SARS-CoV-2 3CL and PLpro: Key Protease Targets for COVID-19

Coronaviruses provide two attractive targets for small-molecule therapeutic intervention: the Papain-Like Protease (PLPro) and the 3C-like protease (3CL or “Main”).  Both enzymes are non-structural, cysteine proteases that are essential to the viral life cycle.  Because mammals lack proteases with similar substrate preferences it’s possible that protease inhibitors developed against these viral proteins will have low toxicity.  

PLpro: The Papain-Like Protease

Coronavirus PLPro has a dual function: it’s required for maturation of the viral polypeptide precursor and it antagonizes innate cellular antiviral responses. The latter function stems from the enzyme’s ability to deubiquitinate and de-ISG15ylate proteins in antiviral signaling pathways. A recent report indicated that the PLPro enzyme from SARS-CoV-2 is less immunosuppressive than the PLPro from the SARS virus that caused an outbreak in 2003, which may explain why the original SARS had a higher mortality rate than CoV-2.

Because of our interest in ubiquitin, ubiquitin-like modifiers (including ISG15) and related assays, we compared the in vitro activities of our recombinant PLPro enzymes using ubiquitin-AMC and ISG15-AMC fluorescent substrates. Our basic kinetic assays consisted of 50 nM PLPro enzyme (SARS, MERS or SARS-CoV-2) and 1 µM Ubiquitin-AMC or ISG15-AMC substrate. Reactions were carried out at 25°C and fluorescence increase was monitored for 20 minutes.

Figure 1. Reaction traces for PLPro enzymes with ISG15-AMC substrate (grey) and Ubiquitin-AMC (blue). Enzyme free controls are shown for both ISG15-AMC and Ubiquitin-AMC (yellow and orange, respectively). As seen in Figure 1, the PLPro enzymes have a large preference for the ISG15-AMC substrate vs the Ubiquitin-AMC substrate in the assay conditions used, though both substrates would be useful in high-throughput screening applications.

3CL: The 3C-like or ‘Main Protease’

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.

In Summary: PLpro and 3CL Proteases

In summary, we believe the coronaviral PLPro and 3CL proteases are excellent targets for antiviral intervention. We’ve created the tools to develop and study compound efficacy in vitro, and existing data from clinical trials in cats demonstrate great promise for this approach.

Featured Protease Reagents for Coronavirus Research

More Tools and Resources for COVID-19 Research

• Recombinant Proteins for Coronavirus Research
• Novus Biologicals: SARS-CoV-2 Research Resources
• Simple Plex for COVID-19 Research
• Tocris: Small Molecules for COVID-19 Research