Leukotrienes are key inflammatory mediators associated with pathological states of inflammation in diseases such as asthma and allergic rhinitis and play a pivotal role in normal host defense
]. They have been shown to promote leukocyte chemotaxis and activation, vascular tone and permeability, smooth muscle contractility and immune function. 5-lipoxygenase (5-LO) is the key enzyme of leukotriene biosynthesis and so is a promising target for drug development
5-LO is expressed predominantly in leukocytes and is responsible for the synthesis of both leukotriene A4 (LTA4) and 5(S)-hydroperoxy-6,8,1l,14-(E,Z,Z,Z)-eicosatetraenoic acid (HP)
]. The reaction scheme is given in Figure
. There are two steps in this reaction: oxygenation of arachidonic acid (AA) using O2
to produce HP and the dehydration of the hydroperoxide intermediate, to produce the epoxide, leukotriene A4 (LTA4). HP can be further converted either to 5-hydroxyeicosatetraenoic acid (HT) by glutathione peroxidase (GPx)
]. HT, in turn, can be converted to 5-Oxo-6E,8Z,11Z,14Z-eicosatetraenoic acid (oxoETE) by 5-hydroxyeicosanoid dehydrogenase (HEDH)
]. oxoETE is produced by various cells including neutrophils, eosinophils, and monocytes
] and acts as a potent chemo-attractant for these cell types. For example, 5oxoETE stimulates eosinophil migration and tissue infiltration 30 fold more potently than leukotriene B4 (LTB4)
], and also increases intracellular calcium (Ca2+
) concentration and actin polymerization in eosinophils
Figure 1 Schematic representation of Leukotriene and oxoETE synthesis model (“LOS model”). The reactions occurring with 5-LO are in the dashed red circle. Blue rectangular represents metabolites which are variables of “LOS model”. (more ...)
5-LO activity is controlled by the intracellular Ca2+
concentration and the cellular redox state
]. The redox state modulates activity via the iron (Fe) atom located in the catalytic site of the enzyme. HP and other lipid peroxides are able to oxidize the Fe atom from Fe2+
(ferrous state) to the active Fe3+
(ferric state). Consistent with this the addition of glutathione peroxidase (GPx) inhibits formation of products of 5-LO catalyzed reactions in vitro
]. Similar to other lipoxygenases, 5-LO also demonstrates redox state dependent hydroperoxidase activity
]: ferrous 5-LO reacts with lipid-hydroperoxide to form ferric 5-LO, an oxygen-centered lipid radical and hydroxide ion. Additionally, 5-LO activity, in the presence of Ca2+
, is increased by structural stabilization via ATP (adenosine-5’-triphosphate) without ATP hydrolysis, microsomal membranes or phosphatidylcholine vesicles (PtdCho)
]. Mitogen activated protein MAP kinase cascade activity, nuclear import and export of 5-LO, interaction with coactosin-like protein (CLP), 5-LO activating protein (FLAP) and phosphorylation of the enzyme by protein kinase A, resulting in suppression of 5-LO activity, also have been reported to modulate it’s activity
Most of the known inhibitors of the 5-LO act on the basis of a redox-mechanism or by chelation of the Fe atom of 5-LO
]. Redox inhibitors reduce the Fe atom from active ferric state to the inactive ferrous state. The complexity of 5-LO regulation and the multiple reaction paths suggests that redox and non-redox inhibitors may have qualitatively and quantitatively different effects on the products of 5-LO catalyzed reactions.
Zileuton (Z, N-(l-benzo(b)thien-2-ylethyl)-N-hydroxyurea or Zyflo IR/CR®), is a redox inhibitor of 5-LO currently approved for the daily treatment of asthma in adults and children
]. Zileuton has a sub-optimal pharmacokinetic and pharmacodynamic profile resulting in a high total daily dose (2400mg) and frequent dosing (4 times a day [q.i.d] for Zyflo IR® and twice a day [b.i.d] for Zyflo CR®)
], plus a potential for hepatotoxicity
]. Therefore, the development of a medicine with more convenient dosing regimen may maximise the benefits of inhibiting the leukotriene pathway and provide efficacy superior to that obtained with zileuton. PF-4191834, 4-(3-(4-(1-methyl-1H-pyrazol-5-yl)phenylthio)phenyl)-tetrahydro-2H-pyran-4-carboxamide (PF), is a novel non-iron chelating, non-redox, 5-LO inhibitor under investigation for the treatment of various inflammatory conditions
]. The presence of significant data in the public domain for zileuton and PF suggested that development of a mathematical model would allow insight into the potential differential effects of non-redox and redox inhibitors. To characterize these potential effects it was necessary to attempt to capture the key properties of the entire pathway and its interactions in inflammatory states Therefore, a detailed mathematical model, describing the processes of 5-LO mediated catalysis regulation (self-inactivation, effect of redox state of the medium) was developed.
Several mathematical models of 5-LO have already been reported, for example a model describing the inhibition of lipoxygenase activity by one of the substrates of the enzyme, AA, by Aharony et al.
]. In this model, 5-LO is able to bind 2 molecules of AA simultaneously (one molecule in the catalytic site and other molecule in the additional regulatory site) which renders the enzyme catalytically inactive. However, LTA synthesis and the pseudo-peroxidase reaction have not been taken into account in this model. An alternative model of reticulocyte lipoxygenase was developed with 9,12(Z,Z)-octadecadienoic acid (linoleic acid) as substrate
]. This model did take into account both the activation of the enzyme by product, hydroperoxy derivative of fatty acid, and inhibition of 5-LO with substrate, polienoic fatty acid. However, the substrate inhibition was described in terms of a competitive mechanism, binding to inactive form of 5-LO thus preventing its activation, and the LTA4 synthase activity of the enzyme was not captured. Additionally, none of the models describe the reversible inactivation of 5-LO with HT
] and irreversible inactivation with LTA4
The rate laws for lipoxygenase and LTA4 synthase reactions were derived by Yang et al.
], where the influence of various inhibitors of 5-LO and cyclooxygenase on AA metabolism were determined. These rate equations take into account the inhibition of 5-LO activity by LTA4, HP and HT. However, the inhibition with LTA4 was also described as reversible and the substrate inhibition, product activation and pseudo-peroxidase activity of 5-LO were not taken into account.
Therefore, an opportunity exists to develop a more detailed model of 5-LO activity which describes all the activities of the enzyme and their regulation by substrates and products. The main purpose of this paper is to summarize the development of a detailed mathematical model of 5-LO operation, its application to describe the production of LTA4 and oxoETE, and to study the differences between redox and non-redox inhibitors. The “LOS (Leukotriene-OxoeETE-Synthesis) model” (Figure
) includes four enzymes: 5-LO, cytosolic phospholipase A2 (cPLA2), GPx and HEDH and describes the major interactions between the components of the system (for example, the influence of glutathione concentration on 5-LO activity). We used the “LOS model” to predict the dose-responses of various inflammatory mediators to redox and non-redox inhibitors and provide a mechanistic explanation for the differences between them.