key: cord-0860710-gm0vg53l
authors: Doboszewska, Urszula; Wlaź, Piotr; Nowak, Gabriel; Młyniec, Katarzyna
title: Targeting zinc metalloenzymes in COVID‐19
date: 2020-07-15
journal: Br J Pharmacol
DOI: 10.1111/bph.15199
sha: 34f91f9526a217443f808b6c640f0ec2cb25cf63
doc_id: 860710
cord_uid: gm0vg53l

Several lines of evidence support a link between the essential element zinc and the coronavirus disease 2019 (COVID‐19). An important fact is that zinc is present in proteins of humans and of viruses. Some zinc sites in viral enzymes may serve as drug targets and may liberate zinc ions, thus leading to changes in intracellular concentration of zinc ions, while increased intracellular zinc may induce biological effects in both the host and the virus. Drugs such as chloroquine may contribute to increased intracellular zinc. Moreover, clinical trials on the use of zinc alone or in addition to other drugs in the prophylaxis/treatment of COVID‐19 are ongoing. Thereby, we aim to discuss the rationale for targeting zinc metalloenzymes with regard to COVID‐19.

The coronavirus disease 2019 has emerged in December 2019 in the city of Wuhan, China and has spread worldwide since then. It is caused by severe acute respiratory syndrome-related coronavirus 2 (SARS-CoV-2) (Zhou et al., 2020) . Until now no drugs designed against specific SARS-CoV-2 proteins have been developed. Novel drugs are urgently needed in view of the fact that, for example, the treatment with drugs which are being repurposed for COVID-19, such as chloroquine/hydroxychloroquine, is not safe in patients with cardiovascular comorbidities, constituting a large group of patients dying because of the disease (Kalil, 2020) .

Ananda Prasad recognized the nutritional essentiality of zinc in humans and consequences of zinc deficiency in 1963 in Iran (Prasad, 2012) . Later, they observed recurrent opportunistic infections in patients with acrodermatitis enteropathica (AE), in whom zinc deficiency is due to malabsorption of zinc caused by a mutation in ZIP4, an intestinal zinc transporter. Immune system dysfunction in AE patients has been corrected with zinc supplementation, thus showing that zinc is essential for the function of the immune system (Shankar & Prasad, 1998) . Potential benefits of zinc administration in COVID-19 in terms of improved immunity, which may be foreseen in populations at risk for and zinc deficiency, such as the elderly, have recently been discussed by Derwand and Scholz (2020) , Rahman and Idid (2020) and Skalny et al. (2020) .

Due to anti-inflammatory properties zinc has been suggested to limit the cytokine storm (Skalny et al., 2020) , which might occur in patients with severe COVID-19 (Mehta et al., 2020) . A cytokine storm, also termed macrophage activation syndrome or secondary haemophagocytic lymphohistocytosis, is a potentially fatal systemic hyperinflammation associated with hypercytokinaemia and multiple organ failure (McGonagle, Sharif, O'Regan, & Bridgewood, 2020; Sun et al., 2020) . Noteworthy, a combination of zinc, hydroxychloroquine and azithromycin has been proposed as an early treatment of in the outpatient setting, that would prevent disease progression and hospitalization (Derwand & Scholz, 2020; Risch, 2020) .

In the human body zinc is the second most abundant metal. It plays catalytic, structural and signaling function. The biochemistry of zinc began in 1939 with the observation that the enzyme carbonic anhydrase contains zinc. Moreover, zinc was shown to be indispensable for its enzymatic activity (Lindskog, 1997; Maret, 2013) . Since then, this element has been found in hundreds of other enzymes, which are called zinc metalloenzymes (Haraguchi, 2017; Maret, 2013) . Angiotensin-converting enzyme (ACE) and angiotensinconverting enzyme 2 (ACE2) belong to zinc metalloenzymes (Turner, Hiscox, & Hooper, 2004) . Furthermore, zinc fingers, relatively small protein domains consisting of cysteines (Cys) or Cys and histidines (His) bound to zinc ions, were discovered. It is predicted that 10% of the human genome encodes zinc fingers (Krishna, Majumdar, & Grishin, 2003; Maret, 2013) .

Zinc plays an important role not only in proteins and enzymes of humans and other forms of life, but also in viruses. For example, RNA-dependent DNA polymerase from avian myeloblastosis virus has been demonstrated to be a zinc-dependent enzyme in 1974 (Poiesz, Seal, & Loeb, 1974) . Zinc fingers are present in many viral proteins (Lei, Kusov, & Hilgenfeld, 2018; Ma et al., 2015; Ma-Lauer et al., 2016; Tijms, van Dinten, Gorbalenya, & Snijder, 2001) . Versatile functions of zinc fingers are being increasingly uncovered (Fu & Blackshear, 2017; Jen & Wang, 2016; Laity, Lee, & Wright, 2001) . As structural motifs they are gaining attention as drug targets -the disruption of zinc fingers in viral proteins, which causes destabilization of proteins, has been proposed as a therapeutic approach to treat viral diseases (Abbehausen, 2019; Garcia & Damonte, 2007) .

Our aim is to discuss the possibility of targeting zinc as a therapeutic strategy for COVID-19. We start with background information on potential drug targets for SARS-CoV-2 and classes of compounds which can be collectively termed as drugs targeting zinc. We attempt to integrate data on the effects of agents targeting zinc fingers in viral metalloenzymes (zinc fingers targeting agents), which cause removal of zinc from the proteins, thus destabilizing the proteins and leading to increased intracellular concentration of zinc ions, and other agents which induce changes in intracellular levels of zinc (zinc ionophores), with data on consequences of altered level of intracellular zinc, with a focus on SARS-CoV-2 and related pathogens. Furthermore, we provide examples of compounds targeting zinc which have entered clinical trials in order to demonstrate that investigating zinc drugs may lead to success in the clinic. Finally, we summarize current clinical trials on the use of zinc in the treatment or prophylaxis of COVID-19.

The human pathogen responsible for the outbreak of COVID-19 has a positive-sense RNA genome and has been placed within the Coronaviridae family (Coronaviridae Study

Group of the International Committee on Taxonomy of Viruses, 2020). Because of the relatedness to severe acute respiratory syndrome-related coronavirus (SARS-CoV) (Zhou et al., 2020) , which causes severe acute respiratory syndrome (SARS), the virus responsible for the 2019 outbreak has been designated as SARS-CoV-2 (Coronaviridae Study Group of the International Committee on Taxonomy of Viruses, 2020). SARS-CoV-2, SARS-CoV and Middle East respiratory syndrome-related coronavirus (MERS-CoV), causing Middle East respiratory syndrome (MERS), are three coronaviruses behind major epidemics in the last two decades.

SARS-CoV (Turner, Hiscox, & Hooper, 2004) and SARS-CoV-2 (Shang et al., 2020) use the host zinc metalloenzyme, ACE2, as an entry point to cells. Inside cells, RNA of coronaviruses is translated into two large polyproteins: pp1a and pp1ab. These polyproteins are cleaved by the main protease (M pro , or 3-chymotrypsin-like protease, 3CL pro ) and the papain-like protease (PL pro ) into the nonstructural proteins (nsps . Furthermore, other drug targets such as PL pro or

RdRp are conserved between SARS-CoV-2 and SARS-CoV (Sargsyan et al., 2020; Wu et al., 2020) . Thus, although no specific drugs have been discovered during SARS or MERS epidemics (de Wit, van Doremalen, Falzarano, & Munster, 2016) , the outcomes of studies on drug leads for SARS-CoV may give some insights into the possible treatments for SARS-CoV-2.

For that reason, data on the relationship between zinc and M pro , PL pro or RdRp of SARS-CoV-2, SARS-CoV or MERS-CoV will be discussed in detail in subsequent sections of this review, as they are potentially important for drug discovery.

Ionophores are molecules forming complexes with ions and facilitating ion transport across lipid bilayers. There are ionophores promoting transport of cations (cationophores) and

anion-selective ionophores (anionophores), but the latter are less common (Alfonso & Quesada, 2013) . Cationic ionophores may transfer proton, alkali, alkaline earth, or transition metal ions and may display selectivity for some of them (Alfonso & Quesada, 2013; Freedman, 2011; Riddell, 2002) . Because of the similarities between the elements, it is unlikely that an ionophore will bind one ion at the exclusion of others (Helsel & Franz, 2015) . However, ionophores may be selective in terms of, e.g., kinetics, as they may transport one ion faster than the others (Helsel & Franz, 2015; Riddell, 2002) .

Because the plasma membrane is non permeable for ions, ionophores comprise a lipophilic exterior which facilitates the transport across the membrane and a hydrophilic interior, where an ion is bound (Kaushik, Yakisich, Kumar, Azad, & Iyer, 2018) . When the pH of the extracellular space is higher than the pK a of the ionophore, the compound binds a metal ion. A complex is formed which diffuses across the plasma membrane. When the pH in the intracellular space is lower than the ionophore's pK a , the compound releases the ion. As a result, the intracellular concentration of the ion rises. Thus, some compounds may release ions in the cytosol. Because there are differences in pH between organelles, some compounds may release ions, e.g., in acidic organelles, such as lysosomes (Riddell, 2002) .

With a view of potential clinical administration, compounds with low or moderate metal affinity shall be tested as ionopohores. Such compounds shall bind metals in regions of high concentration, and transport metals to regions where the concentration is lower, thus restoring equilibrium (Bush, 2008; Ding & Lind, 2009 ).

In contrast, the use of chelators may be associated with removal of metal ions from regions where they are essential, which may lead to unwanted effects. Chelators also bind ions, forming complexes, but the functional effect is opposite to ionophores. Traditionally, chelating agents have been used to remove toxic substances (Helsel & Franz, 2015) . Such chelates should be water soluble, thus easily excreted in the urine. For some compounds (e.g., diethyldithiocarbamate, DEDTC, a metabolite of disulfiram, a drug which has been long registered in alcohol use disorder (Kranzler & Soyka, 2018) ) both actions, i.e., an ionophore and a chelator activity (Jones et al., 1980) , have been reported.

The examples of zinc ionophores are given in Table 1 . In these studies, cell cultures were used. The cells were exposed to metals and the tested compounds. Cell membrane permeable fluorescent probes, thus indicating intracellular zinc ions, such as ptoluenesulfonamido-quinoline (TSQ), mag-fura-2 or FluoZin-3 were used (Andersson, Gentry, Moss, & Bevan, 2009; Kim et al., 2000; Reeder et al., 2011; Wiggins et al., 2015; Xue et al., 2014) . In some of the studies, these probes were combined with probes staining lysosomes, such as LysoTracker or

Dextran-Alexa 647 (Wiggins et al., 2015; Xue et al., 2014) , or inductively coupled plasma mass spectrometry (ICPMS) was used to monitor changes in intracellular concentration of zinc ions White et al., 2006) .

Furthermore, it was demonstrated that Cys4 zinc fingers or Cys3His zinc fingers in which zinc-bound Cys has no hydrogen bonds are reactive and may liberate zinc ions, which causes protein unfolding and increases intracellular zinc. A search algorithm based on physical principles has been employed in order to search for such zinc fingers, which have been termed "labile zinc fingers" (Lee, Wang, Duh, Yuan, & Lim, 2013 ). The following terms: "zinc finger targeting agents" and "zinc ejecting agents" or "zinc ejectors" can be found in the literature (Lee, Wang, Duh, Yuan, & Lim, 2013; Supuran, Innocenti, Mastrolorenzo, & Scozzafava, 2004) . For example, disulfiram has been demonstrated to act as zinc ionophore (Wiggins et al., 2015) and as an agent ejecting zinc from zinc fingers Sargsyan et al., 2020) . In order to examine the latter feature, the purified recombinant proteins predicted to contain labile zinc fingers were mixed with disulfiram in the presence of FluoZin-3 probe and the increase in fluorescence was monitored (Sargsyan et al., 2020) .

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Several lines of evidence suggest a link between zinc and COVID-19, including the observation that chloroquine, a drug being repurposed for COVID-19 (Gautret et al., 2020) , is a known zinc ionophore . Studies on zinc ionophores and zinc finger targeting agents as well as zinc in relation to SARS-CoV-2, SARS-CoV, or MERS-CoV will be therefore discussed in detail.

The dramatic outbreak of COVID-19 worldwide prompted to search for possible treatment options within already available drugs (Harrison, 2020) . Chloroquine, an old antimalarial drug (Blount, 1967) , was demonstrated to block virus infection at low micromolar concentration in Vero E6 cells infected with SARS-CoV-2 , thus suggesting the possible use of chloroquine in patients with COVID-19. Moreover, its derivative, hydroxychloroquine, was found to inhibit SARS-CoV-2 infection in vitro . Furthermore, hydroxychloroquine was shown to be more potent than chloroquine at inhibiting SARS-CoV-2 (Yao et al., 2020) .

Mode of chloroquine action has been extensively reviewed (Slater, 1993) . In addition, several mechanisms have been recently proposed with regard to the use of chloroquine for COVID-19 (Devaux, Rolain, Colson, & Raoult, 2020; Shittu & Afolami, 2020; Skalny et al., 2020) , including its activity as zinc ionophore . It was found that administration of zinc and chloroquine to the human ovarian carcinoma cell line, A2780, indicating zinc ions, co-localized with signals of LysoTracker, a cell membrane-permeable probe selective for acidic organelles. These observations suggest that chloroquine is zinc ionophore, which transports zinc to lysosomes ).

An important finding coming from this study is that treatment of cells with zinc chloride alone or with chloroquine alone produced less pronounced increase in intracellular zinc ions, compared to the effects induced by administration of zinc chloride and chloroquine , which suggest that combined treatment with ionophore and zinc is necessary in order to substantially increase the level of zinc inside a cell.

Similar issues to the above-mentioned, which have been examined in relation to chloroquine , have been addressed with regard to disulfiram (Wiggins et al., 2015) . It was shown with the aid of Fluozin-3 probe that disulfiram increases intracellular zinc in MCF-7 and BT474 breast cancer cells. The increase in Fluozin-3 fluorescence in cells treated with disulfiram depended on extracellular zinc, thus supporting the hypothesis that disulfiram acts as zinc ionophore. Moreover, the fluorescence of Fluozin-3 was not observed following disulfiram treatment under low-zinc and copper conditions. Under such conditions, zinc, but not copper, was able to restore the fluorescence, demonstrating that the increase in fluorescence after administration of disulfiram was due to selective interaction with zinc.

Finally, it was shown that disulfiram sequesters intracellular zinc in lysosomes (Wiggins et al., 2015) .

Disulfiram produced a dose-dependent inhibitory effect of both SARS-CoV and MERS-CoV PL pro with IC50 in the micromolar range, as it was measured by the deubiquitination assay , since PL pro has deubiquitinating activity in vitro (Barretto et al., 2005) . Disulfiram was found to be a noncompetitive and competitive (or . It was also demonstrated that mutation of the zinccoordinating Cys caused a significant loss of enzymatic activity of SARS-CoV PL pro . This observation demonstrates that the zinc-binding ability is essential for SARS-CoV PL pro enzymatic function (Barretto et al., 2005) .

Recently, Sargsyan et al. (2020) found labile zinc fingers, thus likely to be targeted and disrupted, in three SARS-CoV-2 proteins, i.e., PL pro , nsp10 and nsp13. In this study, the protease activity was determined using a fluorogenic substrate Dabcy-FTLKGGAPTKVTE-Edans-NH2. Disulfiram and organoselenium compound, ebselen, inhibited SARS-CoV-2 PL pro with an IC50 in the micromolar range. Moreover, incubation of SARS-CoV-2 PL pro with ebselen and disulfiram was associated with increased concentration of zinc ions measured with the aid of Fluozin-3 probe (Sargsyan et al., 2020) .

Furthermore, disulfiram and ebselen are among inhibitors of another crucial SARS-CoV-2 enzyme, i.e., M pro , with an IC50 in the micromolar range . The effects of disulfiram on SARS-CoV-2, SARS-CoV and MERS-CoV enzymes are summarized in Table 2 . In addition, disulfiram and ebselen decreased the number of SARS-CoV-2 viral RNA copies (as it was determined by qRT-PCR analysis) in SARS-CoV-2 infected Vero E6 cells . 

Based on the above-mentioned studies, a chain of events can be hypothesized, which may happen after administration of zinc ionophore (and zinc) and/or a zinc finger targeting drug which causes ejection of zinc from zinc fingers in viral metalloenzymes.

Zinc ionophore and/or zinc finger targeting agent may enter a cell, as does SARS-CoV-2. An agent targeting zinc fingers may bind labile zinc fingers in essential viral enzymes such as PL pro . It may cause ejection of zinc from the enzyme, which will destabilize the enzyme, thus increasing intracellular concentration of zinc ions, as it has been demonstrated for SARS-CoV and MERS-CoV by Lin et al. (2018) and for SARS-CoV-2 by Sargsyan et al.

(2020). Moreover, zinc ionophores such as chloroquine may contribute to increased intracellular concentration of zinc ions . Additionally, compounds such as ebselen may contribute to increased intracellular zinc by releasing zinc from metallothioneins (Jacob, Maret, & Vallee, 1998) , a family of cysteine-rich, low molecular weight, metal binding proteins (Thirumoorthy et al., 2011) . Furthermore, zinc ions may inhibit RdRp, as it has been shown for SARS-CoV by te Velthuis et al. (2010) (Figure 1 ).

In addition to presumed inhibition of RdRp, intracellular zinc may initiate a cascade of events in the host. Intracellular zinc acts as a second messenger and modulates a variety of signaling pathways. All immune cells are affected by intracellular zinc signaling, as it has been comprehensively reviewed by Maywald et al. (2017) . Thus, treatment strategies based on targeting zinc in viral enzymes, leading to increased intracellular zinc, or other approaches increasing intracellular zinc, will have potential consequences for many functions of the immune system (Read, Obeid, Ahlenstiel, & Ahlenstiel, 2019; Skalny et al., 2020) .

based on their ability to chelate zinc in matrix metalloproteinases (MMPs) (Sodhi & Etminan, 2020) . MMPs are another group of zinc metalloenzymes. They are endopeptidases, which are involved in the degradation of proteins in the extracellular matrix (ECM) (Cui, Hu, & Khalil, 2017) . However, recent evidence demonstrates that MMPs are multitasking proteins working both in the extracellular and intracellular compartments. Most of MMPs substrates are non-ECM proteins and include chemokines, cytokines, cell surface receptors and proteins involved in immune signaling (Chopra, Overall, & Dufour, 2019) .

It has been demonstrated in vitro that the neurotropic strain JHM.SD of the murine coronavirus -mouse hepatitis virus uses an unidentified batimastat-sensitive metalloprotease for both viral entry and virus-mediated cell-cell fusion. Batimastat is a potent, broad spectrum MMPs inhibitor. Thus, this study suggests the importance of MMPs for JHM.SD infection (Phillips, Gallagher, & Weiss, 2017) . Moreover, coronavirus HCoV-229E infection of primary monocytes was associated with increased production of MMP-9 (Desforges, Miletti, Gagnon, & Talbot, 2007) .

Tetracyclines are well known MMPs inhibitors (Boelen et al., 2019; Castro, Kandasamy, Youssef, & Schulz, 2011) , but the direct relationship between MMPs and SARS-CoV-2 has yet to be examined.

Targeting metal homeostasis with the aid of chelators or ionophores have been suggested as a therapeutic strategy in a variety of diseases, e.g., cancer (Ding & Lind, 2009; Vaden et al., 2019) , diseases of the central nervous system (Doboszewska et al., 2017; Doboszewska et al., 2019; Weekley & He, 2017) and infectious diseases, such as malaria (Bharti, Singal, Raza, Ghosh, & Nag, 2019 ). An important fact is that there are ongoing clinical trials, in which metal binding compounds are being tested because of their influence on metal homeostasis. For example, activation of procaspase-3 by procaspaseactivating compound 1 (PAC-1) was shown to be dependent on the chelation of zinc (Sarkar et al., 2016) . There are several ongoing clinical trials on the use of PAC-1 in cancer patients (ClinicalTrials.gov Identifiers: NCT03927248, NCT03332355, NCT02355535).

In relation to cancer, there are ongoing clinical trials on the use of disulfiram (NCT04265274, NCT03950830, NCT03714555, NCT03363659, NCT03323346, NCT03151772, NCT02715609, NCT02671890).

Clioquinol was a registered drug worldwide until its use was associated with the occurence of subacute myelo-optic neuropathy, a condition primarily endemic to Japan.

Today, in view of new information that may explain this phenomenon, clioquinol serves as a drug lead to treat cancer (Perez, Sklar, & Chigaev, 2019) .

PBT2 is a next generation derivative of clioquinol, which is characterized by higher solubility and increased blood-brain barrier permeability. These features, together with its activity as a zinc-copper ionophore, make it a possible disease-modifying drug for Alzheimer's disease . According to the metal hypothesis of Alzheimer's disease, in the brain there is a failure in endogenous regulatory mechanisms, which leads to an unbalance of two metals: zinc and copper, resulting in their toxic excess in some compartments and deficit in others (Sensi, Granzotto, Siotto, & Squitti, 2018) . Moreover, deposition of amyloid-β has long been regarded as a leading hypothesis on Alzheimer's disease pathology (Hardy & Higgins, 1992) . The proposed mechanism of action of PBT2 is related to its ability to react with zinc and copper ions in oligomerized and precipitated form of amyloid-β, thus promoting the soluble form of amyloid-β. PBT2 transports also ions captured from the amyloid-β oligomers into the nearby cells .

PBT2 was well tolerated and significantly improved executive function as well as lowered cerebrospinal fluid levels of amyloid-β in patients with Alzheimer's disease, in a phase II a double-blind, randomized, placebo-controlled trial (Lannfelt et al., 2008) . In addition, a phase II double-blind, randomized, placebo-controlled trial of patients with

Huntington's disease revealed that PBT2 was generally safe and well tolerated, although it was concluded that the therapeutic potential on cognition needs to be confirmed in larger studies. The suicidal ideation was higher in patients with Huntington's disease taking PBT2, which urges careful observation of suicidality in future studies with this compound (Huntington Study Group Reach2HD Investigators, 2015) . Nevertheless, these clinical results

show that novel drugs targeting metal ions can be successfully developed.

In regard to zinc fingers, zinc finger nuclease technology is a tool in the field of genome editing, which is being increasingly developed and has entered clinical trials (Lee et al., 2020; Mullard, 2017; Paschon et al., 2019; Tebas et al., 2014) . Zinc finger nucleases are enzymes that selectively bind, cleave and enable the repair of DNA. Zinc finger nuclease drugs are currently in clinical trials for mucopolysaccharidosis (e.g., clinicaltrials.gov identifier: NCT02702115) and HIV-infection (e.g., NCT04201782). Azodicarbonamide was the first compound targeting zinc fingers (Rice et al., 1997) , which was in clinical trials for HIV infection (Goebel et al., 2001) . A few compounds which replace zinc in zinc fingers by another metal ion have also entered clinical trials (Abbehausen, 2019) . In addition, a registered anti-cancer drug cisplatin (Dasari & Tchounwou, 2014) , was found to interact with zinc fingers and to eject zinc (Castiglione Morelli, Ostuni, Cristinziano, Tesauro, & Bavoso, 2013) .

Clinical trials on the use of zinc in COVID-19 are associated with repurposing of chloroquine/hydroxychloroquine. The outcomes of clinical trials with chloroquine in a variety of acute or chronic viral diseases have recently been discussed (Touret & de, X, 2020) . Generally, it was not effective in humans in prevention or treatment of acute viral diseases in randomized trials (Touret & de, X, 2020) . In relation to  hydroxychloroquine was demonstrated to be effective in a small, open-label, non-randomized clinical trial (Gautret et al., 2020) . Currently there is no evidence coming from randomized controlled trials supporting the use of chloroquine/hydroxychloroquine in patients with COVID-19. Therefore, no such registration has been made by the Food and Drug

Administration so far (Mahase, 2020) . Clinical trials using these medications have been registered, including, e.g., the SOLIDARITY study, a large-scale, multicenter, randomized clinical trial to evaluate the safety and efficacy of treatments for patients diagnosed with COVID-19.

In some of the registered clinical trials on hydroxychloroquine repurposing, zinc will be administered as an adjuvant treatment to hydroxychloroquine therapy, both in the prophylaxis and treatment of COVID-19. Studies NCT04377646 (COVID-Milit) and

NCT04384458 will examine the effects of combined treatment with hydroxychloroquine and zinc in healthcare professionals providing care for patients with COVID-19, as a prophylactic strategy. Two studies will assess the impact of combined treatment with hydroxychloroquine, zinc, vitamin C and vitamin D in the prophylaxis of COVID-19 in health care professionals (NCT04326725, NCT04335084).

Several clinical trials will explore the combination of hydroxychloroquine, azithromycin and zinc in the treatment of patients with the diagnosis of COVID-19. Awaiting the outcomes of the clinical trials, a combination of zinc, hydroxychloroquine and azithromycin has been proposed as an early treatment of COVID-19 in the outpatient setting.

Early outpatient treatment can prevent disease progression and hospitalization (Derwand & Scholz, 2020; Risch, 2020) . Table 3 contains data on clinical trials in regard to zinc and COVID-19 and information whether they are scheduled in the inpatient or outpatient setting.

The study NCT04334512 (HAZDpaC) will examine the efficacy of quintuple therapy compromising hydroxychloroquine, azithromycin, zinc, vitamin D and vitamin C in the treatment of adult patients with the diagnosis of COVID-19. The international ALLIANCE study (NCT04395768) will investigate the treatment with hydroxychloroquine, azithromycin, zinc, vitamin D, vitamin B12 with or without vitamin C. The study NCT04392427 will assess the effects of the combination of nitazoxanide, ribavirin, ivermectin and zinc in children or adults. The study NCT04373733 will compare treatment with hydroxychloroquine, azithromycin and zinc vs. favipiravir.

Moreover, the study NCT04370782 will examine the effects of hydroxychloroquine and zinc in combination with either azithromycin or doxycycline in COVID-19 patients. With regard to doxycycline, the study NCT04371952 (DYNAMIC Study (DoxycYcliNe AMbulatoIre COVID-19)) is aimed to compare a treatment with doxycycline vs a placebo.

Chelation of zinc in MMPs of the host by tetracyclins is a rationale for this study.

Furthermore, two studies have been registered in order to assess the effects of combination of zinc and vitamin D or zinc and vitamin C in the treatment in patients with COVID -19: institutionalized elderly patients or in adult outpatients (NCT04351490, ZnD3CoVici; NCT04342728, COVIDAtoZ). Noteworthy is the fact, that the study NCT04342728 (COVIDAtoZ) includes a group of patients who will receive only zinc gluconate (without vitamins). Inclusion of this group will allow to draw conclusions regarding the role of zinc in the treatment of COVID-19.

Finally, the study NCT04323228 aims at assessing immunonutrition in patients with COVID-19. Immunonutrition is a concept of nutrition which has an impact on the immune system. This strategy is often used in critical ilnesses (Calder, 2003) . For example, a metaanalysis of sixty-one randomized controlled trials on immunonutrition in cancer patients has shown that immunonutrition was associated with reduced risk of postoperative infectious complications, including reduced risk for respiratory tract infection (Yu et al., 2019) , compared to standard nutrition . In the study on immunonutrition in COVID-19, patients with confirmed SARS-Cov-2 infection, who do not require intensive care unit admission, will receive oral nutrition supplement (ONS) enriched in eicosapentaenoic acid (EPA), gammalinolenic acid (GLA), vitamin A, vitamin C, vitamin E, selenium and 5.7 mg zinc (Oxepa, Abbott Nutrition, Abbott Laboratories) or iso-caloric-isonitrogenous product (prepared by the same manufacturer). The ONS or control product will be administered in the morning.

In addition, the study NCT04407572 is an observational study aimed at measuring serum zinc, vitamin D and vitamin B12 levels in pregnant women with COVID-19. Another observational study (NCT04412746) will assess the prevalence of diabetes among hospitalized patients with COVID-19 receiving hydroxychloroquine, azithromycin and zinc or lopinavir/ritonavir.

Many lines of evidence suggest the relationship between zinc and COVID-19 and support the hypothesis that targeting zinc may lead to the development of new drugs for COVID-19. Increasing intracellular zinc is among mechanisms of action of chloroquine, a drug being repurposed for COVID-19 . It is plausible that the therapeutic mechanisms induced by chloroquine in patients with COVID-19 at least in part result from its impact on zinc levels. Disulfiram (Sargsyan et al., 2020) and tetracyclines (Sodhi & Etminan, 2020 ) are among already known drugs which have been proposed to combat COVID-19

based on their effects on zinc. Disulfiram increases intracellular zinc, similarly to chloroquine (Sargsyan et al., 2020; Wiggins et al., 2015) .

Although the above-mentioned agents apparently have many mechanisms of action, the entrance of other metal binding compounds into clinical studies raises the possibility that investigating zinc binding drugs may lead to the development of pharmacotherapy. An example of such successful metal binding drug is PBT2, which was safe and well tolerated in clinical trials (Huntington Study Group Reach2HD Investigators, 2015) .

The principles of a potential strategy of combating SARS-CoV-2 with the aid of zinc targeting agent would be similar to the mechanism of action of PBT2 in Alzheimer's disease.

In the case of Alzheimer's disease amyloid-β is enriched in zinc, which is taken away and redistributed by PBT2. With regard to COVID-19, a novel drug would target labile zinc fingers in SARS-CoV-2 proteins, thus destroying the proteins and producing an increase in intracellular concentration of zinc ions.

The design of therapeutic agents selectively binding a labile zinc finger motif in viral protein is theoretically feasible (Huang et al., 1998) and would be a solution to overcome the problem of binding of such agent to host's proteins (Garcia & Damonte, 2007) . The time is ripe for the design, synthesis and evaluation of new zinc binding drugs, which may be helpful during this and future pandemics.

As intracellular zinc signaling is critically involved in antiviral immunity (Read, Obeid, Ahlenstiel, & Ahlenstiel, 2019) , increased intracellular zinc following administration of zinc and/or zinc ionophore and/or a labile zinc finger targeting drug, will affect function of the immune system. A question arises what level of intracellular zinc will be beneficial and detrimental, since also zinc excess may produce changes in immune cell number and function (Maywald, Wessels, & Rink, 2017) . On the other hand a question is whether disruption of Figure 1 . The possible mechanism of action of zinc metalloenzymes targeting drugs in COVID-19. A drug targeting zinc fingers in zinc metalloenzymes would bind zinc in PL pro (or another essential enzyme of SARS-CoV-2). Such drug would remove zinc from the enzyme, thus destabilizing the enzyme, and produce an increase in intracellular zinc concentration. Zinc administered together with its ionophore would contribute to increased intracellular zinc. Intracellular zinc would inhibit RdRp of the virus. RdRp, RNA-dependent-RNA-polymearse; PL pro , papain-like protease; SARS-CoV-2, severe acute respiratory syndrome-related coronavirus 2 

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Metal ionophores -an emerging class of anticancer drugs

Zinc signaling and epilepsy

Zinc in the monoaminergic theory of depression: its relationship to neural plasticity

Coronaviruses: an overview of their replication and pathogenesis

Ionophores in planar lipid bilayers

RNA-binding proteins in immune regulation: a focus on CCCH zinc finger proteins

Zn finger containing proteins as targets for the control of viral infections

Hydroxychloroquine and azithromycin as a treatment of COVID-19: results of an This article is protected by copyright. All rights reserved

open-label non-randomized clinical trial

Phase I/II dose escalation and randomized withdrawal study with add-on azodicarbonamide in patients failing on current antiretroviral therapy

Metallomics: the history over the last decade and a future outlook

The IUPHAR/BPS Guide to PHARMACOLOGY in 2018: Updates and expansion to encompass the new guide to IMMUNOPHARMACOLOGY

Alzheimer's disease: the amyloid cascade hypothesis

Coronavirus puts drug repurposing on the fast track

Pharmacological activity of metal binding agents that alter copper bioavailability

Anti-HIV agents that selectively target retroviral nucleocapsid protein zinc fingers without affecting cellular zinc finger proteins

Safety, tolerability, and efficacy of PBT2 in Huntington's disease: a phase 2, randomised, double-blind, placebocontrolled trial

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Ebselen, a selenium-containing redox drug, releases zinc from metallothionein

Zinc finger proteins in cancer progression

Structure of M(pro) from COVID-19 virus and discovery of its inhibitors

Dithiocarbamate chelating agents for toxic heavy metals

Treating COVID-19-off-label drug use, compassionate use, and randomized clinical trials during pandemics

Ionophores: potential use as anticancer drugs and chemosensitizers

Pyrithione, a zinc ionophore, inhibits NF-kappaB activation

Biphasic effects of dithiocarbamates on the activity of nuclear factor-kappaB

Pyrrolidine dithiocarbamate induces bovine cerebral endothelial cell death by increasing the intracellular zinc level

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Diagnosis and pharmacotherapy of alcohol use disorder: a review

Structural classification of zinc fingers: survey and summary

Zinc finger proteins: new insights into structural and functional diversity

Safety, efficacy, and biomarker findings of PBT2 in targeting Abeta as a modifying therapy for Alzheimer's disease: a Phase IIa, double-blind, randomised, placebo-controlled trial

Recent advances in genome editing of stem cells for drug discovery and therapeutic application

Identification of labile Zn sites in drug-target proteins

Nsp3 of coronaviruses: structures and functions of a large multi-domain protein

Disulfiram can inhibit MERS and SARS coronavirus papain-like proteases via different modes

Structure and mechanism of carbonic anhydrase

Hydroxychloroquine, a less toxic derivative of chloroquine, is effective in inhibiting SARS-CoV-2 infection in vitro

Structural basis and functional analysis of the SARS coronavirus nsp14-nsp10 complex

p53 down-regulates SARS coronavirus replication and is targeted by the SARS-unique domain and PLpro via E3 ubiquitin ligase RCHY1

Covid-19: six million doses of hydroxychloroquine donated to US despite lack of evidence

Zinc biochemistry: from a single zinc enzyme to a key element of life

Zinc signals and immunity

The role of cytokines including interleukin-6 in COVID-19 induced pneumonia and macrophage activation syndrome-like disease

COVID-19: consider cytokine storm syndromes and immunosuppression

First in vivo gene-editing drugs enter the clinic

Diversifying the structure of zinc finger nucleases for high-precision genome editing

Clioquinol: to harm or heal

Neurovirulent murine coronavirus JHM.SD uses cellular zinc metalloproteases for virus entry and cell-cell fusion

Reverse transcriptase: correlation of zinc content with activity

Discovery of human zinc deficiency: 50 years later

Can Zn be a critical element in COVID-19 treatment?

The role of zinc in antiviral immunity

Zinc pyrithione inhibits yeast growth through copper influx and inactivation of iron-sulfur proteins

Azodicarbonamide inhibits HIV-1 replication by targeting the nucleocapsid protein

Structure, conformation, and mechanism in the membrane transport of alkali metal ions by ionophoric antibiotics

Early outpatient treatment of symptomatic, high-risk Covid-19 patients that should be ramped-up immediately as key to the pandemic crisis

Multitargeting of functional cysteines in multiple conserved SARS-CoV-2 domains by clinically safe Zn-ejectors

Molecular evidence of Zn chelation of the procaspase activating compound B-PAC-1 in B cell lymphoma

Copper and zinc dysregulation in Alzheimer's disease

Structural basis of receptor recognition by SARS-CoV-2

Zinc and immune function: the biological basis of altered resistance to infection

Improving the efficacy of Chloroquine and Hydroxychloroquine against SARS-CoV-2 may require Zinc additives -A better synergy for future COVID-19 clinical trials

SARS-CoV replication and pathogenesis in an in vitro model of the human conducting airway epithelium

Zinc and respiratory tract infections: perspectives for COVID-19 (review)

Chloroquine: mechanism of drug action and resistance in Plasmodium falciparum

Therapeutic potential for tetracyclines in the treatment of COVID-19

Cytokine storm intervention in the early stages of COVID-19 pneumonia

Antiviral sulfonamide derivatives

Zn(2+) inhibits coronavirus and arterivirus RNA polymerase activity in vitro and zinc ionophores block the replication of these viruses in cell culture

Gene editing of CCR5 in autologous CD4 T cells of persons infected with HIV

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A review of metallothionein isoforms and their role in pathophysiology

A zinc fingercontaining papain-like protease couples subgenomic mRNA synthesis to genome translation in a positive-stranded RNA virus

Of chloroquine and COVID-19

ACE2: from vasopeptidase to SARS virus receptor

A cancerselective zinc ionophore inspired by the natural product naamidine A

SARS-coronavirus replication/transcription complexes are membrane-protected and need a host factor for activity in vitro

Remdesivir and chloroquine effectively inhibit the recently emerged novel coronavirus (2019-nCoV) in vitro

Developing drugs targeting transition metal homeostasis

Degradation of the Alzheimer disease amyloid beta-peptide by metal-dependent up-regulation of metalloprotease activity

Disulfiram-induced cytotoxicity and endo-lysosomal sequestration of zinc in breast cancer cells

Analysis of therapeutic targets for SARS-CoV-2 and discovery of potential drugs by computational methods

Chloroquine is a zinc ionophore

In vitro antiviral activity and projection of optimized dosing design of hydroxychloroquine for the treatment of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)

Immunonutrition vs standard nutrition for cancer patients: a systematic review and meta-analysis (part 1)

2015) dithiocarbamates (e.g., DEDTC, pyrrolidine dithiocarbamate) Ionophore: zinc, copper Cell culture, mag-fura 2

Pyrithione Ionophore: zinc, copper Cell culture

Clioquinol Ionophore: zinc, copper Cell culture

SARS-CoV-2 Pl pro inhibitor (µM) (Sargsyan et al., 2020) SARS-CoV-2 M pro inhibitor (µM