Targeting the glycans: A paradigm for host‐targeted and COVID‐19 drug design

Abstract There is always a need for new approaches for the control of virus burdens caused by seasonal outbreaks, the emergence of novel viruses with pandemic potential and the development of resistance to current antiviral drugs. The outbreak of the 2019 novel coronavirus‐disease COVID‐19 represented a pandemic threat and declared a public health emergency of international concern. Herein, the role of glycans for the development of new drugs or vaccines, as a host‐targeted approach, is discussed where may provide a front‐line prophylactic or threats to protect against the current and any future respiratory‐infecting virus and possibly against other respiratory pathogens. As a prototype, the role of glycans in the coronavirus infection, as well as, galectins (Gal) as the glycan‐recognition agents (GRAs) in drug design are here summarized. Galectins, in particular, Gal‐1 and Gal‐3 are ubiquitous and important to biological systems, whose interactions with viral glycans modulate host immunity and homeostatic balance.


| INTRODUC TI ON
of the SGP S has been identified which was found fully conserved among clinical isolates of SARS-Cov-2 worldwide. 1,3,5,6 Out of a dozen or more potential drugs to treat  infection are already in clinical trials. One of them is the wellknown antimalarial drug, chloroquine (CLQ), and its alternative | 5843 POURRAJAB hydroxychloroquine (CLQ-OH), which have attained notifications in clinics. In vitro and infected patients, both compounds displayed that can efficiently inhibit severe acute respiratory system Cov-2 (SARS-Cov-2) infection. The mechanism that has been revealed until now is through increasing the low pH of intracellular endosomes (required for virus fusion), interfering with the host glycosylation process, as well as, masking sialoglycans at the cellular surface where blocking virus-host receptor interaction and viral entry (needed for subsequent viral replication and syncytial formation). [7][8][9] Identification of this new mechanism of action of CLQ and CLQ-OH (interfering with the host glycosylation and masking sialoglycans) provides supports for the use of these repositioned drugs to cure patients infected with SARS-Cov-2. 3,5 There are reports for the prevention of influenza by intranasal targeting of host receptors using engineered proteins masking the cell surface glycans. Studies have designed multivalent biologics, engineered carbohydrate-binding agents (CBAs) specific for sialic acid that use sialic acid as a receptor and mask the cell surface receptors. The biologics mask the cell surface receptors recognized by the influenza virus (IV) and could protect mice from a challenge with a lethal dose of 2009 pandemic H1N1 IV. However, there was sufficient virus replication to establish an immune response, potentially protecting the animal from future exposure to the virus. [10][11][12] Additionally, there are reports for antiviral activity of iminosugars (eg in the case of HIV-1), whose mechanism of action is through inhibiting ER glucosidases and glycosylation process of cell surface receptors. The ER-glucosidase activity is required for glycosylation of cellular and viral receptors. The iminosugars potently inhibit the release of infectious virion particles and the number of infected cells, however, a barrier to their clinical application, is that they show off-target effects whereby impeding host gut glucosidases leading to diarrhoea and abdominal pains. Additionally, viral spike glycoproteins (SGPs) are glycosylated by the host cell as able to hijack cellular glycosylation. 6,13 It is, therefore, possible that by the inhibition of the host cell glycosylation process or interfering with glycan interactions at the cellular surface, the SGPs are no longer able to bind to the host glycans. 3,5 Some mechanisms and features show the important role of glycans in viral infection, as well as, in the development of prophylactics and treatments especially for the development of new vaccines. 6,12,13 Furthermore, the host-targeted approach could provide a frontline prophylactic that has the potential to protect against any current and future respiratory-infecting virus and possibly against other respiratory pathogens.

| RECEP TOR RECOG NITI ON MECHANIS MS OF CORONAVIRUS E S
The large spike (S) glycoprotein (GPS) (~200-kDa) on the envelope of CoV virions binds to host-specific receptors whereby mediate virus entry, tissue tropism, and host range; and can affect virus virulence.
CoV GPS is class I viral fusion protein, like IV hemagglutinin (HA), human immunodeficiency virus-1 (HIV-1) envelope GP (Env), Ebola virus GP and paramyxovirus F GPs. 14,15 There are structural similarities between GPS of SARS-CoV-2, SARS-CoV and MERS-CoV that suggest the possibility for them to share receptors. In SARS-CoV and SARS-CoV-2, the GPS exhibits similarity in structure and sequence of the RBD and the identity in residues critical for ACE2 binding, the majority of which are either highly conserved or shared similar side chain properties. SARS-CoV and SARS-CoV-2 GPS exhibit an overall sequence identity of about 76%, with only 51% identity in NTD, 64% in RBD and 90% in the S2 fusion domain. [16][17][18] In comparison, SARS-CoV-2 and MERS-CoV share lower sequence identity in their spikes (~35%), RBDs, or RBM, and yet they recognize the same receptor dipeptidyl peptidase-4 (DPP4). The GPS of the SARS-CoV-2 gene is longer than SARS-CoVs and there are three short insertions in the N-terminal domain, which may confer a sialic acid-binding activity like as MERS-CoV GPS. 1,[16][17][18] CoV GPS is separated into two subunits, called S1 and S2, by cellular proteases (host or producing-cell). It first binds to a receptor on the host cell surface through its S1 subunit and then fuses viral and host membranes through its S2 subunit. 14,15 The S1 subunit of GPS contains two distinctive domains, the N-terminal domain (S1-NTD) and the C-terminal domain (S1-CTD), both of which can function as receptor-binding domains (RBDs). S1-NTD is a carbohydrate-binding domain, (CBD) responsible for recognizing and binding to sugars, while S1-CTD binds protein receptors such as dipeptidyl peptidase-4 (DPP4), angiotensin-converting enzyme 2 (ACE2) and APN, and function as the functional-binding domain (FBD). [1][2][3]19,20 In the case of the SARS-CoV-2 and MERS-CoV receptor, the human GP CD26 or DPP4 is a key immune-regulatory factor for hijacking and virulence and is widely expressed on epithelial cells in the kidney, alveoli, small intestine, liver, and prostate, and activated leucocytes. These viruses might deregulate antiviral T cell responses due to the stimulation of T cell apoptosis. 1,21 In the case of the SARS-CoV receptor, the membrane-associated aminopeptidase ACE2 is highly expressed in the lung, vascular endothelia, renal and cardiovascular tissue, and epithelia of the small intestine and testes. 22 ACE2 plays a crucial role in elderly people by regulating the RAS via opposing the actions of Ang II because it has a beneficial role in many diseases such as hypertension, diabetes and cardiovascular disease. 11 Through S1-CTD, SARS and MERS-CoVs bind to ACE2 and DPP4, respectively, while the novel SARS-CoV-2 S1-CTD seems able to utilize both receptors to infect host cells. 1,2,20,23 The S1 subunit is further divided into four β-rich subdomains, designated as A, B, C and D, with subdomains A and B acting as RBDs in different coronaviruses. 17,24 In SARS-CoV-2 and MERS-CoV, the S1B subdomain recognizes the host receptors ACE2 and DPP4, respectively, while viral initial binding and primary attachment is through the interaction between the S1A subdomain and host α2,3/6-linked Sia.
Herein, the S1B subdomain has a major influence on the virus-host range and tissue tropism, and its receptor tissue localization varies between species. 1,2,5,25 Whereas, the S1A subdomain which participating in the early phase of viral attachment and infection shares conserved structural folds and sugar-binding sites in different viral lectins. 4,5,24,25 Studies reveal that the S1-NTD resembles the structural folds and sugar-binding sites of human galectin-3 (Gal-3), 1,20,24 whereas the S1-CTD is aggressively evolving and exploits novel protein receptors to determine the viral specificity of receptor binding and host tropism. 14,15,26 It appears to be a successful strategy for viruses to share a lectin structure as a sugar receptor and acquire RBDs with novel specificity or altered specificity for a protein receptor. 14,16,25,27

| Glycans enrolled to participate in virus-host interaction
Several articles have focused on glycosylation patterns that oc- Additionally, alteration of N-linked glycans on the surface of host cells impairs their ability to support the transduction of an infectious virus such as CoVs and IV. 6,7,29 In the case of virus infectivity, the glycosylation pattern can be compositionally different across different species and individuals, an important feature of viral interspecies/ inter-individual transmission potential where influences viral tropism. 6,13 Additionally, differential organization of viral glycosylation across viral GPs influences not only individual glycan compositions but also the immunological pressure across the viral protein surface that put them principal targets of the neutralizing humoral immune response. 17,23 In SARS-CoV-2, SARS and MERS-CoV, the overall structure of GPS can resemble each other, but the density and compositions of glycan shielding are markedly different (Figure 1). Marked differences exist between the residues 40-318 located at the subdomain S1A in S1-NTD. Moreover, densely glycosylated spike proteins be- The SGP S structure can be divided into the S1 and S2 domains, and the structural domains in the spike protein are located in the order (from C to the N terminus) as: transmembrane (TM), heptad repeats (HRs) in the S2 domain, C-terminal domain (CTD), and N-terminal domain (NTD) in the S1 domain as well as the signal peptide (SP). The S1-CTD is divided into three subdomains SD-SB, while S1-NTD contains subdomain SA. SD-SA is accounted as receptor-binding domain (RBD). (B-D) Glycosylation sites for oligomannose, hybrid and complex-type N-glycans are coloured in green, blue and red, respectively. Unique glycosylation sites for SARS-CoV-2 are dashed in violet, green and blue, the dual recognition of gangliosides and angiotensin-converting enzyme-2 (ACE-2) by SARS-CoV-2 spike (S) protein. The viral protein displays two distinct domains, the tips of which are available for distinct types of interactions (S1-NTD). The receptor-binding domain (S1-CTD) binds to the ACE-2 receptor, and the N-terminal domain (NTD) binds to the ganglioside/sialoglycan-rich domain of the plasma membrane. Lipid rafts, which are membrane domains enriched in gangliosides and cholesterol, provide a perfect attractive interface for adequately positioning the viral S protein at the first step of the infection process sulphates provides a measurable effect on virus adhesion and is required for viral attachment. [33][34][35][36] Tissue-specific glycosylation is a key determinant of interspecies viral transmission potential and can lead to the targeting of vulnerable tissues within a host. For example, the capacity of the avian influenza virus (AIV) HA to interact with both α2,3-and α2,6-linked Sia has been shown to facilitate cross-species barrier of the virus from birds to humans. Besides, the differential expression of these Sia structures between the upper and lower respiratory tracts in humans can shape the distribution of influenza infection within an individual. 6,13,28 Sialic acid-widespread distribution of the human respiratory tract predisposes as a potential receptor and binding site for human and zoonotic viruses, as well as, their transmission. 37 There is enough evidence that shows sialoglycans play a critical role in human zoonotic virus biology, and broaden the therapeutic options to block the replication of viruses attacking the respiratory system such as pandemic COVID-19 and AIV epidemics. 1,7,10,13 Remarkably, shifting in the host of swine IVs during their adaptation is mediated by shifts in the HA Sia-binding affinity, just as an early adaptation step of avian H9N2 strains. Only two mutations in the H9N2 AIV HA (at positions A190V and T212I), led to its adaptation to the respiratory epithelium of pigs and enhances Sia-binding activity and virulence. 29,38 Or, a single point mutation in the highly patho- Among the enveloped viruses that recognize Sia-containing receptors are members of the families Coronaviridae, Paramyxoviridae and Orthomyxoviridae. 4 CoV GPSs share structural features in their sugar-binding sites (S1-NTD) resembling the galectin-3 folding structure in humans. 24  The key saccharide-binding residues are located in the S1-NTD subdomain A which determines the host tropism and viral attachment at the early phase of viral infection. 2,3,24 These residues should be strictly conserved in the Gal3-like fold of domain A (eg Tyr162, Glu182, Trp184 and His185 in BCoV) that involved in interacting with sialoglycans in a similar way. 26 The sialyl-lactose binding domain of the COVID-19 agent is identified at residues 111-158, which is fully conserved among clinical isolates worldwide, and shows attachment to membrane lipid rafts, and facilitates contact with the ACE-2 receptor ( Figure 2). 3,41 The increased transmissibility of the COVID-19 agent in comparison with MERS and SARS-CoV is addressed to its engagements with sialoglycans in lipid rafts; glycoproteins and gangliosides. 3,8 Human

sialoglycan distribution and viral infection
The N-glycome of the human lung contains extremely large complex-type N-glycans with linear poly-N-acetyllactosamine (PL) n extensions, which are predominantly terminated in α2,3-linked Sia. There are smaller N-glycans lack PL while are enriched in α2,6linked Sia. There are also large glycosphingolipid-glycans, which also consist of linear PL, terminating in mainly α2,3-linked Sia. 42 The  Increased expression of Siglecs in human alveolar monocytes is accompanied by an enhanced intracellular level of IL-1β and IL-10.

F I G U R E 3 Engagement of innate immune receptors causes phagocytosis and engulfment of the virus which facilitates and/or augments infection, independently of peptidase receptors. Virus engulfment by phagocyte cells and innate immune infection through C-type lectins. A, Virus binding and infecting an erythrocyte through gangliosides/sialogylcans on the erythrocyte, 1) Erythrocyte infection leads to changes in the glycoprofile and influences 2 & 3) phagocytosis of the infected cells, through 3) the asialoglycoprotein receptors (sialic
Siglec-1 interacts specifically with sialylated viral envelope proteins and gangliosides, but not with host membrane gangliosides. This mechanism likely leads to an increased viral capture and thus prolonged exposure to the cell surface receptors CD4 and CCR5 on the macrophage surface ( Figure 3). 28,31,32 Erythrocyte membranes are constituted with lipids, some of which are also known to promote viral fusion in T cells and virus enrichment. These lipids include sphingolipids and their glycosylated derivatives that form part of the blood group milieu of the red blood cell. In reports, lipid rafts comprising of glycosphingolipids and cholesterol were considered to be sufficient for viral fusion without the need for co-receptors. A Sia-binding virus that manages to make its way into the bloodstream would immediately encounter this extensive cell surface and can bind to RBCs wherein getting appropriate mechanisms to allow latter invasion and replication. 47,48 Erythrocytes are enriched in α2,6/3-Sia residues whereby can be Importantly, the ganglioside-binding domain in SARS-CoV-2 S1-NTD could selectively interact with the lipid rafts of human erythrocytes and enrich infectious CoV virions in the body. The tip of S1-NTD contains ~290 amino acids, particularly targets gangliosiderich microdomains in cell membranes, such as lipid rafts in RBCs.
There is an over-representation of aromatic and basic residues in the 100-175 region of S1-NTD including segment; 129-K VCE F Q F CNDP F LGV YY H K NN K S W MESE FR -158, which also contains Gly, Pro, and/or Ser residues. CLQ and CLQ-OH drugs by binding to ganglioside-binding motifs can efficiently prevent viral S1-NTD interaction with lipid rafts of the host cell ( Figure 4). 3,32 Virus binding to erythrocytes leads to erythrophagocytosis (via asialyllactose-binding receptors or DC-SIGN or L-SIGN) ( Figure 3).

As a result, SARS-CoV-2 infection causes reduced levels of RBCs
and haemoglobin in the blood, while leads to increased plasma concentration of free iron and hyperferritinemia. 9

| TARG E TING G LYC AN S IN THER APEUTI C S DE VELOPMENT IN VIR AL B I O LO GY
Accordingly, the glycom of the viral envelope and the host cellular surface has a crucial role in enabling efficient transmission of the pathogen and/or entry into its susceptible target cells. 6,28 Antiviral agents that interact with the surface glycans may, therefore, compromise the efficient entry of the virus into its sus- Collectively, data indicate that Gal-1 preferentially has anti-IV activity by binding to viral HA and inhibiting its infectivity. Thus, Gal-1 may be further explored as a novel therapeutic agent for influenza. 12  virus to the body is inhibited. In masking the Sialo-galacto-glycans, the spike GPs cannot bind the host receptors that could block virus attachment and human respiratory epithelium infection. 15,24,40,58 From the literature, it is interpreted that intranasal administration of Gal-3 before or during the early phase of virus infection would reduce viral load, accompanying inflammation, tissue damage and mortality in the susceptible host. 11,12,59 In a mouse model for IAV infection, Gal-3 was discharged on the airway epithelia and caused the release of pro-inflammatory mediators that induce the expression of antiviral genes, including IFNs (β & γ), to inhibit IAV replication. 11 As intranasal administration of recombinant Gal-1 in infected mice led to reducing viral load, accompanying inflammation, tissue damage and mortality in the murine model infected with influenza virus. 15,58 The protective effect of Gal-3 at the airway epithelia against roles that they play in the innate immune system. The diverse, at times, paradoxical roles that they may play in the innate immune system are related to their different interactions at the molecular level which is dependent on their structure-activity relationship and needs to be dissected. 5,6,11,21 Thereby, surface glycans as an important class of microbial signature that is recognized by a variety of host cell lectins can function as both recognition and effectors against viral and bacterial infection. In therapeutic design, surface glycans can 'subvert' viral adhesion to or cause to gain entry into the host cells. Herein, host-intranasal administration of a galectin, before or during the early phase of virus infection, is proposed that recognize specific glycan ligands and subverting viral adhesion and promoting host immune response. Data suggest that galectins (here in Gal-1 and -3) can potentially be used as viral therapeutic targets or antagonists.

CO N FLI C T O F I NTE R E S T
There is no conflict of interest to be declared.