©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
The Environment of the Lipoxygenase Iron Binding Site Explored with Novel Hydroxypyridinone Iron Chelators (*)

(Received for publication, August 25, 1995; and in revised form, January 25, 1996)

Rajeewa D. Abeysinghe (1)(§) Pamela J. Roberts (1)(¶) Chris E. Cooper (2)(**) Kirsteen H. MacLean (1) Robert C. Hider (3) John B. Porter (1)(§§)

From the  (1)Department of Clinical Hematology, University College London Medical School, WC1E 6HX London, United Kingdom, the (2)Department of Paediatrics, University College London Medical School, WC1E 6JJ, London, United Kingdom, and the (3)Department of Pharmacy, Kings College London, Manresa Road, London SW3, United Kingdom

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

The mechanisms of lipoxygenase inhibition by iron chelators have been investigated in human neutrophils and in isolated soybean lipoxygenase. Their Fe(III)-containing active sites have been targeted by synthesizing novel bidentate chelators from the hydroxypyridinone family sufficiently small to gain access through the hydrophobic channels of lipoxygenase. In stimulated human neutrophils, release of [^3H]arachidonate-labeled eicosanoids is dependent on the lipid solubility of hydroxypyridinones, but larger hexadentate chelators have no effect on this or on total cellular leukotriene B(4) production. Lipophilic hydroxypyridinones inhibit 5-lipoxygenase at equivalent concentrations to the established inhibitor, piriprost, and show additional but minor anti-phospholipase A(2) activity. Soybean 15-lipoxygenase inhibition is also dependent on the lipid solubility and coordination structure of chelators. Inhibition is associated with the formation of chelate-iron complexes, which are removed by dialysis without restoration of enzyme activity. Only after adding back iron is activity restored. Electron paramagnetic resonance studies show the removal of the iron center signal (g = 6) is concomitant with formation of Fe(III)-chelator complexes, identical in spectral shape and g value to 3:1 hydroxypyridinone Fe(III) complexes. Removal of iron is not the only mechanism by which hydroxypyridinones can inhibit lipoxygenase in intact cells, however, as a lipophilic non-iron-binding hydroxypyridinone, which shows no inhibition of the soybean lipoxygenase activity, partially inhibits 5-lipoxygenase in intact neutrophils without inhibiting neutrophil phospholipase A(2).


INTRODUCTION

Lipoxygenases are non-heme iron-containing enzymes that catalyze the site-specific oxygenation of polyunsaturated fatty acids to produce hydroperoxides. In the human neutrophil, the principal lipoxygenase is 5-LPO, (^1)which catalyzes the first two steps in the conversion of its substrate arachidonic acid to leukotriene A(4) (recently reviewed in (1) ). Leukotriene A(4) is the precursor of the potent inflammatory mediator leukotriene B(4) (LTB(4)). Together with 15-lipoxygenase, 5-lipoxygenase also plays a central role in the formation of lipoxins(2) . Inhibitors of 5-lipoxygenase are of therapeutic interest as anti-inflammatory agents (3, 4) and fall broadly into three categories: first, competitive lipid substrate inhibitors; second, redox-type inhibitors, which act by chelation or reduction of the Fe(III) or by interacting with the fatty acid radical intermediate produced during the catalytic step; and third, indirect inhibitors, which prevent the translocation of 5-lipoxygenase from the cytosol to the nuclear membrane or interact with FLAP, a protein essential for the functioning of 5-lipoxygenase in vivo(5) .

Although it has been known for decades that other lipoxygenases such as the soybean 15-lipoxygenase (sbLPO) contain non-heme Fe(III) essential for their catalytic activity(6, 7, 8) , its presence and function in human 5-LPO was only demonstrated recently(9) . Despite the lack of definitive evidence for an iron center in 5-LPO, compounds with chelating moieties have been used successfully as 5-LPO inhibitors(10, 11) . The Fe(III) center of lipoxygenases plays a central catalytic role, probably by the generation of a fatty acid radical, which subsequently reacts with molecular oxygen to produce the hydroperoxide (12) . Molecules that inhibit human 5-LPO by reduction of Fe(III) to Fe(II), such as naphthols(13) , or by chelation of the iron, such as catechols(14) , are increasingly inhibitory as they become more hydrophobic, although it is not clear whether iron is removed from the active site. Structure-activity studies with non-chelators (15) also indicate that lipid solubility has an important bearing on potency. Apart from lipid solubility, the interaction of Fe(III)-binding chelators with the lipoxygenase iron center has not been studied in detail. In particular, studies with small Fe(III)-chelating molecules that can access the iron center have not been undertaken simultaneously in cellular and cell-free systems to investigate the effects of the iron binding constant, the coordination structure, and hydrophobicity on the hydrophobic lipoxygenase iron center. Further, their effects on enzymes upstream of 5-LPO such as phospholipase A(2) (PLA(2)), the inhibition of which may also inhibit eicosanoid production in cellular systems, have not been investigated in detail. To examine these mechanisms, we have synthesized a series of hydroxypyridinone and hydroxypyranone iron chelators, investigated their inhibitory activity on human neutrophil 5-LPO, and compared it with the inhibition of purified sbLPO.

The hydroxypyridinones and hydroxypyranones have been selected so that the effects of Fe(III) affinity constant (beta(3)) and lipid solubility on lipoxygenase activity can be examined systematically. Their general structures are shown in Fig. 1. Each of these classes are bidentate chelators, three molecules of which coordinate one Fe(III) atom in free solution. However, only a single molecule is required to form ternary complexes with an enzyme-bound Fe(III) atom. The hydroxypyridin-4-ones (HP-4-ones) possess a higher iron binding constant than the hydroxypyridin-2-ones (HP-2-ones), which in turn have higher affinities than the hydroxypyranones (Table 1). By varying the lengths of the alkyl chain substituents in the 1 position of the nitrogen ring, compounds with varying lipid solubility but with identical stability constants for iron (III) have been synthesized (16) . Some hydroxypyridinones also inhibit platelet lipoxygenases at high (millimolar) concentrations(17) , but no formal structure-activity studies have been reported. A non-iron-binding HP-4-one has also been synthesized by substituting one of the iron-binding hydroxyl groups with an ether (Fig. 1). This molecule retains all the properties common to HP-4-ones except the ability to bind iron and forms an useful internal control to dissociate those inhibitory activities that are dependent on iron chelation from those that are independent.


Figure 1: The general structures of the 3-hydroxypyridin-4-ones, 3-hydroxypyridin-2-ones, and 3-hydroxypyranones and the structure of the non-iron-binding HP-4-one analogue Ome-25 are shown. The 3:1 coordination of the iron by bidentate chelators is also illustrated using the HP-4-ones as an example.






EXPERIMENTAL PROCEDURES

Chelating Agents and 5-LPO Inhibitors

The hydroxypyridinone and hydroxypyranone chelators were synthesized as described previously (16, 18) and their purity confirmed by elemental analysis, ^1H NMR, and reversed-phase HPLC. Lipophilicity is quantified by the measurement of the partition (distribution) coefficient (K) of the chelators, which is the ratio of chelator that is extracted into the organic phase when added to an organic/aqueous mixture(16) . The K values of the hydroxypyridinones are shown in Table 1. Desferrioxamine (Desferal) was purchased from Ciba-Geigy (Basel, Switzerland). Piriprost was a kind gift of Dr. M. Bach of Upjohn Ltd. (Kalamazoo, MI). Oxine (8-hydroxyquinoline) and tropolone were from BDH (Poole, UK) and Aldrich (Gillingham, UK), respectively.

Other Reagents

The calcium ionophore used in all the experiments described was A23187 (Sigma). All other reagents were of commercial analytical grade.

Neutrophil Purification

Blood from healthy volunteers was taken into EDTA (2.5 mM), erythrocytes were sedimented with dextran, and neutrophils were isolated by density centrifugation as described previously(19) . The purified neutrophils were suspended in PBS containing CaCl(2) (0.9 mM), MgCl(2) (0.5 mM), D-glucose (5 mM) (PBSG). Neutrophils thus obtained were 98% pure and were 100% viable by trypan blue exclusion.

Extracellular Release of 5-LPO-derived [^3H]Arachidonate-labeled Eicosanoids

The labeling of purified neutrophils with [5,6,8,9,11,12,14,15-^3H]arachidonic acid and the measurement of eicosanoid release was essentially as described previously(20) . Aliquots of labeled purified neutrophils (0.5 ml, 1 times 10^6 ml) were prewarmed to 37 °C (5 min) and incubated with either compounds or their respective diluents for 15 min (or for different times for time courses). All chelators except CP26, (^2)CP84, and oxine were made up in PBS; CP26 and CP84 were made up in dimethyl sulfoxide and added to cells from a 500-fold concentrated stock; oxine was added from a 500-fold stock in ethanol. Eicosanoid synthesis was stimulated with calcium ionophore (1 µM) for 10 min, and the reaction was stopped by placing tubes on ice for 30 min. The samples were spun at 12000 times g for 4 min, and 0.4 ml of supernatant was taken to determine radioactive release by beta-scintillation. Under these conditions the release of radioactivity from unstimulated control cells was less than 5% of total counts up to 4 h of incubation at 37 °C.

PLA(2) Assay

A radiometric assay was used for the quantification of PLA(2), and its validity was confirmed by thin layer chromatography. These methods are described elsewhere(21) .

HPLC Quantification of Total Cellular Eicosanoids

Purified neutrophils (4.5 times 10^6 ml, unlabeled) were incubated for 15 min with chelator or diluent at 37 °C. The reaction was stopped, and the cells were lysed in a single step by the addition of 2 volumes of ice-cold ethanol. Samples were extracted using a procedure specific for the extraction of eicosanoids other than peptidoleukotrienes. This method is essentially identical to the method of Powell (22) with the following modifications. Prostaglandin B(2) (400 ng) (Cascade Biochem Ltd., Reading, UK) was added to the samples before extraction as the internal standard, and Sep-Pak C(18) minicolumns were replaced by those from Alltech Associates (Carnforth, UK). The extracted eicosanoids were reconstituted in the mobile phase (water/methanol/acetic acid (33:67:0.1) and chromatographed by reversed-phase HPLC using a 10-cm C(18) column (ChromSpher, Chrompack Ltd., London) under isocratic conditions (0.2 ml min)(22) .

Measurement of Superoxide Generation by Neutrophils

Purified neutrophils (1 times 10^6 ml) were prewarmed for 5 min at 37 °C in 1-ml plastic spectrophotometer cuvettes and preincubated with chelators for 20 min. Cells were stimulated either with 12-O-tetradecanoylphorbol-13-acetate (TPA) (500 ng ml) or with arachidonic acid (12.5 µM) for 10 min, and the superoxide generated was quantified as an end-product assay of superoxide dismutase-inhibitable reduction of ferricytochrome c(19) .

Inhibition of Soybean Lipoxygenase

Spectrophotometric Studies

sbLPO (type 1B) and its preferential substrate linoleic acid were purchased from Sigma. The inhibition of the enzyme was monitored by a change in the absorption of the product linoleic hydroperoxide at 234 nm in a 3.0-ml quartz cuvette according to the ``low ethanol'' protocol supplied by the manufacturer. Briefly, sbLPO was prepared as a 10,000 units ml stock (^3)in 0.2 M borate buffer, pH 9.0, and linoleic acid as a 3.21 M stock in ethanol. The enzyme (final concentration 500 units/3.0-ml reaction volume) was preincubated with chelator or respective diluent for the appropriate times, and the reaction was initiated by the addition of linoleic acid (667 µM in borate buffer). The rates of reactions in the absence and presence of various compounds were determined using the molar absorption coefficient of the hydroperoxide (e = 23600 M cm)(11) .

Spectrophotometry was also employed to study the formation of iron-chelate complexes and their removal from sbLPO. Hydroxypyridinones and their 3:1 iron complexes have characteristic absorption spectra (23) . The formation and removal of hydroxypyridinone and desferrioxamine iron complexes were monitored against paired blanks, and the iron complexes were quantified using the respective molar absorption coefficients (hydroxypyridinone-iron complex: (max) = 450 nm, = 5200 M cm; ferrioxamine: (max) = 430 nm, = 2640 M cm).

EPR Studies

sbLPO (30 µM) was dissolved in PBS, pH 7.4, and preincubated with the compound of interest (300 or 100 µM iron-binding equivalents) in the presence of linoleic acid (100 µM) at room temperature. After 2 h, aliquots were placed in quartz EPR tubes, frozen in a liquid nitrogen-cooled methanol bath, and stored in liquid nitrogen until analysis. Spectral data were obtained using a spectrometer (Bruker ESP 300) fitted with a liquid helium flow cryostat. Quantification of the amount of iron-chelate complex formed was performed by comparing the size and double integral of the g = 4.3 signal to a standard solution of the iron-chelator complex under identical EPR conditions. The EPR spectra illustrated were taken from sbLPO samples frozen in PBS, pH 7.4; at the higher pH of 9.0 used in the activity assay, it was difficult to quantitate the amount of iron-chelate complex formed due to significant broadening of the g = 4.3 signal. However, the effects of the chelators on the g = 6.0 signal from high spin iron in the sbLPO were identical at pH 7.4 and 9.0.


RESULTS

Effect of Iron Binding Constant on Extracellular Release of Neutrophil 5-LPO-derived [^3H]Arachidonate-labeled Eicosanoids

The effect of a variety of chelators with Fe affinity constants ranging from log beta(3) = 28 to log beta(3) = 37 on eicosanoid release is shown in Table 1. All the 2-methyl-3-hydroxypyridinones and 3-hydroxypyranones tested showed inhibition of eicosanoid synthesis to varying degrees, but the more bulky 2-ethyl-3-hydroxypyridinone CP102 was inactive. The chelators that were the most effective inhibitors were the HP-4-ones CP26, CP25, and CP84, tropolone, and oxine. These five chelators are all bidentate ligands and possess high affinities for Fe (log beta(3) > 35).

Effect of Iron Chelation on 5-LPO-derived Total Cellular Eicosanoid Synthesis in Neutrophils

The inhibition of the extracellular release of [^3H]arachidonate-labeled eicosanoids by HP-4-ones was confirmed by the measurement of total cellular eicosanoid synthesis by HPLC as described under ``Experimental Procedures.'' Under these conditions, the production of total cellular eicosanoids in calcium ionophore-stimulated control cells was 27.2 ± 5.7 pmol/10^6 cells (mean ± S.E., n = 4). There were no detectable levels of eicosanoids in unstimulated cells. In cells preincubated with CP26, CP25, or the positive control piriprost (300 µM, 15 min), followed by ionophore stimulation, no eicosanoids were detected (n = 3). This is illustrated for CP25 in Fig. 2. Under similar conditions, the non-iron-binding analogue Ome-25 produced partial inhibition of 21.74 ± 3.1 pmol/10^6 cells ± S.D. (n = 3). This is consistent with the observations made using radiolabeled neutrophils (Table 1). At lower concentrations (33 µM), CP26-treated cells again produced complete eicosanoid inhibition, but CP25-treated cells produced intermediate inhibition of 11.2 ± 4.1 pmol/10^6 cells (mean ± S.E., n = 3). No inhibition was observed with DFO even at 100 µM (26.3 and 32.6 pmol/10^6 cells, respectively, n = 2).


Figure 2: Inhibition of total cellular eicosanoid synthesis in control and CP25-treated neutrophils analyzed by reversed-phase HPLC. The chromatograms shown on the left were recorded at 280 nm for neutrophils pretreated with diluent (A) or CP25 (300 µM, 100 µM iron-binding equivalents) (B) for 15 min before firing for 10 min with calcium ionophore (1 µM). Spectral scans (220-320 nm) of the eluent peaks are shown on the right. The peaks shown are 2-COOH-LTB4 (a), prostaglandin B(2) internal standard (b), leukotrienes of unknown identity (c and d); LTB4 (e), and an unidentified non-leukotriene peak present in chelator-treated cells (f).



Effect of Lipophilicity on [^3H]Eicosanoid Synthesis and Linoleic Hydroperoxide Production

After incubation for 15 min with 300 µM (100 µM iron-binding equivalents) chelator, the greatest inhibition of the calcium ionophore-stimulated synthesis of eicosanoids in neutrophils (Table 1) and of the production of linoleic hydroperoxide by sbLPO (Fig. 3) was obtained with the lipophilic HP-4-ones CP26 and CP25. Under the same conditions, the non-iron-binding HP-4-one Ome-25 had significant inhibition in the cellular (5-LPO) system (55% of control, Table 1) but not in the cell-free (sbLPO) system (92% of control, Fig. 3). This lack of inhibition by Ome-25 in the cell-free system could not be overcome with longer preincubation with the enzyme up to 24 h; under identical conditions significant inhibition was obtained with iron-binding HP-4-ones (Fig. 4). Dose-response studies in the cellular system show that below 300 µM (100 µM iron-binding equivalents) the extremely lipophilic CP26 is a significantly more potent inhibitor of eicosanoid synthesis than the less lipophilic CP25 (Fig. 5). Comparison of eicosanoid inhibition by CP26 with that by the lipoxygenase inhibitor piriprost shows their patterns of inhibition to be very similar in the range 3.6-300 µM, similar concentrations being required to achieve 90% inhibition (IC of CP26 and piriprost = 8.0 and 8.8 µM, respectively; Fig. 5). This pattern is likely to be similar below this range as well, as indicated by 50% inhibition also being achieved by similar concentrations of CP26 (IC = 2.5 µM) and piriprost (IC = 2.0 µM; (25) ). In contrast, much higher concentrations are required to achieve the same inhibition with CP25 (IC = 185 µM, IC = 28 µM; Fig. 5).


Figure 3: Relationship of lipophilicity to short term inhibition by HP-4-ones of soybean lipoxygenase. sbLPO (10,000 units ml) was preincubated for 15 min with HP-4-ones (300 µM, 100 µM iron-binding equivalents) or the non-iron-binding analogue Ome-25 (300 µM). The reaction was initiated by the addition of linoleic acid (667 µM), and the production of linoleic hydroperoxide was monitored spectrophotometrically at 234 nm. The data are the mean ± S.E. of the maximum linear rate from four experiments.




Figure 4: Long term effects of hydrophilic chelators on soybean lipoxygenase. sbLPO (1000 units ml) was preincubated with chelators (300 µM, 100 µM iron-binding equivalents) or the non-iron-binding analogues (300 µM) for the times shown, and the reaction was followed under conditions similar to those in the legend to Fig. 3.The data are the mean ± S.D. of seven observations.




Figure 5: Inhibition of eicosanoid synthesis by CP25 and CP26. Purified [3H] arachidonic acid-loaded neutrophils in PBSG (0.5-1.0 times 106 ml) were incubated for 15 min with CP25, CP26 (0.39-300 µM), or diluent at the concentrations shown and stimulated with calcium ionophore (1 µM) for 10 min. The released eicosanoids were quantified by beta-scintillation counting. The dose response for piriprost is also shown in the range 1.2-300 µM. The data are the mean ± S.E. from three to six experiments performed using neutrophils from different donors.



Electron Paramagnetic Resonance Studies

The failure of the non-iron-binding analogue Ome-25 to inhibit sbLPO ( Fig. 3and 4) is strong evidence that the inhibition of this enzyme is dependent on iron chelation. In order to investigate the interaction of the HP-4-ones with the iron center of sbLPO directly, studies were performed in which sbLPO treated with CP25, CP26, or Ome-25 (300 µM) was analyzed by EPR as described under ``Experimental Procedures.''

The high spin ferric iron present in the active site of sbLPO (11) has a distinct EPR spectrum (at g = 6.0) compared with that bound to hydroxypyridinones (at g = 4.3). This enables a direct measure to be made of the amount of iron removed from the enzyme by these chelators. The results in Fig. 6show that in the absence of linoleic acid there is no detectable EPR signal from sbLPO, consistent with previous reports (24) that, as purified, the active-site iron is in an EPR-silent state, presumably Fe (spectrum a). The small signal observed at g = 4.3 under these conditions represents less than 5% of the total iron content of the enzyme and is probably due to nonspecifically bound high spin ferric iron in the solution. Following incubation with the substrate (spectrum b) a clear signal is seen at g = 6.0, consistent with high spin ferric iron in the active site of the enzyme(11) . The non-iron-binding analogue, Ome-25, has no effect on the EPR spectrum (c) at g = 6.0 or at g = 4.3, consistent with its lack of enzyme inhibition and its inability to bind iron (spectrum g). However, preincubation of the enzyme with CP25 (spectrum d) or CP26 (spectrum e) for 2 h completely removes the g = 6.0 signal, with a concomitant increase in the g = 4.3 region (note change of scale in spectra d, e, and f). The signals at g = 4.3 (spectra d and e) are identical in spectral shapes and g values of known 3:1 chelator-iron complexes (e.g. spectra f). Ternary complexes of hydroxypyridinones with sbLPO Fe(III) would have at least two different coordinating ligands compared with the pure hydroxypyridinone-Fe complex, and it is highly unlikely that such a complex would have an identical EPR spectrum. All the iron initially present in sbLPO could be accounted for quantitatively by the formation of iron-chelate complexes. This suggests that the mechanism of inhibition of these chelators is by removal of iron from the active site of the enzyme, resulting in the formation of free iron-chelate complexes in solution.


Figure 6: EPR spectra of the Fe center of sbLPO after preincubation with HP-4-ones. sbLPO (30 µM) was dissolved in PBS, pH 7.4, and incubated with respective compounds (300 µM, 100 µM iron-binding equivalents) for 2 h in the presence of bubstrate (linoleic acid, 100 µM). The samples were frozen in liquid nitrogen and analyzed as described under ``Experimental Procedures.'' Spectrum a, sbLPO only; spectrum b, sbLPO and substrate; spectrum c, sbLPO and Ome-CP25; spectrum d, sbLPO and CP25; spectrum e, sbLPO and CP26. The spectrum and the authentic Fe-CP25 complex (64:1920 µM) is shown in spectrum f, and the spectrum of Ome-025 (1920 µM) in the presence of iron (64 µM) in spectrum g. ESR parameters were as follows: power, 20 mW; temperature 5 K: spectral amplitudes of spectra d, e, and f have been attenuated 10-fold with respect to the others.



Size of Ligand and Nature of Coordination of Iron

Comparison of Fig. 3and Fig. 4shows that the inhibition of sbLPO by the bidentate HP-4-one CP40, though modest at 15 min, increases progressively with time (up to 24 h), unlike that of the hexadentate DFO. CP40 and DFO are both hydrophilic chelators with similar K values (Table 1), but the former is a small molecule (molecular weight 206), whereas the latter is larger (molecular weight 657). This suggests that access to the iron in sbLPO is favored by small bidentate molecules over large hexadentate ones.

Of all the hydroxypyridinones studied, with exception of the non-chelating analogue Ome-25, there is a progressive increase in the formation of chelate complexes with time as monitored spectrophotometrically at 450 nm. Preincubation of 60 µM sbLPO with highly lipophilic CP26, moderately lipophilic CP25, or hydrophilic CP40 all led to a progressive increase in the appearance of the corresponding ferric complex as follows: CP26, t(0) (8.2 µM), 15 min (13.6 µM), 24 h (20.2 µM); CP25, t(0) (11.1 µM), 15 min (16.1 µM), 24 h (20.9 µM); CP40, t(0) (5.0 µM), 15 min (7.5 µM), 24 h (20.5 µM). The removal of iron at 24 h by all these chelators determined by spectrophotometry is in close agreement with the total iron content of the enzyme determined by atomic absorption spectroscopy (18.0 µM). In contrast, incubation of the enzyme with DFO showed no increase in the appearance of the ferrioxamine complex at 15 min and even after 24 h there was only a moderate increase (2.2 µM and 5.3 µM, respectively). The increased removal of iron from sbLPO by lipophilic HP-4-ones (CP26 and CP25) compared with hydrophilic HP-4-ones (CP40) at short time intervals (15 min) and its convergence to similar levels at longer times (24 h) are in agreement with their inhibitory effects on the function of the enzyme at these times ( Fig. 3and Fig. 4, respectively).

Further evidence to support the direct interaction of hydroxypyridinones with the iron center of lipoxygenase by removal of iron was obtained by investigating the formation and removal of chelate complexes of the HP-4-ones. Following the incubation of soybean lipoxygenase (60 µM) with CP25 (300 µM), there is a progressive formation of iron-chelator complexes with characteristic UV absorbance ((max) = 450 nm, = 5200 M cm). The iron complexes formed by the incubation of CP25 (300 µM) with sbLPO (100 µM) for 24 h were quantitated spectrophotometrically at 450 nm and then removed by dialysis against 300 µM CP25 for 24 h followed by further dialysis against borate buffer (0.2 M pH 9.0) for 24 h. The concentration of chelate complexes fell from 27.5 µM ( = 0.143 ± 0.006, n = 3) prior to dialysis, reaching a final concentration of 8 µM ( = 0.042 ± 0.003, n = 3) after dialysis. In three independent experiments using sbLPO at 10,000 units ml and linoleic acid substrate at 1 mM (as described under ``Experimental Procedures''), enzyme activity remained completely inhibited after the dialysis. Addition of iron back to this solution (as ferrous ammonium sulfate, 20 µM final concentration) restored enzyme activity to levels above those seen before treatment with chelators (600 ± 59%, n = 4, expressed as percentage of activity of native enzyme) and equal to the activity seen with native enzyme to which iron was added under the same conditions (635 ± 35%, n = 2). This increased activity after the addition of iron is compatible with the relatively low iron saturation of the native enzyme before treatment with chelators.^3

The Effect of Hydroxypyridinones on Phospholipase A(2)

The liberation of arachidonic acid from membrane phospholipid by PLA(2) is a prerequisite for the synthesis of eicosanoids by 5-LPO. Therefore, in principle, an agent that inhibits PLA(2) can also inhibit eicosanoid biosynthesis. In order to investigate whether chelators have differential inhibitory effects on PLA(2) and 5-LPO, simultaneous measurements of PLA(2)-derived arachidonate release and 5-LPO-derived eicosanoid release were undertaken in neutrophils obtained from the same donor.

These results (Table 2) show that there is no significant inhibition of PLA(2) by DFO, the non-iron-binding analogue Ome-25, or the hydrophilic HP-4-one CP20 at 300 µM (100 µM iron-binding equivalents). The lipophilic N-hexyl derivative CP25, which has considerable 5-LPO inhibitory activity also has no effect on PLA(2) at this concentration (Table 2). In contrast, the extremely lipophilic N-octyl derivative CP26, which shows nearly complete inhibition of 5-LPO, also shows significant inhibition of PLA(2) (Table 2). However, dose-response data show that at lower concentrations it is inhibitory to a greater extent in the 5-LPO assay (IC = 2.5 µM) than in the PLA(2) assay (IC 40 µM) (Fig. 7). Interestingly, the 5-LPO inhibitor piriprost also shows considerable inhibition of PLA(2) at 300 µM, suggesting that its inhibitory effects on eicosanoid biosynthesis may be mediated via inhibition of both enzymes (Table 2).




Figure 7: The dose-dependent inhibition of 5-LPO and PLA2 by CP26 and inhibition of PLA2 by CP25. 5-LPO and PLA2 activities were measured in [^3H]arachidonic acid-loaded neutrophils as described under ``Experimental Procedures.'' The data are from two to three experiments with CP26 and a single experiment with CP25. The mean ± S.E. is given where n = 3, and the range is given where n = 2.



Effect of Chelators on Superoxide Generation in Neutrophils

TPA-stimulated Neutrophils

To investigate whether the inhibitory activity of the hydroxypyridinones on 5-LPO could be attributed to any toxic effects of these agents on neutrophils, the effects of hydroxypyridinones on neutrophil NADPH oxidase were measured by the superoxide dismutase-inhibitable reduction of cytochrome c as described under ``Experimental Procedures.''

The hydroxypyridinones used in the 5-LPO studies including the non-iron-binding analogue do not, in general, have adverse effects on the TPA-induced superoxide generation. Superoxide production in neutrophils fired for 10 min with TPA in the absence of chelator was 56.6 ± 7.4 nmol/10^6 cells (n = 4). In chelator-treated cells superoxide production expressed as a percentage of the appropriate diluent control was as follows: CP20, 93.3 ± 8.4 (n = 5); CP84, 105.5 ± 8.3 (n = 3); CP25, 109.0 ± 10.5 (n = 5); CP26, 95.6 ± 10.9 (n = 4); Ome-25, 110.1 ± 14.1 (n = 3); DFO, 113.8, 97.4 (n = 2). (^4)

Arachidonic Acid-stimulated Neutrophils

Further experiments were performed to investigate whether CP26 had significant 5-LPO inhibitory activity independent from its effects on PLA(2). Exogenous arachidonic acid stimulates the rapid production of superoxide by purified neutrophils(26) . In the absence of 5-LPO inhibitors the arachidonic acid is rapidly metabolized via the 5-LPO pathway(27) . However, when the metabolism of arachidonic acid is inhibited there is a significant enhancement of the respiratory burst due to the prolonged availability of the agonist. Using this approach, 5-LPO inhibition was demonstrated in experiments in which neutrophil superoxide production was stimulated with arachidonic acid (12.5 µM) in the absence and presence of CP26. As predicted, superoxide production was much greater when cells were preincubated with 300 µM CP26 (40.7 ± 2.7 nmol/10^6 cells, mean ± S.E., n = 3) compared with control cells (8.0 ± 4.1 nmol/10^6 cells, mean ± S.E., n = 3). Experiments performed with CP25 also produced similar results (data not shown), demonstrating the effectiveness of these HP-4-ones in the inhibition of arachidonic acid metabolism via the 5-LPO pathway.


DISCUSSION

Several lines of evidence suggest that direct access of Fe(III) chelators to the iron center of lipoxygenases is required for inhibition to occur. At least four of the five most inhibitory chelators of neutrophil 5-LPO (CP25, CP26, CP84, oxine, and tropolone) have the highest affinity constant for iron (log beta(3) = 37, Table 1). Lower affinity chelators such as the HP-2-ones (log beta(3) = 32) and hydroxypyranones (log beta(3) = 28) inhibit the enzyme to a far lesser extent (Table 1). Furthermore, the non-iron-binding analogue of CP25 (Ome-25) has no inhibition of sbLPO either by spectrophotometry up to 24 h (Fig. 4) or by EPR up to 2 h (Fig. 6), and it is significantly less inhibitory of neutrophil lipoxygenase than CP25. EPR data show that inhibition of sbLPO by iron-binding HP-4-ones CP25 and CP26 is associated with decline of the g = 6.0 iron signal and the concurrent quantitative formation of Fe(III)-chelator complexes (g = 4.3) identical to those obtained for 3:1 complexes of each respective HP-4-one. Additionally, incubation of sbLPO with HP-4-ones inhibits enzyme activity with the concurrent formation of iron-chelate complexes monitored spectrophotometrically. After these complexes are removed by dialysis, the enzyme remains inhibited with restoration of activity only upon readdition of iron. The most likely explanation for these findings, taken with the EPR results, is that iron is removed from the active site by HP-4-ones (with the formation of 3:1 complexes). Alternatively single HP-4-one molecules might coordinate iron in situ within the enzyme without removing the iron. However if this were so, the removal of the chelator by dialysis would be associated with a return of enzyme activity, which is not the case. Furthermore, contrary to what we have shown, the subsequent addition of iron to the enzyme would not restore enzyme activity.

Experiments examining effects of lipid solubility also support direct access to the iron center as being important to lipoxygenase inhibition and are consistent with current knowledge about lipoxygenase structure. There are two hydrophobic regions in lipoxygenases that are highly conserved (amino acids 352-402 and 543-562). Some of these have been proposed (28) and subsequently confirmed (7, 29, 30) as ligands coordinating the active site iron. Although x-ray crystallographic data are not yet available for 5-LPO, the existence of the iron-coordinating ligands in a hydrophobic region of the enzyme is strong evidence of the occurrence of the iron in this region too. In sbLPO, crystallographic studies (31) have shown the occurrence of two large internal cavities or channels that are lined by the side chains of hydrophobic amino acids and that connect the iron center to the outside of the protein. These structures are consistent with a model in which the rate of movement of highly lipophilic chelators through these hydrophobic regions is favored while that of less lipophilic or hydrophilic hydroxypyridinones is retarded and is supported by the findings in this paper. For example, appreciable inhibition of sbLPO by the relatively hydrophilic HP-4-ones, CP40 and CP20, is seen only after the enzyme has been incubated with these chelators for many hours, whereas with lipophilic HP-4-ones (e.g. CP25 or CP26), inhibition is seen within minutes ( Fig. 3and Fig. 4). Incubation of HP-4-ones with neutrophils (Table 1) as well as with sbLPO (Fig. 3) shows that inhibition is dependent upon chelator lipophilicity as quantified by the partition coefficient (K) (Table 1). This relationship also holds for the related 3-hydroxypyranones as illustrated by ethylmaltol and CP166 (Table 1).

Molecular size and the nature of coordination of the iron also appear to be important determinants of lipoxygenase inhibition, again consistent with chelators needing direct access to lipoxygenase iron center. DFO, a hexadentate chelator with a molecular weight about three times that of the bidentate CP40, would be predicted to be too large to pass through the channels of sbLPO. Thus, although both CP40 and DFO have similar lipid solubilities (Table 1), DFO has no effect on sbLPO inhibition, whereas CP40 inhibits to the same extent as lipophilic HP-4-ones by 24 h (Fig. 4).

Indirect support for the ability of bidentate HP-4-ones, but not of DFO, to penetrate the hydrophobic channels of sbLPO comes from experiments showing iron removal from ferritin by HP-4-ones to be significantly faster than DFO(32) . The protein shell of ferritin is composed of 24 subunits, which form channels about 7 Å wide (33) resembling those in sbLPO and which allow access to the internal cavity containing the storage iron. The diameter (7 Å) and length (13 Å) of the 1:1 iron complex of CP22 (34) is less than the maximum size of the hydrophobic channels reported for sbLPO(31) , but the sizes of the 2:1 and 3:1 complexes are greater(34) . The ability of bidentate chelators to form 1:1 complexes with iron, in addition to the usual 3:1 complexes, may therefore be a key property enabling them to access lipoxygenase iron. The evidence from spectrophotometric and iron add-back experiments suggests, however, that iron is removed from sbLPO in this process rather than forming 1:1 complexes in situ.

The inhibition of eicosanoid biosynthesis by Ome-25 indicates that mechanisms other than iron chelation may be involved in neutrophils. During activation, 5-LPO is translocated from the cytosol to the nuclear membrane, where it binds to its activating protein FLAP(35, 36) . Concurrently its substrate arachidonic acid is liberated from membrane phospholipid by the action of PLA(2). Therefore, compounds that inhibit translocation of 5-LPO (37, 38) or interfere with the 5-LPO-FLAP interaction (5) as well as those that inhibit PLA(2)(39) will also inhibit eicosanoid biosynthesis. Ome-25 has no effect on PLA(2) inhibition (Table 2). Short term (15 min, Fig. 3) and longer (up to 24 h, Fig. 4) incubations of iron-binding HP-4-ones with sbLPO show similar degrees of inhibition to cellular 5-LPO, contrasting with known translocation inhibitors, which are considerably less active in cell-free compared with the cellular systems(37, 40) . Translocation-type inhibition is unlikely to be a major mechanism with iron chelators but could account for some of the effects of Ome-25. It is unlikely that the inhibition of eicosanoid synthesis with CP26 and CP25 is due to interaction with the lipid-rich membranes as reported for other lipophilic inhibitors (15) because there are no adverse effects on neutrophil NADPH oxidase, a membrane-associated enzyme(41) , at chelator concentrations that inhibit eicosanoid synthesis.

The simultaneous measurements of PLA(2) and 5-LPO activities in neutrophils (Table 2) indicate that the only HP-4-one producing appreciable PLA(2) inhibition at high concentrations is the N-octyl derivative, CP26. However, preferential inhibition of 5LPO by CP26 at low chelator concentrations (Fig. 3) suggests that PLA(2) inhibition is not the sole inhibitory mechanism of eicosanoid biosynthesis and specific inhibitory effects on 5LPO independent from PLA(2) have been demonstrated. In contrast to CP26, CP20 and DFO inhibit PLA(2) insignificantly, suggesting this is not a general effect of iron chelators.

These studies show that the direct interaction of iron-chelating molecules with the iron center of lipoxygenase is influenced by the lipid solubility, iron binding constant, and coordination structure of chelators, provided that the molecular size is sufficiently small to allow access to this site. Our data further suggest that iron is removed from this site by HP-4-ones. Compounds with a maximal diameter of <8 Å (31) such as the bidentate hydroxypyridinones are likely to access the active site iron via the two hydrophobic channels. These studies additionally suggest that, while direct chelation is the major mechanism of enzyme inhibition with hydroxypyridinones, within intact cells inhibition of translocation and/or PLA(2) inhibition may play additional roles with some molecules.


FOOTNOTES

*
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Supported by National Institutes of Health Grant R01-HL-42800-01. Present address: Dept. of Biochemistry, Michigan State University, East Lansing, MI 48824-1319.

Supported by the Kay Kendall Leukemia Fund.

**
Supported by a Medical Research Council research fellowship.

§§
To whom correspondence and requests for reprints should be addressed: Dept. of Clinical Hematology, University College Medical School, 98 Chenies Mews, London WC1E 6HX, United Kingdom. Tel.: 44 171 209 6224 or 44 171 209 6230; Fax: 44 171 209 6222.

(^1)
The abbreviations used are: 5-LPO, 5-lipoxygenase; sbLPO, soybean 15-lipoxygenase; FLAP, 5-lipoxygenase activating protein; PLA(2) phospholipase A(2); LTB(4), leukotriene B(4); TPA, 12-O-tetradecanoylphorbol-13-acetate; HP-4-one, 3-hydroxypyridin-4-one; HP-2-one, 3-hydroxypyridin-2-one; DFO, desferrioxamine; PBS, phosphate-buffered saline; PBSG, phosphate-buffered saline containing calcium, magnesium and glucose; HPLC, high pressure liquid chromatography.

(^2)
Systematic names of the hydroxypyridinone and hydroxypyranone chelators are as follows: 1-(hydroxyethyl)-2-methyl-3-hydroxypyridin-4-one (CP40); 1,2-dimethyl-3-hydroxypyridin-4-one (CP20); 1-ethyl-2-methyl-3-hydroxypyridin-4-one (CP21); 1-propyl-2-methyl-3-hydroxypyridin-4-one (CP22); 1-butyl-2-methyl-3-hydroxypyridin-4-one (CP24); 1-phenyl-2-methyl-3-hydroxypyridin-4-one (CP84); 1-pentyl-2-methyl-3-hydroxypyridin-4-one (CP77); 1-hexyl-2-methyl-3-hydroxypyridin-4-one (CP25); 1-octyl-2-methyl-3-hydroxypyridin-4-one (CP26); 1(hydroxyethyl)-2-ethyl-3-hydroxypyridin-4-one (CP102); 1-ethyl-3-hydroxypyridin-2-one (CP02); 6-(methoxybutyl)-3-hydroxypyridin-4-one (CP166); 1-hexyl-2-methyl-3-(methoxy)pyridin-4-one (Ome-25).

(^3)
One unit will cause an increase in A of 0.001 per min at pH 9.0 at 25 °C when linoleic acid is used as substrate. Reaction volume is 3.0 ml (1-cm light path). The protein content was 60%, the rest being sodium chloride and stabilizer. The iron content by atomic absorption spectroscopy (18 µM at 60 µM sbLPO) suggested of the binding sites were saturated. The protein content has been taken into account in the calculation of enzyme molarities.

(^4)
Shown are the S. E. where n geq 3; the individual values are given where n = 2.


ACKNOWLEDGEMENTS

We thank Professor R. Cammack and Dr J. Shergill (Metals in Biology and Medicine Centre, Kings College London) for the use of the EPR spectrometer and T. B. Weir (Department of Chemical Pathology, University College London Hospitals) for atomic absorption spectroscopy measurements.


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