Interactions of staphyloxanthin and enterobactin with myeloperoxidase and reactive chlorine species.

When neutrophils engulf bacteria, myeloperoxidase converts hydrogen peroxide to hypochlorous acid, which is toxic to all micro-organisms. It has been suggested that some pathogens have virulence factors that target myeloperoxidase to dampen the oxidative reactions of neutrophils. These virulence factors include staphyloxanthin, the golden pigment of Staphylococcus aureus, and enterobactin - a siderophore released by gram-negative bacteria. We investigated the potential of staphyloxanthin and enterobactin to shield bacteria from hypochlorous acid and related chloramines. Clinical strains of S. aureus with high levels of staphyloxanthin and related carotenoids were in general more resistant to low doses of hypochlorous acid than non-pigmented bacteria. But some non-pigmented strains were also resistant to the oxidant. Doses of reactive chlorine species that killed bacteria also bleached their carotenoids. Hypochlorous acid, NH2Cl, and NHCl2 bleached purified staphyloxanthin. When S. aureus were phagocytosed by neutrophils there was no discernible loss of staphyloxanthin. These data suggest that staphyloxanthin is capable of protecting bacteria from low doses of reactive chlorine species formed inside phagosomes. Enterobactin was not an inhibitor of myeloperoxidase. We conclude that staphyloxanthin may protect some bacterial strains against oxidative killing by neutrophils, but enterobactin will not inhibit the production of hypochlorous acid.

A multi-substrate assay for finding physiologically effective inhibitors of myeloperoxidase.

Myeloperoxidase, an abundant neutrophil enzyme, promotes oxidative damage during inflammation by generating hypohalous acids and free radicals. Currently, there are no selective drugs to inhibit its adverse activity. This short-coming is partly due to the lack of screening assays that mimic the complex enzymatic activities of myeloperoxidase in vivo. We have developed an assay for myeloperoxidase activity that includes its major physiological substrates - chloride, thiocyanate, tyrosine, and urate. The multi-substrate assay monitors bleaching of 5-thio-2-nitrobenzoic acid and measures total oxidant production when hydrogen peroxide activates the enzyme. Known suicide inhibitors and tight-binders tested positive in the assay, whereas compounds that merely convert myeloperoxidase to reducible enzyme intermediates were poor inhibitors. The new assay revealed that some aromatic compounds, including tryptamine, inhibit myeloperoxidase by binding reversibly to the enzyme. Our multi-substrate assay is selective for physiologically relevant inhibitors and has potential for identifying new classes of myeloperoxidase inhibitors.

Measuring chlorine bleach in biology and medicine.

BACKGROUND: Chlorine bleach, or hypochlorous acid, is the most reactive two-electron oxidant produced in appreciable amounts in our bodies. Neutrophils are the main source of hypochlorous acid. These champions of the innate immune system use it to fight infection but also direct it against host tissue in inflammatory diseases. Neutrophils contain a rich supply of the enzyme myeloperoxidase. It uses hydrogen peroxide to convert chloride to hypochlorous acid. SCOPE OF REVIEW: We give a critical appraisal of the best methods to measure production of hypochlorous acid by purified peroxidases and isolated neutrophils. Robust ways of detecting it inside neutrophil phagosomes where bacteria are killed are also discussed. Special attention is focused on reaction-based fluorescent probes but their visual charm is tempered by stressing their current limitations. Finally, the strengths and weaknesses of biomarker assays that capture the footprints of chlorine in various pathologies are evaluated. MAJOR CONCLUSIONS: Detection of hypochlorous acid by purified peroxidases and isolated neutrophils is best achieved by measuring accumulation of taurine chloramine. Formation of hypochlorous acid inside neutrophil phagosomes can be tracked using mass spectrometric analysis of 3-chlorotyrosine and methionine sulfoxide in bacterial proteins, or detection of chlorinated fluorescein on ingestible particles. Reaction-based fluorescent probes can also be used to monitor hypochlorous acid during phagocytosis. Specific biomarkers of its formation during inflammation include 3-chlorotyrosine, chlorinated products of plasmalogens, and glutathione sulfonamide. GENERAL SIGNIFICANCE: These methods should bring new insights into how chlorine bleach is produced by peroxidases, reacts within phagosomes to kill bacteria, and contributes to inflammation. This article is part of a Special Issue entitled Current methods to study reactive oxygen species - pros and cons and biophysics of membrane proteins. Guest Editor: Christine Winterbourn.

Potent reversible inhibition of myeloperoxidase by aromatic hydroxamates.

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 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 a 5 nm concentration of a trifluoromethyl-substituted aromatic hydroxamate, HX1. The hydroxamates were specific for MPO in neutrophils and more potent toward MPO compared with a broad range of redox enzymes and alternative targets. Surface plasmon resonance measurements showed that the strength of binding of hydroxamates to MPO correlated with the degree of enzyme inhibition. The crystal structure of MPO-HX1 revealed that 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 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 and oxidation of uric acid in gout: implications for the clinical consequences of hyperuricaemia.

OBJECTIVES: The aims of this study were to establish whether, in patients with gout, MPO is released from neutrophils and urate is oxidized to allantoin and if these effects are attenuated by allopurinol. METHODS: MPO, urate, allantoin and oxypurinol were measured in plasma from 54 patients with gout and 27 healthy controls. Twenty-three patients had acute gout, 13 of whom were receiving allopurinol, and 31 had intercritical gout, 20 of whom were receiving allopurinol. Ten additional gout patients had samples collected before and after 4 weeks of allopurinol. RESULTS: Plasma MPO and its specific activity were higher (P < 0.05) in patients with acute gout not receiving allopurinol compared with controls. MPO protein in patients' plasma was related to urate concentration (r = 0.5, P < 0.001). Plasma allantoin was higher (P < 0.001) in all patient groups compared with controls. In controls and patients not receiving allopurinol, allantoin was associated with plasma urate (r = 0.62, P < 0.001) and MPO activity (r = 0.45, P < 0.002). When 10 patients were treated with allopurinol, it lowered their plasma urate and allantoin (P = 0.002). In all patients receiving allopurinol, plasma allantoin was related to oxypurinol (r = 0.65, P < 0.0001). Oxypurinol was a substrate for purified MPO that enhanced the oxidation of urate. CONCLUSION: Increased concentrations of urate in gout lead to the release of MPO from neutrophils and the oxidation of urate. Products of MPO and reactive metabolites of urate may contribute to the pathology of gout and hyperuricaemia. At low concentrations, oxypurinol should reduce inflammation, but high concentrations may contribute to oxidative stress.

2-thioxanthines are mechanism-based inactivators of myeloperoxidase that block oxidative stress during inflammation.

Myeloperoxidase (MPO) is a prime candidate for promoting oxidative stress during inflammation. This abundant enzyme of neutrophils uses hydrogen peroxide to oxidize chloride to highly reactive and toxic chlorine bleach. We have identified 2-thioxanthines as potent mechanism-based inactivators of MPO. Mass spectrometry and x-ray crystal structures revealed that these inhibitors become covalently attached to the heme prosthetic groups of the enzyme. We propose a mechanism whereby 2-thioxanthines are oxidized, and their incipient free radicals react with the heme groups of the enzyme before they can exit the active site. 2-Thioxanthines inhibited MPO in plasma and decreased protein chlorination in a mouse model of peritonitis. They slowed but did not prevent neutrophils from killing bacteria and were poor inhibitors of thyroid peroxidase. Our study shows that MPO is susceptible to the free radicals it generates, and this Achilles' heel of the enzyme can be exploited to block oxidative stress during inflammation.

An activating mutation in the transmembrane domain of the granulocyte colony-stimulating factor receptor in patients with acute myeloid leukemia.

To date, constitutively activating point mutations reported in hematopoietic growth factor receptors in patients with acute myeloid leukemia (AML) have been restricted to receptors with intrinsic tyrosine kinase activity such as c-kit and FLT3. We describe here a Thr617Asn mutation in the transmembrane domain of the non-tyrosine kinase receptor for granulocyte colony-stimulating factor (G-CSF) in the blast cells of two out of 555 AML patients examined. The mutant receptor conferred growth factor independence on factor-dependent Ba/F3 cells. In the absence of ligand, immunoblotting showed weak phosphorylation of JAK2, STAT3, ERKs 1 and 2 and the receptor itself, and there was approximately 70% of maximal growth in a proliferation assay. All signals were significantly enhanced in the presence of G-CSF. Retroviral transduction of mutant receptor into primary hematopoietic CD34+ cells induced G-CSF independent myeloid differentiation as assessed by the development of neutrophils and surface expression of CD11b and CD14. These results confirm the importance of the transmembrane domain for receptor function and suggest that introduction of an asparagine residue can cause sufficient stabilization of helix-helix interactions in the absence of ligand to activate downstream signaling pathways involved in directing proliferation and differentiation.

Isoniazid as a substrate and inhibitor of myeloperoxidase: identification of amine adducts and the influence of superoxide dismutase on their formation.

Neutrophils ingest Mycobacteria tuberculosis (Mtb) in the lungs of infected individuals. During phagocytosis they use myeloperoxidase (MPO) to catalyze production of hypochlorous acid (HOCl), their most potent antimicrobial agent. Isoniazid (INH), the foremost antibiotic in the treatment of tuberculosis, is oxidized by MPO. It rapidly reduced compound I of MPO [k = (1.22 ± 0.05) × 10(6) M(-1) s(-1)] but reacted less favorably with compound II [(9.8 ± 0.6) × 10(2) M(-1) s(-1)]. Oxidation of INH by MPO and hydrogen peroxide was unaffected by chloride, the physiological substrate for compound I, and the enzyme was partially converted to compound III. This indicates that INH is oxidized outside the classical peroxidation cycle. In combination with superoxide dismutase (SOD), MPO oxidized INH without exogenous hydrogen peroxide. SOD must favor reduction of oxygen by the INH radical to give superoxide and ultimately hydrogen peroxide. In both oxidation systems, an adduct with methionine was formed and it was a major product with MPO and SOD. We show that it is a conjugate of an acyldiimide with amines. INH substantially inhibited HOCl production by MPO and neutrophils below pharmacological concentrations. The reversible inhibition is explained by diversion of MPO to its ferrous and compound III forms during oxidation of INH. MPO, along with SOD released by Mtb, will oxidize INH at sites of infection and their interactions are likely to limit the efficacy of the drug, promote adverse drug reactions via formation of protein adducts, and impair a major bacterial killing mechanism of neutrophils.