The expression of hACE2 receptor protein and its involvement in SARS-CoV-2 entry,pathogenesis,and its application as potential therapeutic target

1 Introduction

Idiopathic pneumonia cases in Wuhan, Hubei province, China were reported to the World Health Organization on 31st of December 2019 [1]. The pathogen was then reported to be a novel coronavirus designated severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) that causes Corona virus disease 2019 (COVID-19) [1, 2]. It was a severe blow to the world since the disease was transmitted like a wave all the way to the Americas from China. Therefore, WHO declared the COVID-19 outbreak a “Public Health Emergency of International Concern” [2]. As of 21^st of June 2021, 178 million confirmed COVID-19 cases and 3.86 million deaths have been reported by WHO [2].

The initial spread of the virus was linked to Huanan seafood market in Wuhan resulting in subsequent human to human transmission [3]. Bats were believed to be the natural host of SARS-CoV-2 virus that crossed the species barriers via an intermediate host to infect humans. It was thought that beside pangolins and snakes, turtles (Pelodiscus sinensis, Chrysemys picta bellii, and Chelonia mydas) are also believed to be a potential intermediate hosts of SARS-CoV-2 [4].

SARS-CoV-2 is the seventh discovered coronavirus to infect humans. Coronaviruses are members of the Coronavirinae subfamily that ramify from the family Coronaviridae (International Committee on Taxonomy of Viruses). Coronavirinae subfamily comprises 4 genera (Alphacoronavirus, Betacoronavirus, Gammacoronavirus, and Deltacoronavirus). Only 7 viruses of Alphacoronavirus, and Betacoronavirus genera infect humans causing respiratory illness. However, SARS-CoV, MERS-CoV, and SARS-CoV-2 are three tremendously pathogenic ß coronaviruses that cause severe respiratory syndrome in humans [5].

SARS-CoV-2 is commonly known to mediate entry into the host cells through the ACE2 receptor [6]. The ACE2 receptor is known to be expressed on the alveolar epithelial cells in the lungs. Binding of the viral spike protein to the ACE2 receptor leads to subsequent fusion of the viral envelope and host cell membrane thereby allowing successful viral entry into the host cells [7, 8]. This binding of the virus to the ACE2 receptor leads to the endocytosis of lung alveolar epithelial cells causing irreversible damage to the lung tissue [9]. Following initial viral entry, cleavage between the S1 and S2 subunits of the viral glycoprotein takes places. This step is regulated by the receptor transmembrane protease serine 2 (TMPRSS2) that is a member of the Hepsin/TMPRSS subfamily [10]. Detachment of the S1 subunit from S2 causes the latter to undergo a conformational change that promotes and completes the fusion between the viral and host-cell membrane [11]. This fusion is a requisite for viral internalization, release of the viral content, replication, and subsequent infection of other cells. Therefore, due to the critical role hACE 2 binding with SARS-CoV-2, it is postulated that targeting of hACE2 by various mechanisms such as utilizing ACE inhibitors, blocking of S protein binding to ACE-2 by soluble recombinant human ACE2 protein, blocking ACE-2 receptor by antibodies or small molecules, inhibition of transmembrane protease serine 2 (TMPRSS2) activities, and generation of spike based protein vaccine could serve as a helpful therapeutic strategy.

In this review we aim to focus on the tissue distribution of hACE2 and its association with risk factors, structural binding mechanism of SARS-CoV-2 S1subunit binding to human ACE2 that facilitates viral internalization, possible immunogenic sequences of spike protein, effect of ACE2 polymorphism on viral binding and infectivity/ susceptibility to disease. Furthermore, targeting of ACE2 will be discussed to understand its role in therapeutics.

2 Tissue distribution of human angiotensin converting enzyme 2 (hACE2)

It is of importance to elucidate the expression of ACE2 surface receptor in human organs to identify the potential routes of infection and possible passage processes of SARS-CoV-2 throughout the body. Although ACE2 mRNA expression was found in about 72 human tissues, ACE2 protein receptor was found to be remarkably expressed in limited number of cell types [12, 13]. ACE2 receptor was found to be localized within the cilia of epithelial cells lining the airways as well as in the alveolar epithelial cells, thus suggesting the initial site of SARS-CoV-2 entry via respiratory transmission [13, 14]. Furthermore, ACE2 surface receptor within the digestive system is abundantly expressed in salivary gland, epithelial cells of the oral mucosa, and small intestine enterocytes, which might facilitate viral entry and contagiousness [13, 15, 16]. ACE2 receptor was also found to be highly expressed in the cholangiocytes of the liver as well as in the gallbladder epithelial cells [13, 17]. In addition, ACE2 expression was observed across several tissues in the kidney as in nephron epithelial cells, epithelial cells, endothelial cells, mesangial cells, and podocytes thus revealing kidneys susceptibility to COVID 19 infection and its disease-related complications [18, 19]. Reproductive system of both males and females showed high ACE2 expression particularly in the testes, spermatids, ovaries, fallopian tubes, placenta, uterus, and vagina thus revealing another mode of viral transmission [20, 21]. Moreover, ACE2 expression in the superficial ocular layers of conjunctiva, limbus, and cornea have shown high SARS-CoV-2 tropism in COVID 19 patients thus proposing novel routes of viral entry and transmission [22–24]. It is astonishing that adipose tissue comprises high ACE2 expression among all tissues which in turn explains why obesity is considered to be one of the risk factors for SARS-CoV-2 infection and disease progression [25]. On the other hand, high ACE2 expression in vascular endothelial cells and arterial smooth muscle cells could potentiate spreading of the virus to other body parts [13].

2 Association between risk factors/ACE2 expression and susceptibility to SARS-COV-2

3 Structural binding of human ACE2 receptor to S1 protein

ß Coronaviruses are enveloped viruses with four structural proteins known as Membrane glycoprotein (M), Spike protein (S), Nucleocapsid protein (N), and Envelope protein (E) [75]. Virion surface proteins such as the M, E, and S proteins in SARS-CoVs are well conserved [76, 77]. It is thought that E and M proteins are responsible for viral entry, replication, and particles assembly within the host cells [78]. However, viral infection is initiated via binding of viral particles to cellular surface receptors of the host cells where Spike glycoprotein (S) of SARS-CoV-2 virus recognize and bind to human angiotensin converting enzyme 2 (hACE2) [76].

Spike protein priming is essential for host cell entry and virus infectivity of SARS-CoV-2. Host TMPRSS2 mediates priming of the trimeric S protein thus cleaving it into S1 and S2 subunits at the S1/S2 furin-like multibasic cleavage site that harbors multiple arginine residues indicating high cleavability [34, 79]. The S1 subunit of the spike protein was found to be responsible for binding host cell receptor hACE2 while S2 subunit contributes to viral and cellular membranes fusion [35, 80]. S1 subunit of the spike protein comprises both, C terminal domain (CTD) and N terminal domain (NTD). SARS-CoV-2 virus recognizes the hACE2 receptor via the CTD also known as receptor binding domain (RBD) [81].

Similar to other studied beta coronaviruses CTD structure shows two conserved subdomains [82]. One is the core subdomain that is composed of 5 inverse parallel beta strands (ß1, ß2, ß3, ß4, and ß7) structure with a disulfide bond between ß2 and ß4 strands. The other subdomain is a conserved extended insertion of (ß5 and ß6 strands, ∝4 and ∝5 helices, and loops) to the core subdomain forming the external subdomain that contains the receptor binding motif (RBM) which holds most of the binding residues of SARS-CoV-2 and hACE2 [76, 83].

There are 3 pairs of cysteine residues (Cys336 –Cys361, Cys379 –Cys432, and Cys391 –Cys525) in the RBD forming disulfide bonds to help in stabilizing the ß sheet structure and 1 pair (Cys480 –Cys488) that facilitates connecting loops in the distal end of the RBD (Fig. 1a) [83].

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Fig. 1

SARS-CoV-2/hACE2 structure and detailed binding: a. Overall SARS-CoV-2/hACE2 complex monomer. (b, c, d, e, and f) detailed structure of SARS-CoV-2 binding to human ACE2 receptor [76]. Human angiotensin converting enzyme (hACE2) in green, spike protein (S1) of SARS-CoV-2 core subdomain in orange, and spike protein (S1) of SARS-CoV-2 receptor binding motif (RBM) in blue.

The C terminal receptor binding domain of SARS-CoV-2 S1 subunit binds the N terminal subdomain I of hACE2 [84]. The N terminal subdomain I of hACE2 has 2 lobes where the external subdomain of SARS-CoV-2 binds the small loop, with a concave structure of RBM accommodating the N terminal helix of the hACE2 [83].

Two lysine residues (31 and 353) on hACE2 are essential for binding to SARS-CoV-2 which are known as binding hotspots found in the SARS-CoV-2 / hACE2 interface. Hotspot Lys31 forms a salt bridge with Glu35 and hotspot Lys353 forming a salt bridge with Asp38. Both salt bridges are weak due to the relatively long distance between both residues. However, the energy of salt bridges is enhanced when buried in a hydrophobic environment upon viral binding. This process is facilitated via hotspots interactions with adjacent RBD residues. The salt bridge in hotspot 31 breaks apart and each of Lys31 and Glu35 residues forms a hydrogen bond with Gln493 from SARS-CoV-2 RBM (Fig. 1b). In addition, the Asn501 residue within the SARS-CoV-2 RBM forms a hydrogen bond to the main chain of RBM to slightly stabilize hotspot Lys353 with the presence of Lys353-Asp38 salt bridge (Fig. 1c) [85]. Several hydrophilic residues within SARS-CoV-2 / hACE2 interface were implicated in forming strong hydrogen bonds and salt bridge interactions. Those polar interlinkages include residue Ala475 of SARS-CoV-2-CTD interacting with Ser19 on hACE2, Asn487 with Gln24 (Fig. 1d), Glu484 with Lys31 (Fig. 1e), and Tyr453 with His34 (Fig. 1f) [76, 85]. The Phe486 and Tyr 489 of SARS-CoV-2 RBM interacts with Phe28, Leu79, Met82, and Tyr83 residues on hACE2 forming a hydrophobic pocket thus increasing the affinity of the virus to hACE2 receptor [76, 83, 85].

The affinity between the ligand (SARS-CoV-2 S1) and the receptor (hACE2) reported in several studies by measuring the “dissociation constant K_d” [76, 83]. Different dissociation constant values were reported due to variations in materials and methods used. K_d Values were ∼15 nM, 44.2nM, 94.6 + 6.5 nM, and 4.7 nM [76, 83, 85, 86].

3.1 Comparison between SARS-CoV-2 and SARS-CoV binding affinity to ACE2 receptor

Overall studies revealed approximately four folds higher binding affinity between SARS-CoV-2 S1 subunit and hACE2 compared with SARS-CoV/hACE2. Cryo electron microscopy and X-ray crystallography techniques facilitated the discovery of viral receptor binding domain/ human ACE2 complex structure [76, 83, 85]. Although, receptor binding mode was in general similar in both ligands; SARS-CoV-2 RBM forms more atomic interactions with hACE2 thus increasing the binding affinity compared to the ligand between SARS-CoV and ACE2 receptor. The ß5 and ß6 loop harbors the most variable region that facilitates stronger ionic and aromatic-aromatic interactions between SARS-CoV-2 and hACE2 compared to SARS-CoV loop [76].

When comparing the molecular interaction within the binding interface of the viral CTD, among the 24 residues of hACE2 that forms van der Waals forces (vdw) with residues of the virus CTD, 15 residues exhibit more contacts with SARS-CoV-2 CTD. Moreover, there are more residues on SARS-CoV-2 that binds hACE2 compared to SARS-CoV (21 versus 17) that can form vdw interlinks (288 versus 213) in addition to higher hydrogen (H) bonds interactions with SARS-CoV-2 than SARS-CoV (16 versus 11) [76]. The location that harbors the residues Leu455/Tyr442, SARS-CoV-2 Leu455 as SARS-CoV Tyr442 interact with the same residues on hACE2 Asp30, Lys31, and His34 (Fig. 2-a1) [76]. Hot spots Lys31 and Lys353 are critical in the binding of coronavirus RBD to hACE2. Lysine residues are stabilized where Asn501 on SARS-CoV-2 stabilizes hotspot 353/ Gln493 stabilize hotspot 31 which subsequently increase binding affinity of the novel virus [8]. Asn501 of SARS-CoV-2 and Thr487 of SARS-CoV, both interact with Tyr41, Lys353, Gly354, and Asp355 residues in hACE2 receptor (Fig. 2-a2) [83].

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Fig. 2

Comparison between SARS-CoV-2 and SARS-CoV binding hACE2 receptor. a. Interactions within the RBM of SARS-CoV-2 and SARS-CoV with hACE2 receptor. b. Variations in the K417/V404 position [76, 120].

Phenylalanine residue Phe 486 in SARS-CoV-2 RBD interacts with residues (Gln24, Leu79, Met82, and Tyr83) on hACE2 forming a hydrophobic pocket that increases the binding affinity while in SARS-CoV the corresponding residue is a leucine Leu472 side chain that provides a weaker interaction with (Leu79 and Met82) residues on hACE2 receptor (Fig. 2-a3) [76, 83, 85].

Glutamine residue Gln493 in SARS-CoV-2 interacts with Glu35, Lys31, and His 34 on hACE2 and forms a hydrogen bond with Glu35 while in SARS-CoV corresponding residue Asn479 interacts only with His34 on hACE2 (Fig. 2-a4) [83].

Finally, there is a unique residue Lys417 harbored in an external location outside SARS-CoV-2 RBM that forms a salt bridge link with hACE2 Asp30 (Fig. 2b) [83].

Coronavirus entry into host cells is mediated by the transmembrane spike (S) glycoprotein which plays a major role in the stimulation of the anti-viral immune response [87]. This S glycoprotein includes 1255 amino acids (aa) [GenBank submission ID: AAP13441.1] [88] in SARS-CoV and 1273 aa in SARS-CoV-2 [GenBank submission ID: QHD43416.1][89]. In addition, S glycoprotein comprises two functional subunits; S1 and S2 subunits, which are respectively responsible for the binding to the host cell ACE2 receptor and for the fusion of the viral and host cellular membranes. Indeed, the S1 subunit contains the Receptor Binding Domain (RBD) which mediates the virus-host cell interaction through binding to the cell’s ACE2 receptor [35]. Interestingly, it has been demonstrated that SARS-CoV and SARS-CoV-2 S glycoproteins show 25.2%dissimilarity in the amino acids sequences [90]. In particular, a difference of 28%was observed at the Receptor Binding Domain residues [91]. Indeed, this RBD is located at the 318–569 aa sequence in SARS-CoV [GenBank submission ID: AAP13441.1][88] and at the 329–538 aa region in SARS-CoV-2 [GenBank submission ID: QHD43416.1][89]. This structural divergence between the two S glycoproteins might trigger changes in the antigenic properties of the two viruses leading to a differential modulation of the specific immune response.

Indeed, it has been demonstrated that most of the antibodies against SARS-CoV do not have a cross-neutralization activity toward SARS-CoV-2 [92]. Yet, the SARS-CoV viral epitopes targeted by the neutralizing antibodies are situated at the RBD [93]. Furthermore, a potent ACE2-blocking anti-SARS-CoV neutralizing antibodies showed limited cross-binding and cross-neutralizing activities against SARS-CoV-2 [90]. In addition, two monoclonal antibodies m396, CR3014, developed against SARS-CoV did not show evident binding to SARS-CoV-2 RBD [92]. Another monoclonal antibody CR3022 against SARS-CoV has shown cross-reactivity against the RBD region of SARS-CoV-2 [92]. Crystal structure and interaction studies were performed using CR3022 and the SARS-CoV-2 RBD in order to determine the cross-reactive epitope at the RBD region in SARS-CoV and SARS-CoV-2. Interestingly, the mapping of the interacting region has shown that out of 28 residues, 24 (86%) are conserved between SARS-CoV-2 and SARS-CoV. Thus, the presence of highly conserved sequences would explain the cross-reactivity of CR3022 between SARS-CoV-2 and SARS-CoV. However, in vitro binding affinity and micro-neutralization assays revealed that the antibody had higher affinity to SARS-CoV RBD and do not show any cross-neutralization activity between SARS-CoV-2 and SARS-CoV [94]. Interestingly, a recent study has identified a novel human monoclonal antibody called 47D11 that neutralizes SARS-CoV and SARS-CoV-2 in vitro [95]. However, the neutralization assay was not confirmed in vivo and the mechanism of anti-viral activity is not fully described, yet it seems to be independent from receptor-binding interference [95].

The antigenic difference between SARS-CoV and SARS-CoV-2 spike glycoproteins has also been confirmed using patients’ serum. Interestingly, cross-reactivity of antibodies isolated from plasma samples of SARS-CoV and SARS-CoV-2 infected patients was mostly observed at the non-RBD regions and especially at the Spike protein ectodomain. Despite the presence of cross-reactivity in binding, cross-neutralization activity was not detected in any of the plasma samples [96]. For instance, the SARS-CoV-2 epitopes targeted by antibody repertoires remain uncharacterized. Therefore, the identification of SARS-CoV-2 immunogenic epitopes, that induce the secretion of specific neutralizing antibodies, will present an opportunity to develop targeted therapeutic strategies that would enhance the patients’ clinical prognosis and contain the spread of the virus. Indeed, immune response to viral pathogens is essential to recovery and an effective serotherapy may be the best tool to prevent the spread of COVID-19. However, the development of specific assays for the evaluation of the quality and the quantity of viral specific antibodies and the characterization of neutralizing antibodies is very critical to provide a successful passive immunization protocol such as convalescent plasma therapy. Convalescent plasma therapy is an immunotherapeutic strategy which was successfully used for the prevention and treatment of various infectious diseases including MERS and SARS-CoV. Indeed, plasma from patients who have recovered from COVID-19 contains high titers of neutralizing antibodies which would be used as promising treatment option for COVID-19 rescue [97]. For instance, Mapping of antibodies epitopes based on computational biology enabled the identification of formulated sequence-based epitopes scores in spike proteins of SARS-CoV and SARS-CoV-2 [98]. It has been shown that SARS-CoV-2 had significantly higher antibody epitope score compared with SARS-CoV. Moreover, sequence alignment studies revealed that the non-conserved regions had significantly higher antibody epitope score as well as higher surface epitope accessibility confirming that these domains of the spike proteins are more antigenic and more available for antibody recognition [98]. In addition, it has been shown that SARS-CoV-2 RBD presents higher affinity towards the ACE2 receptor than that of SARS-CoV. This is linked to the presence of a furin-like cleavage site restricted to SARS-CoV-2 [79].

Viral evolution has resulted in the appearance of new SARS-CoV-2 variants presenting mutations within the Spike glycoprotein, thus, increasing its binding affinity to ACE2 receptor. Indeed, a recent study reported that N501Y S protein mutation of the B.1.1.7 variant and L452R, E484Q mutations in B.1.617 have increased the binding energy of S protein/ACE2 binding when compared to wild type [99]. Altogether, different structural and functional characteristics could contribute to the increased infectivity of SARS-CoV-2 compared to SARS-CoV.

5 Potential therapeutic targeting of angiotensin-converting enzyme 2

ACE-2, having a critical role in the entry, replication, and pathogenicity of SARs-CoV-2, has been considered as a potential target for therapeutics. A number of clinical trials and pre-clinical studies focusing on various strategies including blocking of S protein binding to ACE-2 by soluble recombinant human ACE2 protein, blocking ACE-2 receptor by antibodies or small molecules, inhibition of transmembrane protease serine 2 (TMPRSS2) activities, utilizing ACE inhibitors and generation of spike based protein vaccine are underway worldwide (Table 1) [104].

Several studies have documented promising results targeting S protein binding of ACE-2 by using recombinant human ACE2 (rhACE2) [105–108]. In vitro studies with soluble rhACE2 in cell culture and engineered replicas of human blood vessels/kidneys in organoids have shown that rhACE2 forms high affinity binding with receptor-binding domain of SARS-CoV-2 [106]. This leads to neutralization of S protein on the SARS-CoV-2 surface thus reducing viral cellular entry and thereby protecting the host from developing severe ARDS or acute lung injury (ALI) facilitated by its peptidase-dependent function [105, 106]. On the other hand, ACE2 antibodies or peptides that selectively block the interaction site of SARS-CoV-2 with ACE2 have also been identified as potential therapeutic candidates [76]. In this, it has been proposed that the generation of ACE2 fusion protein with extracellular domain of the ACE2 to a human immunoglobulin G Fc domain could be used to facilitate SARS-CoV-2 Spike protein binding to this protein, thus blocking viral entry and replication [109, 110]. Furthermore, it has been suggested that if the effector function of Fc domain is, by some means, retained in the molecule, it could also allow recruitment of immune cells thus facilitating rapid activation of the host antiviral immune response and elimination of the virus. However, this ACE2-Fc strategy could have challenges such as mutating RNA virus may escape neutralization and increased levels of extracellular ACE2 could have implications in the host [111].

Recently, several studies have documented the role of antibodies interfering with Spike protein-ACE-2 interactions [112, 113]. A study by Ge et al., described the structural and functional analyses of monoclonal antibodies from SARS-CoV-2 infected individuals. The authors observed that an antibody, labelled P2C-1F11, closely mimicked the binding of receptor ACE2 and was found to exhibit the most potent neutralizing activity in vitro. Furthermore, in Ad5-hACE2-sensitized mice, this antibody conferred strong protection against SARS-CoV-2 infection and triggered rapid and extensive shedding of S1 from the cell-surface expressed spike glycoprotein indicating its potency for neutralization via disruption of the critical steps for SARS-CoV-2 cell entry[114]. Another study by Chen et al., generated an ACE2-targeting monoclonal antibody,3E8, by immunizing BALB/c mice with Fc-tagged human ACE2 protein and screening the sera for binding to ACE2 and blocking SARS-CoV-2-S1-subunit/ACE2 interaction. It was observed that antibody 3E8 was the most potent neutralizing antibody and efficient blocker of S1-subunit/ACE2 binding indicating that 3E8 is a promising therapeutic candidate targeting human ACE2 molecule [115].

Inhibition of transmembrane protease serine 2 (TMPRSS2) activity is also considered an important strategy for blocking viral entry and replication [107]. Therefore, serine protease inhibitors of TMPRSS2 such as Camostat mesylate have been utilized in in vitro studies to study the effect on SARS-CoV-2 [116]. A recent study showed that Camostat mesylate significantly reduced the infection of Calu-3 lung cells by SARS-CoV-2 [34]. Previous studies based on SARS and MERS have also shown similar results indicating that inhibition of TMPRSS2 may facilitate reduction of infection of lung cells and therefore may be a suitable therapeutic target for SARS-CoV-2 [117, 118]. Furthermore, development of synthetic inhibitors of TMPRSS2 can also be used to inhibit spike protein activation by TMPRSS2. However, limitations of this candidate is that molecular inhibitors of TMPRSS2 require validation of their specificity against other serine protease and need toxicological studies [119].

Utilizing ACE inhibitors (ACEIs/ARBs) is a topic of major interest for targeting of SARS-CoV-2. ACEIs/ARBs increase ACE2 activation subsequently leading to RAS deregulation. This deregulation may facilitate reduction in acute lung injury, heart injury, and renal damage induced by SARS-CoV-2 [53] However, clinical trials are on-going to provide evidence for the utility of ACE inhibitors for SARS-CoV-2.

Many studies are being published every day indicating various aspects related to therapeutic candidates against SARS-CoV-2. However, since COVID-19 is a new disease, any therapeutics targeting ACE-2 is deliberated until evidence of its efficacy via clinical trials sheds more light on its applicability.

ACE2 has been identified as a key mediator of entry and subsequent manifestation of pathogenesis in SARS-CoV-2. Several factors such as binding affinity, differential antigenic profiles in spike glycoprotein, ACE2 mutations and risk factors that influence susceptibility to disease have been postulated and implicated in influencing expression and functional activity of ACE2. Furthermore, identification of these factors and their importance in investigating the therapeutic efficacy of ACE2 in SARS-CoV-2 have been highlighted in this review. It is concluded that further large-scale studies keeping these factors in perspective would allow better understanding/management of SARS-CoV-2. Of utmost importance is to focus on studies to understand the role of ACE2 with respect to variation in disease outcome. Stratification of patients with respect to ACE2 and the disease dynamics can then be utilized for personalized therapy with input of sophisticated clinical algorithms to be used for predictive modelling. This approach would allow identification of novel avenues for therapeutic modulation for COVID-19 and future viral diseases.