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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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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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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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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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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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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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REFERENCES

1. Halliwell, B., and Gutteridge, J. M. C. (1999) Free Radicals in Biology and Medicine, 3rd ed.,

Oxford University Press, Oxford

2. Davies, M. J., Hawkins, C. L., Pattison, D. I., and Rees, M. D. (2008) Mammalian heme

peroxidases: from molecular mechanisms to health implications. Antioxid. Redox Signal. 10,

1199-1234

3. van der Veen, B. S., de Winther, M. P. J., and Heeringa, P. (2009) Myeloperoxidase:

Molecular mechanisms of action and their relevance to human health and disease. Antioxid.

Redox Signal. 11, 2899-2937

4. Hernandez-Mijares, A., Rocha, M., Apostolova, N., Borras, C., Jover, A., Banuls, C., Sola, E.,

and Victor, V. M. (2011) Mitochondrial complex I impairment in leukocytes from type 2

diabetic patients. Free Radic. Biol. Med. 50, 1215-1221

5. Kettle, A. J., and Winterbourn, C. C. (1991) Mechanism of inhibition of myeloperoxidase by

anti-inflammatory drugs. Biochem. Pharmacol. 41, 1485-1492

6. Jantschko, W., Furtmuller, P. G., Zederbauer, M., Neugschwandtner, K., Lehner, I.,

Jakopitsch, C., Arnhold, J., and Obinger, C. (2005) Exploitation of the unusual

thermodynamic properties of human myeloperoxidase in inhibitor design. Biochem.

Pharmacol. 69, 1149-1157

7. Sliskovic, I., Abdulhamid, I., Sharma, M., and Abu-Soud, H. M. (2009) Analysis of the

mechanism by which tryptophan analogs inhibit human myeloperoxidase. Free Radic. Biol.

Med. 47, 1005-1013

8. Rees, M. D., Bottle, S. E., Fairfull-Smith, K. E., Malle, E., Whitelock, J. M., and Davies, M. J.

(2009) Inhibition of myeloperoxidase-mediated hypochlorous acid production by nitroxides.

Biochem. J. 421, 79-86

9. Queiroz, R. F., Vaz, S. M., and Augusto, O. (2011) Inhibition of the chlorinating activity of

myeloperoxidase by tempol: revisiting the kinetics and mechanisms. Biochem. J. 439, 423-

431

10. Hsuanyu, Y., and Dunford, H. B. (1999) Oxidation of clozapine and ascorbate by

myeloperoxidase. Arch. Biochem. Biophys. 368, 413-420

11. Meotti, F. C., Jameson, G. N. L., Turner, R., Harwood, T., Stockwell, S., Rees, M. D.,

Thomas, S. R., and Kettle, A. J. (2011) Urate as a physiological substrate for

myeloperoxidase: Implications for hyperuricemia and inflammation. J. Biol. Chem. 286,

12901-12911

12. Chapman, A. L. P., Mocatta, T. J., Shiva, S., Seidel, A., Chen, B., Khalilova, I., Paumann-

Page, M. E., Jameson, G. N. L., Winterbourn, C. C., and Kettle, A. J. (2013) Ceruloplasmin is

an endogenous inhibitor of myeloperoxidase. J. Biol. Chem. 288, 6465-6477

13. Kettle, A. J., Gedye, C. A., and Winterbourn, C. C. (1997) The mechanism of inactivation of

myeloperoxidase by 4-aminobenzoic acid hydrazide. Biochem. J. 321, 503-508

14. Tiden, A.-K., Sjogren, T., Svensson, M., Bernlind, A., Senthilmohan, R., Auchere, F.,

Norman, H., Markgren, P.-O., Gustavsson, S., Schmidt, S., Lundquist, S., Forbes, L. V.,

Magon, N. J., Paton, L. N., Jameson, G. N. L., Eriksson, H., and Kettle, A. J. (2011) 2-

Thioxanthines are mechanism-based inactivators of myeloperoxidase that block oxidative

stress during inflammation. J. Biol. Chem. 286, 37578-37589

15. Koelsch, M., Mallak, R., Graham, G. G., Kajer, T., Milligan, M. K., Nguyen, L. Q.,

Newsham, D. W., Keh, J. S., Kettle, A. J., Scott, K. F., Ziegler, J. B., Pattison, D. I., Fu, S.,

Hawkins, C. L., Rees, M. D., and Davies, M. J. (2010) Acetaminophen (paracetamol) inhibits

myeloperoxidase-catalyzed oxidant production and biological damage at therapeutically

achievable concentrations. Biochem. Pharmacol. 79, 1156-1164

16. Forbes, L. V., Furtmueller, P. G., Khalilova, I., Turner, R., Obinger, C., and Kettle, A. J.

(2012) Isoniazid as a substrate and inhibitor of myeloperoxidase: Identification of amine


http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/


11

adducts and the influence of superoxide dismutase on their formation. Biochem. Pharmacol.

84, 949-960

17. Arnhold, J., Furtmuller, P. G., Regelsberger, G., and Obinger, C. (2001) Redox properties of

the couple compound I/native enzyme of myeloperoxidase and eosinophil peroxidase. Eur.J.

Biochem. 268, 5142-5148

18. Furtmuller, P. G., Arnhold, J., Jantschko, W., Pichler, H., and Obinger, C. (2003) Redox

properties of the couples compound I/compound II and compound II/native enzyme of human

myeloperoxidase. Biochem. Biophys. Res. Commun. 301, 551-557

19. Uetrecht, J. P. (1992) The role of leukocyte-generated reactive metabolites in the pathogenesis

of idiosyncratic drug reactions. Drug Metab. Rev. 24, 299-366

20. Jiang, X., Khursigara, G., and Rubin, R. L. (1994) Transformation of lupus-inducing drugs to

cytotoxic products by activated neutrophils. Science 266, 810-813

21. Davies, B., and Edwards, S. W. (1989) Inhibition of myeloperoxidase by salicylhydroxamic

acid. Biochem. J. 258, 801-806

22. Rich, P. R., Wiegand, N. K., Blum, H., Moore, A. L., and Bonner, W. D. (1978) Studies on

mechanism of inhibition of redox enzymes by substituted hydroxamic acids. Biochim.

Biophys. Acta 525, 325-337

23. Kettle, A. J., Gedye, C. A., Hampton, M. B., and Winterbourn, C. C. (1995) Inhibition of

myeloperoxidase by benzoic acid hydrazides. Biochem. J. 308, 559-563

24. Ikeda-Saito, M., Shelley, D. A., Lu, L., Booth, K. S., Caughey, W. S., and Kimura, S. (1991)

Salicyhydroxamic acid inhibits myeloperoxidase activity. J. Biol. Chem. 266, 3611-3616

25. Davey, C. A., and Fenna, R. E. (1996) 2.3 A resolution X-ray crystal structure of the

bisubstrate analogue inhibitor salicylhydroxamic acid bound to human myeloperoxidase: A

model for a prereaction complex with hydrogen peroxide. Biochemistry 35, 10967-10973

26. Hope, H. R., Remsen, E. E., Lewis, C., Heuvelman, D. M., Walker, M. C., Jennings, M., and

Connolly, D. T. (2000) Large-scale purification of myeloperoxidase from HL60

promyelocytic cells: Characterization and comparison to human neutrophil myeloperoxidase.

Protein Express. Purif. 18, 269-276

27. Dypbukt, J. M., Bishop, C., Brooks, W. M., Thong, B., Eriksson, H., and Kettle, A. J. (2005)

A sensitive and selective assay for chloramine production by myeloperoxidase. Free Radic.

Biol. Med. 39, 1468-1477

28. Wolff, S. P. (1994) Ferrous ion oxidation in presence of ferric ion indicator xylenol orange for

measurement of hydroperoxides. Methods Enzymol. 233, 182-189

29. Auchere, F., and Capeillere-Blandin, C. (1999) NADPH as a co-substrate for studies of the

chlorinating activity of myeloperoxidase. Biochem. J. 343, 603-613

30. Prutz, W. A., Kissner, R., Koppenol, W. H., and Ruegger, H. (2000) On the irreversible

destruction of reduced nicotinamide nucleotides by hypohalous acids. Arch. Biochem.

Biophys. 380, 181-191

31. Ferrari, R. P., Laurenti, E., Ghibaudi, E. M., and Casella, L. (1997) Tyrosinase-catecholic

substrates in vitro model: Kinetic studies on the o-quinone/o-semiquinone radical formation.

J. Inorg. Biochem. 68, 61-69

32. Connolly, S., Aberg, A., Arvai, A., Beaton, H. G., Cheshire, D. R., Cook, A. R., Cooper, S.,

Cox, D., Hamley, P., Mallinder, P., Millichip, I., Nicholls, D. J., Rosenfeld, R. J., St-Gallay,

S. A., Tainer, J., Tinker, A. C., and Wallace, A. V. (2004) 2-aminopyridines as highly

selective inducible nitric oxide synthase inhibitors. Differential binding modes dependent on

nitrogen substitution. J. Med. Chem. 47, 3320-3323

33. Weaver, R., Graham, K. S., Beattie, I. G., and Riley, R. J. (2003) Cytochrome P450 inhibition

using recombinant proteins and mass spectrometry/multiple reaction monitoring technology in

a cassette incubation. Drug Metab. Dispos. 31, 955-966

34. Noguchi, M., Miyano, M., Kuhara, S., Matsumoto, T., and Noma, M. (1994) Interfacial

kinetic reaction of human 5-lipoxygenase. Eur. J. Biochem. 222, 285-292


http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/


12

35. Auerbach, B. J., Kiely, J. S., and Cornicelli, J. A. (1992) A spectrophotometric microtiter-

based assay for the detection of hydroperoxy derivatives of linoleic-acid. Anal. Biochem. 201,

375-380

36. Böyum, A. (1968) Isolation of mononuclear cells and granulocytes from human blood.

Isolation of mononuclear cells by one centrifugation, and of granulocytes by combining

centrifugation and sedimentation at 1 g. Scand. J. Clin. Lab. Invest. 21, 77-89

37. Dahlgren, C., and Karlsson, A. (1999) Respiratory burst in human neutrophils. J. Immunol.

Methods 232, 3-14

38. Thornalley, P. J., Battah, S., Ahmed, N., Karachalias, N., Agalou, S., Babaei-Jadidi, R., and

Dawney, A. (2003) Quantitative screening of advanced glycation endproducts in cellular and

extracellular proteins by tandem mass spectrometry. Biochem. J. 375, 581-592

39. Leslie, A. G. W. (1999) Integration of macromolecular diffraction data. Acta Crystallogr.,

Sect D 55, 1696-1702

40. Collaborative Computational Project, N. (1994) The CCP4 Suite: Programs for protein

crystallography. Acta Crystallogr., Sect D 50, 760-763

41. Fiedler, T. J., Davey, C. A., and Fenna, R. E. (2000) X-ray crystal structure and

characterization of halide-binding sites of human myeloperoxidase at 1.8 angstrom resolution.

J. Biol. Chem. 275, 11964-11971

42. Jones, T. A., Zou, J. Y., Cowan, S. W., and Kjeldgaard, M. (1991) Improved methods for

building protein models in electron-density maps and the location of errors in these models.

Acta Crystallogr., Sect A 47, 110-119

43. Marquez, L. A., and Dunford, H. B. (1995) Kinetics of oxidation of tyrosine and dityrosine by

myeloperoxidase compounds I and II. Implications for lipoprotein peroxidation studies. J.

Biol. Chem. 270, 30434-30440

44. Kettle, A. J. (1996) Neutrophils convert tyrosyl residues in albumin to chlorotyrosine. FEBS

Lett. 379, 103-106

45. Ochs, R. S. (2000) Understanding enzyme inhibition. J.Chem. Educ. 77, 1453-1456

46. Abu-Soud, H. M., and Hazen, S. L. (2000) Nitric oxide modulates the catalytic activity of

myeloperoxidase. J. Biol. Chem. 275, 5425-5430

47. Singh, A. K., Singh, N., Sharma, S., Singh, S. B., Kaur, P., Bhushan, A., Srinivasan, A., and

Singh, T. P. (2008) Crystal structure of lactoperoxidase at 2.4 angstrom resolution. J. Mol.

Biol. 376, 1060-1075

48. Brooks, J. L. (1983) Stimulation of peroxidase reactions by hydroxamates. Biochem. Biophys.

Res. Commun. 116, 916-921

49. Samuni, Y., Samuni, U., and Goldstein, S. (2012) The mechanism underlying nitroxyl and

nitric oxide formation from hydroxamic acids. Biochim. Biophys. Acta 1820, 1560-1566

50. Abusoud, H. M., Wang, J. L., Rousseau, D. L., Fukuto, J. M., Ignarro, L. J., and Stuehr, D. J.

(1995) Neuronal nitric-oxide synthase self-inactivates by forming a ferrous-nitrosyl complex

during aerobic catalysis. J. Biol. Chem. 270, 22997-23006

51. Reisz, J. A., Bechtold, E., and King, S. B. (2010) Oxidative heme protein-mediated nitroxyl

(HNO) generation. Dalton Trans. 39, 5203-5212

52. Farmer, P. J., and Sulc, F. (2005) Coordination chemistry of the HNO ligand with hemes and

synthetic coordination complexes. J. Inorg. Biochem. 99, 166-184


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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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