Reversible inhibition of myeloperoxidase 1 Potent Reversible

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  • Reversible inhibition of myeloperoxidase

    1

    Potent Reversible Inhibition of Myeloperoxidase by Aromatic Hydroxamates*

    Louisa V. Forbes1, Tove Sjgren

    2, Franoise Auchre

    1, David W. Jenkins

    3A, Bob Thong

    3B, David

    Laughton3B

    , Paul Hemsley3C

    , Garry Pairaudeau3C

    , Rufus Turner1, Hkan 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 Mlndal, Sweden

    3Bioscience and Medicinal Chemistry, AstraZeneca R&D Charnwood, Loughborough, Leicestershire

    LE11 5RH United Kingdom

    4AstraZeneca R&D Sdertlje, SE-151 85 Sdertlje, 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: louisa.forbes@otago.ac.nz

    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.

    by guest on April 6, 2018

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  • Reversible inhibition of myeloperoxidase

    2

    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

    neutrophils 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

    Alzheimers disease, Parkinsons 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

    enzymes 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 enzymes 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 hydroxama