Myeloperoxidase (MPO) is a major oxidant-producing enzyme produced by activated neutrophils. It is released extracellularly after agonist activation, and it can cause tissue damage at sites of inflammation and even in the absence of infection.

MPO-derived oxidants have been implicated in many pathological conditions, including cardiovascular disease, rheumatoid arthritis, atherosclerosis, kidney disease, and cystic fibrosis. These findings have led to an increasing interest in MPO as a target for the development of therapeutics.

Inhibition of MPO

Myeloperoxidase (MPO) is a mammalian heme peroxidase enzyme that produces hypohalous acids, e.g., hypochlorous acid (HOCl), in the presence of halides (X-). Excessive MPO-mediated production of these hypohalous oxidants is associated with many inflammatory diseases including cardiovascular, respiratory and neurological syndromes. Inhibiting MPO-mediated oxidant production could therefore be a therapeutic strategy to prevent or treat these diseases.

A number of inhibitors have been developed to target MPO, a class that includes thioxanthines and indole derivatives. These inhibitors can covalently modify the heme groups of MPO and decrease HOCl formation in vivo. These compounds have shown a wide range of clinical activity, from reducing blood pressure to inhibiting tumor growth in animal models of cancer.

One thioxanthine-based inhibitor, AZM198, has demonstrated clinical effectiveness as a cardioprotective agent in a high fat diet (HFD)-induced obesity mouse model with cardiac phenotypes (200). The thioxanthine derivative reduces MPO-mediated oxidation of HDL, atherosclerotic plaque instability and pulmonary arterial hypertension in this model by blocking the conversion of HOCl to nitrosative products such as NO2Tyr and 4-HNE. Its mechanism of action is complex, but it is thought that AZM198 interferes with the first step of MPO metabolism, Compound I.

Compound I is formed via one-electron reduction of the ferric (Fe2+) form of MPO to give the adenosine triphosphate analog, ADP. The ADP-ADP tetraphosphate intermediate can be further reduced to the adenosine monophosphate (AMP) form. This is the first step in the AMP-dependent catalytic cycle of MPO, or “peroxidase cycle.” The second step involves the reaction of ADP with the O2* radical anion to give the quinoline (Q) species and subsequent addition of the O2* molecule.

Several other indole derivatives have also been found to exhibit potent anti-inflammatory properties. These include indole-3-acetic acid (IAA), indole methyl ketone (IMK) and indole-3-acetaminopropane (IAAAP).

Inhibitors of MPO have been studied in a variety of inflammatory disease models, including MPO FA cardiovascular and respiratory disorders, as well as autoimmune encephalomyelitis (EAE). The inhibition of MPO by these agents has been reported to promote reversal of oxidized proteins and inflammation, restore BBB function, improve neuronal proliferation and differentiation, protect the brains of EAE mice, and reduce neuropathic pain. KYC inhibition of MPO has also been shown to improve the clinical outcomes of patients with stroke by decreasing pro-inflammatory microglia/macrophages and promoting adult neurogenesis in the post-stroke brains (Yu et al., 2018).

Inhibition of HOCl-induced HDL modification

Myeloperoxidase (MPO) is a member of the heme peroxidase family and plays a key role in innate immunity by contributing to cellular damage. MPO is also found within atherosclerotic plaques and contributes to disease progression through oxidative stress, impairing HDL function, inhibiting nitric oxide (NO) production and stimulating leukocyte recruitment into the plaque. In addition to these pathophysiological effects, MPO is also an important inflammatory mediator in atherosclerosis, triggering the production of extracellular matrix (ECM)-derived nitric oxide, inducing endothelial dysfunction (ED), activating TIMP-1 and promoting platelet aggregation and adhesion to the ECM.

MPO is a potent oxidoreductase that produces HOCl by halide oxidation, with tyrosine residues of LDL particles being chlorinated. It is thought that the oxidation of MPO-modified LDL by HOCl is responsible for most of the oxLDL produced in atherosclerotic lesions.

HOCl-modified LDL is a major contributor to the formation of atherosclerotic plaques and the development of cardiovascular disease. It is also the most common oxidized LDL, and is found in both vascular cells and extracellular spaces in atherosclerotic lesions. The oxidation of MPO-modified HDL is important in atherosclerosis because it can bind to the apolipoprotein E (ApoE) gene and induce phosphorylation of ADAMTS1 and other enzymes involved in plaque stability (133).

Inhibition of MPO with compounds that covalently modify heme groups inhibits synthesis of MPO and subsequently reduces HOCl production. Several such compounds have been used clinically to reduce the production of MPO in various inflammatory conditions including arthritis, chronic obstructive pulmonary disease and peptic ulcer.

However, the mechanisms of HOCl-induced HDL modification are still not fully understood. The enzyme MPO can be diverted from HOCl production by supplementation with alternative substrates, such as NO2- or a SCN- anions. This may provide an avenue for the development of non-toxic alternative oxidants.

Alternatively, a thioxanthine compound called AZM198 can inhibit MPO activity and decrease HDL production in vivo. AZM198 is currently being investigated in clinical trials for the treatment of hyperlipidemia.

MPO is secreted by neutrophils and macrophages and contributes to the initiation and progression of atherosclerosis by oxidizing low-density lipoprotein (LDL) and high-density lipoprotein (HDL). It also exacerbates inflammation and inflames the endothelium, thereby contributing to atherosclerotic plaque formation.

Inhibition of HOCl-induced oxidative stress

During chronic inflammatory conditions, excess MPO release can result in the formation of HOCl which is responsible for damage to cells. As such, HOCl-induced oxidative stress has been associated with various pathologies including cardiovascular disease (50, 265) and pulmonary disorders, such as cystic fibrosis and asthma.

MPO produces a broad spectrum of oxidants that are capable of altering protein structure and enzyme activity, including the ability to bind to heme. These include hypochlorous acid, oxalate, thiocyanate, and hydroxylamine. These oxidants can also induce protein aggregation, which may further impair the function of certain proteins.

As such, MPO-mediated oxidative stress is a key contributor to inflammation and has a critical role in the development of disease. Therapeutic modulation of MPO can be beneficial in the treatment of a wide range of inflammatory diseases, with interest being focused on its involvement in cardiovascular and respiratory conditions.

Inhibition of HOCl-induced oxidative stress is an important pharmacological strategy in the treatment of MPO-mediated oxidants. Several MPO inhibitors have been evaluated in this regard. The most promising and effective ones are thiocyanate and ABAH. MPO FA These compounds are capable of limiting MPO activity and have been shown to inhibit the formation of HOCl.

However, they are toxic and may not be suitable for use in patients with compromised immune systems, such as those with cystic fibrosis. Furthermore, their use may only reduce the extent of MPO-induced oxidant production, not its actual toxic effects.

This is why it is important to identify and monitor MPO chlorination activity using probes that are not consumed by the oxidants OCl-, H2O2 or Cl-. This is possible when using a fluorophore-based probe, such as COH or NBD-TSO. The fluorescent products are stable under MPO assay conditions (Supplementary Table S1).

The stoichiometry of the probes used to monitor the MPO chlorinating activity is carefully controlled, and the concentrations of both MPO-derived HOCl and OCl- induced fluorescence product are determined over time. This allows for a highly sensitive monitoring system, without the need of any developing reagent. This is particularly useful in the context of MPO chlorination assays based on the reaction of MPO with an fluorescent resorufin.

Inhibition of HOCl-mediated platelet aggregation

Despite the importance of MPO in human health, there is evidence of its inflammatory properties (see Table 1). The peroxidase has been associated with many inflammatory pathologies, including fibrotic disorders such as hepatocellular carcinoma (HCC) and chronic obstructive pulmonary disease (COPD) (253). In inflammatory conditions, MPO can promote oxidative tissue damage via a range of mechanisms. These include: cytotoxicity and oxidative stress, platelet aggregation and release of reactive oxygen species (ROS), nitrite formation and hypersensitivity responses, and apoptosis. In addition, the peroxidase is a major HOCl producer, releasing Cl-Tyr and causing oxidative DNA damage in cultured cells and tissue. This oxidative DNA damage, mediated by MPO, is thought to play a significant role in the development of numerous inflammatory diseases, as well as accelerating the progression of atherosclerosis in atherosclerosis-prone mice.

The peroxidase catalytic activity of MPO is primarily mediated by Compounds I and II, which have heme-iron ligands that stabilize the Fe3+ heme form. Covalent modification of the heme groups by sulfonium ions destabilizes the Fe3+ form and increases the activity of Compounds I and II in the resting state, in a two-step mechanism that can be blocked by inhibitors of Compounds I and II (253). Thioxanthines are effective HOCl inhibitors and have been shown to inhibit MPO activity in vivo and in vitro (200).

Treatment of human platelets with TG and Iono induces SOCE through the activation of Ca2+ ATPase (Rosado et al., 2004b). Since these agents are both inhibitors of the Ca2+ ATPase, depletion of cellular calcium stores causes SOCE, which is also accompanied by release of hydrogen peroxide (Rosado & Frye, 2006; Rosado et al., 2010). In contrast, activation of TG+Iono by MPO did not cause depletion of cellular calcium stores and, instead, potentiated SOCE induced by ADP by 26+-9% (P0.05; n = 3), indicating that a redox reaction is involved in this process.

Hence, it is plausible that the depletion of Ca2+ intracellular stores that occurs during platelet depletion by TG+Iono is due to a direct interaction between ATPase and the actin cytoskeleton, as the latter regulates both the initiation of Ca2+ entry in the cell and its subsequent release through SOCE (Rosado et all, 2000; 2002). MPO treatment enhances SOCE and therefore inhibits TG+Iono-induced platelet aggregation.

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