reversible inhibition of myeloperoxidase 1 potent reversible
TRANSCRIPT
Reversible inhibition of myeloperoxidase
1
Potent Reversible Inhibition of Myeloperoxidase by Aromatic Hydroxamates*
Louisa V. Forbes1, Tove Sjögren
2, Françoise Auchère
1, David W. Jenkins
3A, Bob Thong
3B, David
Laughton3B
, Paul Hemsley3C
, Garry Pairaudeau3C
, Rufus Turner1, Håkan Eriksson
4,
John F. Unitt3B
and Anthony J. Kettle1
1From the Centre for Free Radical Research, Department of Pathology, University of Otago Christchurch,
Christchurch 8140, New Zealand
2Discovery Sciences, AstraZeneca R&D Pepparedsleden 1, 43181 Mölndal, Sweden
3Bioscience and Medicinal Chemistry, AstraZeneca R&D Charnwood, Loughborough, Leicestershire
LE11 5RH United Kingdom
4AstraZeneca R&D Södertälje, SE-151 85 Södertälje, Sweden
(Current addresses: ANovartis Institute for Biomedical Research Inc., Cambridge, MA 02139, United
States of America; BSygnature Discovery Ltd, Nottingham NG1 1GF, United Kingdom;
CAstraZeneca
R&D Macclesfield, Cheshire SK10 4TF, United Kingdom)
*Running title: Reversible inhibition of myeloperoxidase
To whom correspondence should be adressed: Louisa V. Forbes, Centre for Free Radical Research,
Department of Pathology, University of Otago Christchurch, P.O. Box 4345, Christchurch, New Zealand.
Tel.: +64 3 364 0590, E-mail: [email protected]
Keywords: myeloperoxidase; reversible inhibition; hydroxamate; crystal structure; surface plasmon
resonance.
_____________________________________________________________________________________
Background: Myeloperoxidase causes oxidative
damage in many inflammatory diseases.
Results: New substituted aromatic hydroxamates
are identified as potent, selective, and reversible
inhibitors of MPO.
Conclusion: Binding affinities of hydroxamates to
the heme pocket determine the potency of
inhibition.
Significance: Compounds that bind tightly to the
active site of myeloperoxidase have potential as
therapeutically useful inhibitors of oxidative
stress.
SUMMARY
The neutrophil enzyme myeloperoxidase
(MPO) promotes oxidative stress in numerous
inflammatory pathologies by producing
hypohalous acids. Its inadvertent activity is a
prime target for pharmacological control.
Previously, salicylhydroxamic acid (SHA) was
reported to be a weak reversible inhibitor of
MPO. We aimed to identify related
hydroxamates that are good inhibitors of the
enzyme. We report on three hydroxamates as
the first potent reversible inhibitors of MPO.
The chlorination activity of purified MPO was
inhibited by 50% by 5 nM of a trifluoromethyl-
substituted aromatic hydroxamate, HX1. The
hydroxamates were specific for MPO in
neutrophils and more potent toward MPO
compared to a broad range of redox enzymes
and alternative targets. Surface plasmon
resonance measurements showed the strength
of binding of hydroxamates to MPO correlated
with the degree of enzyme inhibition. The
crystal structure of MPO-HX1 revealed the
inhibitor was bound within the active site cavity
above the heme and blocked the substrate
channel. HX1 was a mixed-type inhibitor of the
halogenation activity of MPO with respect to
both hydrogen peroxide and halide. Spectral
analyses demonstrated that hydroxamates can
act variably as substrates for MPO and convert
the enzyme to a nitrosyl ferrous intermediate.
This property was unrelated to their ability to
inhibit MPO. We propose that aromatic
http://www.jbc.org/cgi/doi/10.1074/jbc.M113.507756The latest version is at JBC Papers in Press. Published on November 5, 2013 as Manuscript M113.507756
Copyright 2013 by The American Society for Biochemistry and Molecular Biology, Inc.
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Reversible inhibition of myeloperoxidase
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hydroxamates bind tightly to the active site of
MPO and prevent it from producing
hypohalous acids. This mode of reversible
inhibition has potential for blocking the activity
of MPO and limiting oxidative stress during
inflammation.
_____________________________________ Myeloperoxidase (MPO) is a vital component
of host defense. This heme-enzyme produces
hypochlorous acid (HOCl) as part of the
neutrophil’s microbicidal attack on invading
organisms. It is apparent, however, that MPO
activity exacerbates many inflammatory diseases
including atherosclerosis, glomerulonephritis,
multiple sclerosis, rheumatoid arthritis, asthma
and cystic fibrosis (1). Evidence is also mounting
for its role in promoting oxidative stress in
Alzheimer’s disease, Parkinson’s disease, diabetes
mellitus and some cancers (2-4). Therefore, MPO
inhibitors may be useful for the treatment of a
broad range of human diseases. Despite the
growing understanding of its complex enzymology
and pharmacology, few therapeutically suitable
inhibitors have been discovered that specifically
target MPO.
A number of different inhibitors of MPO
have been reported over the last four decades.
These can be classified into three main categories;
those that promote accumulation of Compound II,
suicide substrates, and those that bind reversibly to
the native enzyme. The first two types of
inhibitors serve as alternative substrates that divert
MPO from its normal catalytic cycle (Fig. 1).
Inhibitors that cause accumulation of Compound II
are poor peroxidase substrates that react well with
Compound I but slowly with Compound II. These
include dapsone (5), tryptamines (6), tryptophan
analogues (7), and nitroxides (8,9). Such
inhibition is unlikely to be effective in a normal
physiological environment, due to an abundance
of better peroxidase substrates such as ascorbate
(10) or urate (11) that will efficiently convert any
accumulated Compound II back to the active
native MPO state. The plasma protein
ceruloplasmin is an endogenous inhibitor of MPO
that also acts by promoting accumulation of
Compound II (12). However, it also prevents
reduction of Compound II so MPO becomes
trapped in this redox state.
Suicide substrates, or mechanism-based
irreversible inhibitors, of MPO include 4-
aminobenzoic acid hydrazide (13) and 2-
thioxanthines (14). Oxidation of these inhibitors
by MPO promotes inactivation either by
destruction or covalent modification of the
enzyme’s heme prosthetic groups. Other redox-
based inhibitors include paracetamol (15) and
isoniazid (16). They are reversible inhibitors that
divert MPO from its halogenation cycle. In the
process they produce radical intermediates. With
all of the substrate-based inhibitors, whether
irreversible or reversible, there is possible
generation of undesirable, reactive by-products of
the oxidized inhibitor. As MPO is a heme
peroxidase with extremely powerful oxidizing
abilities (17,18), it is indeed not surprising that the
majority of known inhibitors are oxidized by the
enzyme. Reactive radicals formed during
inhibition may promote local toxic chain reactions
or lead to hapten formation in vivo (16,19,20).
This feature places major restrictions on the
feasibility of inhibitors as therapeutic agents.
However, the problem is minimized for the most
potent 2-thioxanthine compounds because they
inactivate MPO within a single turnover of the
enzyme (14).
Reversible inhibitors that bind to the native
enzyme differ from the substrate-based inhibitors,
in that they compete with MPO substrates by
occupying the heme binding pocket. As an
alternative mechanism, this is an attractive means
of inhibition because the enzyme’s oxidizing
capability is simply blocked, without permanent
changes to the enzyme or production of unwanted
by-products. Salicylhydroxamic acid (SHA) was
identified as a reversible inhibitor of MPO (21)
after earlier observations of broad peroxidase
inhibition by substituted aromatic hydroxamates
(22). However, SHA performed poorly in MPO
inhibition assays in comparison to benzoic acid
hydrazides (23).
Proof of the competitive nature of SHA-
enzyme binding (24) and the subsequent crystal
structure of the MPO-SHA complex (25), spawned
the hypothesis that modified hydroxamates could
be identified as new, more potent reversible
inhibitors of MPO. For this type of inhibitor, the
critical feature is the docking of the molecule in
the heme binding pocket of MPO. In this study,
we aimed to explore different substituted aromatic
hydroxamates to identify compounds with stronger
binding affinities, and improved specific inhibition
of the halogenation activity of MPO. Our results
show that the strength of hydroxamate-MPO
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Reversible inhibition of myeloperoxidase
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binding correlated with the inhibition of MPO
activity. We have solved the crystal structure of
the MPO-hydroxamate complex, and have
determined the mechanism of inhibition by heme
spectral analysis and substrate competition
kinetics. We present new compounds, in
particular hydroxamate HX1, as highly potent and
reversible inhibitors of MPO.
EXPERIMENTAL PROCEDURES Materials - Human MPO (EC 1.11.1.7)
purified from human blood (purity index
(A430/A280) > 0.84) was purchased from Planta
(Wien, Austria). Human recombinant thyroid
peroxidase (TPO, purity >95% by SDS-PAGE)
was purchased from RSR Ltd (Cardiff, UK).
Bovine lactoperoxidase (LPO, purity index
(A412/A280) > 0.88) was purchased from Sigma
(Poole, UK). For structural characterization of
complexes between MPO and inhibitors, MPO
was purified from HL-60 cells which were
obtained from American Type Culture Collection
(Manassas, VA). Cells were grown in
DMEM/F12 (Invitrogen) plus 5% fetal calf serum
and 5 mM glutamine in a 50 litre reactor to a cell
density of 1.7 x 106/ml. The purification was a
modification of the protocol described previously
(26). In the modified protocol the ammonium
sulfate precipitation steps were excluded and the
final purification was achieved using Superdex
200 (GE Health Care) size exclusion
chromatography. Purity and identity of MPO was
determined by 10% SDS-PAGE and N-terminal
sequencing. Hydroxamates were prepared by the
Department of Medicinal Chemistry, AstraZeneca
R&D Charnwood. Pronase was from Roche
Diagnostics (Germany), and human serum
albumin was the clinical product Albumex 4 from
CSL Ltd (Australia). All other reagents were of
the highest purity commercially available and
were from Sigma unless otherwise stated.
Myeloperoxidase assays -
Enzyme activity
was determined as the production of hypochlorous
acid (HOCl) via accumulation of taurine
chloramine, which was detected using iodide-
catalyzed oxidation of 3,3’,5,5’-
tetramethylbenzidine (27). Assays were
performed at 22°C with 2 nM MPO and 10 µM
hydrogen peroxide (H2O2) in 20 mM NaH2PO4
buffer, pH 6.5, containing 140 mM NaCl, 10 mM
taurine and 1 mM L-tyrosine. Inhibitor
compounds were preincubated with MPO for 15
min prior to the addition of H2O2, and the
accumulation of taurine chloramine was
determined after 1 min. Inhibitory effects of
compounds are expressed as % control activity in
the absence of compound, and curves were fitted
to data using Origin 7.5 (Origin Labs, USA). The
concentration of inhibitor giving 50% of the full
enzyme activity measured in the absence of
inhibitor is the IC50 value.
The consumption of H2O2 by MPO was
measured with the ferrous oxidation of xylene
orange (FOX) assay (28). The assay was modified
to imitate the protein-rich environment of plasma
with the inclusion of 200 µM urate, 50 µM
tyrosine, 50 µM tryptophan and 1 mg/ml albumin
in 50 mM phosphate buffer, pH 7.4, containing
140 mM NaCl, and 1 mM methionine to scavenge
any HOCl. Reactions were performed at room
temperature in Eppendorf tubes, and started by
adding 20 µM H2O2 to 5 nM MPO in the presence
or absence of inhibitor. Each 200 µl reaction was
stopped after 15 min by addition (on vortex mixer)
of 1/3 volume (67 µl) of FOX developer (400 µM
xylene orange, 1mM ferrous ammonium sulfate,
400 mM sorbitol, in 200 mM H2SO4). Aliquots
(200 µl) were transferred to a microtitre plate and
the absorbance was measured after 45 min at
560 nm. Time course experiments showed
approximately 10 µM H2O2 was consumed in 15
min (not shown). Each reaction was blanked
against a control without MPO, and inhibition was
expressed as a ratio of the change in absorbance in
the presence of inhibitor to that in the absence of
inhibitor.
Reversibility of inhibition was determined by
immobilizing MPO (10 g/ml in 100 mM sodium
carbonate buffer, pH 10) onto the well surface of
protein immobilizer plates (Exiqon, Vedbaek,
Denmark) as per the manufacturer’s instructions
and assessing HOCl production by taurine
chloramine assay prior to and after extensive
washing in enzyme assay buffer.
The halogenation of NADH by MPO was
monitored to determine the kinetics for competing
substrates. This assay, detecting the initial rate of
bromohydrin production at λ 275 nm using ε275
11800 M-1
cm-1
, is a direct measure of the
formation of hypohalous acid by MPO (29).
Bromide was chosen due to faster reaction rates
compared to other halides (30). Briefly, 20 nM
MPO was incubated at room temperature in 20
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Reversible inhibition of myeloperoxidase
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mM phosphate buffer pH 7.4 containing 100 µM
NADH and varying concentrations of inhibitor and
NaBr. The absorbance changes were monitored
upon addition of H2O2 to start the reaction. Initial
rates were measured over the first minute of
reaction and Km and Vmax were determined using
non-linear regression (Sigma Plot, Jandel
Scientific, USA).
Selectivity assays – The halogenation
activities of LPO (EC 1.11.1.7) and TPO (EC
1.11.1.8) were determined using a modified
method of that described previously (31).
Previously described assays were also used to
measure the activities of nitric oxide synthase
(NOS; EC 1.14.13.39) (32), cytochrome P450
(33), and arachidonate lipoxygenases 5-LO (34)
and 15-LO (35).
Cell assays - Human neutrophils were
purified from peripheral venous blood (36), then
resuspended in 10 mM NaH2PO4 buffer, pH 7.4,
containing 140 mM NaCl, 0.5 mM MgCl2, 1 mM
CaCl2 and 1 mg/ml D-glucose (Hanks buffer).
Production of HOCl was measured by the taurine
chloramine assay (27), using cells at 1.4 x 106/ml
with 5 mM taurine included in the buffer, and
stimulated with 30 ng/ml of 12-phorbol myristate
13-acetate (PMA) for 40 min at 37°C. Superoxide
production was measured as the rate of
cytochrome c reduction (37), using PMA-
stimulated cells as above with 2.5 mg/ml
cytochrome c added to the buffer. Absorbance
readings were taken at 550 nm, at 1 min
intervals for 15 min, at 37°C.
Neutrophils (2 x 106/ml in Hanks buffer) were
stimulated with PMA (100 ng/ml) in the presence of
human serum albumin (0.5 mg/ml) and the
chlorination of tyrosine residues was measured by
mass spectrometry. After 40 min at 37°C, cells
were pelleted and the supernatant was removed
and spiked with internal standards, including 1
nmol 13
C6-tyrosine and 500 fmol 13
C9-3-
chlorotyrosine. The samples were then
lyophilized prior to Pronase digestion in 100 mM
Tris pH 7.5 containing 10 mM CaCl2 for 18 h with
a 5:1 excess of protein to protease. Samples
(approximately 100 µg protein) were lyophilized
again and reconstituted in 10 mM phosphate
buffer at pH 7.4 for detection of 3-chlorotyrosine
and tyrosine by liquid chromatography with mass
spectrometry (LCMS).
3-Chlorotyrosine measurement by LCMS/MS - The method of analysis was similar to that
published previously (38) with additional
monitoring of 3-chlorotyrosine by the 3:1 ratio of its 35
Cl and 37
Cl isomers. High performance liquid
chromatography (HPLC) was performed on a
Dionex Ultimate 3000 pump with 3 µm Hypercarb
column, 250 x 2.1 mm, with an identical guard
column and a SDS guard cartridge (all Thermo
Scientific). Detection was on an Applied
Biosystems (Ontario, Canada) 4000 QTRAP
electrospray mass spectrometer via stable isotope
multiple reaction monitoring for tyrosine and its
chlorinated derivatives. Use of the internal
standards 13
C6-tyrosine and 13
C9-chlorotyrosine
enabled complete quantification as well as
monitoring any artifactual chlorination of tyrosine.
For tyrosine, the fragment transitions that were
monitored had m/z values of 182 to 136, 188 to 142
and 191 to 144 for 12
C-tyrosine, 13
C6-tyrosine and 13
C9-tyrosine, respectively. Correspondingly, for 3-
chlorotyrosine the transitions had m/z values of 216
to 170, 222 to 176, and 225 to 178 for the 35
Cl
isotope of each species, and 218 to 172, 224 to 178,
and 227 to 180 for the 37
Cl isotopes. Standard
curves were generated using known standards and
results were calculated as moles of 3-chlorotyrosine
per 1000 moles of tyrosine (Cl-Y/1000Y).
Measurement of compound binding kinetics -
Binding kinetics were determined by surface
plasmon resonance (SPR) using a Biacore S51
(Biacore, Sweden). MPO (50 g/ml, dissolved in
10 mM sodium acetate, pH 5.0) was immobilized
onto the surface of CM5 sensor chips (Biacore)
using surface amine coupling. One of the spots on
the sensor surface was left without MPO to control
for non-specific binding. The signal observed in
response to analyte binding was, as expected,
linearly related to the amount of immobilized
ligand, and 10 000 response units (RU) was
routinely used to characterize compound binding
(data not shown). Compounds were dissolved in
binding buffer (10 mM HEPES, pH 7.4, 150 mM
NaCl, 3 mM Na2EDTA, 0.005% (w/v) surfactant
p20, 1% (v/v) DMSO final) and association was
assessed during 60-210 s injections. After this
time, analyte injection was terminated and the chip
surface was perfused with binding buffer at 30
l/min for 4-12 min to monitor compound
dissociation.
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Reversible inhibition of myeloperoxidase
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Compound binding responses were
determined as a change in signal following solvent
correction and subtraction of baseline responses
using the Biacore S51 Evaluation program.
Specific saturation binding data was fitted to a
logistic equation assuming an interaction of the
compound with a single class of binding site to
obtain estimates of the dissociation constant pKD (-
log10KD where KD=kd/ka). Kinetic data were fitted
to single exponential association and dissociation
equations using Biacore S51 evaluation (Biacore,
Uppsala, Sweden). An interaction with a single
population of binding sites was assumed and
estimates of association and dissociation rates (ka
and kd), and the half-life for dissociation (t1/2 =
0.69/kd) were obtained. Mean values were
generated by taking the arithmetic mean of the
individual estimates.
Crystal structure determination - For
crystallization, the protein buffer was exchanged
with 20 mM sodium acetate buffer pH 5.5,
containing 50 mM ammonium sulfate and 2 mM
CaCl2, and the MPO sample was concentrated to
about 10 mg/ml. The compound was added to the
protein sample to a final concentration of
approximately 1 mM in 2% DMSO. After 6 h
incubation, excess ligand precipitate was removed
by centrifugation. Crystals were obtained by the
hanging drop vapor diffusion technique. The
protein sample (1 µl) was mixed with 1 µl of a
well solution containing 18% PEG3350 and 0.1 M
NaCl. The drop was allowed to equilibrate over a
reservoir containing well solution. Prior to data
collection the drops containing crystals were
supplied with glycerol as a cryoprotectant. The
crystals were then quickly removed from the drop
and flash-cooled in liquid nitrogen. Data were
collected at beam line ID14 EH4 at a wavelength
of 0.939 Å. The data were processed using
MOSFLM (39) scaled and further reduced using
the CCP4 suite of programs (40), for statistics see
Table 1.
Initial phasing was done by molecular
replacement using a high resolution ligand-free
structure of MPO (protein data bank PDB id code
1CXP (41)), as a starting model. The Fo-Fc
difference map showed positive residual density in
the distal heme cavities in each half of the
molecule corresponding to the bound HX1 (see
Fig. 6B). Although the difference map allowed
unambiguous modeling of the HX1 molecule it
was clear that the site was not fully occupied and it
was possible to outline the solvent structure of the
ligand-free enzyme superimposed on the ligand
structure. When the ligands were refined
assuming full occupancy of the ligand, the B
factors were refined to values almost twice the
average B factor for protein atoms and the
occupancy was therefore set to 0.5. Model
rebuilding was performed within O (42) and
refinement was performed using REFMAC5 (40).
For statistics for the final models see Table 1. The
final model is deposited in the PDB, id code
4C1M.
Spectrophotometric analyses – UV-visible
absorbance spectra were recorded on an Agilent
(CA, USA) 7500 diode array spectrophotometer
operated at room temperature. Spectra between
190 and 1100 nm at 1 nm intervals were recorded
every 30 s (and 250-700 nm reported) for analysis
of spectral changes due to the MPO-HX
interaction. On this spectrophotometer, each
spectrum is the average of 10 readings taken over
1 s.
RESULTS Inhibition of MPO by aromatic hydroxamates
- We set out to design more potent & selective
ferric state MPO inhibitors based initially on SHA.
Three substituted aromatic hydroxamates have
been named HX1, HX2 and HX3 (Fig. 2).
Inhibitor potency against production of HOCl was
routinely estimated in the presence of 10 M H2O2
and 1 mM tyrosine. The inclusion of tyrosine
ensures optimal turnover of the enzyme and
prevention of Compound II accumulation as it is a
good peroxidase substrate (43). SHA was found
to be a weak inhibitor of HOCl production, and the
other inhibitors evaluated were significantly more
potent (Fig. 3A). HX1 was by far the most potent
inhibitor (IC50 5 nM) with the following rank order
of potency obtained across the group: HX1 > HX2
> HX3 > SHA.
The selectivity of the inhibitors for MPO was
tested against a panel of enzymes (Table 2)
including the closely related heme peroxidases
lactoperoxidase (LPO) and thyroid peroxidase
(TPO). Generally, the new inhibitors all showed
high selectivity for the heme peroxidases over the
other redox enzymes and the more disparate
targets tested. HX1, HX2, and HX3 were tested in
a panel of more than 30 standard in vitro activity
assays# covering a diverse range of receptors, ion
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Reversible inhibition of myeloperoxidase
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channels, transporters and enzymes, at a single
concentration of 10 µM in duplicate, to explore
their pharmacological profile at MDS Pharma
Services (currently Eurofins Panlabs).
Compounds were inactive in all assays except the
arachidonate lipoxygenases (results shown in
Table 2). HX1 was the most selective inhibitor
identified with >300-fold higher potency against
MPO compared to any of the other targets
investigated.
Next we sought to test the efficiency of HX1
as an inhibitor of MPO in an assay that more
closely resembled the physiological environment.
This assay would demonstrate whether effective
inhibition could be achieved in the presence of
protein and other potential substrates of the
enzyme. We measured the consumption of
hydrogen peroxide in a modified FOX assay (28)
that included typical plasma concentrations of
urate, tyrosine, tryptophan and albumin (Fig. 3B).
Hydrogen peroxide consumption was linear and
dependent on the presence of MPO. HX1 showed
strong inhibition of MPO in this system with an
IC50 of 50 nM. Inhibition was immediate and
constant over time (not shown). Under the same
conditions, TX1, the 2-thioxanthine that
irreversibly inhibits MPO (14), was required at 10-
fold higher concentration to achieve the same
inhibition.
Reversibility of inhibition - The inhibition of
MPO displayed by all the compounds under
evaluation was shown to be fully reversible.
Representative data for HX1 and SHA are given in
Figure 3C. In this assay, MPO was immobilized
onto an ELISA plate and its chlorination activity
determined in the presence of inhibitor. The plate
was then washed to remove the inhibitor and the
chlorination activity remeasured. The chlorination
activity of the immobilized MPO was lowered
upon incubation with inhibitor, in line with
previously determined IC50 values. However,
upon extensive washing of the plate, enzyme
activity was restored, indicating that the
interaction and inhibition were reversible.
Inhibition of neutrophil MPO by
hydroxamates - Inhibitor potencies for blocking
the production of HOCl by neutrophils are
presented in Table 3. Peripheral blood neutrophils
were incubated with increasing concentrations of
inhibitor, and the effect on PMA-stimulated
oxidant production was measured. The inhibitors
blocked HOCl generation with the following rank
order of potency as determined by IC50 values:
HX1 > HX2 > HX3 > SHA. To test for specificity
of inhibition in the cell environment, compounds
HX1 to HX3 were also assessed for their effect on
superoxide production. There was no significant
effect on superoxide production by neutrophils
with concentrations well in excess (5-200 fold) of
the IC50s for HOCl production.
To test the effect of HX1 on chlorination of
tyrosine residues in proteins, neutrophils were
stimulated in the presence of human serum
albumin under conditions that produce substantial
formation of 3-chlorotyrosine in proteins (44).
When proteins in the supernatant of stimulated
neutrophils were digested with Pronase, it was
apparent that 3-chlorotyrosine was formed because
a product of the requisite mass co-eluted with the
stable isotopes of authentic 13
C 3-chlorotyrosine
(Fig. 4A). The product also co-eluted with a
second product that was two mass units higher and
was present at a third of its abundance, which is
characteristic of chlorine isotopes. The formation
of 3-chlorotyrosine was quantified by a sensitive
LCMS/MS method which demonstrated that under
the reaction conditions, stimulated neutrophils
chlorinated approximately three tyrosine residues
per 1000 and that this was almost completely
inhibited by 1 µM HX1 (Fig. 4B inset). HX1
inhibited chlorination of tyrosine residues in a
dose dependent manner having an estimated IC50
of 150 nM (Fig. 4B). No artifactual chlorination
during sample handling was detected. Also, HX1
did not inhibit chlorination by reagent HOCl (data
not shown), indicating that it inhibited MPO rather
than scavenging HOCl.
Inhibitor-enzyme binding characteristics - The ability of the hydroxamate analogues to bind
to ferric MPO was investigated by surface
plasmon resonance (SPR). HX1, HX2 and HX3
all caused concentration dependent, saturable
increases in SPR responses (Fig. 5) and the rank
order of affinity was the same as the rank order of
potency as enzyme activity inhibitors (Table 4).
The observed increase in binding affinity from
HX3 to HX1 was reflected by significantly slower
off rates and on rates. For example, the
dissociation phase for HX3 was much faster than
that of HX1. In contrast, the calculated on rate
constants were similar (within 4-fold) for HX2 and
HX3. This suggests that inhibitor optimization
(decreasing KD) from HX3 to HX1 is largely
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driven by increasing the stability of the bound
complex.
Crystal structure of the ferric MPO–inhibitor
complex - To investigate the nature of the
interaction between ferric MPO and HX1, we
determined the X-ray crystal structure of the
complex to 2.0 Å resolution. The overall structure
of the protein chain in the inhibitor complex was
identical to the one found in the ligand-free
enzyme (41). HX1 was unambiguously modeled
into the electron density (Fig. 6). The pyridine
ring of HX1 is positioned almost parallel to the
plane of pyrrole ring D of the MPO heme, and the
trifluoromethyl-aromatic ring is bent up occupying
the hydrophobic pocket at the entrance of the
active site. The hydroxamic acid group lies in the
centre of the distal cavity and both the carbonyl
and hydroxyl oxygens form hydrogen bonds with
amino acid side chains in the distal cavity. One of
the pyrimidine nitrogens provides the main
interaction with the heme propionate group while
the oxygen of the hydroxyl group is well
positioned for a hydrogen bond with Arg 239. The
trifluoromethyl-aromatic ring “tail” of the
molecule extends towards the surface of the
enzyme. There are no hydrogen bonds to the
protein outside the active site but the
trifluoromethyl groups is likely contributing weak
electrostatic interactions with Thr 238 (C-O…
F-C
distance around 3.4 Å). The carbonyl oxygen
binding site overlaps with the previously identified
halide binding site (41).
Kinetic studies of the inhibition of MPO
halogenation activity - To examine the kinetics of
the inhibition of halide oxidation by MPO, the
effect on the initial rate of the bromination of
NADH was assessed at varying concentrations of
bromide and hydrogen peroxide. The NADH
bromohydrin has a distinctive absorption spectrum
that gives this assay high sensitivity with respect
to the halogenation activity of MPO. HX1
inhibited the formation of NADH bromohydrin
with an IC50 of 70 nM, using optimal
concentrations of 50 µM H2O2 and 10 mM NaBr.
At 100 nM, HX1 had a marked effect on the rate
of formation of the bromohydrin with both
increasing concentration of halide (Fig. 7A) or
hydrogen peroxide (Fig. 7B). Kinetic constants
were obtained from these curves (Table 5) and
showed that the inhibitor decreased the catalytic
production of bromohydrin, kcat, by 82% and 78%
for halide and hydrogen peroxide, respectively.
The ratio indicating the catalytic efficiency,
kcat/Km (45), of MPO in this system also decreased
significantly with respect to the two substrates; by
43% and 59% for bromide and hydrogen peroxide
respectively. This indicates that the hydroxamate
HX1 acts as a mixed-type inhibitor with respect to
both halide and hydrogen peroxide.
Spectral changes upon MPO-hydroxamate
interaction - To further understand the mechanism
of inhibition of MPO by these compounds, their
interaction was studied spectrophotometrically.
MPO was incubated with hydroxamate HX1 or
HX2 (Fig. 8). The inhibitors had no effect on the
absorption spectrum of ferric MPO. However,
upon adding hydrogen peroxide there were
changes in the heme spectrum of MPO. In the
case of HX1 there was a decline in absorbance at
430 nm and a slight increase in absorbance
between 450 and 500 nm, followed by slow decay
back to the ferric spectrum (Fig. 8A). The
difference spectrum of the transient form revealed
peaks at 466 nm and 632 nm (Fig. 8B). The
spectral changes were much more pronounced for
the interaction with HX2, showing a stable shift to
a spectral form with peaks at 468 nm and 637 nm
(Fig. 8C). The spectrum also showed changes in
the far UV region consistent with oxidation of the
inhibitor HX2 (Fig. 8D). The effect on the heme
spectrum, although different in magnitude for
HX1 and HX2, gave a common result. The
interaction between MPO and these hydroxamates
gave rise to the characteristic spectrum of a
nitrosyl complex with ferrous MPO with Soret
maxima 467 and 635 nm (46). Together with the
UV spectral changes, the formation of nitrosyl
ferrous MPO indicates that these hydroxamates
are, to some extent, metabolized by MPO.
Compound HX2 showed greater formation of this
complex than HX1, which is the inverse of their
inhibitory potency. Hence, the loss of activity
does not correlate with the degree of the formation
of the nitrosyl-complex of MPO.
DISCUSSION We have identified three aromatic
hydroxamates that have unprecedented high
potency as reversible inhibitors of the
halogenation activity of MPO. Of particular
interest is the trifluoromethyl-substituted
compound HX1, which is by far the best inhibitor
of MPO currently known. Its physical occupancy
of the active site is the defining feature by which it
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inhibits MPO. The new hydroxamates showed
specific inhibition of MPO when screened against
other human enzymes, and offer the highest seen
reversible inhibition, with an IC50 of 50 nM, under
physiological conditions. Our findings
demonstrate that this type of inhibitor has potential
as a therapeutic agent against the detrimental
activity of MPO and should also provide useful
information about the active site of heme
peroxidases.
The hydroxamates inhibit MPO by
binding to the active site of ferric MPO and
blocking the access of substrates. This mode of
action is supported by our findings that
hydroxamates bind tightly to the enzyme, occupy
the active site and perturb binding of substrates,
cause reversible inhibition and act as mixed-type
inhibitors with respect to hydrogen peroxide and
halides. Also, their abilities to inhibit and bind to
MPO were directly related. Inhibition of this type
is likely to occur in vivo because the hydroxamates
not only exhibited potent inhibition of the purified
enzyme, but also consistently inhibited MPO in
more physiological systems including a multi-
substrate MPO assay. With PMA-stimulated
neutrophils, they were effective inhibitors of
HOCl production as well as chlorination of
tyrosine residues in proteins. The IC50 values in
the latter systems were significantly higher than in
the simple assay with purified enzyme but the
trend in potency remained unchanged with HX1 >
HX2 > HX3 > SHA. The raised IC50 values in the
more physiological assays are consistent with the
inhibitors being affected by interactions such as
protein binding, as well as competition for the
enzyme by physiological substrates. Our binding
and inhibition studies indicate that the
predominant interaction between the
hydroxamates and MPO is the formation of an
inactive enzyme-hydroxamate complex. This is
depicted by the non-cycling MPO complex (MPO
FeIII
–RC(O)NHOH) in Figure 9, that prevents
turnover of the enzyme in both its halogenation
and peroxidation cycles.
The crystal structure of the complex
formed by ferric MPO and HX1 was solved to
high resolution. Although HX1 binds tightly to
MPO (KD 15 nM) the occupancy was only
approximately 0.5. It is likely that the
crystallization conditions have influenced ligand
binding. In particular, the low pH of
crystallization (pH 5.5) may partially protonate
His95, which is involved in hydrogen bonding to
HX1, and this would have an effect on ligand
occupancy. Nevertheless, the crystal structure
provides clear evidence of the nature of the
interactions between HX1 and MPO. The
hydroxamate is located in the substrate binding
pocket of MPO without any significant
conformational changes to the enzyme’s native
structure. The position of the hydroxamic acid
group in the distal cavity is similar to that
previously described for the MPO-SHA complex
(25). However the planar tilt angle differs by
approximately 20○ due to additional interactions
between the pyridine ring of HX1 and the heme
propionate group. Also the second ring of HX1
with its trifluoromethyl groups creates a
hydrophobic tail that contributes to improved
affinity over SHA and provides additional steric
hindrance for substrate access to the active site.
The second ring system also enhances selectivity
for MPO over other heme peroxidases. That is,
for SHA the potency of inhibition is more than 10-
fold lower for MPO than for TPO and LPO,
whereas HX1 is greater than two orders of
magnitude more potent toward MPO than both
TPO and LPO. The structure of TPO is not
known, but the improved selectivity for MPO over
LPO can be rationalized by comparing the shape
of the cavity adjacent to the active site. The loop
corresponding to residues 407-415 in MPO adopts
a different conformation in the structure of both
caprine and bovine LPO (47) which would prevent
binding of HX1.
The reversibility of the inhibition
displayed by HX1 and SHA (Fig. 3C) confirms
that hydroxamate inhibitors simply dock at the
active site of MPO, unlike the 2-thioxanthine
inhibitors that become irreversibly covalently
bound to the heme (14). Notably the 2-
thioxanthine series of potent inhibitors also
features multi-ring structures with a bent tail that
extends into the hydrophobic pocket.
Another aspect of the interaction between
hydroxamates and MPO that is of interest to their
pharmacology, is that they are also potential
peroxidase substrates. We found spectral evidence
of hydroxamate oxidation by MPO in the case of
HX2, with discernible losses in the far UV region
attributable to MPO metabolizing this substrate. It
is long established that hydroxamates such as SHA
can serve as redox substrates of peroxidases such
as horse-radish peroxidase (22,48) as well as MPO
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(21,24). Hydroxamates can undergo oxidation to a
transient nitroxide radical (RC(O)NHO●) (49) and
therefore are potential reductants in the classical
peroxidase cycle. The reactions of hydroxamates
(RC(O)NHOH) with MPO intermediates is
summarized in Figure 9, and can be regarded as
secondary to the inhibitory complex formation.
Our spectral analyses with the new
substituted aromatic compounds revealed the
formation of a nitrosyl-adduct form of ferrous
MPO (Fig. 9; NO:FeII). This was previously
discovered by the direct reaction of gaseous NO
with MPO, yielding Soret maxima 467 and 635
nm (46). We observed this NO:FeII spectrum after
reaction of MPO with hydrogen peroxide in the
presence of HX1 or HX2 (Fig. 8). Ferric nitrosyl
MPO with maxima at 433 and 630 nm (46) was
not observed. The formation of a ferrous nitrosyl
heme intermediate occurs in normal catalytic
activity of nitric oxide synthase (50) but has not
been reported with other substrates of MPO. We
have proposed a mechanism for its production via
oxidation of hydroxamates with concomitant
formation of nitric oxide and ferrous enzyme (Fig.
9). The extent to which the NO:FeII complex is
formed depends on the ease of oxidation of a
particular hydroxamate to the nitroxide radical
(RC(O)NHO●). This radical promotes the
reduction to ferrous MPO, and upon hydrolysis of
its oxidized form will also lead to HNO and NO
(49,51). NO binds reversibly to ferrous MPO (46)
and in our aerobic conditions was stable for a few
minutes only (Fig. 8). Alternatively, the ferrous
NO complex could form by reaction of nitroxyl
with the ferric enzyme (52). There was a
significant difference between the spectral changes
seen for HX1 and HX2. It was evident that HX2
formed the most NO:FeII, but it was not as potent a
binder or inhibitor of MPO as HX1. Therefore the
formation of NO:FeII is counter-inhibitory and
indicates inhibitor instability. These results have
implications for development of inhibitors as
robust pharmaceutical agents regarding
mechanisms of drug breakdown. The propensity
of hydroxamates to act as NO-donors is a
recognized problem in the generation of all
hydroxamate-based drugs (49).
The most potent inhibitors of MPO
previously reported are the 2-thioxanthine family
of suicide substrates (14). These compounds are
notable in that they are mechanism-based
inhibitors that do not release reactive free radicals
from the active site of MPO. The production of
unwanted side products is also avoided by
reversible inhibitors provided there is no
concurrent oxidation of the bound inhibitor. This
issue of inhibitor metabolism by the enzyme is a
potential shortcoming of the new aromatic
hydroxamates. Another limitation of these
compounds is that they undergo slow hydrolysis
which decreases their pharmacological efficacy.
However, their mode of binding to MPO and their
extreme potency signifies that reversible inhibition
is potentially the best strategy for limiting the
activity of MPO in vivo.
We conclude that modified hydroxamates
have proven to be highly potent and specific
reversible inhibitors of MPO. The differently
substituted double-ring hydroxamates have
achieved higher potency due to increased polar
interactions with the MPO heme and a modified
bent shape suited to filling the active site cavity.
This leads to stronger binding at the heme and
better interference of the access of substrates to the
active site. These new potent reversible inhibitors
demonstrate a valuable alternative mechanism for
MPO inhibition to that of irreversible mechanism-
based inhibitors exemplified by the 2-
thioxanthines (14). Without permanently crippling
the enzyme or generating multiple radical chain
reactions and by-products, this reversible type of
inhibitor should be ideal for therapeutic inhibition
of unwarranted MPO activity and oxidative
damage. In the case of inflammatory disorders
characterized by episodes of heightened neutrophil
attack such as cystic fibrosis, this benign but
efficient type of reversible inhibitor could be
administered to control transient fluxes of released
MPO. Development of pharmacologically stable
inhibitors that bind reversibly to the active site of
MPO but are not substrates, would be of great
value for the treatment of inflammatory diseases.
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Acknowledgments - We thank Alice Flaherty for her medicinal chemistry expertise in the design and
synthesis of the molecules described in this paper, and Robert Björnestedt for support with expression
and purification of MPO. We are very grateful to Anna-Karin Tiden, Philip Mallinder and Anders
Broo for their support.
FOOTNOTES
*D.W.J., B.T., D.L., P.H., G.P., T.S., H.E., and J.F.U. were employed by AstraZeneca when involved
in this study. Where applicable, the authors have a current address listed after the address where the
reported work was undertaken. A.J.K. received financial support from AstraZeneca to conduct this
research.
Current addresses: ANovartis Institute for Biomedical Research Inc., Cambridge, MA 02139, United
States of America; BSchool of Graduate Entry Medicine and Health, University of Nottingham, Royal
Derby Hospital, Derby DE22 3DT, United Kingdom; CSygnature Discovery Ltd, Nottingham NG1
1GF, United Kingdom; DAstraZeneca R&D Macclesfield, Cheshire SK10 4TF, United Kingdom)
To whom correspondence should be adressed: Louisa V. Forbes, Centre for Free Radical Research,
Department of Pathology, University of Otago Christchurch, P.O. Box 4345, Christchurch, New
Zealand. Tel.: +64 3 364 0590, E-mail: [email protected]
# Targets for selectivity screening: G protein coupled receptors; LTD4, muscarinic M2, muscarinic
M3, ETA, ETB, adrenergic α2A, dopamine D2L, histamine H1, nicotinic acetylcholine receptor,
adrenergic β1, opiate mu, 5-HT1, 5-HT2A. Ion channels; L-type calcium channel, sodium channel (site
2). Enzymes; 5-LO, 15-LO, PDE4, ERK2, thromboxane synthetase, XO, acetyl cholinesterase, MMP-
1, MMP-2, MMP-3, MMP-7, MMP-9, COX-1, COX-2, LTA4 hydrolase, LTC4 synthase, lipid
peroxidase, MAO-A. Also norepinephrine transporter and estrogen receptor-α.
Abbreviations used: MPO, myeloperoxidase; SHA, salicylhydroxamic acid; HX, hydroxamate;
HEPES, N-(2-hydroxylethyl)piperazine-N’-(2-ethane-sulfonic acid); DMSO, dimethylsulfoxide;
EDTA, ethylenediaminetetraacetic acid; SDS-PAGE, sodium dodecyl sulfate polyacrylamide gel
electrophoresis; NADH, nicotinamide adenine dinucleotide; ELISA, enzyme-linked immunosorbent
assay; LCMS, liquid chromatography and mass spectrometry; SEM, standard error of the mean.
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FIGURE LEGENDS
FIGURE 1. Normal catalytic cycling of myeloperoxidase. The native state of the enzyme, Ferric
MPO, reacts with hydrogen peroxide to form the redox intermediate Compound I. Compound I either
oxidizes chloride to regenerate ferric MPO via the halogenation cycle (a), or will oxidize an organic
substrate (RH) to a free radical (R●), forming the redox intermediate Compound II which can be
reduced back to the native state via the peroxidation cycle (b).
FIGURE 2. Chemical structures of aromatic hydroxamates (RC(O)NHOH) that inhibit MPO.
The structures are shown for salicylhydroxamic acid (SHA), 2-(3,5-bis-trifluoromethyl-benzylamino)-
6-oxo-1H-pyrimidine-5-carbohydroxamic acid (HX1), 4-benzyl-2-hydroxy-benzenecarbohydroxamic
acid (HX2) and 2-(benzylamino)-6-oxo-3H-pyrimidine-5-carbohydroxamic acid (HX3).
FIGURE 3. Inhibition of HOCl production by aromatic hydroxamates. A, MPO (2 nM) was
preincubated with either SHA, HX1, HX2 or HX3 for 15 min prior to the addition of H2O2 (10 M).
HOCl production was determined after 1 min by the taurine chloramine assay, in the presence of 1
mM tyrosine. Data is presented as % control HOCl production determined in the absence of inhibitor
and represents the mean SEM of 3-51independent experiments. B, MPO (5 nM) was incubated at
room temperature in 50 mM phosphate buffer pH 7.4, containing 140 mM NaCl, 200 µM urate, 50
µM tyrosine, 50 µM tryptophan, 1 mg/ml albumin,1 mM methionine and with or without inhibitor
HX1 (●) or TX1 (○). Reactions were started by adding 20 µM H2O2 and the consumption of H2O2
was measured after 15 min. Inhibition of H2O2 consumption was measured relative to the full system
lacking added inhibitor, in which approx. 10 µM H2O2 was consumed. Data are means ± range of
duplicates and are representative of 2-3 separate experiments. C, MPO was immobilized on protein
immobilizer plates (Exiqon) and incubated with either SHA (■) or HX1 () for 15 min prior to the
addition of 10µM H2O2 substrate. HOCl production was determined after 1 min by the taurine
chloramine assay. After extensive washing with assay buffer, a further 10µM H2O2 was added and
HOCl production was re-determined (post-wash SHA(□); HX1 (○)). Data are presented as % control
HOCl production determined in the absence of compounds, and represents the mean SEM of 3
independent experiments.
FIGURE 4. Chlorination of tyrosine residues by stimulated neutrophils and inhibition by HX1.
Neutrophils were incubated with human serum albumin and stimulated with PMA. The proteins were
digested with Pronase and then analyzed for their content of 3-chlorotyrosine. A, A typical
chromatogram of 3-chlorotyrosine showing the characteristic 3:1 isotopic ratios for the internal
standard (Cl-Y13
C9) and chlorinated tyrosine (Cl-Y12
C) produced by neutrophils. B inset, The 3-
chlorotyrosine content of proteins from the supernatant of neutrophils stimulated with PMA in the
absence or presence of 1 µM HX1. Data are means ± SEM of 3-7 measurements taken over three
separate experiments. B, Inhibition of 3-chlorotyrosine formation by PMA-stimulated neutrophils
with increasing concentration of HX1 relative to the full system without HX1. Data are plotted as
mean ± range of duplicates and are representative of three experiments. Experimental details are
described in Experimental Procedures.
FIGURE 5. Determination of binding kinetics of MPO inhibitors using SPR. MPO was immobilized
to a CM5 sensor chip and compound binding was evaluated for HX1, HX2 and HX3. Figures show
specific binding traces representative of 3-6 separate experiments. Each figure shows the overlay of
multiple serial sensogram traces for 9 different compound concentrations in series of 3-fold dilutions
ranging from 0.3 µM to 30 pM for HX1 and HX2, and from 30 µM to 3 nM for HX3. From t=0
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compound was continuously perfused over the sensor chip leading to a clear net association of
compound to the immobilized MPO. Subsequently compound perfusate was replaced with buffer at
t=60-210 s, leading to a loss of response due to net compound dissociation.
FIGURE 6. X-ray crystal structure of the MPO-HX1 complex and electron density maps. A, The
enzyme-inhibitor complex has HX1 (orange stick representation) bound in the active site pocket above
the MPO heme group (green). MPO side chains are shown for residues within 5 Å from the ligand in
a stick representation (yellow). Hydrogen bonds between HX1 and MPO and solvent are indicated
with dashed lines. The pink surface outlines the solvent accessible area of the active site. Red atoms
indicate the oxygen of water molecules. B, Electron density maps for HX1; the Fo-Fc omit map
contoured at 2.5 and calculated in the absence of HX1, and the 2Fo-Fc map contoured at 1 and
calculated for the final model where occupancy of HX1 was set to 0.5.
FIGURE 7. Inhibition of the rate of NADH bromohydrin formation. MPO (20 nM) was incubated at
room temperature in 20 mM phosphate buffer pH 7.4, containing 100 µM NADH with either A, 50
µM H2O2 ± 0.1 µM HX1 with varying concentrations of NaBr, or B, 5 mM NaBr ± 0.1 µM HX1 with
varying concentrations of H2O2. The formation of NADH bromohydrin was detected by absorbance at
275 nm and initial rates were determined within the first minute. Data shown are means ± SEM of
triplicates in the absence (●) or presence (○) of HX1, and are representative of three separate
experiments.
FIGURE 8. Effect of HX1 and HX2 on the absorption spectrum of MPO. A, MPO (1.8 µM) was
incubated with 10 µM HX2 in 50 mM phosphate buffer pH 7.4 (gray). H2O2 (40 µM) was then added
and spectra recorded at 10 s (black), and 5 min (dashed). B, The difference spectrum between the first
observable spectrum after H2O2 (A, black) and ferric MPO (A, gray). C, MPO (2.75 µM) was
incubated with 62.5 µM HX2 in 50 mM phosphate buffer pH 7.4 (gray). H2O2 (50 µM) was added
and the spectral changes recorded (black). The new spectrum with peaks at 468 and 637 nm formed
within 30 s and was stable for approx. 3 min. D, Spectral changes in the UV region after adding H2O2
to MPO and HX2. The arrows indicate the direction of the spectral changes observed at 30 s intervals.
All results are typical of three experiments.
FIGURE 9. Proposed mechanism for the inhibition of MPO by hydroxamates, and their concurrent
oxidation. Ferric MPO is bound by hydroxamate (RC(O)NHOH) forming an inactive complex (top
left) thereby abrogating the cycling of ferric MPO via Compounds I and II. This is the inhibition
pathway. Hydroxamates can, to varying degrees, also serve as substrates of MPO Compound I to
form transient nitroxide radical RC(O)NHO●. This in turn can reduce ferric MPO to ferrous MPO
(FeII) to yield nitrosyl-ferrous MPO (NO:Fe
II) upon binding of released NO. The oxidized product of
RC(O)NHO● is subject to hydrolysis (dotted line), yielding the carboxylic acid and HNO, a source of
NO.
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TABLES
TABLE 1 (For Methods section)
Crystallography data collection and
refinement statistics
Rmerge = ∑|I-〈I〉|/∑ I
R factor = ∑||Fo|-k|Fc|| /∑ |Fo| where Fo and Fc are the observed
and calculated structure factor amplitudes, respectively.
Free R factor = R factor calculated for a test set of 5% of the
measured reflections which were excluded from refinement.
*Assuming occupancy of 0.5
Data collection
Space group P21
Unit cell parameters a=111.3 Å, b=63.4 Å
c=92.4 Å, β=97.4°
Resolution range (Å) 30-2.0 (2.11-2.00)
No. Reflections (total/unique) 291795 / 81456
Redundancy 3.7 (3.6)
Data completeness (%) 94.4 (93.8)
Average I/σI 6.2 (2.6)
Rmerge (%) 9.3 (27.4)
Statistics for the final model
No.of nonhydrogen atoms 10168
R factor (%) 22.2
Free R factor (%) 27.8
Wilson B factor (Å2) 16.2
Average B factors (Å2):
All atoms 14.1
Protein (chains A,C/B,D) 11.5 / 15.9
Heme (A / B) 6.0 / 7.8
HX1 (A / B)* 6.4 / 12.3
RMSD bond length (Å) 0.015
RMSD bond angles (°)
Ramachandran outliers
Ramachandran favored
1.941
0.0% (0/1127 residues)
97.2% (1095 residues)
PDB code 4C1M
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TABLE 2
Selectivity of aromatic hydroxamates as inhibitors of MPO Enzyme inhibition data represents the mean IC50 (µM) ± range or SEM for the number of
determinations in parentheses. N.T.: not tested, N.A.: not active < 30% inhibition at 100 µM
compound. a Data is result for all three NOS isozymes tested.
b Data is result for all five P450
isozymes tested. c Effect of 10µM compound on activity of arachidonate lipoxygenases 5-LO and 15-
LO; % inhibition is indicated.
Assay SHA HX1 HX2 HX3
MPO 25 ± 1 (4)
0.00501 ± 0.00002 (51)
0.0251 ± 0.0003 (9)
0.79 ± 0.01 (7)
TPO 2.00 ± 0.04 (2) 1.59 ± 0.03 (8)
0.063 ± 0.001 (4)
1.59 ± 0.01 (4)
LPO 0.40 ± 0.01 (2) 6.3 ± 0.1 (3) 0.040 ± 0.001 (3) 2.00 ± 0.04 (4)
NOSs a N.T. N.T. N.A. (2) N.A. (2)
P450s b > 10 > 10 (2) > 10 > 10 (2)
LOs c N.T. 5-LO: 75%
15-LO: 72%
N.T. 15-LO: 72%
TABLE 3
Inhibitory effects of aromatic hydroxamates on
HOCl production by human neutrophils
Data represent the mean ± range or SEM for the number
of determinations in parentheses.
Compound IC50 (µM) for HOCl production
SHA 40 ± 2 (2)
HX1 0.0501 ± 0.0006 (12)
HX2 2.00 ± 0.03 (8)
HX3 6.31 ± 0.04 (5)
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TABLE 4
Affinity and kinetic estimates for hydroxamates binding to ferric MPO
Dissociation constants (KD) were derived from parameter logistic curve fitting to [compound] versus
binding measurements. Association and dissociation rate constants (ka and kd) were measured by SPR
from which the half-life for dissociation (t1/2) was calculated. Data are presented as mean SEM from
3-6 independent experiments. N.D.: not determined.
Compound KD (µM) ka (M-1
s-1
) kd (s-1
) t1/2 (s)
HX1 0.0158 0.0001 1.8 0.2 x 105 3.5 0.4 x 10
-3 275
HX2 0.063 0.001 6.9 1.1 x 105 3.0 0.1 x 10
-2 23
HX3 2.00 0.04 N.D. N.D. < 10
TABLE 5
Kinetic parameters for the formation of NADH bromohydrin by MPO in the absence and
presence of HX1 Values for Vmax and Km (± standard errors generated by Sigma Plot) were determined by curve fitting
to initial rate plots (Fig. 7), from which the catalytic production of bromohydrin, kcat (Vmax/[enzyme])
was calculated. Data shown are representative of three separate experiments.
kcat (s-1
)
kcat /Km (M-1
s-1
)
Substrate -HX1 +HX1 -HX1 +HX1
Bromide 35.1 ± 1.2 6.3 ± 0.8 11.3 ± 1.4 x 103 6.4 ± 3.9 x 10
3
H2O2 28.6 ± 0.4 6.2 ± 0.4 4.4 ± 0.3 x 106 1.8 ± 0.6 x 10
6
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FIGURES
Figure 1 (For Introduction section)
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Figure 2
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Figure 3
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Figure 4
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Figure 5
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Figure 6
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Figure 7
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Figure 8
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Figure 9 (For Discussion section)
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and Anthony J. KettleLaughton, Paul Hemsley, Garry Pairaudeau, Rufus Turner, Håkan Eriksson, John F. Unitt
Louisa V. Forbes, Tove Sjögren, Françoise Auchère, David W. Jenkins, Bob Thong, DavidPotent Reversible Inhibition of Myeloperoxidase by Aromatic Hydroxamates
published online November 5, 2013J. Biol. Chem.
10.1074/jbc.M113.507756Access the most updated version of this article at doi:
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