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British Journal of Pharmacology (2007) 152, 838854

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Myeloperoxidase: a target for new drug
E Malle1, PG Furtmu
ller2, W Sattler1 and C Obinger2

Center of Molecular Medicine, Institute of Molecular Biology and Biochemistry, Medical University of Graz, Graz, Austria
and 2Division of Biochemistry, Department of Chemistry, BOKU University of Natural Resources and Applied Life Sciences,
Vienna, Austria

Myeloperoxidase (MPO), a member of the haem peroxidase-cyclooxygenase superfamily, is abundantly expressed in

neutrophils and to a lesser extent in monocytes and certain type of macrophages. MPO participates in innate immune defence
mechanism through formation of microbicidal reactive oxidants and diffusible radical species. A unique activity of MPO is its
ability to use chloride as a cosubstrate with hydrogen peroxide to generate chlorinating oxidants such as hypochlorous acid, a
potent antimicrobial agent. However, evidence has emerged that MPO-derived oxidants contribute to tissue damage and the
initiation and propagation of acute and chronic vascular inflammatory disease. The fact that circulating levels of MPO have
been shown to predict risks for major adverse cardiac events and that levels of MPO-derived chlorinated compounds are
specific biomarkers for disease progression, has attracted considerable interest in the development of therapeutically useful
MPO inhibitors. Today, detailed information on the structure of ferric MPO and its complexes with low- and high-spin ligands
is available. This, together with a thorough understanding of reaction mechanisms including redox properties of intermediates,
enables a rationale attempt in developing specific MPO inhibitors that still maintain MPO activity during host defence and
bacterial killing but interfere with pathophysiologically persistent activation of MPO. The various approaches to inhibit enzyme
activity of MPO and to ameliorate adverse effects of MPO-derived oxidants will be discussed. Emphasis will be put on
mechanism-based inhibitors and high-throughput screening of compounds as well as the discussion of physiologically useful
HOCl scavengers.
British Journal of Pharmacology (2007) 152, 838854; doi:10.1038/sj.bjp.0707358; published online 25 June 2007

Keywords: MPO-H2O2-halide system; hypochlorite; inflammation; neutrophils, atherosclerosis; lipid oxidation; protein
Abbreviations: 2-ClHDA, 2-chlorohexadecanal; ABAH, 4-aminobenzoic acid hydrazide; BHA, benzylhydroxamic acid;
E10 , standard reduction potential; HCN/CN, hydrocyanic acid (hydrogen cyanide)/cyanide; HOCl/OCl,
hypochlorous acid/hypochlorite; HOSCN/OSCN, hypothiocyanous acid/hypothiocyanate; HSCN/SCN,
thiocyanous acid/thiocyanate; HOX/X, hypohalous acid/halide; MCD, monochlorodimedon; MPO,
myeloperoxidase; PDB, Protein Data Bank; ROOH, lipid-hydroperoxide; SHA, salicylhydroxamic acid
(2-hydroxybenzohydraxamic acid); TauNH2, taurine; TauNHCl, taurine chloramine; TMB, 3,30 ,5,50 -tetra-
methylbenzidine; TNB, 5,50 -dithiobis(2-nitrobenzoic acid)


An intensely green protein containing iron that had it is entirely located in azurophil (primary) granules, resulted
peroxidase activity was originally isolated from canine pus in renaming of this protein, verdoperoxidase, to myeloper-
and purulent fluid of patients with tuberculous empyema oxidase (MPO) (Yamada and Kurahashi, 1984; Koeffler et al.,
(Agner, 1941). Observations that expression of this enzyme is 1985). Promyelocytes and promyelomonocytes actively
restricted to the myeloid series of haematopoietic cells where synthesize MPO during granulocyte differentiation in the
bone marrow. MPO levels in neutrophilic polymorpho-
nuclear leucocytes (neutrophils) make up in between 2 and
Correspondence: Dr E Malle, Center of Molecular Medicine, Institute of
Molecular Biology and Biochemistry, Medical University of Graz, Harrachgasse 5% of the total cellular protein or 2 to 4 mg per 106 cells
21, Graz A-8010, Austria. E-mail: ernst.malle@meduni-graz.at or (Schultz and Kaminker, 1962; Bos et al., 1978). Human
Dr C Obinger, Division of Biochemistry, Department of Chemistry, BOKU monocytes also contain MPO-positive granules although
University of Natural Resources and Applied Life Sciences, Vienna A-1190,
they are fewer in number than in neutrophils and lost during
Austria. E-mail: christian.obinger@boku.ac.at
Received 22 April 2007; revised 30 May 2007; accepted 31 May 2007; differentiation into tissue macrophages (for a review see:
published online 25 June 2007 Klebanoff, 2005).
Inhibition of myeloperoxidase activity
E Malle et al 839

Observations that destruction of microorganisms occurs in cytes, MPO colocalizes with HOCl-modified epitopes on the
the phagosome of neutrophils and that MPO is among the endothelial layer lining the blood vessel (Malle et al., 2006b)
enzymes discharged into these phagocytic vacuoles from and contributes to endothelial dysfunction (Eiserich et al.,
cytoplasmic granules suggest an important role of MPO in 2002).
bacterial killing (Klebanoff, 2005). Among the antimicrobial HOCl has also been reported to react with nucleobases
systems present in the phagosome, a major proportion resulting in the formation of 5-chlorouracil, a marker for
consists of MPO, hydrogen peroxide (H2O2, formed during DNA damage during inflammation, which is enriched in
the respiratory burst), and a halide (X), particularly chloride human atherosclerotic tissue (Takeshita et al., 2006).
(Cl) (Hampton et al., 1998). The initial product of the MPO Another indicator for MPO-catalysed oxidation and spe-
H2O2Cl system is the potent antimicrobial oxidant hypo- cific protein-associated biomarker, formed either directly
chlorous acid/hypochlorite (HOCl/OCl, pKa 7.53) (Figure 1). by HOCl attack or via intermediate formation of chlorine
However, under pathological conditions, persistent activa- gas from HOCl (Hazen et al., 1996), is 3-chlorotyrosine.
tion of the MPOH2O2 system of activated phagocytes 3-Chlorotyrosine was identified in human atherosclerotic
may adversely affect tissues. HOCl is able to initiate lesion and lipoproteins extracted from lesions (Hazen and
modification reactions targeting lipids, DNA and (lipo)pro- Heinecke, 1997; Zheng et al., 2004), in respiratory tract
teins, including halogenation, nitration and oxidative cross- disease (Buss et al., 2003; Kettle et al., 2004), and neutrophil-
linking (Figure 1). induced liver injury (Gujral et al., 2004).
Chlorohydrins are formed by addition of HOCl to double Immunocytochemistry and immunohistochemistry with
bonds present either on cholesterol or various unsaturated specific monoclonal antibodies generated against HOCl-
ester- and ether-phospholipid species (Malle et al., 2006a; modified proteins/epitopes (Malle et al., 1995) provides an
Leig et al., 2007); however, the usefulness of chlorohydrins immunological tool to identify chlorinated biomarkers in
to be used as biomarkers is limited. Most importantly, the kidney disease (Malle et al., 1997, 2003; Gro ne et al., 2002),
remaining lyso-phospholipid remnants formed by HOCl- ischaemia reperfusion (Hasegawa et al., 2005), Parkinsons
mediated attack of ester-/ether-phospholipids (plasmalo- disease (Choi et al., 2005), and atherosclerosis (Hazell et al.,
gens) may adversely affect membrane dynamics and cause 1996; Malle et al., 2000). Fractionation of human plaque
cell lysis. In contrast to ester-phospholipids, HOCl-mediated homogenate by density gradient centrifugation and subse-
attack of ether-phospholipids (present in biological mem- quent immunoblot analysis of the low-density lipoprotein-
branes and lipoprotein particles) results in formation of bouyant fraction revealed that apoB-100, the major apolipo-
2-chlorohexadecanal (2-ClHDA), a chlorinated fatty alde- protein of low-density lipoprotein is modified by the MPO
hyde that is enriched in human atherosclerotic lesions H2O2Cl system of activated phagocytes in lesion material
(Thukkani et al., 2003) and infarcted myocardium (Thukkani (Hazell et al., 1996). In vitro studies have shown that
et al., 2005). 2-ClHDA is a potent chemoattractant in vitro lipoproteins, modified by HOCl (added as reagent or
initiating recruitment of circulating neutrophils to areas of generated by the MPOH2O2Cl system), display a number
inflammation and promotes endothelial dysfunction of pathophysiological effects on phagocytes and vascular
(Marsche et al., 2004). After release from activated phago- cells, contributing to initiation and maintenance of the

+ pathogens Host defense,

bacterial/viral killing Physiology

+ unsaturated Lysophospholipids,
lipids -halo-fatty aldehydes;
e.g. 2-ClHDA Cardiac dysfunction

+ DNA Atherosclerosis
+ proteins Ischemia/reperfusion
+ X-
+ NH2
Haloamines Diabetes
+ SCN- decay Respiratory tract
MPO Radical products diseases
H2O2 + NO2- CNS-diseases
Initiation of new lipid
radicals able to promote
+ Tyr lipid peroxidation

primary MPO-products secondary MPO-products

Figure 1 Primary and secondary MPO-reaction products and (patho)physiology. Adapted and modified from Malle et al. (2006a). CNS,
central nervous system.

British Journal of Pharmacology (2007) 152 838854

Inhibition of myeloperoxidase activity
840 E Malle et al

inflammatory process during atherosclerotic lesion develop- contradictory. It is the aim of this review to provide a
ment (Daugherty et al., 1994; Malle et al., 2006a, b). In systematic overview about potential inhibitors and their
particular, the uptake and degradation of HOCl-modified mode of action that is based on our present knowledge about
lipoproteins by monocyte-derived macrophages via scaven- structurefunction relationship and (patho)physiological
ger receptor-mediated pathways (Marsche et al., 2001, 2003) relevance of MPO.
is a leading event in the formation of cholesterol-enriched
foam cells (Malle et al., 2006a), representing the hallmark of
fatty streaks and the earliest recognisable lesion of athero- MPO transcription, translation, processing and release
sclerosis. HOCl-modified (lipo)proteins are also high-affinity MPO is encoded by a single gene (approximately 14 kb in
signalling transducing ligands for RAGE (Marsche et al., size), composed of 11 introns and 12 exons, and located on
2007a, b), a multiligand receptor that, originally reported to the long arm of chromosome 17 in segment q1224. The
bind advanced glycation end products, promotes an array of primary 80 kDa translation product preproMPO is processed
inflammatory complications. in the endoplasmic reticulum undergoing cotranslational
In general, oxidation of amino-acid side chain appears to N-glycosylation (resulting in an 90 kDa apoproMPO), tran-
play a specific role during HOCl-mediated protein backbone sient interaction with molecular chaperones, and haem
fragmentation. The first step involves chloramine formation incorporation to generate enzymatically active proMPO that
(probably from lysine side chains), giving rise to nitrogen- is exported into the Golgi compartment (for a review see:
centred radicals in a time- and HOCl-concentration-depen- Hansson et al., 2006). After exiting the Golgi, the propeptide
dent manner. These radicals may further form carbon- is removed before final proteolytic processing in azurophilic
centred radicals at peptide bonds thereby promoting protein granules. The processed MPO protein is a glycosylated,
fragmentation and/or promote lipid peroxidation (Hawkins predominantly a-helical cationic 146 kDa dimer with a single
and Davies, 1998). disulphide bridge between symmetry-related halves (73 kDa),
Although several groups have detected accumulation of each containing two polypeptides of 108 (14.5 kDa, light
lipid peroxidation products in HOCl-modified liposomes chain) and 466 amino acids (58.5 kDa, heavy chain). Dimeric
and lipoproteins, the mechanisms of lipid-hydroperoxide MPO is found in neutrophils and monocytes. All available
(ROOH) formation is not entirely clear (Malle et al., 2006a). structural data refer to this mature dimeric MPO (Zeng and
Panasenko et al. (1997) have suggested that this requires the Fenna, 1992; Fiedler et al., 2000; Blair-Johnson et al., 2001)
presence of preformed ROOH able to induce further lipid and the majority of studies on MPO inhibitors have been
peroxidation. During reaction with hydroperoxide, HOCl performed with this protein.
induces the formation of singlet molecular oxygen and However, some proMPO escapes granule targeting and
comparable observations were reported for fatty acid and becomes constitutively secreted to the extracellular environ-
phospholipid hydroperoxides (Miyamoto et al., 2006). The ment as a monomer (Hansson et al., 2006). There is also
reaction of HOCl with preformed ROOH may be of particular evidence of de novo MPO synthesis in certain subtypes of
importance at sites of inflammation where both, MPO/HOCl macrophages and it has been suggested that foam cells newly
and ROOH concentrations are significantly elevated leading engaged in MPO gene transcription lack the cellular
to the formation of secondary lipid/protein oxidation machinery to proteolytically process proMPO into subunits
products. of mature MPO. As a result, only proMPO enters the
In the absence of physiological Cl concentrations, secretory pathway and is released in the extracellular
formation of tyrosyl radicals promotes protein crosslinks environment. It is important to note that recombinant
via dityrosine formation, whereas nitrogen dioxide radical MPO overexpressed in Chinese hamster ovary cell lines
generates nitrated lipids. In addition, both radical species are (Moguilevsky et al., 1991) resembles monomeric and par-
able to induce lipid peroxidation (Figure 1). tially unprocessed proMPO. Nevertheless, several spectral
As MPO levels of leucocyte- and blood-MPO are associated and kinetic investigations clearly demonstrated that mature
with the presence of coronary artery disease (Baldus et al., MPO and recombinant proMPO exhibit identical enzymatic
2003; Brennan et al., 2003) and MPO considerably con- features (Jacquet et al., 1991; Moguilevsky et al., 1991;
tributes to plaque vulnerability (for a review see: Nicholls Furtmu ller et al., 2001), indicating identical molecular
and Hazen, 2005), quantitation of MPO mass and activity, as architecture of the haem cavity and substrate channel. Thus,
well as analysis of MPO-derived biomarkers (secondary MPO recombinant proMPO represents a valid model for inhibitor
products, Figure 1) is of prognostic value to follow disease studies and it is reasonable to assume that the action of
progression. inhibitors on mature dimeric MPO and monomeric (recom-
Development of therapeutic intervention strategies aiming binant) proMPO is identical.
at efficient MPO inhibition can be envisaged at different
levels: (i) active site blockade of MPO, (ii) diversion of MPO
from the chlorination cycle, (iii) irreversible suicide inhibi- Structural information about MPO
tion by use of oxidized inhibitors, and (iv) application of The overall protein fold of MPO was first revealed by a 3 A
HOCl scavengers to prevent initiation and propagation of resolution crystal structure of the canine enzyme (Protein
diseases with an inflammatory component. All these strate- Data Bank (PDB) code: 1MYP) (Zeng and Fenna, 1992). By
gies have already been followed, but in a limited and non- now, the structure of human MPO at 2.3 A resolution is
systematic way. The provided information is distributed in solved and refined to 1.8 A using X-ray data recorded at
journals of varying disciplines and often fragmentary or even 1801C (1CXP) (Fiedler et al., 2000). The structure of MPO

British Journal of Pharmacology (2007) 152 838854

Inhibition of myeloperoxidase activity
E Malle et al 841

and its interaction with bromide (Br) (1D2V) and thiocya- or by measuring the oxidation of yellow 5-thio-2-nitroben-
nate (SCN) (1DNU) (Fiedler et al., 2000) as well as of the zoic acid (e412 14 000 M1 cm1) to colourless 5,50 -dithio-
MPO-cyanide (MPOCN) complex (1D5L) and its interac- bis(2-nitrobenzoic acid) (TNB) by TauNHCl (Weiss et al.,
tion with Br (1D7W) and SCN (1DNW) was published 1982). This is an extremely sensitive assay and can accurately
(Blair-Johnson et al., 2001). In addition, there is one report measure concentrations of HOCl as low as 5 mM.
on the crystal structure of salicylhydroxamic acid (SHA) Alternatively, I can be used to detect TauNHCl produced
bound to human MPO (Davey and Fenna, 1996), but by MPO (Dypbukt et al., 2005). TauNHCl oxidizes I to HOI,
unfortunately no entry in the PDB exists. Additionally, which finally oxidizes 3,30 ,5,50 -tetramethylbenzidine (TMB)
computational analysis of indole derivative binding to to a strongly absorbing blue product or dihydrorhodamine
MPO by computational docking is reported (Hallingback to highly fluorescent rhodamine, respectively (Dypbukt
et al., 2006). et al., 2005). The advantage over the TNB assay is that in
this assay a signal is produced rather than quenched, which
enhances its sensitivity and accuracy and offers its applica-
tion to microtitre plate format (Dypbukt et al., 2005).
Enzymatic properties and activity assays
Besides MCD and TauNH2, ascorbate (Chesney et al., 1991)
and TMB (Suzuki et al., 1983) have been used as the basis for
Chlorination activity
assaying the chlorination activity of MPO. The loss of
The principal reaction catalysed by MPO under physiological
ascorbate (e267 15 000 M1 cm1) can be related to its
conditions is the oxidation of X, for example Cl and Br as
oxidation by HOCl because the reaction is inhibited by
well as SCN to the corresponding hypohalous acids (HOX)
methionine. However, results must be interpreted with
and hypothiocyanus acid (HOSCN) according to equation 1
caution because ascorbate can act directly as peroxidase
(halogenation activity). HOX/HOSCN can participate in
substrate (equation 2). Similarly, oxidation of TMB
subsequent nonenzymatic reactions such as oxidation and
(e285 2.1  104 M1 cm1) by HOCl or via the peroxidation
halogenation of compounds present in the immediate
cycle of MPO gives a blue product with an absorbance
environment (Figure 1). There is no well-defined pH
maximum (e652 3.9  104 M1 cm1) (Josephy et al., 1982).
optimum for MPO-catalysed chlorination because it depends
Generally, the TMB assay is extremely sensitive and has been
on the relative concentrations of Cl and H2O2. However, at
used to quantitate the total utilization of H2O2 by MPO.
constant concentrations of these reaction partners, the rate
However, under usual assay conditions, the lack of inhibi-
of HOCl release increases with decreasing pH (Andrews and
tion by methionine indicates that the peroxidation activity
Krinsky, 1982). It is important to realize that at high
completely overrides the chlorination activity.
concentrations, H2O2 inactivates MPO. To minimize inacti-
vation, the concentration should be kept at p100 mM.
Because amine-containing buffer systems are potent scaven-
gers of HOCl activity, measurements must be performed in Peroxidase activity
phosphate buffers. MPO is also able to catalyse typical peroxidation reactions
(equation 2) with AH2 representing the peroxidase substrate
H2 O2 X H ! HOX H2 O 1 oxidized to the corresponding radical (KAH). Principally,
each peroxidase assay described in the literature can also be
Two assays are routinely used to measure the chlorination performed with MPO.
activity of MPO. The first assay involves chlorination of
monochlorodimedon (MCD) by HOCl, a process that is H2 O2 2AH2 ! 2 AH 2H2 O 2
accompanied by a decrease in absorbance (e290 1.99 
104 M1 cm1) (Kettle and Winterbourn, 1988). However, Oxidation of tyrosine to dityrosine can be followed spectro-
MCD cannot be regarded as an inert detector of HOCl fluorimetrically using an excitation wavelength of 325 nm
production by MPO because it has been shown to function as and an emission wavelength at 405 nm (Marquez and
competitive electron donor (Kettle and Winterbourn, 1988). Dunford, 1995). As already mentioned, TMB functions both
Thus, the MCD assay underestimates the chlorinating as scavenger of HOCl and as peroxidase substrate. In the
activity (Kettle and Winterbourn, 1994). Nevertheless, this absence of Cl, the TMB assay can be used to monitor the
method is useful for detecting HOCl, as shown by its peroxidatic activity of MPO (Marquez and Dunford, 1997).
complete inhibition by methionine, and thus is applicable Another assay, following the oxidation of guaiacol to
in inhibitor studies. tetraguaiacol (e470 2.6  104 M1 cm1) (Maehly and Chance,
The second assay involves chlorination of nitrogen 1954) is also often used to follow MPO activity. Furthermore, a
compounds, particularly primary amines. The reaction of luciferin derivative (2-methyl-6-(p-methoxylphenyl)-3,7-dihydro-
HOCl with these compounds yields chloramines containing imidazol[1,2-a]pyrazin-3-one) has also been used for the
covalent nitrogen-chlorine bonds. The taurine chloramine determination of singlet oxygen produced from the reaction
assay is based on the reaction of HOCl with taurine (TauNH2) of HOBr with H2O2 (Nakano and Koga, 1994).
to produce taurine chloramine (TauNHCl). Usually, chlor-
amines are unstable and decompose to the corresponding
aldehydes and nitriles. By contrast, TauNHCl is relatively Hydrogen peroxide electrode assay
stable and its formation can be either followed spectro- The loss of H2O2 catalysed by MPO during halogenation and
photometrically [e252 429 M1 cm1] (Thomas et al., 1986) peroxidatic cycles (equations 1 and 2) can be continuously

British Journal of Pharmacology (2007) 152 838854

Inhibition of myeloperoxidase activity
842 E Malle et al

monitored using a H2O2 electrode (Kettle and Winterbourn, (Tarpey et al., 2004). Caution is advised when horseradish
1994). In the presence of Cl (up to 100 mM) as the only peroxidase and a peroxidase substrate is used for H2O2
electron donor, this method allows direct assessment of the visualisation, because the inhibitor of MPO could also impair
chlorination activity without the need for a detector the activity of horseradish peroxidase. Alternatively,
compound that might potentially interfere with MPO. The I-mediated oxidation of TMB or dihydrorhodamine by
formation of HOCl accounts completely for the loss of H2O2. TauNHCl can be used to elucidate the effect of putative
It is recommended to include TauNH2 (10 mM) or methio- inhibitors.
nine (500 mM) in the reaction buffer to scavenge HOCl and
prevent it from inactivating MPO (Kettle and Winterbourn,
1994). Redox intermediates of MPO

To understand the underlying mechanism of MPO inhibi-

Assays for inhibitor studies tion it is necessary to investigate the interaction of potential
In our opinion screening for putative MPO inhibitors should inhibitors with the individual reaction steps both in the
include both, the use of the H2O2 electrode and the TauNHCl halogenation (equation 1) and peroxidation (equation 2)
assay, respectively. Polarographic monitoring of the loss of reactions. Firstly, the inhibitor could block the access of
H2O2 should be followed either in the presence of Cl or a H2O2 or its binding and reduction. As a consequence it
peroxidase substrate (for example, tyrosine). This approach would inhibit both cycles because both start by reaction of
allows a first conclusion of (i) whether the inhibitor the Fe(III) form of MPO with H2O2 to form compound I,
specifically interferes with the chlorination and/or peroxi- which contains two oxidizing equivalents more than the
datic cycle, (ii) whether the inhibitory mode is reversible or resting enzyme (Figure 2, reaction 1). The reaction occurs at
irreversible, and (iii) whether the putative inhibitor simply the distal haem cavity and amino acids His95 and Arg239
acts as a scavenger for HOCl or TauNH2. In any case, (Figure 3b) plays critical roles in the binding/orientation/
measuring inhibition of only peroxidative activity is an activation and finally heterolytic cleavage of H2O2. Com-
inappropriate strategy in searching for inhibitors of HOCl pound I is an oxoiron(IV) intermediate [Fe(IV) O] contain-
production because many reversible inhibitors act by divert- ing a porphyrin p-cation radical (PorK ) (Dolphin et al.,
ing MPO from the chlorinating cycle to the peroxidase cycle 1971).
where they act as competitive electron donors for com- Once produced, compound I of MPO is a strong oxidant
pounds I and II (see below). that mediates both one- and two-electron oxidation reac-
In high-throughput screenings, the polarographic method tions. Depending on the apparent bimolecular rate constant
is inapplicable. Here, remaining H2O2, which has not been and donor concentration, the enzyme follows either the
consumed either in the chlorination (equation 1) or halogenation (Figure 2, reactions 1 and 2) or the peroxidase
peroxidase activity (equation 2) due to inhibitory effects, cycle (Figure 2, reactions 1, 3 and 4). In the halogenation
can be monitored photometrically or fluorimetrically cycle X and SCN directly reduce compound I to the Fe(III)

Figure 2 General reaction scheme of MPO. In the first step H2O2 is used for compound I formation (reaction 1). Compound I is two oxidizing
equivalents above the native enzyme with a porphyrin p-cation radical in combination with an oxoiron(IV) centre. Compound I can react with
halides (X) reducing the enzyme back to the ferric state (reaction 2, halogenation activity). In the peroxidase reaction, compound I is
transformed in the first one-electron reduction to compound II, which contains an oxoiron(IV) centre (reaction 3). Compound II is finally
reduced back to ferric peroxidase in a second one-electron reduction (reaction 4). Compound III (oxyperoxidase) is formed either from ferric
peroxidase with superoxide anion (reaction 9), from ferrous MPO with O2 (reaction 8) or from compound II with H2O2 (reaction 6).
Compound III is a complex of ferrous-dioxygen in resonance with ferric superoxide. Ferrous [Fe(II)]MPO can be formed from ferric [Fe(III)]MPO
by reducing radicals produced in the peroxidase cycle (reaction 11).

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Inhibition of myeloperoxidase activity
E Malle et al 843

oxoiron(IV) species (Figure 2, reactions 3 and 4). Many

inorganic and organic substrates were found to act as
electron donors for both compounds I and II (for a review
see: Dunford, 1999; Furtmu ller et al., 2006). MPO compound I
is an extremely strong one-electron oxidant, because the
standard reduction potential of the couple compound I/
compound II is 1.35 V; the standard reduction potential for
the couple compound II/native enzyme is only 0.97 V
(Furtmu ller et al., 2003). This marked difference in the
oxidation capacity of MPO compounds I and II is strongly
reflected by the actual reaction rate constants of these
intermediates with substrates of one-electron reduction
potentials 40.95 V, for example indole and tryptamine
derivatives (Jantschko et al., 2005). The latter molecules are
good electron donors for compound I but do not react with
compound II thus diverting MPO from the chlorination
Owing to its strong oxidation capacity, MPO compound I
is responsible for drug bioactivation (Yang et al., 2006)
including oxidation of phenytoin (Uetrecht and Zahid,
1988; Kubow and Wells, 1989; Mays et al., 1995), carbama-
zepine (Furst and Uetrecht, 1993), L-dopa (Nappi and Vass,
2001), clozapine (Uetrecht et al., 1997; Williams et al., 1997),
trimethoprim (Lai et al., 1999), fluperlapine (Lai et al., 2000)
and procainamide (Uetrecht and Zahid, 1991) to reactive
metabolites/intermediates (for example, nitroso, nitrogen
free radical and iminium species). Diclofenac (Miyamoto
et al., 1997) undergoes p-hydroxylation by MPO, resulting in
p-aminophenols, which can be further oxidized to reactive
quinoneimines. However, MPO itself also oxidizes many
potential inhibitors. The present knowledge concerning
their binding and electron transfer site(s) is summarized
With respect to inhibitory mechanisms further redox
intermediates have to be discussed, which neither participate
in the halogenation nor in the peroxidase cycle, namely
the Fe(II) form of MPO and compound III, the latter being
the ferrous-dioxy/ferric-superoxide anion complex of MPO
(Figure 2). Similar to higher oxidation states the reduction
potential of Fe(III)/Fe(II) of MPO is significantly higher
Figure 3 (a) The hydrogen bonding network and locations of five (5 mV) (Battistuzzi et al., 2006) than that of other haem
water molecules (W1W5) at the distal haem cavity of ferric high- peroxidases. Thus, MPO is very susceptible to reduction to its
spin MPO. (b) The non-planar porphyrin ring in MPO and its Fe(II) form, that is by radical intermediates formed during
covalent attachments to the protein via two ester bonds (Glu242
and Asp94) and one sulfonium ion linkage (Met243). In addition, the peroxidase cycle (Figure 2, reaction 11). In the presence
the proximal His336 and the distal catalytic residues His95, Arg239, of oxygen Fe(II), MPO is readily converted into compound III
and Gln91 are shown, the latter being important in halide binding. (Figure 2, reaction 8). Alternatively, compound III can be
The figure was constructed using the coordinates deposited in the formed by reaction of ferric MPO with superoxide anion
Protein Data Bank (accession code 1CXP).
(Figure 2, reaction 9) or of compound II with H2O2 (Figure 2,
reaction 6). It is important to note that compound III should
be regarded as a complex that can decay to ferric MPO and
form of MPO (Figure 2, reaction 2) releasing the correspond- superoxide anion (Figure 2, reaction 10) or ferrous MPO and
ing HOX/HOSCN. The standard reduction potential of the oxygen (Figure 2, reaction 7).
couple compound I/native MPO is quite high ( 1.16 V)
(Arnhold et al., 2001, 2006) enabling the enzyme to oxidize
Cl. The putative binding site(s) of these small anionic Substrate and inhibitor binding sites
inorganic two-electron donors and the impact of inhibiting
substances on compound I reduction by X/SCNare To be able to rationally design therapeutically useful MPO
described below. inhibitors, a detailed knowledge of the structural features of
In the peroxidase cycle, compound I is reduced by two the enzyme is essential. Each monomer or catalytic domain
successive one-electron steps via compound II, a protonated of dimeric MPO or monomeric proMPO contains one iron

British Journal of Pharmacology (2007) 152 838854

Inhibition of myeloperoxidase activity
844 E Malle et al

atom (present as covalently bound ferri-protoporphyrin IX to be inaccessible for larger anions like Br or I. However, it
derivative) and one Ca2 ion. The Ca2 -binding site has is possible that the W2 position could accommodate Cl
typical pentagonal bipyramidal coordination (Zeng and equally well in both native MPO and its CN complex,
Fenna, 1992; Fiedler et al., 2000). Because one of its ligand, because Cl (1.81 A ) is significantly smaller in radius than
Asp96, is in close vicinity to the distal His95 (Figure 3b),  
Br (1.96 A) or I (2.2 A ). The sterical hindrance of Br and I
Ca2 is important in maintaining the distal architecture but to bind at the W2 position in compound I might be reflected
does not participate immediately in substrate and/or in- by the fact that with increasing radius of the anionic
hibitor binding. A unique structural feature of MPO concerns substrates, the increase in the rate of compound I reduction
the modification of the 1- and 5-methyl groups on pyrrole by these donors at acidic pH is less pronounced compared to
ring A and C of the haem group (Figures 3a and b) enabling Cl oxidation (Furtmu ller et al., 1998). Binding of Cl at
formation of ester linkages with the carboxyl groups of an position W2 together with the MPO typical distal water
Asp94 and Glu242. In addition the b-carbon of the vinyl network of low mobility could be crucial in fixing the
group on pyrrole ring A forms a covalent bond with the position of Cl and in favouring the transfer and incorpora-
sulphur atom of methionine 243, giving rise to a sulpho- tion of the oxyferryl oxygen into HOCl. In any case at
nium ion linkage (Figures 3a and b) (Zeng and Fenna, 1992; neutral pH, Cl is bound weakly (KD4100 mM) and needs a
Fiedler et al., 2000), which is responsible for the peculiar rigid hydrogen-bonding network, which is easily destabilized
redox properties of MPO (Arnhold et al., 2006; Zederbauer by peroxidase substrates or inhibitor binding at the haem
et al., 2007b). As a consequence, the haem porphyrine ring is edge (see below).
considerably distorted from planarity (Figure 3b). Although There is evidence from kinetic studies that the Cl level
pyrrole rings B and D are nearly coplanar, ring A and, to a (2 mM) used to obtain the X-ray structure of the MPOCl
lesser extent ring C are tilted toward the distal side, resulting complex could be insufficient to occupy the distal binding
in a bow-shaped haem structure. The proximal haem ligand site (Proteasa et al., 2007). Moreover, based on the biphasic
is a histidine, which is hydrogen bound with an asparagine. behaviour of nitric oxide binding to MPO in the presence of
The haem of MPO is located in a crevice, about 15 A in increasing Cl concentrations, two distal Cl binding sites of
depth, with access to the solvent via an open channel, different affinities have been proposed (Proteasa et al., 2007).
approximately 10 A in diameter (Figure 5). It discharges into In the X-ray structure, the closest haem atom of X bound
the distal cavity that accommodates the amino-acid triade at the position of W2 is the d-methine bridge between
histidine, arginine and glutamine (Figure 3b). The distal pyrrole rings A and D (Figures 4a and b) at a distance of 3.8 A ,
His95 is hydrogen bonded to a water molecule (W1), which 
and the haem iron is at a distance of 5.0 A. A X bound at the
is positioned approximately mid-way between the Ne atom position of W2 is also in vicinity to the side chain of Glu242,
of His95 and the haem iron (Figures 3a and b). Its distance which is only 3.5 A away from W2 in the native enzyme.
from both the histidine nitrogen and the iron atom suggests Although Met243 is relatively distant and not involved in
that it is hydrogen bonded to the histidine and not strongly positioning of W2 in native MPO or Br in the MPOBr
coordinated to the haem iron. Four additional water complex (data not shown), its exchange has a tremendous
molecules (W2W5) form hydrogen bonds with His95, effect on the Cl affinity. The affinity is decreased by
Arg239, Gln91, the haem pyrrole ring C propionate and 100-fold, compared to recombinant proMPO (Kooter et al.,
between themselves (Figure 3a). Water W2 is hydrogen 1997). In contrast, the exchange of Glu242 and Asp94 by
bonded to the Ne atom of Gln91, W3 to NH2 of Arg239, W4 242Gln and 94Val in MPO mutants is relatively small
to haem propionate of pyrrole ring C and W5 having no (Zederbauer et al., 2005, 2007a). A specific role of Met243 in
additional hydrogen bonding (Figure 3a) (Fiedler et al., X binding cannot be inferred by inspection of the structures
2000). Generally, the covalent haem attachment in MPO but might be again related to its role in maintaining a rigid
causes small solvent reorganisation effects in the reduction structural environment at the distal site that enables the
reaction of Fe(III) MPO (Battistuzzi et al., 2006), suggesting a binding of small anionic ligands (Battistuzzi et al., 2006).
high rigidity of the hydrogen bond network involving water In the MPOCNBr double complex (Figure 4b) (Blair-
molecules in the substrate channel leading to the distal Johnson et al., 2001) the Br binding site is close to the
haem site. d-methine bridge carbon and does not appear to form any
readily identifiable electrostatic interactions with the
enzyme, the haem, or the bound CN. The closest amino
Halide binding site acid is Glu242 and the nearest neighbouring haem atom is
In the MPOBr complex (data not shown), Br replaces the pyrrole ring D methyl carbon (Blair-Johnson et al., 2001).
water molecule W2, which is hydrogen bonded to the amide The close proximity of Glu242 to positions of both W2 and
of Gln91 in the proximity of the Ne atom of His95. W5 is underlined by the fact that compound I reduction by
Unfortunately, there is no structure of a Cl bound to the X is generally decreased in Glu242Gln mutants irrespective
distal haem cavity. Figures 4a and b allow a comparison of of the nature of the X (Zederbauer et al., 2005). By contrast,
the distal cavities of the MPOCN complex with the MPO disruption of the haem to Asp94 ester linkage affected the
CNBr double complex. In the MPOCNBr double oxidation of Cl and Br but not of I and SCN (Zederbauer
complex, which could be regarded as a model for compound I et al., 2007b).
with bound Br, W2 is present and Br binds in place of W5 In the MPOSCN complex (data not shown), SCN is
thereby preventing direct hydrogen bonding to protonated arranged almost parallel with the plane of the haem
His95. When CN is bound to the iron, the W2 site appears replacing water molecules W2 and W5 in the native enzyme.

British Journal of Pharmacology (2007) 152 838854

Inhibition of myeloperoxidase activity
E Malle et al 845

Figure 4 (a) Structure of the myeloperoxidase-cyanide (MPOCN) complex (accession code 1D5L), which can be regarded as a model of
compound I. In the CN-complex water molecule W1 is displaced by CN. (b) Structure of the MPOCNbromide (Br) double complex
(accession code 1D7W). CN and Br are located at positions of W1 and W5. Note that in the MPOBr complex (data not shown), the halide
is binding at W2. (c) Structure of the MPOCNthiocyanate (SCN) double complex (accession code 1DNW). In this complex CN displaces
W1, whereas SCN displaces both W2 and W5.

The nitrogen of SCN, at the W2 position, forms hydrogen Aromatic substrate/inhibitor binding site
bonds with the amide NH2-group of Gln91 and water Binding studies have indicated that aromatic molecules bind
molecule W2 as well as a weak interaction with the Ne atom near to the distal haem pocket in MPO (Hori et al., 1994). On
of distal His95, whereas the sulphur makes van der Waals the basis of electron paramagnetic spectroscopy and mole-
contacts with Cg of Glu242, Cd of Arg239, and the haem cular modelling it was proposed that the hydroxamic acid
pyrrole ring D methyl carbon. SCN in the distal cavity of side chains of benzylhydroxamic acid (BHA) and SHA
the MPOCN complex (Figure 4c) (Blair-Johnson et al., interact with the haem iron in MPO (Ikeda-Saito et al.,
2001) occupies the same position as in the native enzyme, 1991). Finally, the 2.3 A resolution X-ray structure of the
except that it is inclined E151 from its former orientation MPO-SHA complex was published (Davey and Fenna, 1996)
parallel with the haem. In the presence of CN, the nitrogen showing the aromatic ring of SHA binding to a hydrophobic
atom in SCN can form only a single hydrogen bond with region at the entrance to the distal haem pocket between
Gln91. pyrrole ring D and the side chain of Arg239 (Figure 6a). The

British Journal of Pharmacology (2007) 152 838854

Inhibition of myeloperoxidase activity
846 E Malle et al

Figure 5 View through the access channel to the active site

(complexed with cyanide) showing exposure of the haem pyrrole
ring D with its 8-methyl and of the d-methine bridge (that is,
electron transfer site). The figure was constructed using PyMol
Viewer (version 0.99).

hydroxamic acid moiety is hydrogen bonded to both the

distal His95 and Gln91 amide group but is not coordinated
to the haem iron. Although SHA binding displaces three
water molecules from the distal cavity (W1, W2 and W3), no
significant conformational differences between the active
site regions of the complex and the native enzyme became
The hydrophobic region at the entrance to the distal cavity
is conserved among mammalian peroxidases (Figure 5)
(Furtmu ller et al., 2006). The aromatic ring of SHA
(Figure 6a) is tilted about 201 with respect to that of the
haem pyrrole ring D which forms the lower surface of the
hydrophobic cavity, whereas the b, g and d carbons of Arg239
form the upper surface (Davey and Fenna, 1996). In the
MPOSHA complex, the aromatic ring of SHA is almost
centred above the pyrrole ring D 8-methyl group. Additional
van der Waals interactions occur with adjacent phenylala-
nines, that is Phe99, Phe366 and Phe407, each of which has
an aromatic ring within 4.24.6 A of the aromatic ring of the
SHA molecule.
It is reasonable to assume that substrate binding to
compounds I and II (Figure 2, reactions 3 and 4) is identical. Figure 6 Docking models for SHA (a) and melatonin (b) bound to
The proposed electron transfer most probably occurs near MPO. The positions of the three oxygen atoms of SHA are close to
the positions occupied by the three water molecules W1W3 in the
the d-methine bridge carbon (Davey and Fenna, 1996). native enzyme (see Figure 3a). Both models were calculated using
Mutation of neighboured Glu242Gln decreases the overall LigandScout 1.03 (Wolber and Langer, 2005) from Inte:Ligand
rate of guaiacol and tyrosine oxidation compared to non- GmbH (www.inteligand.com). SHA, salicylhydroxamic acid.
mutated recombinant proMPO (Zederbauer et al., 2005).
Similar to the negative effect on X oxidation, disruption of
the neighboured Glu242 ester bond seems to decrease the close enough to the D pyrrole ring to achieve stacking
electron transfer between the donor molecule and the haem (Figure 6b). The distances of the closest indole atom to the
moiety. Interestingly, oxidation of the hydrophilic donor for both substrates. The
centre of the D ring were about 3.4 A
ascorbate by both compounds I and II was enhanced in ligand side chain was never directed towards the distal
Glu242Gln mutated compared to non-mutated recombinant cavity, whereas the indole substituent at position 5 was close
proMPO (Zederbauer et al., 2005), suggesting that the to the haem centre (Hallingback et al., 2006). In detail,
described binding scenario fits to aromatic but not to the distance between the oxygen of the 5-methoxy or
hydrophilic one-electron donors. 5-hydroxyl substituent on the indole ring and the haem
Recently, the binding of melatonin (N-acetyl-5-methoxy- iron ion was calculated to be around 3.0 A .
tryptamine) and serotonin (5-hydroxytryptamine) to MPO Computational docking of compounds I and II states of
in its Fe(III) compounds I and II state was studied by MPO (data not shown) demonstrated, that both, melatonin
computational docking (Hallingback et al., 2006). Both and serotonin were pushed about 1 A away from the ferryl
indole derivatives could be docked into all three forms with oxygen, which abolished the 5-substituent to point to the
their indole rings oriented parallel to the haem plane and haem centre thus favouring an alternative binding mode

British Journal of Pharmacology (2007) 152 838854

Inhibition of myeloperoxidase activity
E Malle et al 847

with a rotated indole still parallel to pyrrole ring D both the chlorination and peroxidation activity of
(Hallingback et al., 2006). MPO irreversibly. Oxidation of isoniazid or hydrazine
sulphate between pH values 6.5 and 7.8 by the MPOH2O2
system caused irreversible loss of haem absorbance asso-
MPO inhibitors ciated with compound III formation (van Zyl et al., 1989a).
Finally, during a study using a series of benzoic acid
Hydroxamic acids hydrazides, it was demonstrated that these are general
Hydroxamic acids [RCNOHOH or RC(O)NHOH] are known inhibitors of the peroxidation and chlorination activity of
to act as reducing substrates for compounds I and II of haem MPO (Kettle et al., 1995). Unsubstituted benzoic acid
peroxidases (Figure 2, reactions 3 and 4) and can also hydrazide and its 4-chloroderivative were poor inhibitors,
substitute for H2O2 in compound I formation (reaction 3) whereas derivatives with substitutents containing oxygen or
(Schonbaum and Lo, 1972; Schonbaum, 1973). SHA has been nitrogen were much better inhibitors. Benzoic acid hydra-
reported to inhibit the luminol-dependent chemilumines- zides are readily oxidized by compound I (Figure 2, reaction 3)
cence of human neutrophils stimulated by phorbol and the rate constants for these reactions are relatively
12-myristate 13-acetate or the chemotactic peptide N-formyl- insensitive to the substituents on the aromatic ring (Burner
methionyl-leucyl-phenyl-alanine (Davies and Edwards, 1989). et al., 1999). By contrast, reduction of compound II (Figure 2,
Furthermore, SHA had no inhibitory effect on the kinetics of reaction 4) by benzoic acid hydrazides strongly correlated
superoxide anion generation or O2 uptake during the with the substitution patterns and to the Hammett rule, and
respiratory burst of phagocytes, but inhibited the peroxidase there were also significant correlations with BrownOkamoto
activity of purified MPO (IC50 value of 35 mM). SHA also substituent constants (Hansch and Leo, 1979; Burner et al.,
prevented the formation of compounds I and II but only 1999). This indicates that the rates of these reactions
at low H2O2 concentrations, suggesting that it may compete were simply determined by the redox potential of the
for the H2O2 binding site on the enzyme (Davies and substrates and that the incipient free radical carried a
Edwards, 1989). positive charge, which is delocalized via resonance between
Binding of SHA and BHA to dimeric leucocytic MPO, as the substituent on the aromatic ring and the hydrazide
studied by optical absorption (Ikeda-Saito et al., 1991) and group.
electron paramagnetic spectroscopy (Hori et al., 1994), 4-Aminobenzoic acid hydrazide (ABAH) was the best
showed dissociation constants of 2 mM (SHA) and 5 mM electron donor of compound II and also the most potent
(BHA) as well as the involvement of an ionizable group on inhibitor of peroxidation. ABAH irreversibly inhibited HOCl
the protein with a pKa of approximately 4. In the SHAMPO production of MPO isolated from leucocytes with an IC50
inhibitory complex determined at 2.3 A resolution (Davey value of 0.3 mM. The kinetic analysis of the inactivation
and Fenna, 1996), it is clearly shown that this residue is the conformed to that for a mechanism-based inhibitor (Kettle
distal histidine, because the hydroxamic acid moiety is et al., 1997). ABAH is oxidized in the peroxidase cycle by
hydrogen bonded to His95 (Figure 6b). The ability of compounds I and II and the produced radicals readily
the three SHA oxygen atoms to closely duplicate the convert the enzyme into ferrous MPO (Figure 2, reaction 11)
hydrogen-bonding pattern of W1W3 in the native state and compound III (Figure 2, reaction 8). Compound III is
(Figure 3a) could account for the strong binding of SHA to neither part of the peroxidase nor of the chlorination cycle
MPO. The salicyl-ring hydroxyl oxygen participates in thus impeding oxidation of reducing substrates reversibly. Its
hydrogen bonding to the guanidinium group of Arg239 turnover is slow and ABAH or an ABAH radical may reduce it
and to water molecule W2 that remains hydrogen bonded to in analogy to the reduction of oxyhaemoglobin by phenyl-
the pyrrole ring C propionate carboxyl group. As described hydrazine (Misra and Fridovich, 1976). In the absence of
above, a separate hydrophobic interaction occurs between oxygen, ferrous MPO accumulates and irreversible inactiva-
the aromatic ring and a hydrophobic region at the entrance tion is exacerbated by destruction of the haem prosthetic
to the distal haem cavity suggesting that oxidation occurs group (Kettle et al., 1997). Protection by dioxygen suggests
at the haem edge, in the vicinity of the d-meso carbon and that ferrous MPO, permanently formed by reactions 11 and 7
the 8-methyl group on pyrrole ring D (Davey and Fenna, (Figure 2) (that is, dissociation of the dioxygenFe(II)
1996). complex), is the actual target of irreversible inactivation
In any case, SHA plays dual inhibitory roles in peroxidase but the detailed mechanism is still unknown (Burner et al.,
catalysis by inhibiting hydroperoxide binding to the iron 1999).
and by competing with other donors in reactions with With neutrophils stimulated with opsonized zymosan or
higher oxidation states. However, this inhibition is not phorbol myristate acetate, ABAH inhibited HOCl production
irreversible and strongly depends both on H2O2 and by up to 90% and corresponding IC50 values were 16 and
competing electron donor concentration. 2.2 mM, respectively. However, in the presence of superoxide
dismutase, these values decreased to 6.4 and 0.6 mM, indicat-
ing that superoxide anion similar to molecular oxygen
Benzoic acid hydrazides protects MPO to some extent from irreversible inactivation.
Hydrazines (RNHNH2) and hydrazides (RCONHNH2) have ABAH was shown to have no effect on superoxide anion
been reported to be suicide substrates of other haem production and degranulation of neutrophils, nor did it
peroxidases than MPO (Ator et al., 1987). The antitubercu- inhibit catalase or glutathione peroxidase (Kettle et al.,
lous agent isonicotinic acid hydrazide (isoniazid) inhibits 1995).

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Inhibition of myeloperoxidase activity
848 E Malle et al

Indoles and tryptamines mefenamic acid, sulphapyridine, quinacrine, primaquine

Indole derivatives act as one-electron donors of compounds I and aminopyrine were found to induce 50% inhibition
and II of haem peroxidases (Figure 2, reactions 3 and 4) of the initial rate of H2O2 loss at concentrations of
(Jantschko et al., 2002). These molecules have been shown approximately 1 mM or less. Phenylbutazone, piroxicam,
to act as reversible inhibitors of MPO when they exhibit salicylate, olsalazine, benzocaine, sulfasalazine (Kettle and
a one-electron reduction potential E10 (AK, H /AH)4E10 Winterbourn, 1991), diclofenac (Zuurbier et al., 1990),
(compound II/ferric MPO) 0.97 V (Jantschko et al., 2005). chlorpromazine (van Zyl et al., 1990), acetaminophen
Generally, tryptamines were more effective in inhibition of (van Zyl et al., 1989b), deferoxamine (Klebanoff and
chlorination than indoles and this strongly correlated with Waltersdorph, 1988) were also effective inhibitors (IC50
the determined ratios of rates of compound I to compound II o30 mM). However, inhibition of MPO by all these drugs
reduction. 5-Fluorotryptamine and 5-chlorotryptamine were was reversed by ascorbate, which is known to reduce
found to act as the most effective chlorination inhibitors compound II to ferric MPO (Figure 2, reaction 4) (Hsuanyu
during this study. and Dunford, 1999). Additionally, the inhibitory effects of
The putative binding mode of indole derivatives has been these molecules were significantly diminished in the pre-
described above (Hallingback et al., 2006). In ferric MPO, the sence of a superoxide anion generating system, because
indole ring binds parallel to the pyrrole ring D and the 5-OH superoxide anion acts as electron donor for compound II
group (serotonin, an excellent substrate) and 5-OCH3 group (Kettle et al., 1993).
(melatonin, a poor substrate) most probably points to the The mechanism of inhibition of HOCl production, which
haem centre. In both cases, the indole ring seems to be in relies on MPO being trapped as inactive compound II, was
the same place. The orientation of the side chain constitutes also supported by the findings that dapsone and indometha-
the largest difference between melatonin and serotonin. In cin are competitive inhibitors with respect to I and Cl
case of serotonin it points out into the access channel, (Stendahl et al., 1978; Shacter et al., 1991). All these drugs are
whereas in case of melatonin it points into a pocket efficiently oxidized by compound I but have a limited
along the periphery of the distal cavity (Hallingback et al., capacity to reduce compound II. This indicates that they are
2006). The ring substituent determines the actual rates unable to compete with high-affinity peroxidase substrates
of compounds I and II reduction with electron-donating indicating that measurement of inhibited peroxidative
groups by increasing the aromatic ring reactivity (Jantschko activity is an inappropriate strategy to screen for potential
et al., 2005). Electron-withdrawing substituents, for example inhibitors of HOCl production by MPO.
Cl and F atoms, will increase the redox potential of the The IC50 values are influenced by the electrochemical
corresponding indole derivatives and, as a consequence, properties of these compounds. Their reduction potential
disqualify them to act as a peroxidase substrate. Owing to determines their reactivity towards MPO compounds I and II
the extremely positive reduction potential of the redox and thus their effectiveness as inhibitors of HOCl produc-
couple compound I/compound II (Arnhold et al., 2006), tion. Compounds with reduction potentials 41.1 V could be
these substituents can still serve as electron donor for relatively specific for MPO because they would react poorly
compound I but are unreactive towards compound II with other haem enzymes. For all these reversible inhibitors
(Jantschko et al., 2005). As a consequence, compound II it is unlikely that size or hydrophobicity have a major impact
accumulates and MPO is diverted from its chlorination cycle on inhibition properties. This is best demonstrated
(Figure 2). by salicylate analogues, sulphasalazine and olsalazine,
In effect these indole and tryptamine derivatives are poor which have similar IC50 values as salicylate, even though
peroxidase substrates and reversible inhibitors. Under in vivo they are much larger and more hydrophobic (Kettle and
conditions compound II can easily be reduced by alternative Winterbourn, 1991).
electron donors thus diminishing the efficacy of these In a recent study, recombinant proMPO has been shown to
reversible-inhibiting compounds. This has been demon- be convenient for pharmacological purposes (Neve et al.,
strated by the effect of tryptophan on formation of HOCl 2001). Fourteen drugs representative of a family of various
by human neutrophils (Kettle and Candaeis, 2000). Using nonsteroidal anti-inflammatory drugs were tested for their
neutrophil-derived MPO, IC50 values increased from 5 to ability to inhibit the MPO-mediated chlorination of TauNH2.
80 mM, because physiological substrates such as ascorbate, Most of them induced a dose-dependent inhibition of
tyrosine, urate or superoxide anion are able to reduce TauNH2 and IC50 values could be calculated. The most
compound II enabling the enzyme to cycle. efficient inhibitors (IC50o40 mM) were found to be flufe-
namic acid, diclofenac, niflumic acid, tenoxicam and
piroxicam. In all cases, these drugs interacted with recombi-
Drugs that promote compound II formation nant proMPO in the same way as already described for
As outlined above, hydroxamic acid, indole and tryptamine dimeric MPO from neutrophils (Kettle and Winterbourn,
derivatives promote compound II formation of MPO thus 1991).
diverting the enzyme from the chlorination cycle. Kettle In any case, within the phagosome inhibition of intracel-
and Winterbourn (1991) have demonstrated that many lular HOCl formation by reversible inhibitors will be limited
anti-inflammatory drugs that are already in clinical applica- by superoxide anion and other electron donors of compound
tion, follow the same reaction pattern. By using the H2O2 II. To achieve more efficient inhibition, drugs that irrever-
electrode to assess the ability of these compounds to sibly inhibit MPO (like benzoic acid hydrazides, see above)
inhibit the conversion of H2O2 into HOCl, dapsone, are needed.

British Journal of Pharmacology (2007) 152 838854

Inhibition of myeloperoxidase activity
E Malle et al 849

Drugs that promote compound III formation lactoperoxidase could be protected against inactivation. This
Compound III is positioned outside both of the chlorination clearly demonstrates that methimazole is oxidized to thiyl
and peroxidation cycle of MPO (Figure 2). Thus, drugs that radicals, which finally could be responsible for irreversible
promote compound III formation divert MPO also from modification of the haem cavity and inhibition of lactoper-
HOCl production. Many drugs and xenobiotics are oxidized oxidase (Bandyopadhyay et al., 1993, 1995). Inhibition of
by MPO via the peroxidase cycle (Figure 2, reactions 1, 3 and MPO-mediated chlorination by methimazole is also based on
4) but in some cases the kinetics of oxidation cannot the oxidation of methimazole but in contrast to lactoperox-
adequately be explained by this pathway alone. Hydroqui- idase inhibition of MPO seems to be only reversible (McGirr
nones are known to be excellent substrates for MPO et al., 1990; Sayo and Saito, 1991).
compounds I and II (Kettle and Winterbourn, 1992; Burner Propylthiouracil (also used as antithyroid drug) was also
et al., 2000). Semiquinone radicals are formed in a high reported to inhibit the peroxidase and chlorination activity
flux, which efficiently reduce ferric MPO to ferrous MPO that of MPO (Lee et al., 1990). The inactivation was dependent on
rapidly binds oxygen to form compound III (Figure 2, H2O2 and was still observed after propylthiouracil and H2O2
reactions 11 and 8). The same reaction pathway seems to were removed by gelfiltration, which suggests an irreversible
be followed in the oxidation of the anticancer drug inactivation mode. With a gastric peroxidase it has been
amsacrine by the MPOH2O2 system (Kettle et al., 1992); in shown that propylthiouracil is a mechanism-based inhibitor
both cases production of HOCl by MPO is inhibited. but with MPO detailed mechanistic studies are lacking.
However, in contrast to hydrazides that follow the same The last group of sulphur-centred compounds with
mechanism but finally irreversibly modify the active site (see inhibitory effects on MPO are thiourea derivatives.
above), the hydroquinone derivatives and amsacrine are Dimethylthiourea (110 mM) has been reported to comple-
reversible inhibitors because compound III is in dynamic tely block formation of HOCl by phorbol myristate acetate-
equilibrium with the native enzyme or ferrous MPO (Figure 2, and zymosan-stimulated neutrophils (Sagone et al., 1989).
reactions 10 and 7) or can even be reduced to the native The compound has been shown to act both as a substrate for
enzyme by ascorbate (Marquez et al., 1990). MPO and a scavenger for HOCl. From these data there is no
evidence that dimethylthiourea mediates an irreversible
inactivation of MPO (Sagone et al., 1989).
Further studies on putative MPO inhibitors
In a comparative study of the effects of nonsteroidal anti-
inflammatory cyclooxygenase 1 and 2 inhibitors on MPO
activity, it has been shown that indomethacin and other Alternative therapeutic options to influence
representative tricyclic aromatics display only poor inhibi- the MPOH2O2chloride system
tion of the peroxidase activity of MPO (EC50 4100 mM) (Rider
et al., 1996). Subjects with profound deficiencies in MPO (approximately
More promising molecules with inhibitory effects on the one out of 4000 subjects) appear to have a significant
TauNH2 chlorination activity of MPO are 2(3H)-benzoxazo- increase in risk for infection (Nauseef et al., 1998; Klebanoff,
lone derivatives. In a recent study 20 o-[2-oxo-3H-benzoxazol- 2005); observations that parallel severe impairment in early
3-yl]-N-phenylacetamide and propionamide derivatives host defence in MPO/ mice against Candida albicans
(carrying substituents of different lipophilic and electrophilic (Aratani et al., 1999) and enhanced inflammation after
nature on the N-phenyl ring) have been synthesized and allogeneic marrow transplantation (Milla et al., 2004). In
shown to exhibit inhibitory effects on the chlorinating contrast, cardiovascular problems appear to be very rare
capacity of MPO (IC50 295 mM) (Soyer et al., 2005). In among MPO-deficient individuals in comparison to their
general, propionamide derivatives with chlorine and nitro- high frequency among the reference population (Kutter et al.,
substituents and non-substituted ones were more active than 2000). Hoy et al. (2001) reported that subjects carrying the
the acetamide counterparts. The authors reported that these A allele of the G-463A MPO polymorphism display higher
compounds were unable to react with HOCl suggesting that levels of triglycerides, total and low-density lipoprotein-
they interact directly with MPO. However, there are no cholesterol, and apoB-100 which are known risk factors for
detailed mechanistic studies available. atherosclerosis. Recent reports demonstrated that lipid-low-
The antithyroid drug methimazole (2-mercapto-1-methyl- ering drugs downregulate MPO on gene (Kumar and
imidazole) (Bandyopadhyay et al., 1993, 1995) and the Reynolds, 2005) and protein level (Stenvinkel et al., 2006;
selenium analogue (Roy and Mugesh, 2005) have been Zhou et al., 2006); statins block 3-hydroxy-3-methylglutaryl-
shown to be irreversible mechanism-based inhibitors of CoA-reductase, the key enzyme of endogenous cholesterol
human peroxidases (Bandyopadhyay et al., 1993, 1995). biosynthesis pathway, thereby apparently blocking produc-
Methimazole is oxidized by thyroid peroxidase, lactoperox- tion of isoprenoid intermediates of the mevalonate pathway,
idase and MPO, because no inactivation occurred in the which is required for human and mouse MPO expression.
absence of H2O2. With lactoperoxidase and a gastric Observations that an Alu receptor response element in the
peroxidase the kinetics of the inactivation process were human MPO promoter is regulated by T0901317 and
described to be consistent with a mechanism-based mode. GW9578, respectively, suggest that MPO regulation is under
With I (good electron donor for lactoperoxidase) and tight control of LXR and PPARa, which normally regulate
addition of the spin-trap 5,50 -dimethyl-1-pyrroline N-oxide, genes involved in cholesterol metabolism and inflammation
but not with Cl (poor electron donor for lactoperoxidase), (Reynolds et al., 2006).

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Inhibition of myeloperoxidase activity
850 E Malle et al

Neutrophils protect themselves from HOCl by maintain- gold compounds in patients treated with aurothiomalate
ing an exceptionally high cytoplasmic concentration of and aurothioglucose is about 50 mM (Lewis et al., 1983).
TauNH2 (approximately 50 mM). TauNH2 reacts with HOCl Interestingly, all four antiarthritic drugs develop their
to yield the relatively innocuous compound TauNHCl beneficial effects over a period of weeks of therapy. Although
that may act as immunomodulator by compensating the therapeutic effect of these antirheumatics may be
adverse effects of primary and secondary MPO-derived multifactorial, scavenging of HOCl and inhibition of MPO
products (Schuller-Levis and Park, 2003). Therapeutic offers an attractive explanation for their mechanism of
approaches using TauNH2 have revealed that this sulphur- action.
containing amino acid may blunt the contribution of Another drug already applied in practice is thiacetazone.
phagocyte activation to acute coronary events (McCarty, It is used to treat tuberculosis and has been reported to be
1999). Consequently, TauNH2 was used as HOCl scavenger in also an effective HOCl scavenger as well as to promote
a number of animal studies. Diminution of atherosclerotic compound III formation of MPO (van Zyl and van der Walt,
lesions has been reported in control and WHHL rabbits 1996).
(lacking functional low-density lipoprotein receptor) (Petty
et al., 1990; Murakami et al., 2002) when dietary-induced
hypercholesterolaemia was accompanied by TauNH2 Future perspectives
supplementation. In the absence of TauNH2, abundant
staining for HOCl-modified (lipo)proteins is present in these MPO inhibitory properties of a variety of compounds were
animals (Malle et al., 2001; Brasen et al., 2003). Although assessed with different methodologies. The information on
endogenous MPO levels in rodents are much lower than in promising MPO inhibiting compounds in the literature is
humans, TauNH2 supplementation leads to diminution and sparse making a comparison and critical evaluation of their
regression of lipid accumulation in murine lesions after actual inhibitory potential difficult. Additionally, only a few
cholesterol feeding (Murakami et al., 1999a, b). Most im- compounds were investigated in detail to clarify the under-
portantly, overexpression of human MPO in murine models lying molecular mechanism of inactivation. Nevertheless,
promoted formation of atherosclerosis (McMillen et al., the knowledge of (i) the three-dimensional structure of
2005; Castellani et al., 2006). Hyperlipidemia-induced human MPO and (ii) binding sites of its endogenous
renal damage in mesangioproliferative glomerulonephritis substrates (that is, two-electron donating anionic halides)
is paralleled by occurrence of HOCl-modified (lipo)proteins and of artificial aromatic one-electron donors/inhibitors (for
in rats (Scheuer et al., 2000). The protective effect of example, SHA or indoles) as well as our knowledge of (iii) its
TauNH2 in rat models of human kidney disease is paralleled reaction mechanisms and redox properties will accelerate the
by a decrease in total proteinuria and albuminuria, preven- process for a rationale design of specific MPO inhibitors in
tion of glomerular hypertrophy, and diminuition of the future.
glomerulosclerosis and tubulointerstitial fibrosis (Malle Almost all known MPO inhibitors are oxidized at least by
et al., 2003). compound I, which is one of the strongest oxidizing enzyme
Another possibility is the application of antioxidants, for intermediates found in vivo. Thus, these inhibitors divert the
example, ambroxol (Cho et al., 1999), dithiocarbamate enzyme from the chlorination cycle and suppress HOCl
(Zhu et al., 2002) and vitamin C (Carr et al., 2000), production. In case the reaction products do not interact
which protect against or even may reverse HOCl- and with the protein, compound II accumulates, which can be
chloramine-dependent modification of proteins by pathways easily reduced by many electron donors found in vivo, for
probably involving reduced glutathione and ATP. Even example superoxide anion, tyrosine or ascorbate. This
phenolic compounds, for example chlorogenic acid (Kono inhibition mode is reversible and the in vivo inhibitory effect
et al., 1995) might be considered as HOCl scavengers. of these drugs on HOCl is questionable.
Chlorogenic acid is approved as food additive (Svetol) and The interaction of MPO with some drugs results in the
used in coffee, chewing gum, and mints to promote weight formation of radicals that either attack the haem prosthetic
reduction. group or functionally important active site residues (me-
Very efficient HOCl scavengers are antiarthritic drugs chanism-based irreversible inactivation). Alternatively, they
containing thiol groups, such as D-penicillamine, tiopronin, can mediate the reduction of ferric MPO to ferrous MPO,
sodium aurothiomalate and aurothioglucose; it has been which is readily converted into its dioxygen complex
suggested that the therapeutic effect of these drugs may be (compound III) under aerobic conditions. In case these
due to the protection of tissues against reactive HOCl radicals are unreactive toward the enzyme this inhibition
released by activated granulocytes at inflamed sites (Cuperus mode is also reversible. With benzoic acid hydrazides the
et al., 1985). D-penicillamine, tiopronin, aurothiomalate or products generated in the peroxidase cycle mediate the
aurothioglucose (1050 mM) are relatively potential inhibitors irreversible destruction of the haem group of MPO in its
of MCD chlorination by HOCl. It has to be mentioned that ferrous form, with ABAH being the most efficient suicide
besides effectively scavenging HOCl, D-penicillamine and substrate (irreversible inhibitor). However, we still do not
tiopronin inhibit the enzyme itself (Cuperus et al., 1983, fully understand the molecular basis of this suicide inactiva-
1985). Extrapolation to in vivo conditions is possible because tion nor do we know the nature of the modification at the
the concentration of D-penicillamine and tiopronin in haem cavity. Understanding these features will facilitate the
serum from patients treated with these drugs is about design of more specific and more potent irreversible MPO
100 mM (van der Korst et al., 1981). The concentration of inhibitors.

British Journal of Pharmacology (2007) 152 838854

Inhibition of myeloperoxidase activity
E Malle et al 851

Acknowledgements Brennan ML, Penn MS, Van Lente F, Nambi V, Shishehbor MH, Aviles
RJ et al. (2003). Prognostic value of myeloperoxidase in patients
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This work was supported by grants from the Austrian Science
Burner U, Krapfenbauer G, Furtmu ller PG, Regelsberger G, Obinger C
Fund (FWF) to EM/WS (P17013-B05, P19074-B05, and (2000). Oxidation of hydroquinone, 2,3-dimethylhydroquinone
F3007-B05) and CO/PGF (P14187-B11, P15660-B10). We and 2,3,5-trimethylhydroquinone by human myeloperoxidase.
thank Inte:Ligand for help with three-dimensional MPO Redox Rep 5: 185190.
Burner U, Obinger C, Paumann M, Furtmu ller PG, Kettle AJ (1999).
structure models.
Transient and steady-state kinetics of the oxidation of substituted
benzoic acid hydrazides by myeloperoxidase. J Biol Chem 274:
Conflict of interest Buss IH, Senthilmohan R, Darlow BA, Mogridge N, Kettle AJ,
Winterbourn CC (2003). 3-Chlorotyrosine as a marker of protein
damage by myeloperoxidase in tracheal aspirates from preterm
E Malle has no conflict of interest. PG Furtmu ller is infants: association with adverse respiratory outcome. Pediatr Res
coinvestigator of a project (protec-TRANS sponsered by the 53: 455462.
Austrian ERP Fonds) executed by Planta Naturstoffe Vertriebs Carr AC, Tijerina T, Frei B (2000). Vitamin C protects against
and reverses specific hypochlorous acid- and chloramine-
GmbH that investigates natural anti-inflammatory sub-
dependent modifications of low-density lipoprotein. Biochem J
stances (January 1, 2005July 31, 2007). W Sattler has no 346: 491499.
conflict of interest. C Obinger is consultant to the Myeloper- Castellani LW, Chang JJ, Wang X, Lusis AJ, Reynolds WF (2006).
oxidase Programme Team of GlaxoSmithKline Research Transgenic mice express human MPO 463G/A alleles at athero-
sclerotic lesions, developing hyperlipidemia and obesity in 463G
& Development Ltd (September 1, 2005August 31, 2007).
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