Nonredox 5-Lipoxygenase Inhibitors Require Glutathione Peroxidase for Efficient Inhibition of 5-Lipoxygenase Activity

Abstract

Nonredox type 5-lipoxygenase (5-LO) inhibitors, such as ZM 230487, its methyl analogue ZD 2138, or the Merck compound L-739,010, suppress cellular leukotriene synthesis of ionophore stimulated granulocytes with IC 50 values of about 50 nM. However, in cell homogenates or in preparations of purified enzyme, up to 150-fold higher concentrations are required for similar inhibition of 5-LO activity. This loss of 5-LO inhibition in cell homogenates was reversed by addition of glutathione or dithiothreitol, which increased the inhibitory potency of ZM 230487 or L-739,010 by about 100 to 150-fold so that 5-LO inhibition was comparable with that of intact cells. In the presence of thiols, addition of hydroperoxide [13(S)-HpODE], glutathioneperoxidase inhibition by iodacetate or selenium-deficiency lead to impaired 5-LO inhibition by ZM 230487 in cell homogenates. Moreover, addition of glutathione peroxidase was required for efficient inhibition of purified human 5-LO by ZM 230487. The data suggest that low hydroperoxide concentrations are important for efficient 5-LO inhibition by ZM 230487. The kinetic analysis revealed a noncompetitive inhibition of 5-LO by ZM 230487 at low hydroperoxide levels, whereas it acted as a competitive inhibitor with low affinity under nonreducing conditions in granulocyte homogenates. No such redox-dependent effects were observed with the 5-LO inhibitor BWA4C, the 5-LO activating protein-inhibitor MK-886 or the pentacyclic triterpene acetyl-11-keto-␤-boswellic acid. These data suggest that physiological conditions associated with oxidative stress and increased peroxide levels lead to impaired efficacy of nonredox type 5-LO inhibitors like ZM 230487 or L-739,010. This could explain the reported lack of activity of this class of 5-LO inhibitors in chronic inflammatory processes.

Inhibition of 5-Lipoxygenase Product Synthesis by Natural Compounds of Plant Origin

“…Nonetheless, representatives out of this class such as the orally active compounds ZD 2138 5 or its ethyl analogue ZM 230 487 6 or L-739.010 8 are highly potent and selective for 5-LO in cellular assays [24]. We found that elevated peroxide levels and/or phosphorylation of 5-LO by MAPKAPK-2 and/or ERKs strongly impaired the potency of non-redox-type 5-LO inhibitors in activated PMNL [74], [75], [76].…”

“…It is still unclear if the bindingsite of these compounds is in fact the AA substrate-binding cleft in the active site. Thus, experimental data from molecular and biochemical studies suggest an allosteric mode of action [74]. Nonetheless, representatives out of this class such as the orally active compounds ZD 2138 5 or its ethyl analogue ZM 230 487 6 or L-739.010 8 are highly potent and selective for 5-LO in cellular assays [24].…”

“…The most-recognized PTs that act on 5-LO are BAs, and many studies addressed the respective molecular interactions (for detailed review see [153], [156]). BAs with an 11-keto moiety, preferably 3-O-acteyl-11-keto-b-BA (AKBA 89, Table 6) are of particular interest, and AKBA 89 is the most efficient derivative with IC 50 values 1.5 ± 15 mM in intact cells [74], [154], [157], [158], [159]. Studies using isolated 5-LO or other types of cell-free assays showed that AKBA 89 is a direct, non-redox-type 5-LO inhibitor (IC 50 values 8 ± 50 mM) [74], [154], [159].…”

“…BAs with an 11-keto moiety, preferably 3-O-acteyl-11-keto-b-BA (AKBA 89, Table 6) are of particular interest, and AKBA 89 is the most efficient derivative with IC 50 values 1.5 ± 15 mM in intact cells [74], [154], [157], [158], [159]. Studies using isolated 5-LO or other types of cell-free assays showed that AKBA 89 is a direct, non-redox-type 5-LO inhibitor (IC 50 values 8 ± 50 mM) [74], [154], [159]. The discrepancies in the potency of AKBA 89 for 5-LO inhibition (1.5 ± 50 mM) reported from different studies may be due to different experimental settings such as cell type, species and enzyme source (purified 5-LO, crude homogenates).…”

Activation of 5-lipoxygenase by cell stress is calcium independent in human polymorphonuclear leukocytes

“…However, experiments with ZM230487, a selective nonredox 5-LO inhibitor that blocks enzyme activity at low concentrations (Ͻ1 M) only in intact cells, 30 revealed that cell stressinduced 5-LO product formation occurred in intact cells and was not related to crude 5-LO activity after cell destruction (data not shown). …”