SARS-CoV-2 Omicron spike H655Y mutation is responsible for enhancement of the endosomal entry pathway and reduction of cell surface entry pathway

The SARS-CoV-2 Omicron variant reportedly displays decreased usage of the cell surface entry pathway mediated by the host transmembrane protease, serine 2 (TMPRSS2) and increased usage of the endosomal entry pathway mediated by cathepsin B/L. These differences result in different cell tropisms and low fusogenicity from other SARS-CoV-2 variants. Recent studies have revealed that host metalloproteases are also involved in cell surface entry and fusogenic activity of SARS-CoV-2, independent of TMPRSS2. However, the involvement of metalloproteinase-mediated cell entry and fusogenicity in Omicron infections has not been investigated. Here, we report that Omicron infection is less sensitive to the metalloproteinase inhibitor marimastat, like the TMPRSS2 inhibitor nafamostat, and is more sensitive to the cathepsin B/L inhibitor E-64d than infections with wild-type SARS-CoV-2 and other variants. The findings indicate that Omicron preferentially utilizes the endosomal pathway rather than cell surface pathways for entry. Moreover, the Omicron variant also displays poor syncytia formation mediated by metalloproteinases, even when the S cleavage status mediated by fusion-like proteases is unchanged. Intriguingly, the pseudovirus assay showed that a single mutation, H655Y, of the Omicron spike (S) is responsible for the preferential entry pathway usage without affecting the S cleavage status. These findings suggest that the Omicron variant has altered entry properties and fusogenicity, probably through the H655Y mutation in its S protein, leading to modulations of tissue and cell tropism, and reduced pathogenicity. Author summaryRecent studies have suggested that the SARS-CoV-2 Omicron variant displays altered cell tropism and fusogenicity, in addition to immune escape. However, comprehensive analyses of the usage of viral entry pathways in Omicron variant have not been performed. Here, we used protease inhibitors to block each viral entry pathway mediated by the three host proteases (cathepsin B/L, TMPRSS2, and metalloproteinases) in various cell types. The results clearly indicated that Omicron exhibits enhanced cathepsin B/L-dependent endosome entry and reduced metalloproteinase-dependent and TMPRSS2-dependent cell surface entry. Furthermore, the H655Y mutation of Omicron S determines the relative usage of the three entry pathways without affecting S cleavage by the host furin-like proteases. Comparative data among SARS-CoV-2 variants, including Omicron, may clarify the biological and pathological phenotypes of Omicron but increase the understanding of disease progression in infections with other SARS-CoV-2 variants.

Metalloproteinase-dependent and TMPRSS2-independnt cell surface entry pathway of SARS-CoV-2 requires the furin-cleavage site and the S2 domain of spike protein

The ongoing global vaccination program to prevent SARS-CoV-2 infection, the causative agent of COVID-19, has had significant success. However, recently virus variants have emerged that can evade the immunity in a host achieved through vaccination. Consequently, new therapeutic agents that can efficiently prevent infection from these new variants, and hence COVID-19 spread are urgently required. To achieve this, extensive characterization of virus-host cell interactions to identify effective therapeutic targets is warranted. Here, we report a cell surface entry pathway of SARS-CoV-2 that exists in a cell type-dependent manner is TMPRSS2-independent but sensitive to various broad-spectrum metalloproteinase inhibitors such as marimastat and prinomastat. Experiments with selective metalloproteinase inhibitors and gene-specific siRNAs revealed that a disintegrin and metalloproteinase 10 (ADAM10) is partially involved in the metalloproteinase pathway. Consistent with our finding that the pathway is unique to SARS-CoV-2 among highly pathogenic human coronaviruses, both the furin cleavage motif in the S1/S2 boundary and the S2 domain of SARS-CoV-2 spike protein are essential for metalloproteinase-dependent entry. In contrast, the two elements of SARS-CoV-2 independently contributed to TMPRSS2-dependent S2 priming. The metalloproteinase pathway is involved in SARS-CoV-2-induced syncytia formation and cytopathicity, leading us to theorize that it is also involved in the rapid spread of SARS-CoV-2 and the pathogenesis of COVID-19. Thus, targeting the metalloproteinase pathway in addition to the TMPRSS2 and endosome pathways could be an effective strategy by which to cure COVID-19 in the future. Author SummaryTo develop effective therapeutics against COVID-19, it is necessary to elucidate in detail the infection mechanism of the causative agent, SARS-CoV-2, including recently emerging variants. SARS-CoV-2 binds to the cell surface receptor ACE2 via the Spike protein, and then the Spike protein is cleaved by host proteases to enable entry. Selection of target cells by expression of these tissue-specific proteases contributes to pathogenesis. Here, we found that the metalloproteinase-mediated pathway is important for SARS-CoV-2 infection, variants included. This pathway requires both the prior cleavage of Spike into two domains and a specific sequence in the second domain S2, conditions met by SARS-CoV-2 but lacking in the related human coronavirus SARS-CoV. The contribution of several proteases, including metalloproteinases, to SARS-CoV-2 infection was cell type dependent, especially in cells derived from kidney, ovary, and endometrium, in which SARS-CoV-2 infection was metalloproteinase-dependent. In these cells, inhibition of metalloproteinases by treatment with marimastat or prinomastat, whose safety was previously confirmed in clinical trials, was important in preventing cell death. Our study provides new insights into the complex pathogenesis unique to COVID-19 and relevant to the development of effective therapies.

Discovery of New Fusion Inhibitor Peptides against SARS-CoV-2by Targeting the Spike S2 Subunit

A novel coronavirus, severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), caused a worldwide pandemic. Our aim in this study is to produce new fusion inhibitors against SARS-CoV-2, which can be the basis for developing new antiviral drugs. The fusion core comprising the heptad repeat domains (HR1 and HR2) of SARS-CoV-2 spike (S) were used to design the peptides. A total of twelve peptides were generated, comprising a short or truncated 24-mer (peptide #1), a long 36-mer peptide (peptide #2), and ten peptide #2 analogs. In contrast to SARS-CoV, SARS-CoV-2 S-mediated cell-cell fusion cannot be inhibited with a minimal length, 24-mer peptide. Peptide #2 demonstrated potent inhibition of SARS-CoV-2 S-mediated cell-cell fusion at 1 µM concentration. Three peptide #2 analogs showed IC50 values in the low micromolar range (4.7-9.8 µM). Peptide #2 inhibited the SARSCoV-2 pseudovirus assay at IC50=1.49 µM. Given their potent inhibition of viral activity and safety and lack of cytotoxicity, these peptides provide an attractive avenue for the development of new prophylactic and therapeutic agents against SARS-CoV-2.

Discovery of New Fusion Inhibitor Peptides against SARS-CoV-2by Targeting the Spike S2 Subunit

A novel coronavirus, severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), caused a worldwide pandemic. Our aim in this study is to produce new fusion inhibitors against SARS-CoV-2, which can be the basis for developing new antiviral drugs. The fusion core comprising the heptad repeat domains (HR1 and HR2) of SARS-CoV-2 spike (S) were used to design the peptides. A total of twelve peptides were generated, comprising a short or truncated 24-mer (peptide #1), a long 36-mer peptide (peptide #2), and ten peptide #2 analogs. In contrast to SARS-CoV, SARS-CoV-2 S-mediated cell-cell fusion cannot be inhibited with a minimal length, 24-mer peptide. Peptide #2 demonstrated potent inhibition of SARS-CoV-2 S-mediated cell-cell fusion at 1 µM concentration. Three peptide #2 analogs showed IC50 values in the low micromolar range (4.7-9.8 µM). Peptide #2 inhibited the SARSCoV-2 pseudovirus assay at IC50=1.49 µM. Given their potent inhibition of viral activity and safety and lack of cytotoxicity, these peptides provide an attractive avenue for the development of new prophylactic and therapeutic agents against SARS-CoV-2.

The anticoagulant nafamostat potently inhibits SARS-CoV-2 infection in vitro: an existing drug with multiple possible therapeutic effects

Although infection by SARS-CoV-2, the causative agent of COVID-19, is spreading rapidly worldwide, no drug has been shown to be sufficiently effective for treating COVID-19. We previously found that nafamostat mesylate, an existing drug used for disseminated intravascular coagulation (DIC), effectively blocked MERS-CoV S protein-initiated cell fusion by targeting TMPRSS2, and inhibited MERS-CoV infection of human lung epithelium-derived Calu-3 cells. Here we established a quantitative fusion assay dependent on SARS-CoV-2 S protein, ACE2 and TMPRSS2, and found that nafamostat mesylate potently inhibited the fusion while camostat mesylate was about 10-fold less active. Furthermore, nafamostat mesylate blocked SARS-CoV-2 infection of Calu-3 cells with an EC50 around 10 nM, which is below its average blood concentration after intravenous administration through continuous infusion. These findings, together with accumulated clinical data regarding its safety, make nafamostat a likely candidate drug to treat COVID-19.

Small Molecule Inhibitors of Middle East Respiratory Syndrome Coronavirus Fusion by Targeting Cavities on Heptad Repeat Trimers

Middle East Respiratory Syndrome Coronavirus (MERS-CoV) is a newly emerging viral disease with fatal outcomes. However, no MERS-CoV-specific treatment is commercially available. Given the absence of previous structure-based drug discovery studies targeting MERS-CoV fusion proteins, this set of compounds is considered the first generation of MERS-CoV small molecule fusion inhibitors. After a virtual screening campaign of 1.56 million compounds followed by cell-cell fusion assay and MERS-CoV plaques inhibition assay, three new compounds were identified. Compound numbers 22, 73, and 74 showed IC50 values of 12.6, 21.8, and 11.12 μM, respectively, and were most effective at the onset of spike-receptor interactions. The compounds exhibited safe profiles against Human embryonic kidney cells 293 at a concentration of 20 μM with no observed toxicity in Vero cells at 10 μM. The experimental results are accompanied with predicted favorable pharmacokinetic descriptors and drug-likeness parameters. In conclusion, this study provides the first generation of MERS-CoV fusion inhibitors with potencies in the low micromolar range.