A novel virus emerged towards the end of 2019 leading to an unprecedented spread to almost all the countries in the world. After the incidence of the outbreak, laboratory studies and sequencing reveal that this is a new strain of corona virus which was initially designated as 2019 novel coronavirus (2019-nCoV) and later reviewed to severe acute respiratory syndrome corona virus 2 (SARS-CoV-2) by the International Committee on Taxonomy of Viruses [1]. It is actually the third corona virus pandemic to emerge after severe acute respiratory syndrome coronavirus (SARS-CoV) in 2002 and the Middle East respiratory syndrome coronavirus (MERS-CoV) around 2012. The symptoms which are flu-like vary from asymptomatic and mild symptoms (dry cough, mild fever, nasal congestion, headache) to severe symptoms (acute pneumonia, acute respiratory distress, as well as the emergence of septic shock and multi organ dysfunction [1-2]. Current reports also indicate that the stability of the virus on different surfaces varies and that it can be inactivated by UV light, heat and disinfectants [3].

With more information emerging about the origin, spread patterns, detection mechanisms and probable treatment of this viral infection, bioscientists are utilizing different approaches including identifying the possible sources, developing possible molecular models surrounding their evolution and infection, carrying out rapid sequencing of its nucleotide genome, understanding the functions of viral protein structures as well as modelling of protein-protein interactions in the bid to develop highly potent antiviral therapies and designing of suitable vaccines against the vir Genomic sequences of coronaviruses have presented huge data that is currently studied to give insight into establishing virus host interaction, mechanisms of viral infections, potential biomarkers and key targets for antiviral therapies for SARS-CoV-2 [4].

Structural Arrangement of SARS-Cov-2 Virus

Corona viruses are a group of RNA viruses with distinctive spike projections, microscopic crown like appearance and distinctively large genomes ranging from between 26 to 32 kilobase pairs. They can be isolated from a variety of animals including camels, bats, mice and cats [2]. The basic morphology of SARS-CoV-2 coronaviruses includes a spherical shape with diameter between (80- 120nm [5], they are composed of structural proteins including surface projections composed of spike glycoprotein (S), nucleocapsid proteins (N) while the envelope is well supported by a membrane glycoprotein (M) and an envelope (E) protein present in small amounts.[6,3]. There are also 16 non-structural proteins designated (nsp 1- 16) with different functions. The structural proteins play different roles from attachment and entry to viron assembly and release. The S proteins functions majorly in the attachment to the host receptors, the M proteins is involved in binding to the nucleocapsid while the E protein functions in the viral assembly and discharge which instigates its virulent activities. The N protein usually binds to the viral RNA genome and is believed to be involved in viral replication [7]. It was suggested that SARS-CoV-2 had evolved into two major types; the L type possesses higher virulence and is more infectious as compared to the S type which is relatively milder [3] The description of key proteins and receptor binding domain in SARS-CoV-2 is presented in figure 1.

Figure 1: The Structure of SARS-CoV-2 

Source [8] Detailed molecular organization of SARS-CoV-2 structure is still at the early stages of being understood, however these structural proteins and some non-structural proteins are believed to play very distinct roles in its host entry, spread, pathogenicity and serve as basic targets for antiviral drugs

Pathogenesis: Mechanisms of Invasion and Virulence Factors

Generally, viral entry, which is the first step to pathogenesis, requires the attachment of virons to the surface of a suitable cell and consequent delivery of the viral nucleic acid to the cell's cytoplasm or nucleus [9-10]. This viral entry and cell invasion activate immune response from the host resulting in the release of antigen-presenting cells (APC) [11]. The APC triggers a cascade of inflammatory activities by performing two main functions which are; exposing CD4⁺ -T-helper (Th1) cells to foreign antigen as well as releasing interleukin-12 to stimulate the Th1 cell thereby enhancing the release of CD8⁺-T-killer (Tk) cells to target any foreign antigen containing cells [12-13]. Consequently, activated Th1 cells stimulate B-cells in order for antibodies to be generated [11]. Current studies are throwing more light into the distinct mode of pathogenesis as well as immune responses peculiar to SARS-CoV-2.

Recent studies utilizing molecular modeling have highlighted the human angiotensin-converting enzyme 2 (ACE2) as the receptor for SARS-CoV-2 as shown in figure 2. It is believed that the replication of corona virus begins with the binding of S protein to the host cell surface receptor(s) [14]. Actually, the first subunit of S protein (S1) binds to receptor on the surface of host cell, leading to a conformational change and subsequent transformation of the second subunit S2 subunit which then translates to a systematic fusion of the virus with the cell membrane and subsequent release of viral nucleoplasid into the cytosol [5].

Figure 2: Mechanism of Viral Entry

Source: [11] The Spike proteins on the external body of the virus fix itself to the enzyme 2 (ACE-2) conversion receptors on the target cell surface A. Type II transmembrane serine protease (TMPRSS2) attaches to the ACE-2 receptor and cleaves it thereby activating the spike protein in the process. B. Viral entry is thus accelerated via the cleaved ACE-2 and the spike protein being activated; therefore, the expression TMPRSS2 enhances the uptake of coronavirus into the cell.

[3], explains that just like SARS-CoV, the S proteins of SARS-CoV-2 binds directly to receptors on the ACE2 to instigate viral entry. There are evidences that the S protein of SARS-CoV-2 binds attach to the human ACE 2 more firmly than SARS-CoV indicating that blocking this binding is a key component in the treatment of viral infection [15]. The SARS-CoV-2 spike protein ectodomain's ACE2-binding affinity was about a score greater than that of the SARS-CoV spike protein [16].The study also suggested that the lower free energy of the S protein of SARS-CoV-2 when compared to SARS-CoV, could confer stability and ability to withstand higher temperature; however it points out that the higher temperature sensitivity of receptor binding domain (RBD) for SARS-CoV-2 when compared to SARS-CoV could be indication of a much more rapid decrease in infectious ability at much higher temperature. The implication is that SARS-CoV-2 might survive high temperature seasons like summer where it could be inactive and then become highly infectious when the temperature drops such as during winte [15].

The most common viral entry route is via the respiratory tract [11], as in the case of SARS-CoV2. The virus spreads from sinfected patients by respiratory droplets that can be deposited in their body [17]. SARS-CoV-2 infection symptoms may be seen 2–14 days after exposure and may result in hitches in airway cell cilium beating and alveolar damage [18]. Coronaviruses affect multiple host-cell pathways that can influence viral replication and virulence in a positive or negative way [19]. Individuals with previous health issues such as basal immune dysfunction in protein-energy malnutrition and sarcopenic obesity may be opened to infection and experience severe symptoms of SARS-CoV-2 [20]. This is because apart from the collapse of alveoli and respiratory failure, the replication of coronavirus leads to systemic effects in the major body organs such as liver, kidneys, brain and gut.

The mutation potential and adaptability of viruses to new host environments adds up to the complexity of viruses, hence the challenge in developing antiviral therapies that specifically target human coronaviruses; the understanding of these protein interactions and other variables will be indispensable in designing a potent antiviral drug directed at disrupting definite stages in the SARS-CoV-2 cycle from assembly to membrane fusion. Studies by [21] used molecular docking to study the interaction between SARS-CoV-2 proteins and already developed antiviral drugs such as those for hepatitis C virus (HCV), Influenza viruses, Picornaviridae viruses and the human immunodeficiency virus (HIV). The report highlighted the potential importance of protease and spike proteins in these interactions. Ribavirin, lopinavir/ritonavir, remdesivir, nelfinavir, arbidol are some antiviral compounds which were generally applied as therapies for patients who came down with SARS-CoV patients in 2002 and there are suggestions that they could be utilized in the management of patients that are positive for covid 19 depending on the symptoms or viral load. Remdesivir was approved by FDA on May, 1^st, 2020, based on faster time recovery of the infected patients. Chloroquine, a zinc ionophore, however presents an interesting proposition as some studies indicate that it potentially interferes the attachment of SARS-CoV with the human angiotensin-converting enzyme 2 (ACE 2), hence there are current recommendations for its use in clinical treatment of SARS-CoV-2 infection [3], but with contraindications in diabetes and heart disease.

Ph Regulated Ionophore

Ionophores are small hydrophobic lipid soluble molecules, which reversibly bind ions that have the capacity of transferring ions through cell membranes. This transmembrane ion concentration gradients are extremely important for the functionality of a lot of biological processes. Ionophores can cause a disorder in the membrane potential through the passage of ions in the absence of a protein pore through a lipid membrane, thus exhibiting cytotoxicity. 

The biological function of this metal-shuttle conjugates is tied to the metal’s dissociation from the complex or its activity as a whole conjugate. In either case, it is termed a ‘‘metal ionophore’’ if this effect is raised by increasing the metal concentration [23]. Ionophores may move ‘to’ and ‘fro’ between the cytoplasm or could as well remain in the plasma membrane as it transports metal ions [24-25].                 

Zinc Ionophore

Zinc and its transport proteins play important roles in many immune responses. Zinc ionophores transport extracellular zinc ions and have been studied for their antiviral and anticancer activities [26]. Examples of zinc ionophores are the quinolone derivatives such as Chloroquine (4-Aminoquinoline), Clioquinol (8-Hydroxyquinoline), Diiodohydroxyquinoline, PBT2 (8-Hydroxyquinolinr analog), Hinokitol and Quercetin. The Zn²⁺/H⁺ ionophore activity moderate the accumulation of intracellular zinc via an exchange of extracellular Zinc for two molecules of intracellular H⁺ in a process of electroneutrality there by maintaining a fairly constant lysosomal pH. The cell membrane structure could to some extent limit the internalization of the zinc ions. Chloroquine, a lysosomal targeting agent, acts as a zinc ionophore by bringing zinc into the cells. In a study of human cancer cells by [23] it was reported that increase in cytotoxicity and subsequent apoptosis was achieved by combining zinc with chloroquine. Similarly, Clioquinol also functions in the transfer of zinc into lysosomes of cancer cells inducing apoptosis [29], reported that zinc finger proteins play proviral and antiviral roles in different viruses. The activity of enzymes such as RNA polymerase have been shown to be inhibited by Zinc in viruses including coronaviruses, rhinoviruses as well as arteroviruses [30-31]. Also indicated that viral replication could be impaired by raising the intracellular concentration of zinc with its ionophores such as pyrithione. In addition, it has been previously suggested that low concentrations of zinc and pyrithione combination could instigate inhibition of SARS-CoV indicating a possible application in SARS-CoV-2.

Zinc ion Transport

The importance of zinc in very important biological functions cannot be overemphasized and various reports have highlighted the key roles it plays in known viral infections. Also important is its role in mammalian protein folding as well as a cofactor in enzyme activity [32]. Also, free Zn²⁺ intracellular concentration is maintained by metallothioneins at a relatively low level [33].This may be attributed to Zn²⁺ functioning as an intracellular second messenger leading to either a decline in the synthesis of proteins or eventual apoptosis. The high intracellular concentrations of Zn²⁺ concentrations coupled with processes that stimulate the import of Zn²⁺ into cells have been found to inhibit RNA virus’ replication by altering the proteolytic processing of viral polyproteins. 

Two Mechanisms Generate Ion Gradients

A primary pump and a secondary active mechanism. ATP hydrolysis supplies energy to the primary pump; while the secondary active mechanism uses Na⁺ to generate Zn²⁺ gradients. In bacteria for instance, active Zn²⁺ transport has been shown to be catalyzed by different forms of p-type ATPases via a zinc pump [34]. There are also indications that Zn²⁺ transmembrane gradient formed in neurons could be facilitated by an active secondary mechanism which is Na⁺-dependent, while some earlier studies that Zn²⁺ extrusion is specifically mediated by neuronal Na⁺/Ca²⁺ exchangers [35]. Recent findings however seem to point to the presence of two major exchangers as shown in Figure 3.

Intracellular zinc homeostasis is achieved through the mediation ZnT SLC 30 proteins. The transportation of zinc from the extracellular fluid into cytoplasm is carried out by the Zip SLC 39A proteins [37].

Figure 3: Diagram Showing the Localization and Direction of Transport of the Zinc Transporter Families, Znt and ZIP in Subcellular Region.

Source: [36] Mobilization of zinc into the cytosol via the ZnT (green) and ZIP (red) proteins is indicated by the arrows. Zinc movement between the outer cellular region and organelles results in a cytosolic net gain of zinc ions.

Hloroquine as a Zinc Ionophore

Chloroquine is an antimalarial drug used for the treatment of malaria in humans and also occasionally used for amoebiasis that is occurring outside the intestine [38]. Chloroquine acts by increasing the vascular pH in organelles such as endosomes and lysosomes. The rise in pH alters the lysosomal and endosomal function leading to autophagosome impairment [23]. Chloroquine also functions in targeting the lysosomes whereby it increases their cytotoxicity to induce apoptosis in a model cell system for human cancer [23]. This allows the influx of zinc into cells, leading to RNA dependent RNA polymerase viral inhibition. Chloroquine binds to extracellular zinc transporting it across the membrane thereby releasing the zinc into the cytosol [39].

Inhibition and Control of SARS-COV-2 Invasion through Ph Regulated Ionophores 

In some eukaryotic cells, certain chemical substances such as ionophores act diversely in that they cause an increase in the vacuolar pH of acidic organelles. This rise in pH affects the acidic potential of the lysosomes which results in weakening of the fusion and degradation of autophagosome [40-41] Different mechanisms have been recognized for the effectiveness of ionophores, for instance, increase in endosomal pH of the host’s intracellular organelles, inhibition of fusion and inactivation of viral enzymes [42]. Ionophores alter the glycosylation of ACE-2, the receptor with which SARS-CoV-2 uses to gain entrance into its host cell [43]. They also interact with enzymes such as acid hydrolases as well, blocking post-translational modifications of the newly synthesized proteins. This is achieved through trans-Golgi networks (TGN) and elevation of lysosomal Ph [44]. Chloroquine, as a zinc ionophore, directs zinc ions to the lysosomes of diseased cells thereby causing lysosome-mediated apoptosis. This action of Chloroquine is supported by [45], who reported that the activity of RNA polymerase in arterivirus and that of coronavirus are restrained in-vitro by zinc ionophore.

Zn Ion Homeostasis and Immune Function 

 Zinc homeostasis (influx, efflux, storage and compartmentalization of zinc) within the cell is very crucial in perturbations of pathogen and the normal functioning of both eukaryotic and prokaryotic cells [46]. Considerable proportion of zinc remains bound to metal-binding proteins (metalloprotein) such as serum albumin or any cysteine-rich proteins capable of binding divalent cations. The metalloprotein is then transported into the cytoplasm where it is been stored or forms a zinc-binding enzyme [47]. The intracellular zinc-protein possesses numerous functions in addition to storage, it is involved in immune responses, heavy metal detoxification and cell apoptosis [48]. 

The homeostasis of zinc is important in immune function, the deficiency of zinc may result in impairment of cell immune response which exposes the body to several types of infections including; inflammatory diseases, pneumonia and autoimmune disease. The manifestation of these infections in zinc deficient patient is associated with the decrease in the level of inflammatory cytokines [49]. Immune and Infected cells secrete are immune-stimulatory cytokines (interferons) which initiate the expression of many antiviral genes [50]. These interferons (IFNs) act either by activation of immune cell or by chemoattraction [51].

Figure 4: The Pathways of Zinc Action on Immune Cells [54].

Zinc was reported to be one of the requirements for gene expression of many cytokine signaling molecules; interferon (IFN-γ) and interleukins-2 (IL-2) [52]. The IL-2 then initiates the activities of T-cytotoxic cells and natural killer (NK) cells [53] IL-12 produced by macrophages also plays a vital role in the killing of viruses, bacteria, and parasites. The immune action mechanism of zinc is illustrated in figure 3. Differentiation and maturation of T-cell is influenced by thymulin, a nonapeptide hormone which has zinc as its important component for its biological function [54]. The thymulin triggers the productions of cytokine signaling molecules (IFN-γ, IL-2 and IL-12) by macrophages-monocytes which are directly involved in killing invaded parasites, viruses, and bacteria [54].

Thymulin is a hormone involved in differentiation, maturation and enhancement of T-cells which has Zinc as its important component. The gene expression of signaling cytokines (IFN-γ and IL-2) is dependent on zinc activity. IL-2 functions in the activation of T cytolytic cells and natural killer cell, while the stimulation of macrophages which is Zinc dependent generates IL-12. The killing of pathogens by macrophages-monocytes is influenced by the collaborative role of IFN-γ and IL-12.

Inhibition and Control Mechanism of Chloroquine 

Chloroquine (a zinc ionophore and a weak base) has been reported to increase the pH of intracellular acidic organelles, e.g. lysosomes and endosomes, which are important for membrane fusion [55]. Chloroquine stimulates an increase in endosomal pH, this increase controls iron metabolism in human cells by reducing the intracellular iron levels. This is achieved by weakening the endosomal releases of iron from ferrated transferrin [56]. This reduction in intracellular iron levels can affect the activity of many enzymes involved in pathways leading to cell DNA replication and various gene expression [57].

A report from [44] stated that the immediate antiviral effects of chloroquine prevent pH-related steps at any stage of viral replication including flavivirus, retrovirus and coronavirus. The movement of endocytosed virus, virus uncoating and transcription of viral nucleic acids are being affected at the early stage while the late stages being affected are post-translational processing of enveloped glycoproteins in the Golgi, movement of enclosed components and newly formed viral particles.

Specific methods of action are suggested to describe the therapeutic effects of chloroquine, most focused on in vitro studies [43]. stated that chloroquine could impair lysosomal and autophagosomal maturation and suppress antigen presentation along the lysosomal pathway, and consequently immune activation. Chloroquine is a nucleic acid inhibitor which recognizes TLR-mediated inflammatory responses (toll-like receptor 5) [58]. Chloroquine has also been involved in inhibiting signaling pathways [43] by inducing changes in endosomal pH and interfering with the processing of TLR9 and TLR7 [59], thus preventing TLR activation on extracellular stimuli by facilitating changes in local pH [60] . In addition to TLR9 signaling, chloroquine can also obstruct the RNA-mediated activation of TLR7 signaling and further delineation at molecular level is required in order to determine the exact modes of action [43].

Earlier studies have also shown that chloroquine has significant in vitro effects against SARS-CoV-1, mainly due to deficiencies in the glycosylation receptors on the surface of the viral cell, which prevents it from being bound to angiotensin-converting enzyme 2 (ACE 2) in some major body organs such as lungs, heart, kidneys and stomach [61]Because SARS-CoV-2 uses the same ACE 2 surface recipient, chloroquine is thought to interact with ACE 2, this prevents SARS-CoV-2 adherence to the target cells thereby decreasing viral replication. In addition, some enzymes such as glycosyltransferases may not function properly as a result of increased pH or structural changes in the Golgi apparatus, hence the glycosylation of SARS coronavirus is inhibited [43]. Furthermore, chloroquine was reported to affect lysosomal pH and consequently inhibits the production of cathepsin [62]. which cleaves the spike protein on SARS-CoV-2 [61] Chloroquine can also induce the absorption of zinc into the cell's cytosol, which can inhibit RNA-dependent RNA polymerase and ultimately stop coronavirus replication in the host cell [57].

The ability of Zinc to inhibit and control SARS-CoV-2 invasion by intracellular pH regulation is presented. There is a provided method to protect against viral invasion, apoptosis, and inflammation of epithelial cells using Chloroquine as a Zinc ionophore. Zinc ion’s immunomodulatory activity, pH regulation pathway and the inhibitory mechanism of chloroquine are also highlighted.

Zinc could be proviral when the concentration is low in the cell, with increased concentration through an ionophore, it becomes antiviral. A Zinc and Chloroquine therapy may be effective at both the early stage and advanced stage of COVID-19 disease, because chloroquine increases cellular concentration of Zinc ion and prevents binding of viruses to ACE 2 through glycosylation of receptor proteins in epithelial tissues such as lungs, heart, kidneys and intestine. But there are contraindications in diabetes and heart disease. 

Different mechanisms attribute to the efficacy; which include the ability of these bases to increase the pH of host intracellular organelle, inhibition of fusion between autophagosome and lysosome and inactivation of RNA dependent RNA polymerases to stop viral replication. At the molecular level, Chloroquine participates in the innate immune pathways by signaling toll receptor (TLR) proteins and the induction of adaptive immune responses. However, further delineation needs to be carried out to understand its specific mode of action. There is a need to further explore other divalent cations and researches on the affinity of chloroquine for binding to various metal ions should also be elucidated.

Declaration of Interest

The authors report no declaration of interest

Availability of data and materials

Data sharing not applicable to this article as no new data were created or analyzed in this study

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