DESIGN, SYNTHESIS AND STUDY OF MYELOPEROXIDASE INHIBITORS IN THE SERIES OF 3-ALKYLINDOLE

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FACULTÉ DE PHARMACIE

École Doctorale en Sciences Pharmaceutiques

DESIGN, SYNTHESIS AND STUDY OF MYELOPEROXIDASE INHIBITORS IN THE SERIES OF 3-ALKYLINDOLE

Jalal Housain SOUBHYE

Thèse présentée en vue de l’obtention du grade de Docteur en Sciences Biomédicales et Pharmaceutiques

Promoteur et co-promoteur

Prof. Jean NEVE, Laboratoire de Chimie Pharmaceutique Organique

Prof. François DUFRASNE, Laboratoire de Chimie Pharmaceutique Organique

Composition du jury :

Prof. Karim AMIGHI (Président) Prof. Stéphanie POCHET (Secrétaire) Prof. Jean-Michel KAUFFMANN Prof. Franck MEYER

Prof. Jean-François LIEGEOIS (Université de Liège)

Prof. Joël PINCEMAIL (Université de Liège)

Année académique 2013-2014

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1. SUMMARY

The imbalance between the oxidant and the antioxidant agents in the body is termed as oxidative stress. It attracted more attention in the last decades and is implicated in many chronic diseases such as atherosclerosis, Parkinson, Alzheimer, schizophrenia, cancer, and many others. While most of the oxidant agents in the body have short half-life, hydrogen peroxide H2O2 and hypochloric acid HOCl are stable and very active oxidants. The last one is produced in the phagosomes of the neutrophils after the phagocytosis of a pathogen. The high oxidative potency of this molecule causes oxidative damages for the biomolecules of the pathogen leading to killing this organism. The main source of HOCl is the heme enzyme myeloperoxidase (MPO) which dismutates H2O2 generating hypohalous acid starting from a halogen or a pseudohalogen. Although the main function of MPO is to produce the antimicrobial agent HOCl inside the phagosomes of the neutrophils, it can be secreted outside the white blood cells causing oxidative damages for the host tissues. Proteins, lipids, lipoproteins, DNA and RNA can be targets of the MPO resulting in many chronic syndromes.

Indeed, a considerable correlation was found between the concentrations of MPO in the blood and the risk of atherosclerosis. The role of MPO in atherosclerosis development has been demonstrated. It is related to its ability to oxidize APO B-100 (the main protein in low-density lipoproteins, LDL) in the intima and at the surface of endothelial cells.

The deleterious effects of MPO make it a new target for medicinal research. The aim of our study is to find promising inhibitors of MPO for using them as starting point of new anti- inflammatory drugs. Depending on previous researches on MPO inhibitors, we selected 5- fluorotryptamine as starting compounds. Using docking experiments, we designed a series of compounds derived from 5-fluorotryptamine. Two modifications were proposed:

 Varying the length of the alkyl side chain between the indole ring and the amino group from 1 to 6 carbons.

 Inserting different alkyl or cyclic substituents on the side chain nitrogen.

The compounds proposed by docking experiments were synthesized and purified. In vitro evaluation was done for these compounds by the taurine chloramine test. It is found that the compounds with 4 and 5 carbons on the side chain have the best activities with IC50= 15 and 8

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7 | P a g e nanomolar respectively. Another test related to the role of MPO in atherosclerosis was done:

the inhibition of the oxidation of LDLs carried out by MPO. Some differences were found between the SAR obtained by this test and the previous one. However, the compounds with 4 and 5 carbons on the side chain also had the best activities in this test and the IC50 values were at low nanomolar range. In order to predict the side effects of these inhibitors, the best compound with 5 carbons on the side chain was injected in 4 groups of rats. Each group received a concentration of the inhibitor. No serious side effect was found in the rats that received 1 and 10 mg/kg.

In addition to the similarity between serotonin and 5-fluorotryptamine structures, some recent papers indicated that the fluorine atom instead of OH group on the indole ring is responsible for inhibition of serotonin transporter receptor (SERT). These findings led us to consider the hybrid compounds that inhibit both MPO and SERT simultaneously. Such compounds can be an important step to find drugs that can be used for treating major depressive disorders (MDD) at the patients who suffer from inflammatory syndromes. To this purpose, our compounds were submitted to the SERT inhibition test. The SAR was studied according to the activity obtained by this test. The most active compound was the derivative with 3 carbons on the side chain and substituted with dimethyl on the amine group of the side chain. The compounds with 4 and 5 carbons on the side chain were among the most active inhibitors with Ki= 6 and 3 nanomolar.

Because the last compounds have the best activity on both MPO and SERT, they appear as promising candidates for hybrid drugs. In order to better document this study, it is important to test the commercial SERT inhibitors on MPO. Among all the SSRIs used, only paroxetine was found to inhibit MPO at nanomolar concentration with IC50= 22 nM. Again this drug was able to inhibit the oxidation of LDLs at nanomolar concentration range.

Although the compounds that inhibit both MPO and SERT open a door for hybrid drugs, we had to develop other compounds that selectively inhibit MPO. These selective MPO inhibitors are very important to avoid the side effects caused by the inhibition of SERT. Again depending on the docking results of 5-fluorotryptamine onto MPO active site, a new series of compounds was designed according to 4 modifications:

Summary

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 Replacement of the fluorine atom with other electron-withdrawing group, or change of its position on the indole ring.

 Introduction of substitution groups on the nitrogen of the side chain.

 Introduction of an electron-donating sulfur atom on the side chain in order to change the redox potential of the compound. This change in redox potential may affect in the behavior of the inhibitor towards the MPO Compound I and II.

 Replacement of the basic amino group of the side chain with other chemical groups.

The proposed compounds were synthesized and subjected to MPO inhibition test and LDL oxidation inhibition test to assess the SAR of these inhibitors. It was found that the fluorine atom on position 7 gives an activity similar to the fluorine on position 5. A carboxyl group on the indole ring caused a loss of activity. The compounds without function on the side chain were not good inhibitors. The best compounds were those with 4 and 5 atoms between the indole and the nitrogen. The compounds that attracted more attention were the compounds with chemical group that are not amines. These compounds were very slightly less active than the compounds with an amine group. All the synthetic compounds were tested by SERT inhibition test to examine the selectivity of the new compounds. Only the compounds with a functional group other than the amine have a high activity on MPO and a low activity on SERT.

Among these selective compounds, the compound with an amide group on the side chain was chosen, and other series of compounds were synthesized derived from this compound to optimize this structure. Changing the position of fluorine, varying the length of the side chain and insertion of alkyl group on the nitrogen of the side chain were assayed. The compound with 3 carbons between the indole and the amide group and fluorine on position 5 had the best inhibition activity on both MPO and LDLs oxidation.

Two compounds were injected in the rats to preliminary determine their toxicity namely the most active compound (compound with fluorine on the position 7 and have 5 atoms between the indole and the amine) and the most selective compound (compound with an amide group and tree carbons on the side chain). No serious side effects were observed upon injecting 10 mg/kg of these compounds in the rats. No mortality was observed in the rats that received 100 mg/kg.

Summary

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9 | P a g e A kinetic study was done for our compounds (including active and non-active ones) to examine the mechanism of the inhibition. The MPO must be oxidized by H2O2 to give MPO Compound I that is able to oxidize the halogen. MPO Compound I can be reduced via one electron process to give MPO Compound II that cannot react with the halogen and considered as the inactive form of MPO. All the derivatives were found to react quickly with MPO Compound I giving Compound II the inactive form of the enzyme. Only the active derivatives very slowly reacted with Compound II leading to the accumulation of this inactive form. These data indicate that our inhibitors are reversible and act by accumulation of MPO Compound II. Paroxetine was subjected to the same test. It reacted very quickly with MPO Compound I but a saturation behavior was observed indicating that the heme group is destroyed. Thus paroxetine is considered as irreversible inhibitor.

In summary, we designed and synthesized new highly potent inhibitors of MPO and determined the mechanism of their action on MPO.

Summary

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2. ABBREVIATIONS

5-HT Serotonin LDL Low-density lipoprotein

ABAH 4-aminobenzoic acid hydrazide LPO Lactoperoxidase

ALA D-aminolevulinic acid LPS Lipopolysaccharide

ANCAs Anti-neutrophil cytoplasmic

antibodies MDD Major depressive disorder

APO A-I Apolipoprotein a-1 MeOH Methanol

APO B-100 Apolipoprotein b-100 MI Myocardial infraction Bil Bilirubin

MIP Macrophage inflammatory

protein

BSA Bovine serum albumin MMP Matrix metalloproteinase

CD Cluster of differentiation moxLDL Myeloperoxidase-modified LDL

CHD Coronary heart disease MPO Myeloperoxidase

CNS Central nervous system MT Metallothionein

CP Ceruloplasmin NOS NO synthase

DIBAL-H Diisobutylaluminium hydride oxLDL Oxidized LDL

DMA Dimethylacetamide RNS Reactive nitrogen species

DMSO Dimethylsulfoxide ROS Reactive oxygen species

EC Endothelial cell SAR Structure activity relationship

EPO Eosinophil peroxidase SHA Salicylhydroxamic acid

EtOAc Ethyl acetate TF Transferrin

EtOH Ethanol TFR Transferrin receptor

GLRXs Glutaredoxins TGF Transforming growth factor

GM-CSF Granulocyte–macrophage

colony-stimulating factor TNF Tumor necrosis factor

GSH Glutathione TPO Thyroperoxidase

HDL High-density lipoprotein VLDL Very low-density lipoprotein hSERT Human serotonin transporter

IFN- Interferon-

IL Interleukin

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3. INTRODUCTION

It is well known that the inflammation is required for healing wounds and infections. It is the initial response of the body to harmful stimuli and it is achieved by the increased movement of leukocytes (especially granulocytes) from blood into injured tissues. Inflammatory mediators, which are responsible for the clinical signs of inflammation, are released in the injured area.

The mediator molecules also alter the blood vessels to permit the migration of leukocytes, mainly neutrophils and monocytes, outside the blood vessels into the tissue. The neutrophils migrate along a chemotactic gradient created by the local cells to reach the site of injury. These neutrophils contain in their granules a large variety of enzymes which perform a number of immunity functions.

Despite of their indispensable role in microbial killing, the inflammation reactions may also cause diseases to a host such as hay fever, atherosclerosis, and rheumatoid arthritis. The enzymes and oxidizing species released during the inflammatory process can cause damages to the host tissues which lead to inflammatory syndromes.

Myeloperoxidase (MPO) which produces oxidizing species through the system MPO/hydrogen peroxide (H2O2)/ (pseudo)halogenous ions (X^-) is one of the enzymes released from neutrophils.

In the inflammatory conditions, a large amount of MPO may be present in the extracellular fluids, this leads to the production of high concentrations of hypo(pseudo)halogenous acid (HOX) which has a high oxidation potential. The very strong correlation between MPO and inflammatory syndromes led several groups to develop therapeutic strategies to inhibit MPO.

One problem encountered in the development of MPO inhibitors might be its impairment in innate host defenses. However, MPO may be relatively protected from inhibition because the enzyme is stored within granules in the leukocytes. After the activation of neutrophil, MPO is released into the phagolysosome or in the extracellular fluid. Because of the location of MPO in granules for its physiological role, the extracellular MPO will be the only target of the inhibitors, so the role of MPO in killing pathogens would not be significantly impeded.

In this manuscript, many compounds were designed by docking approach and synthesized as MPO inhibitors. Structure-activity relationship study of these compounds towards (recombinant) human MPO was carried out. The effect of these new compounds was reported on

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12 | P a g e MPO-mediated taurine chlorination and LDL oxidation. The mechanism of inhibition and the toxicity of the inhibitors were also studied.

Introduction

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Oxidative Stress and Antioxidants

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4. OXIDATIVE STRESS AND ANTIOXIDANTS

4.1. Overview

Oxidative stress is deﬁned as an imbalance between production of free radicals and reactive products, the so-called reactive oxygen species (ROS), and their elimination by protective mechanisms, referred as antioxidants. This imbalance leads to damage of important biomolecules and cells, with potential impact on the whole organism [1]. These biomolecules may be proteins, lipids, or DNA [2] [3] [4]. In humans, oxidative stress may be involved in many diseases including atherosclerosis, Parkinson's disease, heart failure, myocardial infarction, Alzheimer's disease, schizophrenia, cancer, fragile X syndrome [5]. Enzymes such as superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase are cellular defenses against ROS [4]. Many antioxidants are used to prevent and treat some diseases caused by ROS like vitamin C, vitamin E and some minerals such as zinc, selenium and copper that are components of antioxidant proteins [6] [7]. However, the use of antioxidants as supplements to prevent disease is controversial [8], and no experimental data can prove that the antioxidant supplements can prevent chronic diseases [9].

4.2. Reactive Oxygen Species Superoxide anion O2•−

is believed to be the source of ROS (Figure 1 summarizes the network of ROS production). Most O2•−

is generated in cells by the mitochondrial respiratory chain by which the oxygen is reduced to water. However, during respiration 1–2 % of oxygen consumed is partially reduced to superoxide O2•−

instead of being reduced to water. Superoxide produced appears to be at both faces of the inner mitochondrial membrane. When it is produced at the outer face, it is converted to hydrogen peroxide (H2O2) by Cu-Zn-superoxide dismutase (CuZnSOD) and, in the matrix space, by Mn-Superoxide dismutase (MnSOD). H2O2 is a toxic compound. Thus to neutralize this toxicity, it will be a substrate for certain enzymes including catalase (CAT), glutathione peroxidase (GPX) generating H2O and myeloperoxidase (MPO) generating HOCl [10]. H2O2 can give hydroxyl radicals (^•OH) by the Fenton reaction which is catalyzed by free iron or copper ions [10]. The strong oxidant peroxynitrite ONOO^-is formed starting from NO radical which in turn can be obtaind enzymatically by NO synthase (NOS) [11].

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Figure (1): Reactions involved in the production and removal of oxygen free radicals in the cell.

Because of the unstable configuration of ROS, they have short lifes and quickly react with other molecules or biomolecules to achieve a stable configuration. Table 1 illustrates the half-life of the different ROS [12].

Table 1: Major ROS and their half-life [12]

Species Common name Half-life (37°C)

HO^. hydroxyl radical 1 nanosecond

HO2.

hydroperoxyl radical unstable

O2.-

superoxide anion radical 1-4 microsecond

1O2 singlet oxygen 1 microsecond

RO^. alkoxyl radical 1 microsecond

ROO^. peroxyl radical 7 seconds

NO^. nitric oxide radical 1-10 seconds

H2O2 hydrogen peroxide Relatively stable

HOCl hypochlorous acid Relatively stable

Reactive oxygen species play an important role in the inflammatory network. In the case of inflammation or diabetes, ROS also participate in the induction of cell proliferation, migration of white blood cells and increase in extracellular matrix production [13]. ROS generated from p53 activation also play an important role in the apoptotic signaling pathway [14]. Other roles Oxidatiove Stress and Antioxidants

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16 | P a g e of ROS including growth and differentiation, proliferation, and gene expression, acting through both transductional and transcriptional pathways have been also reported [15].

4.3. Defenses against ROS

Since the ROS play a key role in various disorders and diseases, antioxidants have received more attention [16].

The human body has developed a very efficient defense network against oxidative stress. The antioxidants can be defined as substances that significantly inhibit oxidation of substrates when present at low concentrations compared to those of the oxidable substrate [17]. Three essential defense lines against damages caused by ROS were determined in human (Figure 2). The first one is to suppress the generation of ROS, the second is the chemical antioxidants which rapidly remove the ROS at the right time and in the right position in right concentration before they react with biomolecules and the third line defense is the enzymes that repair the oxidative damages caused by ROS [18].

Figure (2): Defense network in vivo against oxidative stress.

Defense network system against oxidative stress in humans consists of antioxidant compounds and enzymes. Many of important antioxidant compounds are delivered from dietary sources such as ascorbic acid, lipoic acid, polyphenols and carotenoids, so when the supply of external antioxidants is weak, the potent of antioxidant network is weakened [19] [20].

Numerous agents can be considered as "antioxidant". Table 2 summarizes these systems:

Oxidatiove Stress and Antioxidants

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Table (2): The classification of antioxidant Systems

Antioxidant System Reference

Enzymatic antioxidants

Superoxide dismutase (SOD) [21] [22]

Catalase (CAT) [23]

Glutathione Peroxidase (GPX) [24]

Glutathione S-Transferases (GST) [25]

Thyroperoxidase (TPO) [26]

Lactoperoxidase (LPO) [27]

Eosinophil peroxidase [28]

MPO [29]

Non enzymatic protein defenses against the oxidative stress

Transferrin (TF) [30]

Ferritin [31] [32]

Lactoferrin [30]

Ceruloplasmin [33]

Albumin [34] [35]

Endogenous low molecular weight antioxidants "scavengers"

Glutathione [36]

Uric Acid [37] [38] [39]

Bilirubin [40] [41]

Melatonin [42]

Melanin [43]

Keto acids [44]

Lipoic acid [45]

Exogenous low weight antioxidants

Ascorbic acid [46] [47] [48]

Vitamin E [49]

Carotenoids [50]

The natural phenolic antioxidants [51] [52]

Oxidatiove Stress and Antioxidants

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Myeloperoxidase – Structure, Synthesis, Activity

and Role in inflammatory

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5. MPO - STRUCTURE, SYNTHESIS AND ACTIVITY.

5.1. Introduction

The heme enzyme myeloperoxidase (EC 1.11.2.2, MPO) is one of the major players in the first line of the unspecific immune defense system. It is important for the microbicidal activity within human neutrophils. This enzyme is packed inside the cytoplasmic azurophilic granules, in relatively high concentration up to 5 % of the dry weight of the cell. The enzyme MPO as the prominent generator of reactive oxidizing species in neutrophils uses H2O2 and (pseudo)halides (X⁻, SCN^-) to produce hypo(pseudo)halous acids (HOX, HOSCN). The reaction takes place preferentially with Cl⁻ because of its high concentration in body fluids (100–140 mM) compared to other anions. Hypochlorous acid is the most powerful neutrophil oxidant. It is extremely cytotoxic and readily reacts with most biological molecules thereby also promoting inflammatory tissue damage caused by neutrophils. Due to deleterious effects of HOCl, MPO is implicated in a growing number of diseases like atherosclerosis, rheumatoid arthritis, lung cancer and many others [53].

Historically, MPO was isolated for the first time in 1941 [54]. This enzyme belongs to the superfamily of peroxidases (EC 1.11) which includes TPO, LPO, GPX, EPO and MPO. They are heme proteins that catalyze the reduction of H2O2 and are found in plants, fungi, bacteria or mammalian [55]. However, their primary and tertiary structure and nature of the prosthetic group are significantly different. Taurog et al. (1999) proposed to name the superfamily of mammalian peroxidases as “animal peroxidases” because of significant homology in the sequence of the protein have been found in invertebrates [56].

5.2. Synthesis of MPO

5.2.1. Overview

Myeloperoxidase is produced in the myeloid cells, which includes any leukocyte that is not a lymphocyte. These cells contain two types of granules, azurophils and specifics, which have separate origins and are different in nature. The large, dense azurophil granules represent a special type of primary lysosome containing peroxidase and lysosomal enzymes. They are produced early in development, during the promyelocyte stage (Figure 3), and arise from the

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20 | P a g e concave face of the Golgi apparatus. The smaller, less dense specific granules represent an entirely different secretory product which contains alkaline phosphatase and lacks lysosomal enzymes and peroxidase. They are produced later in development, during the myelocyte stage, and arise from the opposite or convex face of the Golgi apparatus. The myeloblast is a relatively undifferentiated cell with a large oval nucleus, large nucleoli, and cytoplasm lacking granules. It originates from a precursor pool of elusive stem cells and is followed by two secretory stages:

the promyelocyte and the myelocyte. During each of these stages a distinct type of secretory granule is produced. The metamyelocyte and band cells are nonproliferating, nonsecretory stages which develop into the mature polymorphonuclear leukocytes. The latter is characterized by a multi lobulated nucleus and cytoplasm containing primarily glycogen and granules. Out of every 100 nucleated cells in bone marrow, 2 are myeloblasts, 5 promyelocytes, 12 myelocytes, 22 metamyelocytes and bands, and 20 mature polymorphonuclear leukocytes, giving a total of ≈ 60 % developing polymorphonuclear leukocytes [57].

Figure (3): Diagrammatic representation of polymorphonuclear leukocytes life cycle and stages of polymorphonuclear leukocytes maturation; azurophils (blue forms) formed only during the promyelocyte stage and specific granules (light forms) produced during the myelocyte stage [57].

It is demonstrated that MPO is localized exclusively in azurophil granules of neutrophil in human [58].

MPO - Structure, Synthesis and Activity

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21 | P a g e 5.2.2. MPO gene

The human MPO gene (MPO gene) is located on chromosome 17q21.3-q23 (Figure 4) beside the gene of LPO (LPO gene: 17q23), whereas the mouse MPO gene is located on chromosome 11qc. In humans the external identifications of MPO gene are the following [59]:

RefSeq (mRNA) NM_000250

RefSeq (protein) NP_000241

Start 51,708,013 bp

End 51,718,966 bp

Size 10,954

Orientation minus strand

Number of exon 14 exons

Figure (4): MPO Gene in genomic location: bands according to Ensembl, and genomic region, transcript and product [59].

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22 | P a g e 5.2.3. Protein synthesis

The expression of the MPO gene is regulated by a cell-specific transcription factor AML1. The AML1 binding site is very important for the activity of the MPO proximal enhancer. In addition of its function in regulating MPO gene expression, potential binding sites for the AML1 are located on 5' flanking region of the neutrophil elastase gene. Thus it is concluded that the expression of some azurophil granule proteins occurs by the action of common transcription factors [60]. The MPO gene encodes a single protein precursor from which both subunits of the mature protein are derived. The primary translation product is an 80-kDa protein which undergoes cleavage of the signal peptide followed by cotranslational N-linked glycosylation (Figure 5). The signal peptide is the first 41 amino acids at the N-terminus, followed by a propeptide, a light subunit and a heavy subunit. This glycosylated form is very short-lived and is processed in the endoplasmic reticulum to produce apoproMPO, an enzymatically inactive, heme-free protein. At least two molecular chaperones, calreticulin (CRT) and calnexin (CLN) interact transiently with apoproMPO during its prolonged residence in the endoplasmic reticulum. Subsequently heme is inserted into the peptide backbone of apoproMPO. A calcium- binding protein, CRT, would thus maintain apoproMPO in a conformation allowing the insertion of a heme group, thereby generating the enzymatically active proMPO. After the acquisition of the heme, proMPO undergoes two proteolytic processing events. First, the amino terminal 125 amino acid proregion is cleaved, leaving a 72 to 75-kDa protein containing the light and heavy subunits as a single peptide. Subsequent to the removal of the proregion, there is a second proteolytic event, whereby the light and heavy subunits are generated. The resultant heavy- light protomers dimerize to produce the symmetric form of mature MPO, each molecule being composed of a pair of heavy-light protomers, and the mature enzyme is targeted to the lysosome. It is not known if dimerization is necessary for lysosomal targeting or if dimerization occurs in the lysosome (Figure 6).

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Figure (5): Model of MPO biosynthesis. Myeloperoxidase is synthesized as an 80-kDa primary translation product which is cotranslationally glycosylated (brown points) and associated with CRT. The CRT-apoproMPO complex dissociates and apoproMPO binds to another resident protein in the endoplasmic reticulum, CLN. While associated with CLN, apoproMPO acquires heme (the red form) and becomes the enzymatically active precursor, proMPO.

Subsequently, CLN dissociates from the complex and proMPO exits the endoplasmic reticulum into one of two pathways. Enzyme directed to lysosome undergoes a series of proteolytic steps whereby the 125 amino acids proregion is removed, and the protein is cut into the two subunits of mature enzyme. Subsequently these heavy- light protomers dimerize, either in or en route to the lysosome, to generate the form of mature MPO present in azurophilic granules. ProMPO not destined for the lysosome enters the secretory pathway, wherein passage through the Golgi apparatus results in maturation of the oligosaccharide side chains from the high-mannose type to the complex form [60].

During the formation of mature MPO from proMPO, the amino-terminal propeptide and the peptide between the heavy and light chains (6 amino acids) are excised. In this process a single serine residue of the C-terminal is also removed. The proteolytic trimming of proMPO takes place in pregranule organelles, and perhaps also in matures granules, and is rather slow as heavy and light subunits begin to be visible first after 6-8 hours of biosynthetic radio-labeling.

Intermediate processing forms have been observed, one with a molecular mass of 81 kDa followed in time by another with a molecular mass of 74 kDa, suggesting that the former is a

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24 | P a g e forerunner of the latter. The reduction in size is likely to involve elimination of the propeptide, and cleavage between the light and heavy subunit would generate mature MPO from the 74 kDa intermediate.

The formation of the 74k Da intermediate probably requires an acidic pregranule compartment, whereas the final proteolytic cleavage to generate MPO optimally occurs at a neutral pH. Thus, an acidic pregranule compartment is required only for early but not final proteolytic trimming.

Mature MPO is a dimer, each monomer being composed of 1 heavy and 1 light chain as a pair of protomers linked together by one disulfide bond. ProMPO not destined for the lysosome enters the secretory pathway (Figure 6), wherein passage through the Golgi apparatus results in maturation of the oligosaccharide side chains from the high-mannose complex form [60]

[61]. It is indicated that the inability of induced cells to synthesize MPO is due to the absence of MPO mRNA, thus the synthesis of MPO protein is regulated by MPO mRNA [62].

Figure (6): Schematic model for MPO processing. The primary translation product undergoes cotranslational cleavage of the signal peptide followed by N-linked glycosylation to generate apoproMPO.

Calreticulin facilitates folding of apoproMPO to allow insertion of heme to yield enzymatically active proMPO. This form, proMPO, subsequently has the pro-peptide cleaved and the precursor proteolytically modified to generate heavy (59-kDa) and light (13.5-kDa) subunits, joined together by disulfide bonds. This heavy-light complex dimerizes to generate a symmetric molecule containing a pair of heavy-light protomers and two heme groups, the molecular state of mature, native MPO [60] [61].

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25 | P a g e 5.2.4. Heme group synthesis

It is reported that the heme group of MPO is a derivative of protoporphyrin IX. Starting from - aminolevulinic acid (ALA), the protoporphyrins are synthesized. ALA is in turn synthesized from Gly (a non essential amino acid) and succinyl coenzyme A (an intermediate in the citric acid cycle) in a reaction catalyzed by ALA synthase (ALAS). This reaction requires pyridoxal phosphate (PLP) as a coenzyme, and is the committed and rate-limiting step in porphyrin biosynthesis. The condensation of two molecules of ALA by Zn-containing ALA dehydratase (porphobilinogen synthase) forms porphobilinogen. Four molecules of porphobilinogens condense to produce the linear tetrapyrrole, hydroxymethylbilane, which is isomerized and cyclized by uroporphyrinogen III synthase to produce the asymmetric uroporphyrinogen III.

This cyclic tetrapyrrole undergoes decarboxylation of its acetate groups, generating coproporphyrinogen III. These reactions occur in the cytosol. Coproporphyrinogen III enters the mitochondria, and two propionate side chains are decarboxylated to vinyl groups generating protoporphyrinogen IX, which is oxidized to protoporphyrin IX. The introduction of iron (as Fe²⁺) into protoporphyrin IX spontaneously occurs, but the rate is enhanced by ferro-chelatase (Figure 7) [4].

5.2.5. Incorporation of heme group into the apo-enzyme

Before its incorporation into the protein moiety of the enzyme, the porphyrin IX must undergo a change to allow the formation of covalent bonds with the protein structure, within the endoplasmic reticulum. This modification is the oxidation of methyl groups of rings A and C of the porphyrin to methanol, creating ester links with carboxylate groups of the amino acids of the protein. It seems that this oxidation takes place through an autocatalytic process. Indeed, H2O2 is reported to be essential molecule in heme oxidation process. In addition, it is found also that at least one amino acid would contribute to the heme oxidation reaction. The mechanism of modification of the prosthetic group which leads to esteric bond between heme and protein is concluded and illustrated in Figure 8 [63].

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Figure (7): Pathway of porphyrin synthesis: formation of protoporphyrin IX.

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Figure (8): Proposed mechanism for autocatalytic heme modification and covalent binding in MPO and other mammalian peroxidases [63].

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28 | P a g e 5.3. Structure of mature MPO

Myeloperoxidase is a dimer enzyme with a molecular mass of 140-kDa. Each subunit has two polypeptide chains, where the light chain consists of 108 amino acids while the heavy one consists of 466 amino acids. Each subunit binds covalently with heme group which gives to MPO unusual spectral properties. Mature MPO shows a Soret band significantly red-shifted at 428 nm compared with other heme proteins. Beside this unusual spectral property of MPO, it has also strong absorption bands in the visible region. It is thought that these spectral shifts are responsible for the characteristic green color of the enzyme. Many interchain disulfide bonds have been identified in mature MPO, six of them are found in each unit, five in the heavy chain and one in the light one. One additionnal interchain disulfide bond is found between the two subunits. The latter bond is found at Cys153 in the heavy chain [64] [65].

The overall structure of the enzyme, (Figure 9), is largely α-helical, with very little β-sheet. Each half molecule consists of a central core of five helices and a covalently attached heme. Four of these helices derived from the large polypeptide and the fifth from the small one. The remainder of the large polypeptide folds into four separate domains and a single open loop that surround the central core. The small polypeptide wraps around the surface of the molecule with only its carboxyl-terminal helix penetrating the interior to form part of the central core.

Structure determination has confirmed the presence of a calcium-binding site and five putative sites of asparagine-linked glycosylation (Asn157, Asn189, Asn225, Asn317 and Asn563). If the first and last asparagine-linked glycosylation sites do not appear in crystallography, the glycosylation sites at position Asn189 and Asn225 are well described. In addition, glycosylation at position Asn317 plays a role not only in the dimerization of the enzyme but also in the stabilization of the dimer. The secondary structure also shows the proximal His336 and distal His95 which play a very important role in redox reactions of the enzyme [66].

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Figure (9): Entire MPO dimer, viewed along the molecular dyad axis. The large polypeptides of the two halves are colored red and blue, whereas the small polypeptides are in lighter shades of the same colors and the sugar are yellow structures. Other color coded features include: hemes (green), carbohydrate (orange), calcium (purple), and

chloride (yellow). At the center of the molecule the disulfide linking the two halves is shown in black [66].

The calcium-binding site has typical pentagonal bi-pyramidal form (Figure 10). This form consists of hydroxyl of Ser174, carbonyl oxygen of Phe170, carboxyl oxygen of Asp96, carbonyl oxygen of Thr168 and carboxyl oxygen of Asp172. The latter three ligands make a planar form, while the other two provide the axial ligands. Thr168,Phe170, Asp172 and Ser174 belong to the loop of the heavy polypeptide, while the Asp96 residue is from light polypeptide chain and located beside distal His95 [66] [67]. Van Antwerpen et al [67] have demonstrated that all five N-glycosylation sites (Asn157, Asn189, Asn225, Asn317 and Asn563) are occupied on the large polypeptide. They suggested that N-glycans sites on Asn189, Asn225, and Asn563, are important for the interaction with other proteins while the remaining two sites on Asn157 and Asn317 are important to stabilize the dimer form because they are located at the interface.

They also pointed out that Asn189 is important for the optimal activity of the enzyme [67].

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30 | P a g e

Figure (10): Calcium binding site. The figure is from ‘Protein Data Bank’ code 1cxp.

As previously indicated, the heme group of the mature MPO belongs to protoporphyrin IX structure. It is located in a distal cavity with a narrow oval-shaped opening (Figure 11). Two ester bonds are formed between the modified methyl groups (the modifications that have been implemented on methyl groups were previously discussed) on pyrrole rings A and C and carboxyl groups of Glu242 and Asp94, respectively.

Figure (11): The location of heme group of the active site at the distal cavity with a narrow oval-shaped opening.

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31 | P a g e In addition, another bond between pyrrole ring A of the heme and the protein occurs by the covalent bond between β-carbon of its vinyl group and sulfur atom of Met243 resulting in sulfonium cation. There are also more conventional interactions between the heme of MPO and protein structure. They are ionic bonds between the carboxylic group of the propionate of the rings C and Asp98 and Thr100, and hydrogen bonds between the guanidinium groups of both Arg333 and Arg424 and the ring D [66]. The proximal His336 with its N^links the iron atom together with the four nitrogen atoms of the heme (Figure 12). Moreover, this proximal histidine forms a hydrogen bond between its nitrogen N^ and the oxygen of the amide function of Asn421 [209].

Figure (12): This figure shows the two ester bonds between heme group and Asp91 and Glu242, the sulfur bond between the sulfur atom of Met243 and the β carbon of the pyrrole ring A. The complex of the proximal His336

with the iron atom of heme is also showed in this figure; the figure is from ‘Protein Data Bank’ code 1cxp.

The chloride-binding site consists of the residues Ile324, Ala325, Asn326, Val327, His336 from the amino terminus of the helix, residues Val30, Arg31, Trp32 and Leu33 of the small polypeptide, and Trp436 (Figure 13). Both polypeptides are parallel and bind together by two backbone hydrogen bonds. The chloride has three ligands: Trp32 NH, Val327 NH, and one water

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32 | P a g e molecule which has an additionally hydrogen bond with carbonyl oxygen of Leu33 and the guanidinium group NH of Arg31 [66].

However, the X-ray crystal structure of halide-binding sites of human MPO shows that the enzyme has four halide binding sites in each subunit. Two are inside the distal heme cavity replacing the water molecules and two are on the surface of the protein, which are away from the heme. It was proposed that halide binding at the distal cavity sites inhibit the enzyme by effectively competing with H2O2 for the access to distal His95, whereas the same sites may be the halide substrate-binding sites in the Compound I [66].

5.4. Activity of MPO

5.4.1. Overview

Myeloperoxidase uses H2O2 to oxidize chloride (Cl^-), bromide (Br^-), iodide (I^-), and the pseudohalide thiocyanate (SCN^-) (but not fluoride) to their respective hypo(pseudo)halous acids. The halide oxidation starts by reaction of ferric-MPO with H2O2 to form Compound I, which contains two oxidizing equivalents more than the resting enzyme. Compound I directly oxidizes halide to recover the native enzyme by a two-electron process. These reactions take

Figure (13): Stereo view of the proximal helix chloride-binding site in the native MPO model. Residues 324–327 at the carboxyl terminus of the proximal helix are linked to residues 30–33 via two main chain hydrogen bonds [66].

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33 | P a g e part to the so-called halogenation cycle. During turnover, some Compound I is also converted to Compound II because of the presence of exogenous or endogenous electron donors (AH2).

This reaction initiates the peroxidation cycle. Because of its high concentrations, chloride seems to be the physiological substrate for MPO. Its plasma concentrations are 100−140 mM, whereas it is 20−100 μM for bromide, 0.1−0.6 μM for iodide, and 20−120 μM for thiocyanate.

However, in the case of smokers where the concentration of thiocyanate is 100- 500 µM, the kinetic rate of its oxidation is the same as for chloride [68].

5.4.2. Formation of Compound I

Compound I is generated by oxidation of the native enzyme in the presence of H2O2 (Figure 14).

It is an unstable form of MPO that directly reacts with the halide to oxidize it by a two-electron process, or alternately it is reduced by a one electron donor to generate Compound II. In chlorination reactions, chloride not only acts as a substrate for MPO but also behaves as a competitive inhibitor of H2O2. Chloride appears to bind its protonated form to make an inhibitory chloride complex while H2O2 reacts with the native enzyme when the distal His is unprotonated.

Figure (14): Pathway of the formation of Compound I.

It is demonstrated that the rate constant of oxidation of native MPO to Compound I is 2.3 X 10⁷ M^-1s^-1. It is proposed that the mechanism of the oxidation of native MPO to Compound I starts by the formation of MPO-H2O2 complex which would be in equilibrium with the formation of MPOH⁺-OOH. The O-O bond is then heterolytically broken and an oxygen atom is fixed on the Fe (III) to form the cation-oxy ferryle (Fe (IV)=O) (Figure 15). In this reaction, the absorbance at the wavelength of 430 nm falls over 50 % of the initial value which is for native MPO (Figure 16) [68] [69].

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34 | P a g e

Figure (15): Pathway of the formation of Compound I, at the heme edge (δ-meso bridge between pyrrol rings A and D), substrates bind and donate electrons. Ferric MPO is oxidized by H2O2 to Compound I [69].

The reaction of Compound I with the (pseudo) halogen appears to be second order rate, and the Table 3 summarizes the values of the rate constants of the reaction of Compound I with (pseudo) halogens at pH =7.

Figure (16): UV-visible spectra of MPO and Compound I.

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35 | P a g e

Table (3): Rate constants of the reaction of (pseudo) halogen with Compound I.

(pseudo)halides Rate constant M^-1s^-1 chloride (2.5 ± 0.3) X 10⁴ bromide (1.1± 0.1) X 10⁶ iodide (7.2 ± 0.7) X 10⁶ thiocyanate (9.6 ± 0.5) X 10⁶

The partitioning between the halogenation cycle and the peroxidation cycle depends on the relative rates at which the halides and H2O2 reduce Compound I. In the presence of H2O2 and bromide, iodide, or thiocyanate, MPO mainly exists in the ferric form (native form). However in the presence of H2O2 and chloride, MPO exists as Compound II. Because the reduction of Compound I with H2O2 is known to occur at (8.2 ± 0.1) X 10⁴ M^-1 s^-1 at 25C° and pH 7 whereas the constant rate of the reduction of Compound I by Cl^- is (2.5 ± 0.3) X 10⁴ M^-1s^-1. It is proposed that the reaction of (pseudo)halide with the Compound I occurs in two steps as following:

Compound I + Cl^- → Compound I-Cl^-Eq [1a]

Compound I-Cl^- → native MPO + HOCl Eq [1b]

The first step is the formation of complex between Compound I and the chloride ion. The crystallographic data shows that to date there is no specific binding site for (pseudo)halides in the distal cavity of the native MPO. The second step is the generation of HOCl [68] [70].

5.4.3. Compound II

In the presence of a one electron donor, Compound I is reduced to generate Compound II by a one-electron process (Figure 17). The heme iron of Compound II which is inactive in forming HOCl is probably in the ferryl(IV) state. In this state the iron atom forms a complex with a hydroxyl group as it is indicated in Figure 18 [68] [71].

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36 | P a g e

Figure (17): The formation of Compound I, II, and III. Compound I is generated by oxidation of MPO by H2O2 by two-electron process. In the presence of electron donor HA, Compound I is reduced to Compound II by one- electron process (reaction 3a and 3b). The Compound II may be reduced by other electron donor AH to give native MPO (reaction 4a and 4b). In the high concentration of H2O2, Compound II conversion to Compound III becomes relevant and cycling of MPO then includes the ferric and ferrous state, Compound I, Compound II and Compound III. Upon reaction of Compound III with excess of hydrogen peroxide reactive oxidants might be formed that destroy the heme and oxidize the protein [72] [71].

It is suggested that the reactions catalyzed by MPO depend on the concentration of three elements including native MPO, Cl^-,and H2O2. At high concentration of H2O2, Compound I is reduced to Compound II before a turnover to native MPO by H2O2 which plays an electron donor role in this case [73]. Compound II formation can be also increased in the presence of molecules which can prevent the binding of Cl^- on the MPO and the same effects in the case of Cl^- deficiency can be also seen [74] [68]. On the other hand many compounds can play the role of electron donor including thiols [75], tyrosine (Tyr) [76], guaiacol [77], hydroquinone [78], and some drugs including salicylate, mefenamic acid, dapsone, primaquine, and some others [79].

Although the superoxide O2•-

can reduce Compound I to Compound II by playing an electron donor role, it can enhance the activity of MPO by preventing the accumulation of inactive Compound II in the presence of high amount of O2•-

where it can reduce Compound II by one electron process to form the native enzyme (Figure 17) [72].

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37 | P a g e

Figure (18): Pathway of the formation of Compound II. Compound I is reduced to Compound II [i.e., protonated oxoiron(IV)] by a one-electron donor. Compound II is reduced then to the native state thereby oxidizing a second

substrate molecule [69].

The formation of Compound II can be observed due to its spectral properties, where its spectrum shows significant differences with the spectrum of native enzyme including two maximal absorbance wavelengths at 456 and 625 nm instead of 430 and 560 nm for the native form (Figure 19) [71]. However, there is no significant difference between the spectra of Compound II and that of Compound III [70]. The standard reduction potentials E°’ of all relevant redox couples of MPO have been measured and a significant difference between is observed E°’

(Compound I/Compound II) = 1.35 V and E°’ (Compound II/native MPO-Fe³⁺) = 0.97 V, whereas the value of E°’ (Compound I/native MPO-Fe³⁺) is 1.16 V [68].

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38 | P a g e 5.4.4. Compound III

When the superoxide reacts with the native enzyme Fe³⁺, Compound III will be generated (Figure 17). Compound III, like Compound II, is thought to be inactive with respect to halogenation. Compound II can be formed starting from Compound III through oxidation by H2O2. The inactivation of MPO by high concentrations of H2O2 is proposed to occur via the generation of reactive oxidants when H2O2 reacts with Compound III (Figure 17) [72][71].

5.5. Role of MPO in the chronic diseases

Myeloperoxidase can be released outside the phagocytes producing the most powerful neutrophil oxidant, hypochlorous acid, outside the cells [29]. This production of HOCl contributes to tissue damages. HOCl is able to modify a great variety of biomolecules by chlorination and/or oxidation. It oxidizes sulfhydryl groups in proteins causing their inactivation.

This oxidation can form disulfide bonds that can result in the crosslinking of proteins. However the oxidation of L-cysteine (Cys) by HOCl gives cysteic acid and cystine, this reaction takes place in the free amino acid and also when it is a residue in a protein [80]. Thioether in methionine (Met) can also be selectively oxidized by HOCl to produce a sulfoxide group (Figure 20a) [81].

Hypochlorous acid reacts readily with other amino acids which have an amino side chain resulting in an organic chloramine (Figure 20b) [82].

Figure (19): UV-visible spectra of MPO and Compound II .

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39 | P a g e

Figure (20): (a) Oxidation of cysteine by HOCl. (b) Chlorinating of side chain amino groups of the amino acid residues.

When the amino acid is free, the chloramine group rapidly decomposes, but when it is a residue in a protein, protein chloramine will be generated; this protein-chloramine has a longer half-life and a reduced oxidative capacity. Hypochlorous acid itself cannot attack the amide-nitrogen atom of dipeptides but chloramine groups generated by chlorinating amino groups in protein can attack the peptide bond, resulting in a cleavage of the protein [83]. Other residues of amino acids in some proteins including tryptophan (Trp), glycine (Gly) and tyrosine (Tyr) can be also targets for HOCl. [81].

As HOCl more rapidly reacts with a secondary amine than with a primary amine, it can readily react with guanosine monophosphate (GMP) which has both the heterocyclic secondary amino group and the primary amino group. Thymidine monophosphate (TMP) can also be a target for HOCl through its heterocyclic secondary amino group but less than GMP. Adenosine monophosphate (AMP) and cytosine monophosphate (CMP), that have only less reactive amino groups, are less prone to halogenation [84]. Uridine monophosphate (UMP) reaction with HOCl is slow as compared to the rate of reactions with other nucleotides. These reactions likely take

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40 | P a g e place on DNA and RNA, but they react with HOCl quite slowly comparing with free nucleotides [85].

The reactions of lipids (preferentially unsaturated fatty acids and cholesterol) with HOCl are possible. The reaction of HOCl with lipids of low density lipoprotein (LDL) often yields chlorohydrins (Figure 21) or lipid hydroperoxides (Figure 22), but chlorohydrins are the preferred products of the oxidation of lipids by HOCl [86].

Figure (21): Generation of lipid and Cholesterol chlorohydrins.

Figure (22): Generation of lipid hydroperoxides.

HOCl preferably reacts with the primary amino group in phospholipids which have such as these groups (ex. Phosphatidylethanolamine) resulting in chloramines derivatives rather than chlorohydrins as the major products. Cholesterol can be modified by HOCl to yield large number of oxysterols and cholesterol chlorohydrins. Oxidizing lipids by HOCl seems to depend on the conditions, such as pH and the presence of free radical initiators [87].

Due to the deleterious properties of HOCl, MPO contributes in growing number of diseases such as atherosclerosis, rheumatoid arthritis, lung cancer and many more. Immuno-reactivity and typical MPO oxidation products were even detected in brains of patients diagnosed having

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41 | P a g e Alzheimer's disease and in the central nervous system in multiple sclerosis (MS) patients [70]

[88].

Many biomarkers and modified targets of MPO are reported. Tyr is one of these biomarkers. It can be chlorinated by MPO/H2O2/Cl^- system, but the formation of 3-chlorotyrosine (3-ClTyr) is an unfavorable reaction for HOCl and only 2 % of HOCl produced by MPO is used to convert Tyr residues in albumin to 3-ClTyrs. The MPO-mediated oxidation of thiocyanate contributes to carbamylation of protein lysine residues [89]. Nitration of proteins is often observed during pathophysiological situations where oxidative and nitrative stress conditions simultaneously exist. The most commonly detected marker of biological nitration is 3-nitrotyrosine (NO2Tyr).

Under pro-inflammatory conditions a close relationship between MPO and NO metabolism is shown where the levels of nitration are enhanced by the accumulation of MPO. Indeed, NO and NO2−

are converted to the nitrating reactivespecies NO2•

and NO2•-

. Tyrosine is oxidized to the tyrosyl radical by the MPO/H2O2 system and the phagocytes activated in the environment containing nitrite can generate nitrating species via MPO-dependent mechanism. These mechanisms facilitate the introduction of nitrite on the Tyr residues. This introduction of NO2

group to the o-position of Tyr in proteins increases the net negative surface charge and can induce structural and functional alterations in the affected proteins. Other MPO-related biomarkers are dityrosine and 2-chlorohexadecanal. The detection of MPO metabolites at inflammatory sites proves the implication of MPO in tissue damage. As MPO becomes attached to phosphatidylserine (PS) epitopes on the surface of apoptotic neutrophils and other apoptotic cells, this protein may be recognized as antigen when the apoptotic cells are slowly removed by macrophages. Thus antibodies may be formed such as anti-neutrophil cytoplasmic antibodies (ANCAs) [90].

5.5.1. MPO and the inflammatory syndrome

The inflammatory syndrome.

Inflammation is a normal and essential response to any harmful stimulus that threatens the host and may vary from a localized response to a generalized response. The resulting inflammation cascade can be summarized as follows:

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42 | P a g e 1) initial injury causing release of inflammatory mediators (e.g., histamine, serotonin, leucokinins, slow-reacting substance of anaphylaxis (SRS-A), lysosomal enzymes, lymphokinins and prostaglandins);

2) vasodilation;

3) increased vascular permeability and exudation;

4) leukocytes migration, chemotaxis and phagocytosis;

5) proliferation of connective tissue cells.

The most common source of chemical mediators includes neutrophils, basophils, mast cells, platelets, monocytes/macrophages and lymphocytes.

When an unknown antigen gains access to the patient’s tissues, it combines with an antibody and actives the complement sequences. An antigen-complement-antibody immune complex then stimulates the release of chemical mediators causing the migration of numerous polymorphonuclear leukocytes phagocytizing the immune complexes. The triggering conditions or agents are removed via phagocytic cells that are activated by IL-8, macrophage inflammatory protein (MIP), and interferon- (IFN-). The phagocytized organisms are destroyed in the phagocytic vacuole called phagolysosome resulting from the fusion of the vacuole with granules. Lysosomal and hydrolytic enzymes combined with H2O2, NO, HOCl and O2•- anion contribute to the antigen killing. Lysosomal membranes become fragile and release hydrolytic and oxidative enzymes (e.g. proteases, collagenases, MPO, etc.) from the leukocytes. If this process continues, tissues damage would occurr causing tissues disorders, tissues destruction, collagen polymerization, and modification in some biomolecules (e.g. lipids, proteins, etc.) [91].

Inflammation can be also initiated by tissue injury which causes release of pro-inflammatory mediators including the cytokines such as IL-1 and TNF-, as well as complement activated by the alternate pathway. These mediators induce adhesion molecules on leukocytes, ECs, and epithelial cells.

Two phases of inflammation can be identified:

1) the acute phase which is mediated primarily by neutrophils;

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43 | P a g e 2) the chronic phase which is mediated primarily by lymphocytes and macrophages [92].

The role of MPO in inflammation [90].

Neutrophils are the first cells that rapidly infiltrate from peripheral blood to inflammatory sites, followed later by monocytes. Neutrophils contribute to pathogen defense, regulation of the inflammatory process, activation of T cells, antigen presentation but it is also implicated in tissue injury. Thus during the inflammatory response, neutrophils may function as a double- edged sword, as they combine both beneficial functions and harmful activities. After the apoptosis of neutrophils, they are rapidly eliminated by the macrophages to terminate the inflammatory response. Anti-inflammatory mechanisms are then activated to depress pro- inflammatory and anti-apoptotic pathways, and promote tissue repair. Macrophages, upon phagocytosis of apoptotic neutrophils, produce lipid mediators such as lipoxin A4, resolvin E1 and protectin D1 that exhibit numerous inflammation-resolving properties (Figure 23).

Macrophages can be also triggered by necrotic cells. In this condition macrophages produce pro-inflammatory mediators that further promote the inflammatory process. [90].

Figure (23): Fate of neutrophils at inflammatory sites. After their recruitment from peripheral blood, neutrophils accumulate in inflamed tissue; contribute to pathogen defense, and regulation of the inflammatory process.

Unwanted neutrophils become apoptotic. Rapid clearance of apoptotic PMNs by macrophages triggers the later cells to release anti-inflammatory mediators. In the presence of survival factors, neutrophils are prone to undergo necrotic cell death. Upon phagocytosis of necrotic material, macrophages produce pro-inflammatory mediators [90].

DESIGN, SYNTHESIS AND STUDY OF MYELOPEROXIDASE INHIBITORS IN THE SERIES OF 3-ALKYLINDOLE

FACULTÉ DE PHARMACIE

École Doctorale en Sciences Pharmaceutiques