Cellular electrical impedance to profile SARS-CoV-2 fusion inhibitors and to assess the fusogenic potential of spike mutants

Despite the vaccination campaigns for COVID-19, we still cannot control the spread of SARS-CoV-2, as evidenced by the ongoing circulation of the Omicron variants of concern. This highlights the need for broad-spectrum antivirals to further combat COVID-19 and to be prepared for a new pandemic with a (re-)emerging coronavirus. An interesting target for antiviral drug development is the fusion of the viral envelope with host cell membranes, a crucial early step in the replication cycle of enveloped viruses. In this study, we explored the use of cellular electrical impedance (CEI) to quantitatively monitor morphological changes in real time, resulting from cell-cell fusion elicited by SARS-CoV-2 spike. The impedance signal in CEI-quantified cell-cell fusion correlated with the expression level of SARS-CoV-2 spike in transfected HEK293T cells. For antiviral assessment, we validated the CEI assay with the fusion inhibitor EK1 and measured a concentration-dependent inhibition of SARS-CoV-2 spike mediated cell-cell fusion (IC50 value of 0.13 μM). In addition, CEI was used to confirm the fusion inhibitory activity of the carbohydrate-binding plant lectin UDA against SARS-CoV-2 (IC50 value of 0.55 μM), which complements prior in-house profiling activities. Finally, we explored the utility of CEI in quantifying the fusogenic potential of mutant spike proteins and in comparing the fusion efficiency of SARS-CoV-2 variants of concern. In summary, we demonstrate that CEI is a powerful and sensitive technology that can be applied to studying the fusion process of SARS-CoV-2 and to screening and characterizing fusion inhibitors in a label-free and non-invasive manner. Importance Despite the success of the vaccines against SARS-CoV-2, new variants of the virus are still emerging and spreading, underlining the need for additional effective antiviral countermeasures. An interesting antiviral target for enveloped viruses is the fusion of the viral envelope with host cell membranes, a crucial early step in the life cycle of coronaviruses like SARS-CoV-2. Here, we present a sensitive impedance-based method to monitor in real-time cell-cell fusion elicited by the SARS-CoV-2 spike protein. With this technique we can profile entry inhibitors and determine the inhibitory potential of fusion inhibitors for SARS-CoV-2. In addition, with cellular electrical impedance we can evaluate the fusogenic properties of new emerging SARS-CoV-2 variants. Overall, the impedance technology adds valuable information on the fusion process of circulating coronaviruses and helps unravel the mode of action of new antivirals, opening new avenues for the development of next generation fusion inhibitors with improved antiviral activity.