Drupacine as a potent SARS-CoV-2 replication inhibitor in vitro

Despite the availability of vaccines and antiviral treatments, the continued emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants and breakthrough infections underscores the need for new, potent antiviral therapies. In a previous study, we established a transcription and replication-competent SARS-CoV-2 virus-like particle (trVLP) system that recapitulates the complete viral life cycle. In this study, we combined high-content screening (HCS) with the SARS-CoV-2 trVLP cell culture system, providing a powerful phenotype-oriented approach to assess the antiviral potential of compounds on a large scale. We screened a library of 3,200 natural compounds and identified drupacine as a potential candidate against SARS-CoV-2 infection. Furthermore, we utilized a SARS-CoV-2 replicon system to demonstrate that drupacine could inhibit viral genome transcription and replication. However, <i>in vitro,</i> enzymatic assays revealed that the inhibition could not be attributed to conventional antiviral targets, such as the viral non-structural proteins nsp5 (MPro) or nsp12 (RdRp). In conclusion, our findings position drupacine as a promising antiviral candidate against SARS-CoV-2, providing a novel scaffold for developing anti-coronavirus disease 2019 therapeutics. Further investigation is required to pinpoint its precise target and mechanism of action.