Mapping cellular signaling pathways involved in SARS-CoV-2 spike protein-mediated syncytia formation

Abstract Background: Severe coronavirus disease 2019 is associated with extensive syncytia formation, a process driven by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike (S) protein-mediated cell–cell fusion in infected pneumocytes. This study aims to identify compounds to prevent S-mediated fusion, which may represent a potential therapeutic strategy to limit disease progression. Methods: We developed a bimolecular multicellular complementation assay using NanoLuc binary technology to quantitatively detect cell–cell fusion. A high-throughput screen was conducted against a compound library comprising 16,520 molecules. Candidate inhibitors were classified based on their bioactivities, and selected hits were further assessed for their ability to inhibit infection using pseudotyped virus and authentic SARS-CoV-2 infection in the cell cultures. Results: The screening identified 62 compounds that suppressed S protein-mediated fusion. These included inhibitors of cellular proteases involved in S protein cleavage, ATPase inhibitors linked to endosomal acidification, and modulators of hormone receptors, neurotransmitter receptors, calcium channels, and transmembrane protein 16F. Calcium ions emerged as a common regulatory element across these pathways. However, most hits did not show significant antiviral activity against pseudotyped or authentic SARS-CoV-2 infection. Interestingly, a natural compound trigothysoid N exhibited cell-type-dependent effects, inhibiting fusion in Vero-E6 (half maximal inhibitory concentration (IC 50 ) = 1.70 nM) and HeLa-ACE2 cells (IC 50 = 0.65 nM) but enhancing it in A549-ACE2/TMPRSS2 and 293T-ACE2 cells. Conclusion: The identification of fusion inhibitors helps delineate the complex network of events and signaling pathways involved in S-mediated fusion and supports the development of therapeutics targeting this process. Our findings provide valuable insights for the development of therapeutics targeting syncytia formation. Moreover, the opposite effects of trigothysoid N observed across different cell lines highlight the need for careful evaluation of host-targeted fusion inhibitors, as they may exhibit divergent efficacy among patients.