A Novel Fluorescence Polarization Binding Assay for the Main Protease (M^pro) of SARS-CoV‑2

The COVID-19 pandemic incited a global health crisis that accelerated the development of antiviral therapeutics. One successful avenue for inhibiting SARS-CoV-2 has been through targeting the main protease (M^pro; 3CL^pro), a key enzyme for the viral lifecycle that cleaves at 11 sites in the viral polyprotein pp1a and pp1ab. Although potent inhibitors of M^pro have been discovered, including the FDA-approved drug Paxlovid, the potential emergence of resistant variants requires continued antiviral development efforts. The current methods to characterize binders of M^pro, such as SPR or ITC, are costly and time-consuming. To improve the speed and feasibility of M^pro inhibitor development, we developed a competitive miniaturized fluorescence polarization (FP) binding assay. We repurposed small molecules from a DNA-encoded library screen into FP probes by appending a fluorophore with various linkers. After identifying a probe that exhibited potent M^pro binding (K _D ∼43 nM), we optimized buffer conditions, pH, and additives. Assay validation revealed that our competitive fluorescence polarization assay was robust, with a Z'-score of 0.79 and a signal window of 23. This assay can be used as a single-point assay for high-throughput screening or to triage small molecules by generating K _i values for binding. Efforts from this work resulted in an M^pro binding assay that requires minimal protein consumption, low sample volumes, short incubation times (30 min), and operates at room temperature. In conclusion, we developed a robust FP assay that can be used to rapidly characterize M^pro-binding small molecules to support the development of new SARS-CoV-2 antivirals.