The coronaviral 3CL protease is required for viral maturation, cleaving the translated polyprotein in approximately 11 sites. The druggability of 3CL has already been demonstrated in the context of the domestic cat. Feline Infectious Peritonitis is a lethal infection, causing the death of approximately 700,000 cats a year. The cause of the disease is a coronavirus (FIPV). Recent clinical trials with GC376, a potent inhibitor of the 3CL protease, demonstrate its great efficacy against FIPV, high tolerability and favorable safety profile.
To facilitate development of 3CL protease inhibitors, we generated untagged recombinant enzymes from SARS and SARS-CoV-2 and developed a new fluorescent 3CL substrate. The substrate (Figure 2) uses a quenched rhodamine dye that fluoresces when 3CL proteases cleave the peptide at the glutamine residue. Our reaction setup used 20 nM recombinant 3CL proteases and 10 µM substrate in a kinetic assay. These conditions provided excellent linearity as seen in Figure 3. Finally, we used the assay to measure the inhibition of 3CL proteases with GC376. Both SARS and CoV-2 3CL proteases were fully inhibited by the compound, with IC50’s in the 20-40 nM range (Figure 4).
Figure 2. Rhodamine 110-based 3CL Protease Substrate
Figure 3. Reaction traces for coronavirus 3CL enzymes using our peptide substrate. 20 nM CoV-2 3CL (orange) and 20 nM SARS 3CL (blue) efficiently hydrolyze the peptide which is otherwise stable and has low background fluorescence in the absence of enzyme (grey).
Figure 4. Dose response curves demonstrating GC376 inhibition of SARS 3CL (left panel) and CoV-2 3CL (right panel). Reactions were run in triplicate. IC50’s were determined by plotting compound concentration vs inhibition and fitting data with a 4-parameter logistical fit (Model 205, XLfit). IC50 values were 34 nM and 23 nM for SARS and CoV-2 enzymes, respectively.