotrienes (LTs) from arachidonic acid (AA), and inhibitors of 5-LO are thought to prevent the untowarded pathophysiological effects of LTs. In this study, we present the molecular pharmacological profile of the novel nonredox-type 5-LO inhibitor CJ-13,610 that was evaluated in various in vitro assays. In intact human polymorphonuclear leukocytes (PMNL), challenged with the Ca(2+)-ionophore A23187, CJ-13,610 potently suppressed 5-LO product formation with an IC(50)=0.07 microm. Supplementation of exogenous AA impaired the efficacy of CJ-13,610, implying a competitive mode of action. In analogy to ZM230487 and L-739.010, two closely related nonredox-type 5-LO inhibitors, CJ-13,610 up to 30 microm failed to inhibit 5-LO in cell-free assay systems under nonreducing conditions, but inclusion of peroxidase activity restored the efficacy of CJ-13,610 (IC(50)=0.3 microm). In contrast to ZM230487 and L-739.010, the potency of CJ-13,610 does not depend on the cell stimulus or the activation pathway of 5-LO. Thus, 5-LO product formation in PMNL induced by phosphorylation events was equally suppressed by CJ-13,610 as compared to Ca(2+)-mediated 5-LO activation. In transfected HeLa cells, CJ-13,610 only slightly discriminated between phosphorylatable wild-type 5-LO and a 5-LO mutant that lacks phosphorylation sites. In summary, CJ-13,610 may possess considerable potential as a potent orally active nonredox-type 5-LO inhibitor that lacks certain disadvantages of former representatives of this class of 5-LO inhibitors.

Figure 1

Chemical structures of nonredox-type 5-LO inhibitors. In comparison to ZD2138 or ZM230487, for CJ-13,610 the dihydroquinolinone pharmacophore was replaced by a imidazolylphenyl moiety, the alkoxy group by a carboxamide group, and the methyl ether was replaced by a sulfide group.

Figure 2

CJ-13,610 suppresses 5-LO product formation in intact human PMNL. (a) Freshly isolated PMNL (7.5 × 10⁶ in 1 ml PGC buffer) were preincubated with CJ-13,610 at the indicated concentrations for 15 min at 37°C. Cells were stimulated with ionophore A23187 (2.5 μM) with or without AA at the indicated concentrations. After 10 min at 37°C, 5-LO products were extracted and determined by HPLC as described in the Methods section. Results are given as mean± s.e., n=4. (b) Kinetic analysis of 5-LO product inhibition by 1 μM CJ-13,610 is given as Lineweaver–Burke plot. The AA concentrations were 2, 5, 10, and 30 μM.

Figure 3

Inhibition of 5-LO by CJ-13,610 in whole-cell homogenates of human PMNL. (a) Whole homogenates of human PMNL were prepared as described, preincubated with the indicated concentrations of CJ-13,610 in the presence or absence of 1 mM DTT for 5–10 min on ice. Samples were prewarmed at 37°C for 30 s, then AA (4, 10, or 40 μM) and 2 mM CaCl₂ were added. After another 10 min at 37°C, 5-LO activity was determined as described in the Methods section. Results are given as mean± s.e., n=3–4. (b) Kinetic analysis of 5-LO product inhibition by 1 μM CJ-13,610 is given as Lineweaver–Burke plot. The AA concentrations were 1, 2, 4, 10, and 20 μM.

Figure 4

Inhibition of purified recombinant 5-LO by CJ-13,610. Human recombinant 5-LO was expressed in E. coli and partially purified as described. 5-LO (0.5 μg) was added to a 5-LO reaction mix containing the indicated amounts of CJ-13,610, 1 mM GSH, and 30 mU GPx. After 5–10 min on ice, the samples were prewarmed for 30 s at 37°C and 2 mM CaCl₂ and AA (4 or 40 μM) was added. After 10 min at 37°C, formed 5-LO products were extracted and determined by HPLC. Results are given as mean± s.e., n=3.

Figure 5

Elevation of the cellular peroxide tone impairs the potency of CJ-13,610 in intact PMNL. Freshly isolated PMNL (7.5 × 10⁶ in 1 ml PGC buffer) were preincubated with CJ-13,610 at the indicated concentrations at 37°C. After 15 min, 13(S)-HPODE was added as indicated and cells were subsequently stimulated with (a) 2.5 μM ionophore A23187 plus 10 μM AA or with (b) 2.5 μM ionophore A23187 alone. After 10 min at 37°C, 5-LO products were extracted and determined by HPLC. Results are given as mean± s.e., n=3.

Figure 6

Cell stress does not affect the efficacy of CJ-13,610 in intact cells. Freshly isolated PMNL (7.5 × 10⁶ in 1 ml PGC buffer) were preincubated with CJ-13,610 at the indicated concentrations for 15 min at 37°C. SA (10 μM) and NaCl (300 mM) were added 3 min prior to the addition of 20 μM AA, ionophore A23187 (2.5 μM) was added together with 20 μM AA. After 10 min at 37°C, 5-LO products were extracted and determined by HPLC. Results are given as mean± s.e., n=3.

Figure 7

Effects of CJ-13,610 on nuclear 5-LO translocation in intact PMNL. Freshly isolated PMNL (3 × 10⁷ in 1 ml PGC buffer) were preincubated with CJ-13,610 at the indicated concentrations for 15 min at 37°C. Then, 2.5 μM A23187 was added to the samples and incubated for another 5 min at 37°C. 5-LO was detected in nuclear and non-nuclear fractions by immunoblotting after subcellular fractionation. Similar results were obtained in two additional independent experiments.

Figure 8

Effects of CJ-13,610 on 5-LO product formation in HeLa cells transformed with WT-5-LO and S271A/S663A-5-LO. HeLa cells were transiently transformed with plasmids pcDNA3.1-5LO or pcDNA3.1-5LO-S271A-S663A (10 μg). Cells (2 × 10⁶) were resuspended in 1 ml PGC buffer and CJ-13,610 was added at the indicated concentrations. After 15 min at 37°C, cells were stimulated with 20 μM AA for another 10 min. 5-LO products were extracted and determined by HPLC. Results are given as mean± s.e., n=4.