(Received for publication, August 25, 1995; and in revised form, January 25, 1996)
From the
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
[H]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
production. Lipophilic hydroxypyridinones inhibit
5-lipoxygenase at equivalent concentrations to the established
inhibitor, piriprost, and show additional but minor anti-phospholipase
A
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
.
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, ()which catalyzes the first two steps in the
conversion of its substrate arachidonic acid to leukotriene A
(recently reviewed in (1) ). Leukotriene A
is
the precursor of the potent inflammatory mediator leukotriene B
(LTB
). 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 (PLA
), 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 () 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.
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: = 450 nm,
= 5200 M
cm
; ferrioxamine:
=
430 nm,
= 2640 M
cm
).
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 internal standard (b), leukotrienes of unknown identity (c and d); LTB4 (e), and an unidentified
non-leukotriene peak present in chelator-treated cells (f).
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 (0.5-1.0 106 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
-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.
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.
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 (8.2 µM), 15 min (13.6 µM), 24 h (20.2
µM); CP25, t
(11.1 µM),
15 min (16.1 µM), 24 h (20.9 µM); CP40, t
(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 ( = 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.
These
results (Table 2) show that there is no significant inhibition of
PLA 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
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
(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
assay (IC
40
µM) (Fig. 7). Interestingly, the 5-LPO inhibitor
piriprost also shows considerable inhibition of PLA
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 [H]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.
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 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). (
)
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 = 37, Table 1). Lower affinity chelators such as the
HP-2-ones (log
= 32) and hydroxypyranones (log
= 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. 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
(39) will also inhibit eicosanoid biosynthesis. Ome-25
has no effect on PLA
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 and 5-LPO activities in
neutrophils (Table 2) indicate that the only HP-4-one producing
appreciable PLA
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
inhibition is not the sole inhibitory mechanism of
eicosanoid biosynthesis and specific inhibitory effects on 5LPO
independent from PLA
have been demonstrated. In contrast to
CP26, CP20 and DFO inhibit PLA
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 inhibition may
play additional roles with some molecules.