4  Discussion
Sialic acids linked to glycoproteins and gangliosides are used by a broad range of viruses as receptors and/or attachment factors for cell entry [10]. These viruses include major human pathogens affecting the respiratory tract, such as influenza [21] and coronaviruses [22,23]. The attachment to sialic-acid-containing cell surface structures is mediated by receptor-binding proteins that belong to the viral spike. In the case of coronaviruses, this function is fulfilled by the S glycoprotein [9,24]. SARS-CoV and SARS-CoV-2 interact with the ACE-2 protein, which has been identified as a key determinant of the contagiousness of viruses [8]. However, considering the increased transmissibility of SARS-CoV-2 compared with SARS-CoV, binding to ACE-2 alone might not be enough to ensure robust infection of the upper respiratory tract. Thus, it is likely that SARS-CoV-2 might also bind to other cell surface attachment factors, such as sialic-acid-containing glycoproteins and gangliosides. Consistent with this notion, it has been shown that depletion of cell surface sialic acids by neuraminidase treatment inhibited MERS-CoV entry of human airway cells [25]. These data, which provide direct evidence that sialic acids play a critical role in human coronavirus attachment, broaden the therapeutic options to block the replication of SARS-CoV-2 that is responsible for the COVID-19 pandemic.
Few drugs have shown consistent antiviral efficiency in vitro together with reported efficiency in patients infected with SARS-CoV-2 [3,12]. Of these, CLQ is of interest as its chemical structure is based on a combination of cationic nitrogen atoms and aromatic rings. Both features have been shown to be key determinants of the recognition of sialic acids and gangliosides by proteins [20,26]. Modelling approaches have been used successfully to decipher various molecular mechanisms of protein–sugar interactions accounting for the interaction of virus [27], bacteria [28], membrane [13] and amyloid proteins [20] with cell surface glycolipids. This in-silico strategy was applied to unravel the molecular mechanisms underlying the antiviral mechanisms of CLQ and CLQ-OH against SARS-CoV-2 infection. First, it was shown that CLQ and CLQ-OH bind readily to sialic acids with high affinity, including the typical 9-O-SIA subtype recognized by coronaviruses [23]. Next, it was shown that CLQ and CLQ-OH also bind to sialic-acid-containing gangliosides. Based on these data, the drugs may also recognize the sialic acid residues of glycoproteins. Further studies will help clarify this point.
This molecular modelling study has identified a new type of ganglioside-binding domain in the NTD of the SARS-CoV-2 S protein. This ganglioside-binding domain consists of a large flat interface enriched in aromatic and basic amino acid residues. It covers a stretch of 52 amino acid residues (111–162), which is longer than all linear ganglioside-binding domains characterized to date [29]. However, the new SARS-CoV-2 motif is organized into three distinct regions, including a central aromatic domain and two terminal basic domains (Fig. 10). Thus, this motif displays the typical features that determine optimal binding to gangliosides (i.e. CH-π stacking and electrostatic interactions).
A major outcome of this study is the demonstration that CLQ and CLQ-OH display molecular groups that fully mimic the way in which the S protein binds to gangliosides. Two CLQ (or two CLQ-OH) molecules can bind simultaneously to the polar head group of ganglioside GM1. Interestingly, these simulations indicated that CLQ-OH is more potent than CLQ, in line with the reported increased antiviral activity of CLQ-OH against SARS-CoV-2 [30]. Once bound to GM1, the drugs prevent any access to the Glc and GalNAc units of the ganglioside, which are the critical binding residues for Phe-135 and Asn-137, respectively. This amino acid dyad, as well as all the other residues that mediate ganglioside binding by the SARS-CoV-2 spike, is fully conserved among clinical isolates worldwide. It is also conserved in the bat RaTG13 isolate, which reinforces the hypothesis of bat-to-human transmission. From an epidemiological point of view, it can be hypothesized that the evolution of this motif has conferred an enhanced attachment capacity of human coronaviruses to the respiratory tract through improved S–ganglioside interactions.