Melatonin interferes with COVID-19 at several distinct ROS-related steps

Introduction

In the severe stages of COVID-19 caused by severe acute respiratory syndrome coronavirus 2 (SARS-COV-2) infection, signs of hypoxemia accompanied by a decreased response to oxygen therapy supplementation are present, regardless of the overall preservation of lung mechanics [1,2]. Certain reactions of the immune system during COVID-19 are associated with what has been characterized as a “cytokine storm” in which overwhelming amounts of reactive oxygen species (ROS) including superoxide (O2•-), hydrogen peroxide (H2O2), hydroxyl radical (•OH), and peroxynitrite (ONOO−), as seen in other inflammatory disorders, are produced (Fig. 1). Excessive activation of neutrophils caused by the overactive immune response generates myeloperoxidase (MPO) and contributes to the formation of neutrophil extracellular traps (NETs). NETs are mesh-like DNA fibers that are cast out from neutrophils in response to stimuli including microorganisms, microbial products, and chemokines [3]. The formation of NETs is highly dependent on, and most commonly triggered by, the protein kinase C (PKC) activator phorbol myristate acetate (PMA), a potent neutrophil stimulus that activates O2•- anion producing NADPH-oxidase [4]. PKC activation by PMA triggers the production of intragranular ROS. MPO activity through the generation of HOCl, along with other ROS, works to destroy assaulting pathogens, but may lead to tissue damage when HOCl is produced in excess [[5], [6], [7]] (Fig. 1). HOCl exists in approximate equilibrium with hypochlorite ion (−OCl) at normal body pH 7.4 (pKa 7.59); however only the uncharged molecule can easily penetrate the cell membrane, most likely by passive diffusion through porins, proteins on the cell membrane that form channels [8,9]. MPO can be attached to extracellular NETs, be released at the plasma membrane due to inappropriate trafficking control, bind to the glycocalyx of several cell types and further increase neutrophil recruitment, or otherwise leak from the phagolysosome. All of these events can cause additional catalysis of HOCl [10]. In addition, the production of excessive amounts of ROS can interfere with the catalytic site of many hemoprotein model compounds, ultimately contributing to malfunction and/or destruction of hemoglobin and red blood cells, nitric oxide synthase (NOS), catalase, corrin rings such as in vitamin B12, zinc-Cys clusters, and sulfur‑sulfur bonds, deficiencies of which are characteristic of COVID-19 [8,[11], [12], [13], [14], [15], [16]]. HOCl might also modulate other important pathological mechanisms in COVID-19 including the release of free iron (Fe(II)) from HOCl mediated hemoprotein destruction, which reacts with HOCl directly or through the Fenton reaction generating •OH. Additionally, direct NO consumption by the near diffusion rate reaction of nitric oxide (NO) with O2•- generates ONOO− and leads to vasoconstriction [17,18]. Understanding these mechanisms will help the development of therapeutic strategies to combat SARS-COV-2 and other related inflammatory disorders.



HOCl-associated hemoprotein heme destruction

As an important part of the first immune response, the activation of neutrophils results in the production of ROS; however, when ROS release is excessive due to excess stimulation of neutrophil activity, the result is often physiological damage such as cellular mitochondria poisoning, oxidative phosphorylation uncoupling, and lipid peroxidation. Of commonly appearing ROS, HOCl and H2O2 are long-lived compared to O2•- and •OH, and they can further generate •OH upon reaction with free metal ions, a process that may be enhanced by O2•-. HOCl can also directly react with O2•- to generate •OH [32]. In turn, •OH can react with organic molecules at rates that approach diffusion-limited and contribute to hemoprotein heme modification [33].

MPO catalyzes H2O2-dependent oxidation of Cl− through the formation of a ferryl π cation radical (E − Fe(IV) = O•+π) intermediate, compound I, to form HOCl [5,6]. Alternatively, Compound I can oxidize several organic and inorganic substrates (e.g., NO and nitrite (NO2−) to form nitrosonium cation (NO+) and nitrogen dioxide (NO2), respectively) during which the heme undergoes two sequential one e− reduction steps producing compound II (MPO-Fe(IV) = O) and MPO-Fe(III), respectively [34,35]. Normal MPO levels range from 18 to 39 ng/ml in human plasma, with a significant increase up to 55 ng/ml and 287 ng/ml in inflammatory diseases, making MPO an efficient oxidative stress biomarker. HOCl accumulated by neutrophils have been reported at concentrations up to 25–50 mM/h, but is difficult to measure due to the number of neutrophils present, MPO released, and H2O2 availability [10,36]. Enhanced levels of HOCl are sufficient to substantially damage biomolecules through processes such as thiol oxidation, chloramine formation, aromatic chlorination, and aldehyde generation [37]. Furthermore, HOCl can alter the biological function of hemoproteins through oxidation or destruction of heme (iron-protoporphyrin IX), an essential co-factor involved in multiple biological processes such as oxygen transport and storage, electron transfer, drug and steroid metabolism, signal transduction, and microRNA processing [1,11,12,15,38]. The destructive actions of accumulated HOCl, or other ROS, on hemoprotein heme moieties and other biomolecules, could explain some features of COVID-19 and other similar respiratory disorders such as acute lung injury (ALI)/ acute respiratory distress syndrome (ARDS) [1,[39], [40], [41]]. These features include substantial hypoxemia not sufficiently explained by alveolar-parenchymal pathology, zinc deficiency, vitamin B12 deficiency, microvascular injury, thromboembolism, pulmonary hypertension, and damaged hemoglobin and red blood cell function accompanied by relative unresponsiveness to O2 supplementation. In the absence of an HOCl scavenger, there is a complex and interdependent relationship between levels of self-generated HOCl and MPO catalytic activity during steady-state catalysis. Enhanced self-generated HOCl in the milieu regulates MPO catalytic activity by heme degradation and subsequent free iron release, a process that is attenuated by HOCl scavengers (e.g. methionine) [38]. These studies clearly showed that self-generated HOCl may serve as a ligand for MPO, leading to catalytic inhibition and formation of an MPO–Fe(III)–OCl complex, which subsequently sets the stage for MPO heme destruction and free iron release. The direct reaction between HOCl with MPO–Fe(III) is fast and occurs with a second-order rate constant of 2 × 108 M−1 s−1 (pH 7) [42]. Key targets for HOCl-induced protein damage are various protein side chains, particularly those that are sulfur-containing. Rate constants reported by Davies and Pattison for the reaction of HOCl and different amino acids range from 26 M−1 s−1 to 3.6 × 108 M−1 s−1, a rate comparable to that of the direct reaction of HOCl with MPO [10,43,44]. Regardless of the variation in the rate constants among different amino acids, the final products for these reactions have been detected even for the lowest rate reaction, i.e. halogenated tyrosine has been proposed as a biomarker for detection of HOCl-induced protein damage [45]. Furthermore, an initial rate of 8 × 103 M−1 s−1 was found for the binding of HOCl to hemoglobin, further supporting the possibility of HOCl-mediated hemoglobin damage in a biological setting [12,43]. Similarly, an initial rate constant of 2 × 103 M−1 s−1 was found for the binding of HOCl to a vitamin B12 derivative [8,14]. In a previous study, 200 μM HOCl was found to react with and destroy the heme of hemoglobin in red blood cells [12]. This is consistent with recent work by Elahi et al., in which the harmful effect of COVID-19 on red blood cells is demonstrated [46]. As COVID-19 is known to be a disease of vascular endothelial inflammation, recruited neutrophils will likely be in relative proximity to passing erythrocytes [47]. HOCl and its adducts have been demonstrated to induce lysis of erythrocytes, exposing the erythrocytes' cytoplasmic hemoglobin to further oxidative damage [[48], [49], [50]]. Monochloramines derived from HOCl can also directly oxidize hemoglobin within erythrocytes without hemolysis [51]. Therefore, high concentrations of HOCl can mediate heme destruction of several hemoproteins and corrin ring compounds including hemoglobin and other related hemoprotein model compounds such as lactoperoxidase, myeloperoxidase, catalase, eosinophil peroxidase, and cobalamin derivatives, and more importantly can be prevented in the presence of 1:1 or 1:2 melatonin concentration [11,15,[52], [53], [54], [55]]. Given the toxicity of overwhelming production of HOCl and downstream products such as free iron, HOCl is physiologically regulated. MPO protection against self-inactivation is a necessary process that prevents HOCl mediated MPO heme destruction, therefore allowing the enzyme to function at full capacity in generating HOCl [53]. One potential pathway of preventing MPO auto-inactivation is the rapid consumption of HOCl through rapid reaction with human serum albumin (HSA) and other proteins found in blood and other bodily fluids [56]. However, during COVID-19-associated inflammation, HSA and other HOCl scavengers suffer from decreased effectiveness due to competitive binding of SARS-COV-2, and HOCl production already exacerbated by inflammatory mediators is further unchecked [57,58]. However, HOCl consumption by reaction with these substances does not account for the complete loss of HOCl-dependent signaling and viral killing, suggesting that alternative pathways exist for HOCl depletion. In vitro studies demonstrate that HOCl can react with a variety of hemoprotein model compounds at various oxidation states (e.g., ferric, ferrous, ferrous-deoxy, and ferryl) [11,12,59]. Reactions of HOCl with hemoprotein intermediates that catalyze electron transfer reactions are another potential pathway for HOCl consumption.


















HOCl mediates corrin ring (vitamin B12) destruction

The association between COVID-19 and Vitamin B12 deficiency leading to worse outcomes of respiratory viral infections has been previously established [68]. Furthermore, COVID-19 and vitamin B12 deficiency have shared symptoms, such as increased oxidative stress, increased dehydrogenase, hyperhomocysteinemia, hypercoagulation, and vasoconstriction, particularly of the renal and pulmonary systems [69]. Consistent with this, recent investigation has shown that methylcobalamin supplements not only have the potential to reduce COVID-19-related organ damage but also reduce other symptoms [68,69]. A clinical study has also shown reduced COVID-19 symptom severity in patients who received vitamin B12 (500 μg), vitamin D (1000 IU), and magnesium supplements, displaying a significant reduction in the need for O2 and intensive care support [69,70]. Using a variety of biochemical, physiological, and kinetic techniques, it has been shown that melatonin ceases HOCl-mediated vitamin B12 derivative corrin destruction [8,14]. Vitamin B12 deficiency has been associated with several conditions related to memory loss, immune system disorders, aging, heart disease, male infertility, diabetes, sleep disorders, depression, mental disorders, and inflammation [71]. Patients with one or more of these conditions might be more susceptible to severe SARS-CoV-2 infection [72]. In addition to ROS generated by the cytokine storm, SARS-CoV-2 infection may also interfere with vitamin B12 metabolism, therefore contributing to the pathogenesis of respiratory, gastrointestinal, and central nervous systems infections [69].

The two derivatives most commonly used for the treatment of vitamin B12 deficiency are hydroxocobalamin and cyanocobalamin, with cyanocobalamin being preferred in the United States. However, outside of the U.S., such as in the United Kingdom, hydroxocobalamin is preferred due to its ability to firmly bind to plasma proteins allowing it to remain in the body longer [73]. Although the amount of scientific evidence is limited, the safety of cyanocobalamin supplementation for COVID-19 patients, versus other vitamin B12 derivatives, may be a concern. The biosynthesis of the B12 coenzyme form cyanocobalamin results in the release of CN−, which could lead to acute cyanide poisoning, causing unwanted inflammation. Furthermore, when HOCl levels are high, HOCl-mediated reactions may destroy the corrin ring of vitamin B12, releasing cyanogen chloride (CNC), free active cobalt (Co), and other corrin degradation products. Indeed, our previously published results suggest that the degradation of cyanocobalamin mediated by HOCl is largely modulated by the concentration of HOCl in the reaction milieu. Thus, any dysregulation of neutrophil/macrophage-derived HOCl contributes to inflammation and, similarly, reduction in HOCl consumption could manifest as increased cyanocobalamin destruction and CNCl generation [14]. Therefore, it is of enormous therapeutic and pharmacologic importance to prevent HOCl-mediated damage, especially in chronic inflammation where a higher rate of infiltration of monocytes/macrophages over a longer period leads to pathologic alterations.

4. Mechanism of HOCl mediated tetrapyrrole ring destruction (Heme in hemoprotein and corrin ring in vitamin B12) In hemoprotein, Fe is attached to the nitrogen of four pyrrole rings and a fifth proximal attachment to either the nitrogen of histidine such as in hemoglobin, catalase and mammalian peroxidases or the sulfur of cysteine, as seen in NOS and cytochrome P450. The sixth axial site of Fe can accommodate a variety of diatomic ligands such as O2, NO, CO, CN− and −OCl. The excessive production of ROS in the cytokine storm can result in modification of the heme prosthetic group inhibiting the protein function, whereas, in vitamin B12 derivatives, the structure is based on a corrin ring containing four pyrrole rings attached to a center Co atom, distinguished by two directly attached pyrrole rings. The six coordination sites of Co in these compounds are the four pyrrole nitrogen atoms of the corrin ring, the nitrogen of the 5,6-dimethylbenzimidazole group at the lower (or α-) axial ligand, and naturally occurs with either a cyano-, hydroxy-, aqua-, methyl-, or adenosyl- group at the upper/β-axial ligand site [74]. Here, the overproduction of ROS and HOCl from the cytokine storm nonenzymatically mediates corrin ring destruction and the generation of free Co [14]. Pyrrole ring destruction by HOCl can occur as a result of a direct attack on any of the carbon-methylene bridges between the rings, forming chlorinated adducts [12,14]. These chlorinated intermediates are unstable, quickly releasing Cl− and decaying to an epoxide or aminal. The epoxide implements the buildup of a hydroxylated compound, with the •OH group being connected to the carbon-methylene bridge of the porphyrin ring where the initial attack by HOCl takes place [12,14]. Attack by a second hydroxyl functional group creates vicinal diol, which can be split by either hemolytic cleavage or a transition metal-mediated process (Fe, Co) by the formation of dioxetane intermediate through a heterolytic 2e- process; both processes result in a pair of carbonyl compounds. The aldehydes that are produced may be further oxidized by HOCl to create carboxylic acid through a mechanism previously described [75]. Cleavage of the CC bond can take place at both the carbon-methylene bridge and the terminal CC bond, leading to the generation of formaldehyde [12]. This single carbon aldehyde can be oxidized to formic acid by the electrophilic addition of HOCl. As recently reported, HOCl can alter the tetrapyrrole ring through a mechanism that involves disrupting the axial coordination of the Fe (hemoproteins) or Co (vitamin B12 derivatives) atom, causing ring destruction [8,14]. These alterations of the tetrapyrrole ring geometry might therefore make the ring a more eligible target to HOCl-attack and ring breakage, which is associated with significant Fe/Co-release.


Melatonin is a potent inhibitor of MPO

Studies have shown the severity of the disease is correlated with the level of inflammatory immune response caused by the pro-inflammatory cytokines released in the cytokine storm, with an exceptionally heightened response in those with more severe cases. Neutrophil activity, MPO activity, and ROS generation play an important role in the inflammatory immune response that contributes to the overwhelming production of HOCl; thus, MPO inhibition and elimination of unwanted ROS are undoubtedly important treatment targets in patients with COVID-19. It has been shown that triggering NET formation is dependent upon ROS formation and processing by MPO; therefore, inhibiting MPO could block the formation of NETs [4,83]. Melatonin is not only a reversible inhibitor and important regulator of MPO activity but also plays an important role in detoxifying ROS. These phenomena position the indole as a powerful supplement to fight against early stages and severe SARS-CoV-2 infection. Melatonin-engaging therapies could reduce the stage of virus-associated pathology by controlling the host immune response to viral infection, mainly produced by alveolar macrophages and neutrophil MPO activity and unwanted ROS overproduction. Melatonin inhibits MPO chlorinating activity in a dual mechanism, which includes allosteric binding to the entrance of the MPO heme pocket and accelerating the formation and the decay of MPO compound II, which inhibits the chlorinating activity and slows down the peroxidation activity of the enzyme [23,24]. This allosteric binding to the MPO heme pocket entrance is enhanced by Cl− binding to the halide-binding site, where HOCl is generated and blocks H2O2 from the catalytic site. Thus, melatonin competes with H2O2 and switches the reaction from free to a melatonin-bound enzyme (active to inactive form), and melatonin also competes with the co-substrate, Cl−, and switches the reaction from a 2e− to a 1e− oxidation mechanism [23]. Studies have shown that rapid mixing of MPO preincubated with melatonin and Cl− against the same volume of H2O2 solution caused an immediate buildup of a transient intermediate, compound II, which then decays to MPO − Fe(III) through oxidation of another melatonin molecule, thereby closing the peroxidation cycle [23]. The ability of melatonin to compete with H2O2 and Cl− on the active sites of MPO − Fe(III) and MPO compound I, respectively, is a key feature that drives the enzyme to alter its function to peroxidase activity. This mechanism of competitive inhibition has been shown to occur with tryptophan and several phenolic and aromatic amines [84].


Conclusion

With the discovery of multiple COVID-19 variants spreading around the world, the question remaining is can the current vaccines keep up, or will they become less effective against SARS-CoV-2 mutations? There is a possibility that the cost and time needed to develop a vaccine targeted at new strains may not be met before another spike occurs. In this case, recognition of treatment options is of utmost importance. Melatonin is particularly notable, as its supplementation is safe for use with other treatments, as it has been previously given to COVID-19 patients in combination with the US Food and Drug Administration emergency authorized cocktail, REGEN-COV2 [20,21]. Many of the known clinical signs of COVID-19 could potentially be associated with defects in the function of several key tetrapyrrole and heme proteins, notably hemoglobin, cyanocobalamin, and NO synthases. Here, we have outlined the destructive effects of ROS, especially HOCl, on these biomolecules. As one of the major sources of HOCl is through overactivity of MPO provoked by the cytokine storm of COVID-19, inhibition of MPO and/or elimination of HOCl may play a beneficial role in diverse biological processes by reducing the metal release mediated by HOCl and associated increases in other ROS. Related studies from our lab have shown that MPO and its effects can be inhibited at three points: 1) through heme reduction that causes collapse or narrowing in heme pocket geometry that prevents the access of the substrate to the catalytic site of the enzyme (e.g. ascorbate) [108]; 2) switching the MPO catalytic cycle from peroxidation to catalase-like activity (e.g. melatonin, tryptophan, tryptophan analogs) [23,84,109]; or 3) direct scavenging of HOCl (e.g. lycopene) [75]. The ability of melatonin to act as an antioxidant, anti-inflammatory, and an immunoregulatory agent allows the unique opportunity for its use in a variety of therapeutic approaches, and it has shown promising benefits in cancer, diabetes, infertility, and inflammatory diseases. The outstanding benefits of melatonin provide an accessible, relatively inexpensive, and safe treatment that may ease COVID-19 symptoms commonly seen with the cytokine storm and overactive immune response, leading to improved patient outcomes and reduced suffering. In addition, to use alone, promising evidence is emerging to establish melatonin as a treatment that could potentially be used in combination with current antiviral agents. Further clinical and experimental studies are required to confirm the applied benefits of melatonin supplementation as a therapeutic agent for the treatment of patients infected with SARS-COV2. Olivia G. Camp

(Departments of Obstetrics and Gynecology, The C.S. Mott Center for Human Growth and Development, Wayne State University School of Medicine, Detroit, MI 48201, USA)

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