1  Introduction
The emergence of the novel pathogenic SARS-coronavirus 2 (SARS-CoV-2) has led to a global pandemic disease referred to as coronavirus disease 19 (COVID-19) [1]. In such a health emergency, it is critical to find a cure and stop the pandemic. Among the potential solutions currently under investigation, a combination bitherapy consisting of the antimalarial drug, hydroxychloroquine (CLQ-OH) with the antibiotic, azithromycin (ATM) has received particular attention. Following initial reports from China indicating a potential effect of chloroquine (CLQ) [2], a preliminary clinical trial was implemented in France on a small cohort of COVID-19 patients [3]. A significant viral load reduction was observed in the 20 patients treated with CLQ-OH [3], a CLQ derivative with increased solubility in water and decreased toxicity [4]. With the aim of preventing bacterial super-infection, six additional patients in this trial also received ATM for 5 days. All these patients had undetectable viral load after 6 days [3]. Thus, although these promising data need clinical confirmation with more patients, CLQ-OH/ATM combination therapy already appears superior to CLQ alone as a first-line treatment for COVID-19.
Both CLQ-OH and ATM are repositioned drugs and their antiviral mechanism of action, particularly in combination, remains mostly unclear. In vitro studies have shown that CLQ-OH inhibits SARS-CoV-2 [4], [5], [6]. Far less is known about the antiviral effects of ATM, which has been suggested to interfere with influenza virus internalization [7]. Interestingly, CLQ is also considered as an inhibitor of endocytic pathways through an elevation of endosomal pH [8]. However, several reports indicate that CLQ could also prevent virus attachment through a direct effect on host-cell surface molecules [9,10].
The first step of the replication cycle in human coronaviruses is the attachment of the virus to the host plasma membrane, which is mediated by a membrane protein receptor, i.e., angiotensin-converting enzyme-2 (ACE-2) in the case of SARS-CoV-2 [11]. Moreover, coronaviruses are also dependent upon sialylated membrane components, such as gangliosides, which act as attachment cofactors within lipid raft membrane platforms [12], [13], [14]. As ACE-2 is localized in lipid rafts [15], SARS-CoV-2 infection requires specific targeting to these plasma membrane microdomains, where multivalent interactions between the spike protein and raft components can take place. In line with this notion, lipid raft disruption through cholesterol depletion resulted in a significant reduction of human coronavirus SARS-CoV infection [15]. The recent identification of a potential ganglioside-binding domain in the N-terminal domain (NTD) of the SARS-CoV-2 spike protein, and its potential role in membrane recognition [10], prompted this study of the molecular relationship between this domain, gangliosides, ATM and CLQ-OH. This study involved a molecular modelling strategy that has been successfully applied to unravel the molecular mechanisms of protein binding to raft lipid components, including gangliosides [16,17] and cholesterol [18,19].