Feline Infectious Peritonitis (FIP) is caused by a coronavirus. The 3CLpro of this virus is much like the one in SARS-CoV-2 and is crucial for viral replication. In 2016, Kim and colleagues showed that the compound GC376 blocked viral replication in cats destined to otherwise die (Kim et al. 2016). More recently, there have been exciting reports that this inhibitor also blocks the human 3CLpro found in SARS-CoV-2. We used a 3CL protease biosensor (3CLglow) to monitor GC376 inhibition in living HEK293 cells.
3CLglow: An assay for 3CLpro/Mpro inhibitors
The 3CL protease of SARS-CoV-2, also known as “Main protease” is crucial to viral replication. Drugs that specifically inhibit this enzyme have enormous potential as therapeutics. The structure of the enzyme has been well characterized (Jin et al. 2020; Kneller et al. 2020) making it possible to screen for potential inhibitors in silico through docking (Jo et al. 2020; Sang et al. 2020; Hung et al. 2020; Gordon et al. 2020; Mukherjee et al. 2011; Choudhary et al. 2020).
High throughput assays for 3CLpro inhibitors that rely upon recombinant, purified enzyme and fluorescent substrates can be used to either test compounds identified with in silico screening, or to screen libraries of new compounds. However, these biochemical assays lack cellular context and biological relevance (Vincent et al. 2015). In contrast, 3CLglow reports activity in living cells and captures physiologically relevant dose response information.
The 3CLglow assay can validate hits from biochemical assays, or it can be used in automated primary screening. 3CLglow identifies inhibitors that can enter living cells, block the 3CLpro enzyme (even in a subcellular compartment) without killing the cells, providing an important bridge between biochemistry and animal models. The observation that GC376, a drug that saves cats lives, inhibits SARS-CoV-2 3CL protease is fortuitous as it provides a reliable control for the 3CLglow assay.
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