A Self-Immolative Fluorescent Probe for Selective Detection of SARS-CoV-2 Main Protease

Existing tools to detect and visualize severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) suffer from low selectivity, poor cell permeability, and high cytotoxicity. Here we report a novel self-immolative fluorescent probe (MP590) for the highly selective and sensitive detection of the SARS-CoV-2 main protease (M^pro). This fluorescent probe was prepared by connecting a M^pro-cleavable peptide (N-acetyl-Abu-Tle-Leu-Gln) with a fluorophore (i.e., resorufin) via a self-immolative aromatic linker. Fluorescent titration results show that MP590 can detect M^pro with a limit of detection (LoD) of 35 nM and is selective over interferents such as hemoglobin, bovine serum albumin (BSA), thrombin, amylase, SARS-CoV-2 papain-like protease (PL^pro), and trypsin. The cell imaging data indicate that this probe can report M^pro in HEK 293T cells transfected with a M^pro expression plasmid as well as in TMPRSS2-VeroE6 cells infected with SARS-CoV-2. Our results suggest that MP590 can both measure and monitor M^pro activity and quantitatively evaluate M^pro inhibition in infected cells, making it an important tool for diagnostic and therapeutic research on SARS-CoV-2.