Pharmacophore-based discovery of 2-(phenylamino)aceto-hydrazides as potent eosinophil peroxidase (EPO) inhibitors.

There is an increasing interest in developing novel eosinophil peroxidase (EPO) inhibitors, in order to provide new treatment strategies against chronic inflammatory and neurodegenerative diseases caused by eosinophilic disorder. Within this study, a ligand-based pharmacophore model for EPO inhibitors was generated and used for in silico screening of large 3 D molecular structure databases, containing more than 4 million compounds. Hits obtained were clustered and a total of 277 compounds were selected for biological assessment. A class of 2-(phenyl)amino-aceto-hydrazides with different substitution pattern on the aromatic ring was found to contain the most potent EPO inhibitors, exhibiting IC50 values down to 10 nM. The generated pharmacophore model therefore, represents a valuable tool for the selection of compounds for biological testing. The compounds identified as potent EPO inhibitors will serve to initiate a hit to lead and lead optimisation program for the development of new therapeutics against eosinophilic disorders.

Disruption of the aspartate to heme ester linkage in human myeloperoxidase: impact on ligand binding, redox chemistry, and interconversion of redox intermediates.

In human heme peroxidases the prosthetic group is covalently attached to the protein via two ester linkages between conserved glutamate and aspartate residues and modified methyl groups on pyrrole rings A and C. Here, monomeric recombinant myeloperoxidase (MPO) and the variants D94V and D94N were produced in Chinese hamster ovary cell lines. Disruption of the Asp(94) to heme ester bond decreased the one-electron reduction potential E'(0) [Fe(III)/Fe(II)] from 1 to -55 mV at pH 7.0 and 25 degrees C, whereas the kinetics of binding of low spin ligands and of compound I formation was unaffected. By contrast, in both variants rates of compound I reduction by chloride and bromide (but not iodide and thiocyanate) were substantially decreased compared with the wild-type protein. Bimolecular rates of compound II (but not compound I) reduction by ascorbate and tyrosine were slightly diminished in D94V and D94N. The presented biochemical and biophysical data suggest that the Asp(94) to heme linkage is no precondition for the autocatalytic formation of the other two covalent links found in MPO. The findings are discussed with respect to the known active site structure of MPO and its complexes with ligands.

The vinyl-sulfonium bond in human myeloperoxidase: impact on compound I formation and reduction by halides and thiocyanate.

In human myeloperoxidase (MPO) the heme is covalently attached to the protein via two ester linkages and a unique sulfonium ion linkage between the sulfur atom of Met243 and the beta-carbon of the vinyl ring on pyrrole ring A. Here, we have investigated the variant Met243Val produced in Chinese hamster ovary cells in order to elucidate the role of the electron withdrawing sulfonium bond in compound I formation and reduction. Disruption of this MPO-typical bond causes a blue-shifted UV-vis spectrum and an increase in the heme flexibility. This had no impact on compound I formation mediated by hydrogen peroxide (2.2x10(7) M(-1)s(-1) at pH 7.0 and 25 degrees C). Compared with wild-type recombinant MPO the cyanide association rate with ferric Met243Val was significantly enhanced as were also the calculated apparent bimolecular compound I reduction rates by iodide (>10(8) M(-1)s(-1)) and thiocyanate (>10(8) M(-1)s(-1)). By contrast, the overall chlorination and bromination activities were decreased by 98.1% and 87.4%, respectively, compared with the wild-type protein. Compound I reduction by chloride was slower than compound I decay to a compound II-like species (0.4 s(-1)), whereas compound I reduction by bromide was about 10-times slower (1.3x10(4) M(-1)s(-1)) than the wild-type rate. These findings are discussed with respect to the known crystal structure of MPO and its bromide complex as well as the known redox chemistry of its intermediates and substrates.

Redox thermodynamics of the Fe(III)/Fe(II) couple of human myeloperoxidase in its high-spin and low-spin forms.

Myeloperoxidase (MPO) (donor, hydrogen peroxide oxidoreductase, EC 1.11.1.7) is the most abundant neutrophil enzyme and catalyzes predominantly the two-electron oxidation of ubiquitous chloride (Cl-), to generate the potent bleaching oxidant hypochlorous acid (HOCl), thus contributing to bacterial killing and inflammatory reactions of neutrophils. Here, the thermodynamics of the one-electron reduction of the ferric heme in its ferric high-spin and cyanide-bound low-spin forms were determined through spectroelectrochemical experiments. The E(o)' values for free and cyanide-bound MPO (5 and -37 mV, respectively, at 25 degrees C and pH 7.0) are significantly higher than those of other heme peroxidases. Variable-temperature experiments revealed that the enthalpic stabilization of ferric high-spin MPO is much weaker than in other heme peroxidases and is exactly compensated by the entropic change upon reduction. In contrast to those of other heme peroxidases, the stabilization of the ferric cyanide-bound MPO is also very weak and fully entropic. This peculiar behavior is discussed with respect to the MPO-typical covalent heme to protein linkages as well as to the published structures of ferric MPO and its cyanide complex and the recently published structure of lactoperoxidase as well as the physiological role of MPO in bacterial killing.

Active site structure and catalytic mechanisms of human peroxidases.

Myeloperoxidase (MPO), eosinophil peroxidase, lactoperoxidase, and thyroid peroxidase are heme-containing oxidoreductases (EC 1.7.1.11), which bind ligands and/or undergo a series of redox reactions. Though sharing functional and structural homology, reflecting their phylogenetic origin, differences are observed regarding their spectral features, substrate specificities, redox properties, and kinetics of interconversion of the relevant redox intermediates ferric and ferrous peroxidase, compound I, compound II, and compound III. Depending on substrate availability, these heme enzymes path through the halogenation cycle and/or the peroxidase cycle and/or act as poor (pseudo-)catalases. Based on the published crystal structures of free MPO and its complexes with cyanide, bromide and thiocyanate as well as on sequence analysis and modeling, we critically discuss structure-function relationships. This analysis highlights similarities and distinguishing features within the mammalian peroxidases and intents to provide the molecular and enzymatic basis to understand the prominent role of these heme enzymes in host defense against infection, hormone biosynthesis, and pathogenesis.

Peroxynitrite efficiently mediates the interconversion of redox intermediates of myeloperoxidase.

Nitric oxide-derived oxidants (e.g., peroxynitrite) are believed to participate in antimicrobial activities as part of normal host defenses but also in oxidative tissue injury in inflammatory disorders. A similar role is ascribed to the heme enzyme myeloperoxidase (MPO), the most abundant protein of polymorphonuclear leukocytes, which are the terminal phagocytosing effector cells of the innate immune system. Concomitant production of peroxynitrite and release of millimolar MPO are characteristic events during phagocytosis. In order to understand the mode of interaction between MPO and peroxynitrite, we have performed a comprehensive stopped-flow investigation of the reaction between all physiological relevant redox intermediates of MPO and peroxynitrite. Both iron(III) MPO and iron(II) MPO are rapidly converted to compound II by peroxynitrite in monophasic reactions with calculated rate constants of (6.8+/-0.1) x 10(6) M(-1)s(-1) and (1.3+/-0.2) x 10(6) M(-1)s(-1), respectively (pH 7.0 and 25 degrees C). Besides these one- and two-electron reduction reactions of peroxynitrite, which produce nitrogen dioxide and nitrite, a one-electron oxidation to the oxoperoxonitrogen radical must occur in the fast monophasic transition of compound I to compound II mediated by peroxynitrite at pH 7.0 [(7.6+/-0.1) x 10(6) M(-1)s(-1)]. In addition, peroxynitrite induced a steady-state transition from compound III to compound II with a rate of (1.0+/-0.3) x 10(4) M(-1)s(-1). Thus, the interconversion among the various oxidation states of MPO that is prompted by peroxynitrite is remarkable. Reaction mechanisms are proposed and the physiological relevance is discussed.

Mechanism of interaction of betanin and indicaxanthin with human myeloperoxidase and hypochlorous acid.

Hypochlorous acid (HOCl) is the most powerful oxidant produced by human neutrophils and contributes to the damage caused by these inflammatory cells. It is produced from H2O2 and chloride by the heme enzyme myeloperoxidase (MPO). Based on findings that betalains provide antioxidant and anti-inflammatory effects, we performed the present kinetic study on the interaction between the betalains, betanin and indicaxanthin, with the redox intermediates, compound I and compound II of MPO, and its major cytotoxic product HOCl. It is shown that both betalains are good peroxidase substrates for MPO and function as one-electron reductants of its redox intermediates, compound I and compound II. Compound I is reduced to compound II with a second-order rate constant of (1.5+/-0.1) x 10(6) M(-1) s(-1) (betanin) and (1.1+/-0.2) x 10(6) M(-1) s(-1) (indicaxanthin), respectively, at pH 7.0 and 25 degrees C. Formation of ferric (native) MPO from compound II occurs with a second-order rate constant of (1.1+/-0.1) x 10(5) M(-1) s(-1) (betanin) and (2.9+/-0.1) x 10(5) M(-1) s(-1) (indicaxanthin), respectively. In addition, both betalains can effectively scavenge hypochlorous acid with determined rates of (1.8+/-0.2) x 10(4) M(-1) s(-1) (betanin) and (7.7+/-0.1) x 10(4) M(-1) s(-1) (indicaxanthin) at pH 7.0 and 25 degrees C. At neutral pH and depending on their concentration, both betalains can exhibit a stimulating and inhibitory effect on the chlorination activity of MPO, whereas at pH 5.0 only inhibitory effects were observed even at micromolar concentrations. These findings are discussed with respect to our knowledge of the enzymatic mechanisms of MPO.

Standard reduction potentials of all couples of the peroxidase cycle of lactoperoxidase.

Lactoperoxidase (LPO) is found in mucosal surfaces and exocrine secretions including milk, tears and saliva and has physiological significance in antimicrobial defense. Its predominant physiological role is to convert hydrogen peroxide and thiocyanate in hypothiocyanite. In this study, the standard reduction potentials of all redox couples involved in the halogenation and peroxidase cycle of LPO have been determined by multi-mixing stopped-flow spectroscopy. The standard reduction potentials of the redox couples compound I/native LPO, compound I/compound II of LPO, and compound II/native LPO are (1.09 +/- 0.01) V, (1.14 +/- 0.02) V, and (1.04 +/- 0.02) V, respectively, at pH 7 and 25 degrees C. Thus, for the first time, a full description of these important thermodynamic parameters of lactoperoxidase has been performed, allowing a better understanding in the substantial differences in the oxidation of two- and one-electron donors by LPO and other members of the mammalian heme peroxidase superfamily.

Role of the covalent glutamic acid 242-heme linkage in the formation and reactivity of redox intermediates of human myeloperoxidase.

In human myeloperoxidase the heme is covalently attached to the protein via two ester linkages between the carboxyl groups of Glu242 and Asp94 and modified methyl groups on pyrrole rings A and C of the heme as well as a sulfonium ion linkage between the sulfur atom of Met243 and the beta-carbon of the vinyl group on pyrrole ring A. In the present study, wild-type recombinant myeloperoxidase (recMPO) and the variant Glu242Gln were produced in Chinese hamster ovary cells and investigated in a comparative sequential-mixing stopped-flow study in order to elucidate the role of the Glu242-heme ester linkage in the individual reaction steps of both the halogenation and peroxidase cycle. Disruption of the ester bond increased heme flexibility, blue shifted the UV-vis spectrum, and, compared with recMPO, decelerated cyanide binding (1.25 x 10(4) versus 1.6 x 10(6) M(-)(1) s(-)(1) at pH 7 and 25 degrees C) as well as compound I formation mediated by either hydrogen peroxide (7.8 x 10(5) versus 1.9 x 10(7) M(-)(1) s(-)(1)) or hypochlorous acid (7.5 x 10(5) versus 2.3 x 10(7) M(-)(1) s(-)(1)). The overall chlorination and bromination activity of Glu242Gln was 2.0% and 24% of recMPO. The apparent bimolecular rate constants of compound I reduction by chloride (65 M(-)(1) s(-)(1)), bromide (5.4 x 10(4) M(-)(1) s(-)(1)), iodide (6.4 x 10(5) M(-)(1) s(-)(1)), and thiocyanate (2.2 x10(5) M(-)(1) s(-)(1)) were 500, 25, 21, and 63 times decreased compared with recMPO. By contrast, Glu242Gln compound I reduction by tyrosine was only 5.4 times decreased, whereas tyrosine-mediated compound II reduction was 60 times slower compared with recMPO. The effects of exchange of Glu242 on electron transfer reactions are discussed.

Exploitation of the unusual thermodynamic properties of human myeloperoxidase in inhibitor design.

Myeloperoxidase plays a fundamental role in oxidant production by neutrophils. It uses hydrogen peroxide and chloride to catalyze the production of hypochlorous acid (HOCl), which contributes to both bacterial killing and oxidative injury of host tissue. Thus, MPO is an interesting target for anti-inflammatory therapy. Here, based on the extraordinary and MPO-specific redox properties of its intermediates compound I and compound II, we present a rational approach in selection and design of reversible inhibitors of HOCl production mediated by MPO. In detail, indole and tryptamine derivatives were investigated for their ability to reduce compounds I and II and to affect the chlorinating activity of MPO. It is shown that these aromatic one-electron donors bound to the hydrophobic pocket at the distal heme cavity and were oxidized efficiently by compound I (k3), which has a one-electron reduction potential of 1.35 V. By contrast, compound II (E degrees ' of the compound II/ferric couple is 0.97 V) reduction (k4) was extremely slow. As a consequence compound II, which does not participate in the halogenation cycle, accumulated. The extent of chlorinating activity inhibition (IC50) was related to the k3/k4 ratio. The most efficient inhibitors were 5-fluorotryptamine and 5-chlorotryptamine with IC50 of 0.79 microM and 0.73 microM and k3/k4 ratios of 386,000 and 224,000, respectively. The reversible mechanism of inhibition is discussed with respect to the enzymology of MPO and the development of drugs against HOCl-dependent tissue damage.

Reaction of ferrous lactoperoxidase with hydrogen peroxide and dioxygen: an anaerobic stopped-flow study.

Lactoperoxidase (LPO) is found in mucosal surfaces and exocrine secretions including milk, tears, and saliva and has physiological significance in antimicrobial defense which involves (pseudo-)halide oxidation. LPO compound III (a ferrous-dioxygen complex) is known to be formed rapidly by an excess of hydrogen peroxide and could participate in the observed catalase-like activity of LPO. The present anaerobic stopped-flow kinetic analysis was performed in order to elucidate the catalytic mechanism of LPO and the kinetics of compound III formation by probing the reactivity of ferrous LPO with hydrogen peroxide and molecular oxygen. It is shown that ferrous LPO heterolytically cleaves hydrogen peroxide forming water and oxyferryl LPO (compound II). The two-electron oxidation reaction follows second-order kinetics with the apparent bimolecular rate constant being (7.2+/-0.3) x 10(4) M(-1) s(-1) at pH 7.0 and 25 degrees C. The H2O2-mediated conversion of compound II to compound III follows also second-order kinetics (220 M(-1) s(-1) at pH 7.0 and 25 degrees C). Alternatively, compound III is also formed by dioxygen binding to ferrous LPO at an apparent bimolecular rate constant of (1.8+/-0.2) x 10(5) M(-1) s(-1). Dioxygen binding is reversible and at pH 7.0 the dissociation constant (K(D)) of the oxyferrous form is 6 microM. The rate constant of dioxygen dissociation from compound III is higher than conversion of compound III to ferric LPO, which is not affected by the oxygen concentration and follows a biphasic kinetics. A reaction cycle including the redox intermediates compound II, compound III, and ferrous LPO is proposed, which explains the observed (pseudo-)catalase activity of LPO in the absence of one-electron donors. The relevance of these findings in LPO catalysis is discussed.

Kinetics of interconversion of redox intermediates of lactoperoxidase, eosinophil peroxidase and myeloperoxidase.

Myeloperoxidase, eosinophil peroxidase and lactoperoxidase are heme-containing oxidoreductases, which undergo a series of redox reactions. Though sharing functional and structural homology, reflecting their phylogenetic origin, differences are observed regarding their spectral features, substrate specificities, redox properties and kinetics of interconversion of the relevant redox intermediates ferric and ferrous peroxidase, compound I, compound II and compound III. Depending on substrate availability, these heme enzymes path through the halogenation cycle and/or the peroxidase cycle and/or act as poor (pseudo-) catalases.

Kinetics of oxygen binding to ferrous myeloperoxidase.

Myeloperoxidase (MPO), which is involved in host defence and inflammation, is a unique peroxidase in having a globin-like standard reduction potential of the ferric/ferrous couple. Intravacuolar and exogenous MPO released from stimulated neutrophils has been shown to exist in the oxyferrous form, called compound III. To investigate the reactivity of ferrous MPO with molecular oxygen, a stopped-flow kinetic analysis was performed. In the absence of dioxygen, ferrous MPO decays to ferric MPO (0.04 s(-1) at pH 8 versus 1.4 s(-1) at pH 5). At pH 7.0 and 25 degrees C, compound III formation (i.e., binding of dioxygen to ferrous MPO) occurs with a rate constant of (1.1+/-0.1) x 10(4)M(-1)s(-1). The rate doubles at pH 5.0 and oxygen binding is reversible. At pH 7.0, the dissociation equilibrium constant of the oxyferrous form is (173+/-12)microM. The rate constant of dioxygen dissociation from compound III is much higher than conversion of compound III to ferric MPO (which is not affected by the oxygen concentration). This allows an efficient transition of compound III to redox intermediates which actually participate in the peroxidase or halogenation cycle of MPO.

Direct conversion of ferrous myeloperoxidase to compound II by hydrogen peroxide: an anaerobic stopped-flow study.

Myeloperoxidase (MPO) is one of the essential components of the antimicrobial systems of polymorphonuclear neutrophils. It is unique in having a globin-like standard reduction potential of the ferric/ferrous couple. Here, it is shown that ferrous MPO heterolytically cleaves hydrogen peroxide forming water and oxyferryl MPO (compound II). The two-electron oxidation reaction follows second-order kinetics with the apparent bimolecular rate constant being (6.8+/-0.6)x10(4)M(-1)s(-1) at pH 7.0. After depletion of (micromolar) H(2)O(2) compound II slowly decays to ferric MPO, whereas upon addition of millimolar H(2)O(2) to ferrous MPO, compound III (oxyperoxidase) is formed in a sequence of two reactions involving compound II formation and its direct reaction with H(2)O(2), which also follows second-order kinetics [(78+/-2)M(-1)s(-1) at pH 7.0]. It is discussed how these reactions contribute to the interconversion of compound II and compound III and could explain the catalase activity of MPO.