Inhibition of SARS-CoV-2 infection (previously 2019-nCoV) by a highly potent pan-coronavirus fusion inhibitor targeting its spike protein that harbors a high capacity to mediate membrane fusion

Abstract

<jats:title>Abstract</jats:title><jats:p>The recent outbreak of coronavirus disease (COVID-19) caused by SARS-CoV-2 infection in Wuhan, China has posed a serious threat to global public health. To develop specific anti-coronavirus therapeutics and prophylactics, the molecular mechanism that underlies viral infection must first be confirmed. Therefore, we herein used a SARS-CoV-2 spike (S) protein-mediated cell-cell fusion assay and found that SARS-CoV-2 showed plasma membrane fusion capacity superior to that of SARS-CoV. We solved the X-ray crystal structure of six-helical bundle (6-HB) core of the HR1 and HR2 domains in SARS-CoV-2 S protein S2 subunit, revealing that several mutated amino acid residues in the HR1 domain may be associated with enhanced interactions with HR2 domain. We previously developed a pan-coronavirus fusion inhibitor, EK1, which targeted HR1 domain and could inhibit infection by divergent human coronaviruses tested, including SARS-CoV and MERS-CoV. We then generated a series of lipopeptides and found that the EK1C4 was the most potent fusion inhibitor against SARS-CoV-2 S protein-mediated membrane fusion and pseudovirus infection with IC<jats:sub>50</jats:sub>s of 1.3 and 15.8 nM, about 241- and 149-fold more potent than that of EK1 peptide, respectively. EK1C4 was also highly effective against membrane fusion and infection of other human coronavirus pseudoviruses tested, including SARS-CoV and MERS-CoV, as well as SARSr-CoVs, potently inhibiting replication of 4 live human coronaviruses, including SARS-CoV-2. Intranasal application of EK1C4 before or after challenge with HCoV-OC43 protected mice from infection, suggesting that EK1C4 could be used for prevention and treatment of infection by currently circulating SARS-CoV-2 and emerging SARSr-CoVs.</jats:p>

SARS-CoV-2 Requires Cholesterol for Viral Entry and Pathological Syncytia Formation

“…Upon co-culture, ACE2 and spike RBD cluster at cell-cell interfaces in a binding-dependent manner ( Figure 1B). By contrast, and in agreement with others (Buchrieser et al, 2020;Cattin-Ortolá et al, 2020;Ou et al, 2020;Xia et al, 2020), spike FL/ACE2 interactions drove membrane fusion, with the vast majority of cells joining multinucleated syncytia after a day of co-culture ( Figure 1C).…”

“…Past studies of respiratory syncytial virus (RSV), human immunodeficiency virus (HIV), and others suggest that cell-cell fusion can play key roles in pathogenicity, whether it be in viral replication, or evasion of the host immune response (Frankel et al, 1996;Johnson et al, 2007;Maudgal and Missotten, 1978). Recent studies on SARS-CoV-2 identified similar syncytia (Buchrieser et al, 2020;Cattin-Ortolá et al, 2020;Ou et al, 2020;Papa et al, 2020;Xia et al, 2020), which may or may not be relevant to patient pathology (Bryce et al, 2020;Giacca et al, 2020;Rockx et al, 2020;Tian et al, 2020). It remains an open question if syncytia are related to viral and host cell membrane composition, and whether their formation provides mechanistic insights into membrane-targeting therapeutics directed toward enveloped viruses including SARS-CoV-2 (Daniels et al, 2020;Zhang et al, 2020).…”

Highly Conserved Homotrimer Cavity Formed by the SARS-CoV-2 Spike Glycoprotein: A Novel Binding Site

“…Based on the sequence variability of the Coronavirus, including our findings from MD simulations of the spike S protein, a conserved trimer cavity (HR1, CH and CD domains) is a feature of the S-protein in most Coronaviruses. Consistent with this, previous work has shown that the molecule EK1 exhibited potent inhibitory activity against all hCoVs tested through binding to the Cterminal HR1 domain [37]. Additionally, the 'up' and 'down' conformations of RBD domain observed during MD simulations, supports that concept that the S protein can also be a target of a possible IgG therapeutic [91].…”

“…Binding to the host target destabilizes the pre-fusion homotrimer, which sheds off the S1 subunit, and allows for the transition of the S2 subunit to a highly stable postfusion conformation [18]. Interestingly, protein-mediated cell-cell fusion assays suggest that SARS-CoV-2 S protein displays an elevated plasma membrane fusion capacity when compared to that of SARS-CoV [32,37].…”

SARS-CoV-2 viral budding and entry can be modeled using virus-like particles

“…Until now, SARS-CoV-2 VLPs have only been used to identify M as the driver of viral particle formation [ 13 ] and for vaccine development [ 14 ]; they have yet to be functionalized to study SARS-CoV-2 entry or inhibitors. Instead, the few identified entry inhibitors of SARS-CoV-2 have been evaluated using classical coronavirus assays such as S-mediated cell-cell fusion [ 15 ] or pseudotyped VSV vectors [ 16 ]. VLPs offer a reliable and realistic model of S-mediated fusion and viral entry events in a BSL-2 setting.…”