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

Abstract

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 (Mpro; 3CLpro), a key enzyme for the viral lifecycle that cleaves at 11 sites in the viral polyprotein pp1a and pp1ab. Although potent inhibitors of Mpro 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 Mpro, such as SPR or ITC, are costly and time-consuming. To improve the speed and feasibility of Mpro 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 Mpro 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 Mpro 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 Mpro-binding small molecules to support the development of new SARS-CoV-2 antivirals.