Graphene oxide-based fluorescent biosensor for high-throughput screening to discover SARS-CoV-2 RdRp inhibitors

The COVID-19 pandemic caused by SARS-CoV-2 underscored the global need for rapid, efficient drug discovery platforms to combat emerging viral threats. Conventional antiviral screening methods are often time-consuming and low-throughput, making them insufficient for timely therapeutic development during acute outbreaks. RNA-dependent RNA polymerase (RdRp), a key enzyme in viral replication, represents a validated antiviral target for RNA viruses, including SARS-CoV-2. However, few assays directly monitor RdRp activity in a high-throughput format. To address this gap, we developed a fluorescence-based assay for real-time monitoring of RdRp activity using graphene oxide nanomaterials. Here, we designed a graphene oxide-based RdRp assay that transduces polymerase activity into measurable fluorescence intensity changes. The assay is rapid, homogeneous, and compatible with multi-well plate formats for high-throughput screening. Using this platform, we screened a library of FDA-approved small molecules and identified fingolimod, an immunomodulatory drug for multiple sclerosis, as a potential RdRp inhibitor. <i>In vitro</i> cell-based assays confirmed that fingolimod significantly reduced SARS-CoV-2 replication without cytotoxicity at therapeutic concentrations. This result supports fingolimod's potential as a repurposed direct-acting antiviral agent. The assay's robustness highlights its applicability in antiviral drug discovery, enabling rapid responses to future viral outbreaks. This graphene oxide-based RdRp assay provides a versatile tool for antiviral screening and demonstrates the feasibility of repurposing approved drugs as direct-acting antivirals. The platform's adaptability and rapid readout capability make it well-suited for pandemic preparedness and therapeutic discovery against emerging viral threats.