Abbreviations ACE 2 angiotensin-converting enzyme 2 ACEI angiotensin-converting enzyme inhibitors ADAM17 A disintegrin and metalloprotease 17 AGTR1 angiotensin II receptor type 1 Ang II angiotensin II aPKC atypical protein kinase C AT1 angiotensin II Type 1 AT2 angiotensin II Type 2 BLI bio-layer interferometry CD13 cluster of differentiation 13 (glycoprotein) CD3 cluster of differentiation 3 CD8 cluster of differentiation 8 Cdc42 cell division control protein 42 homologue COVID-19 coronavirus disease 2019 cryo-EM cryogenic electron microscopy DPP4 dipeptidyl peptidase 4 HCoV-229E human coronavirus 229E HCoV-HKU1 human coronavirus Hong Kong University 1 HCoV-NL63 human coronavirus NetherLand 63 HCoV-OC43 human coronavirus organ culture 43 HF heart failure HIV human immunodeficiency virus hrsACE2 human recombinant soluble ACE2 IFN interferon IL-1β interleukin 1 beta IL-6 interleukin 6 ISG interferon-stimulated gene JARID1B jumonji AT-rich interactive domain 1B KDM5B lysine-specific demethylase 5B MERS-CoV Middle East respiratory syndrome coronavirus miRNAs microRNAs MSA measurement systems analysis mTOR mammalian target of rapamycin NF-κB nuclear factor kappa light chain enhancer of activated B cells PAK1 P21-activated kinase 1 PAK2 P21-activated kinase 2 Par3 partitioning-defective protein 3 Par6 partitioning-defective protein 6 PLpro papain-like protease PPIs protein–protein Interactions RAS renin–angiotensin system RBD receptor-binding domain RdRP RNA-dependent RNA polymerase Rho Ras homologous SARS-CoV severe acute respiratory syndrome-related coronavirus SARS-CoV-2 severe acute respiratory syndrome coronavirus 2 SPR surface plasmon resonance TGF-β transforming growth factor beta TMPRSS2 transmembrane serine protease 2 TNF alpha tumour necrosis factor alpha 1 INTRODUCTION

SARS-CoV-2 is a single-stranded positive-sense RNA virus1 that causes acute respiratory distress syndrome, which leads to serious global health issues.2 The SARS-CoV in 2002–3,3 the Mers-CoV in 2012–20134 and the current pandemic of SARS-CoV-2 prove that the diseases distribution is more expansive than previously recognized.5 The glycosylated spike protein (S) is one of several structural proteins encodes by the COVID-19 genome.6 This glycoprotein mediates virus entry by two functional subunits responsible for attachment to host cell receptor (S1 subunit) and viral and cellular membranes (S2 subunit) fusion. S is further cleaved at the S2′ site, by a host transmembrane Serine Protease 2 (TMPRSS2), at immediate upstream of the fusion peptide.7 The resulting cleavage leads to extensive irreversible conformational changes, in which protein is activated for membrane fusion.8, 9 Thus far it is unclear if angiotensin-converting enzyme 2 (ACE2) and TMPRSS2 are required on the same cell, or soluble proteases can activate SARS-CoV-2 S-protein to invade ACE2-single-positive cells.10, 11 Furthermore, it is still ambiguous whether SARS-CoV-2-S may have a furin cleavage site. This potential protease on the spike glycoprotein causes a broad set of host proteases that could mediate S-protein activation.10, 12, 13 An active S-protein has a limited lifetime for finding a target cell membrane. The S-protein's activation timing and cellular location are essential. Effective entries mainly occur in proximity to the plasma membrane.14, 15

Subfamily coronavirinae is divided into four genera; alpha-CoV, beta-CoV, gamma-CoV, and delta-CoV.16, 17 Most SARS-related coronaviruses interact directly with the host ACE2 receptor on the lung and heart cells to enter the target cells.18 The infection efficiency depends on the ability of ACE2 to support viral replication in humans, mice, and rats.19-21 Mutation(s) have occurred in the sequence of the SARS-CoV-2 spike protein that can lead to sustained transmission among humans.22 Acquisition of polybasic cleavage sites in CoV-2 spike is one such example. There are differences between the S1 subunit of the receptor-binding domains (RBDs) of spike that cause a major effect on SARS-CoV-2 spike/ACE2 interaction and decrease the binding energy compared to the one of Bat-CoV to this receptor.23 SARS CoV-1 has six amino acid RBDs essential for interaction. Five of the amino acids are different in CoV-2. Different viral species use distinct domains within the S1 subunit to recognize various attachments and entry receptors.13 Proteolytic processing of SARS-CoV2-S protein in human cells and several arginine residues at the S1/S2 cleavage site of SARS-CoV2-S protein is efficient as oppose to SARS-S protein.24 SARS-S and SARS-2-S proteins share approximately 76% amino acid identity.24

Not only the slow rate of vaccination in low-income countries, but also non-adherence/hesitance to vaccination in high-income countries equally threaten the effectiveness of vaccines. Even while attempting to vaccinate the world's population, the newly emerging variants of COVID19 have negatively affected the efficacy of vaccines. ACE 2, as the essential receptor for binding to SARS-CoV-2, plays a significant role in the occurrence of this deadly disease. Finding treatments that target ACE 2 can be an effective strategy for the treatment of COVID-19. SARS-CoV-2 RBD protein can be used as a viral attachment or entry inhibitor against SARS-CoV-2 because of its ability to block S protein-mediated SARS-CoV-2 pseudovirus entry into its ACE2 receptor-expressing target cell. Human recombinant soluble ACE2 (hrsACE2), as a genetically modified soluble form of ACE2, can reduce cell entry of SARS-CoV-2 since it competes with membrane-bound ACE2. MicroRNAs (miRNAs) can negatively regulate the expression of ACE2/TMPRSS2 and inhibit SARS-CoV2 entry into cells by binding to the target mRNA at the 3′ untranslated regions leading to degradation or translational downregulation of the target. The therapy strategies provided in this article involve ACE2 and the results of the recent studies on ACE2-related potential treatments to encourage and recommend further required research in order to accelerate the quest for a universally effective COVID-19 treatment.

2 ROLE OF ACE2 IN CORONAVIRUS INFECTIONS (SARS-CoV AND SARS-CoV-2) INFECTION

Angiotensin-converting enzyme (ACEI) inhibitors can confront COVID-19 infection by increasing the number of CD3 and CD8 T cells and reducing the viral load and interleukin 6 (IL-6) levels that control SARS-CoV-2 replication via NF-κB.2 There is hope that certain drugs, including SARS-CoV-2 receptor blockers, anti-inflammatory agents (against rheumatic diseases), monoclonal antibodies, anti-IL-1 and anti-IL-6, remdesevir drug (analogue adenosine), and vaccines can provide promising strategies to combat COVID-19.2

Angiotensin II binds to the angiotensin AT1 receptor to cause vasoconstriction, and angiotensin (1−7) elicits vasodilation mediated by AT2.25-27 Manipulation of the ACE2/Ang 1–7 axis can reduce SARS-induced tissue injuries, which can be a potential treatment strategy.28 In vivo studies showed that catalytically active ACE2 mitigates pulmonary damage and vascular damage,29, 30 It also further reduces lung fibrosis, arterial remodelling, and improves ventricular performance.31, 32 In a clinical study, administration of ACE2 lessened systemic inflammation and shifted the RAS peptide balance away from Ang II towards Ang 1–7.33, 34 Ongoing global efforts have focussed on the potential role of the ACE2/Ang 1–7 axis to curtail SARS-CoV-2 infection while trying to highly protect against lung and cardiovascular injury in COVID-19 patients.

ACE2, AT1 receptors, and AT2 receptors are changed during invasion.35 However, according to a hypothesis formed by Sriram & Insel, ACE2 activity and expression are decreased by SARS viruses, which results in an imbalance in the signalling by ACE1 and ACE2 products that increases Ang II/AT1 signalling and is also superimposed on concurrent pathology. Ang II is a crucial mediator of injury in the tissues of the lung and heart. The improved effects and signalling enhancement from co-morbidities can upsurge the severity of COVID-19. The reduction of ACE2 activity duo to SARS viruses can unleash a cascade of injurious effects through a heightened imbalance in the actions of the products of ACE1 versus ACE2 in patients who are more prone to the damaging effects of Ang II36 (Figure 1).

According to the critical role of ACE2 in SARS-Cov-2 cell entry, the Angiotensin-Renin system is affected by the infection. (a) Under the typical situation, Renin converts Angiotensin to Angiotensin I, further converted to Angiotensin II (Ang II) by the Angiotensin-Converting Enzyme 1 (ACE1). Ang II signalling through Ang II type 1 receptor (At1) can lead to organ damages and inflammation. For this reason, ACE2 converts Ang II into Ang 1-7 with an opposite effect through MAS receptor signalling. Thus, ACE2 function balances the At1 signalling pathway by reducing Ang II concentration and neutralizing At1 signalling effects through MAS receptor signalling. (b) During infection, the virus binds to ACE2 through its S Protein, activated earlier by TMPRSS2. This binding causes disfunction in ACE2, leading to an imbalance in the Renin-Angiotensin System. Besides, the virus enters the cell through receptor-intermediated endocytosis and releases its genome into the cytosol. The viral RNA genome expression using the host cell ribosome synthesized two sets of proteins: viral polymerase and accessory proteins. The viral polymerase replicates the viral genome; some break into subgenomic transcriptomes and translate into viral structural proteins, others bind to the nucleocapsid proteins and form the nucleocapsid. Then, the components pack and form the new virus that exits the cell through exocytosis

Blocking the virus from entering cells is the most direct approach to combat SARS-CoV-2.37 Since there is no chance for mutations in the host ACE2 protein, it can be considered a potential drug development strategy.38 Exploring receptors and their targets would be a big step forward to find a remedy for the SARS-CoV-2 infection.39 A genetically modified variant of ACE2, called hrsACE2, can block COVID-19 from entering cells by attaching to the copy instead of the actual cells.40, 41 Drug APN01 is an example of hrsACE2 tested in Phase II trials for lung disease.42 Arbidol 20 is another virus-host cell fusion inhibitor against influenza virus that prevents the virus from entering the host cells. This broad-spectrum antiviral has been considered a clinical trial to treat SARS-CoV-2.24 Losartan is another ACE inhibitor for SARS-CoV-2 pneumonia infections. There are ongoing clinical trials on infected patients.43, 44 The other selective ACE2 inhibitor, DX600, might also be helpful in SARS-CoV-2 infections. Nevertheless, its clinical significance in COVID19 has not been assessed.45, 46

The soluble form of ACE2, which lacks the membrane anchor, can compete with SARS-CoV receptors by inhibiting viral particle binding to the surface-bound, full-length ACE2.6, 47-50 Camostat mesylate is one of these nonspecific TMPRSS2 protease inhibitors that bears the S protein of SARS-CoV-2 in cell culture through pseudovirus.51 Protease inhibitors such as disulfiram, alpha-interferon, lopinavir-ritonavir, and PLpro proteases, have been proposed as potential agents against SARS and MERS. Chloroquine blocks the action of heme polymerase and remdesivir, which also blocks RdRp protease.6, 52 Remdesivir (GS-5734) is a phosphoramidate prodrug of an adenine derivative with a similar chemical structure to the approved tenofovir alafenamide, an HIV reverse transcriptase inhibitor. Remdesivir anti MERS and SARS activities have been reported against RNA viruses in cell cultures and animal models and clinical thus far.53

Another strategy would be using ACE2 inhibitors that inhibit SARS coronavirus spike protein-mediated cell fusion. In a recent study by Huentelman et al., ≈140,000 small molecules were monitored for the highest predicted binding scores for ACE2 enzymatic inhibitory activity to inhibit SARS coronavirus spike protein-mediated cell fusion. Among those small molecules, N-(2-aminoethyl)-1 aziridine-ethanamine has been proposed as a novel ACE2 inhibitor with the highest predicted binding scores.54-57

There is compelling evidence that the colocalization of ACE2 and TMPRSS2 is often found in large number of cells, which can vary with different tissues and in a cell-specific manner.58-61 Specific progenitor cells in the bronchi, which normally develop into the cilia are mainly responsible for producing the coronavirus receptors.62, 63 ACE2 receptor is an interferon-stimulated gene in upper airway epithelial cells. SARS-CoV-2 may increase infection via interferon (IFN)-driven upregulation of ACE2, a key tissue-protective mediator during lung injury.64 Therefore, Antiviral/IFN combination therapy for SARS-CoV-2 infection can assist in balancing host restriction, tissue tolerance, and viral enhancement mechanisms.65-67

SARS coronavirus infection correlates with cell differentiation of airway epithelia and ACE2 expression and localization.68, 69 ACE2 proteins are more abundantly expressed on the apical than the basolateral surface of polarized airway epithelia.70 Among the numerous molecules at the apical membrane, only a few essential molecules are responsible for the identity and epithelial polarity of the apical membrane. These proteins include; Cdc42, atypical protein kinase C, Par6, Par3/Bazooka/ASIP.71-74 Cdc42 has been implicated in numerous functions, including dendritic growth, branching, and branch stability.75-77 Membrane localization principally occurs at the highest concentration of the molecule. When Cdc42 is not present, apical determinants cannot be maintained at the apical membrane. Due to this occurrence, apical identity and polarity are lost.78

It has been shown that the HCoV-229E receptor, aminopeptidase N (CD13), and MERS-CoV receptor DPP4 (CD26) are different from ACE2 receptors of SARS-CoV-2.79 The infectivity of HCoV-229E, HCoV-OC43, HCoV-NL63, and HCoV-HKU1 is comparatively low with slight respiratory symptoms, while SARS-CoV and MERS-CoV, which use ACE2 receptor, cause outbreaks with high mortality.80, 81 According to Cryo-EM structure studies, the binding affinity of SARS-CoV-2 S protein to ACE2 is about 10–20 times stronger than SARS-CoV S protein.6, 82 Therefore, the transmissibility and contagiousness of SARS-CoV-2 is higher compared to SARS-CoV.83

3 POTENTIAL INHIBITOR TARGETING ACE2 RECEPTORS

ACE2 is a type I integral membrane carboxypeptidase that cleaves a single hydrophobic/basic residue from the C-terminus of its substrates.84 The ACE2 open reading frame in humans encodes an 805 amino-acid polypeptide.85 ACE2 protein sequence reveals two hydrophobic regions. A potential 18-amino-acid signal peptide at the N-terminus and a 22-amino-acid—near the Cterminus.86 ACE2 binds to the cell membrane via the hydrophobic region close to the C-terminus of the protein. The active site is positioned on the N-terminal region of ACE2—which faces the extracellular space. ACE2, similar to ACE, is an ectoenzyme located at the cell surface to hydrolyse circulating peptides. ACE2 has six potential N-glycosylation sites in humans, as shown by the presence of the Asn-X-Ser/Thr motif (at positions Asn53, Asn90, Asn103, Asn322, Asn432, Asn546) in its primary structure.87 The ACE2 gene in humans consists of 18 exons with the Ace2 zinc-binding motif (HEXXH) positioned in exon 9.88

Human Cdc42 is a small GTPase of the Rho family. Cdc42 regulates signalling pathways responsible for various cellular functions such as cell morphology, cell migration, endocytosis, and cell cycle progression.89 Rho GTPases are essential for dynamic actin cytoskeletal assembly and rearrangement. These are the basis of cell-to-cell adhesion and migration. Cdc42 activates by conformational changes.90 P21-activated kinases, PAK1 and PAK2 are responsible for regulating cell adhesion, migration, and invasion.91, 92 PAK1 is the major ‘pathogenic’ kinase, whose abnormal activation can lead to cancers, inflammation, malaria, and pandemic viral infection, including influenza, HIV, and COVID-19.93 Natural and synthetic PAK1-blockers such as propolis, melatonin, ciclesonide, ivermectin, and ketorolac have been suggested as potential therapeutics against COVID-19.94 They directly block the replication of this virus in cell culture. However, the lack of binding surface for small molecule targeting of Protein–Protein Interactions (PPIs) involving Cdc42 is yet to be revealed.95 The two small molecules, ZCL278, and AZA197 target Cdc42 to influence PPIs. They can inhibit Cdc42 and suppress proliferative and pro-survival signalling pathways through PAK1-ERK signalling. This pathway can eliminate migration of - colon cancer cells.96, 97

4 ACE2 AND THE RBD

ACE2 is a vital SARS-CoV-2 receptor. A transmembrane protein known for its physiological role and carboxypeptidase activity in the renin-angiotensin system, which is involved in the COVID-19 pathogenesis since it permits viral entry into target cells.98 ACE2 is also the host receptor binding to SARS-CoV's virus S protein. The RBD in SARS-CoV-2 S protein is acknowledged to be a high potential target for developing neutralizing antibodies, virus attachment inhibitors, as well as vaccines.79, 99 Tai et al. have characterized the SARS-CoV-2 RBD that demonstrates strong binding to its cellular and soluble ACE2 receptors originated in bats and humans. The RBD of SARS-CoV-2 spike glycoprotein compared to SARS-CoV RBD presents 10- to 20-fold higher binding affinity to ACE2, which underpins the elevated pathogenesis ability of SARS-CoV-2 infections by blocking the binding, resulting in SARS-CoV RBD and SARS-CoV-2 RBD attachment to ACE2-expressing cells, therefore preventing their infection from hosting cells.100 As a viral attachment or entry inhibitor against SARS-CoV-2, SARS-CoV-2 RBD protein is suggested due to its ability in blocking S protein-mediated SARS-CoV-2 pseudovirus entry into its ACE2 receptor-expressing target cells.100 ACE2, similar to several other cell-surface proteins, undergoes regulated internalization in a clathrin-dependent manner.101, 102 Hence, triggering internalization using identified or designed small molecules that can bind with ACE2 could be a practical approach in reducing the ACE2 cell surface density to block binding and diminish viral entry. RBD from SARS-CoV S protein induces ACE2 internalization by binding to ACE2.103 SARS-CoV RBD-induced antibodies can also cross-react with SARS-CoV-2 RBD and cross-neutralize SARS-CoV-2 pseudovirus infection, which demonstrates the potential application of SARS-CoV RBD-specific antibodies to treat SARS-CoV-2 infection. SARS-CoV-2 or SARS-CoV RBD protein can perform as a candidate vaccine to induce cross-neutralizing or cross-reactive antibodies to inhibit SARS-CoV-2 or SARS-CoV infection.104, 105

The recombinant RBD protein binds strongly to bat ACE2 (bACE2) and human ACE2 (hACE2) receptors and blocks the entry of SARS-CoV and SARS-CoV-2 into their respective hACE2-expressing cells.100 Most monoclonal antibodies neutralize the virus through binding to epitopes in the RBD protein and inhibiting its interaction with ACE2. The antibodies' ability to bind to RBD with high affinity and specificity enables the antagonism of ACE2 binding. Several methods, including surface plasmon resonance, X-ray/cryo-EM, and bio-layer interferometry can be applied to perform characterization.106-109

In research conducted by Oany et al., 14 S proteins have been retrieved and a phylogenetic study has been performed indicating that they were more closely related.57 Moreover, the measurement systems analysis study on RBD sequences from different strains and the structural analysis by Wrapp et al. exposes highly conserved residues in SARS-SOV-2 S RBD compared to SARS-CoV S RBD, that are vital for the binding of ACE2 receptors. Therefore, preventing SARS-COV-2 S protein from binding to human ACE2 receptors appears to be the most promising target for developing an innovative therapeutic drug to come to grips with the current pandemic situation.57, 82

5 HUMAN RECOMBINANT SOLUBLE ACE2

ACE2 is involved in the renin-angiotensin-aldosterone system, which regulates fluid balance, blood pressure, and intestinal functions, and protects organs against inflammatory injuries. ADAM17 and TMPRSS2 are the proteases capable of shedding ACE2. The SARS-CoV binding site is the N-terminal peptidase domain. The cellular form and circulating form are the two types of ACE2 protein, membrane-bound and soluble, respectively. TMPRSS2 and cellular ACE2 are required for positive SARS-CoV-2 infection. ADAM17-shedded ACE2 (circulating ACE2) is considered the major shedding enzyme in protecting the lungs from viral infection. The expression of TMPRSS2 obstructs ADAM17-shedding of ACE2.110-112

TMPRSS2, ADAM17, and cellular ACE2 are expressed on the cell membrane. Soluble ACE2 is first shed by ADAM17 and then released from its full-length form to counteract the consequences of Ang II signalling. Furthermore, cellular ACE2 is shed by TMPRSS2, resulting in the fusion of SARS-CoV-2 cell membrane, releasing SARS-CoV-2 RNA into the cytoplasm, and efficient viral processing replication. The soluble ACE2 has the ability to bind to coronavirus since it comprises the virus binding site. The virus cannot be duplicated without an intracellular environment.35 (Figure 2).

Targeting SARS-CoV-2 entry for the treatment of Covid-19

There is a genetically modified soluble form of ACE2 known as hrsACE2. (Also called hrsACE2) This can reduce SARS-CoV-2 cell entry by competing with the membrane-bound form of ACE2. APN01, currently in a multi-centre, double-blind, randomized, placebo-controlled, interventional trial designed by Apeiron Biologics, is a hrsACE2 that emulates the human ACE2, and reduces SARS-CoV-2 cell entry to decrease lung injury and various organ dysfunctions. A molecular rationalization for the severe lung failure and death caused by COVID-19 was provided by Monteil et al., that suggested the treatment of COVID-19 patients using APN01 due to its prevention abilities in SARS-CoV-2 infections.113-115

In patients with heart failure, chronic kidney diseases, arterial hypertension, and type 1 or type 2 diabetes, circulating ACE2 is over-expressed as a defensive response to counterbalance the adverse effect of Ang II. ACE2 may also regulate immune functions via the Ang-(1-7)-Mas axis since Ang II-AT1 receptor signalling stimulates autoimmune response.35, 116, 117 Therefore, increasing ACE2, mainly circulating ACE2, is an innovative approach to protect organs by reducing SARS-CoV-2-induced severe damage. In experiments using in vitro cell-culture and engineered human blood vessel organoids, it was shown that clinical-grade human soluble ACE2 can reduce SARS-CoV-2 load, thus, lower its infection rate by a factor of 1000–5000 in human kidney organoids. This indicates that ACE2 can effectively neutralize SARS-CoV-2 and block early stages of SARS-CoV-2 infections.114, 118, 119

Soluble ACE2 safety is proven in clinical studies on the treatment of SARS and ARDS.34, 120 The results of the phase-I study of hrsACE2 on 89 healthy volunteers show that APN01 can decrease viremia and viral titres. The phase-II clinical studies of patients with acute respiratory distress syndrome indicate that APN01 reduces the risk of medical complications as well as recovery time. To date, APN01 can be considered as a promising therapeutic drug against COVID-19.119, 121, 122 More potent soluble ACE2 forms have been engineered with computational design, affinity maturation, and deep mutagenesis. Chan et al. discovered sACE22.v2.4 by designing soluble ACE2 utilizing affinity maturation by the mutations of the 117 residues engaged in the binding of S protein. The resilience of sACE22.v2.4 against mutants is demonstrated via its capability in potently neutralizing coronaviruses, such as SARS-CoV-2, SARS-CoV and SARS-like bat coronaviruses, that utilize ACE2 as entry port.123 Linsky and Glasgow et al. have worked successfully on a similar approach.124, 125 CTC-445.2t and CTC-445.2d are the two decoys engineered by Linsky et al., showing SARS viruses' potent neutralization, which protected Syrian hamsters against SARS-CoV-2 with a single prophylactic dose. However, even smaller versions of decoy receptors are able to produce potent neutralization effects.126, 127

APN01 has two theoretical mechanisms of action that should be beneficial in COVID-19 treatment. The first mechanism includes competitively binding the viral spike protein to neutralize SARS-CoV-2 or at least slow viral entry into the host cell, and the second is rescuing cellular ACE2 activity that reduces injury to multiple organs, such as the lungs, heart, and kidneys, due to unabated renin-angiotensin system hyperactivation and amplified angiotensin II concentrations.128-130

Monteil et al., in a recent study examined an innovative approach, combining two different modalities of virus control, and realized by blocking entry using hrsACE2 and preventing intracellular viral RNA replication through remdesivir. Effects can be improved in SARS-CoV-2-infected cells and human stem cell-derived kidney organoids.131

Predominantly, utilizing hrsACE2 did not result in a reduction in the neutralizing antibodies' generation. Abd El-Aziz et al., observed similar data in a patient with severe COVID-19 symptoms treated with two doses of hrsACE2 for one day. The rapid decline of viral load in the serum and the antiviral antibodies' generation were detected in the patient. HrsACE2 diminishes the viral load in the respiratory system. It takes a noteworthy part in slowing or inhibiting the systemic spread of SARS-CoV-2 from the lungs to other organs to minimize the virus attacks on the lining of blood vessels. However, it is unclear if there are any hrsACE2 related side effects that have been reported. Proper precaution should be taken since reduced angiotensin II formation due to the ACE2 overexpression, might lead to hypotension and acute kidney injury. Although Initial clinical observations have been promising, further research is essential to expose the full capacity of hrsACE2 as a proper therapeutic tool.121

6 THERAPEUTIC POTENTIAL OF miRNAs TARGETING ACE2

MicroRNAs (miRNAs) are small endogenous non-coding RNAs consisting of nearly 22 nucleotides, capable of regulating one-third of human gene expression. They are actively involved in adaptive and innate immune responses against coronavirus infections. Also, miRNAs play an essential role in various biological processes such as apoptosis, cellular division, growth and development through the post-transcriptional mechanism, translational repression or mRNA degradation, and numerous protein-coding genes in animals, plants, and some viruses.132, 133 MiRNAs can influence a variety of cell signalling pathways and thus be involved in various pathophysiological conditions. Some viruses express viral miRNAs associated with apoptosis regulation, host-pathogen interplay, and host immune systems modulation that could assure viral survival within infected cells and propel viral pathogenesis.134, 135 Viral miRs can regulate gene expression upon infection.136

MicroRNAs can be an excellent option to negatively regulate the expression of ACE2/TMPRSS2 and inhibit SARS-CoV2 entry into cells. This is done by binding to the target mRNA at the 3′ untranslated regions (3′-UTR) that lead to degradation or translational downregulation of the target. Nersisyan S, et al., Applied the same approach and realized that KDM5B gene encoding lysine-specific demethylase 5B (JARID1B), can repress transcription of hsa-mir-141/hsa-miR-200 and hsa-let-7e/hsa-mir-125a miRNA families, hence, indirectly affecting ACE2/TMPRSS2 expression and impeding virus entry into the host cell.137, 138

MicroRNAs low expression rate allows increased susceptibility to infection while their specific, highly expressed immune response genes can protect the lungs against the viral infection. By investigating miRNAs' role in the host interface with SARS-CoV-2, valuable insights can be provided in detecting promising molecular therapeutic targets to control the pathogenesis ability of SARS-CoV-2.139

ACE2, via epigenetic mechanisms and miRNAs, is subjected to extensive transcriptional and post-transcriptional modulation, with supplementary regulation occurring at the mRNA level. Lambert et al. conducted in vitro research on putative microRNA-binding sites revealed that miR-421 downregulates ACE2, also modulates ACE2 expression through obstructing translation instead of degradation of mRNA transcripts.140, 141 Most of the investigational miRNA-based treatments are directed against the viral S protein-ACE2 receptor checkpoint.142, 143

SARS-CoV-2, as a respiratory infection, targets the airway and lung epithelial cells. After cleavage by TMPRSS2, the SARS-CoV-2 spike protein RBD gets activated and binds to ACE2 receptor of the host cells. Thus, Chauhan et al. suggested that for COVID-19 prevention and management, it is highly helpful to identify and deliver certain miRNAs, as they are responsible for engaging which in blocking the binding or inhibiting the activation of ACE2 or TMPRSS2.144

Hosseini Rad SM & McLellan identified four host miRNAs; hsa-mir-4464, hsa-mir-885-5p, hsa-mir-7107-5p, hsa-mir-1234-3p, which have entirely complementarity within the RBD region of S gene and are able to bind to that RBD. These miRNAs might be related to miRNA-mediated virus attenuation technology applied on SARS-COV-2, since their target sequences are in the critical ACE-2 targeting region, however SARS-CoV-2 target cells do not express these miRNAs.145

Patients with diabetic and cardiac diseases using ACE2 enhancement drugs are more susceptible to infection with SARS-CoV-2. hsa-miRNA-27b, which is also correlated with SARS-CoV-2 Indian origin variant genome, regulating ACE2. The synthesis and replication of viral proteins occur in the host cell. The miRNAs can prevent the target mRNA's translation into the proteins.146-