A Gaussia luciferase reporter assay for the evaluation of coronavirus Nsp5/3CLpro inhibitors

Abstract

Human coronaviruses (hCoVs) infect millions of people every year. Among these, MERS, SARS-CoV-1, and SARS-CoV-2 caused significant morbidity and mortality and their emergence highlights the risks associated with possible future coronavirus outbreaks. Therefore, broadly-active anti-coronavirus drugs are needed. Pharmacological inhibition of the hCoV protease 3CLpro (Nsp5) in COVID-19 patients is clinically beneficial as shown by the wide and effective use of Paxlovid (nirmaltrevir, ritonavir). However, further treatment options are required due to the emergence of drug resistance in some SARS-CoV-2 strains. To facilitate protease inhibitor discovery and evaluation, we developed an assay allowing rapid and reliable quantification of 3CLpro activity under biosafety level 1 conditions. It is based on an ACE2 receptor - Gal4 transcription factor fusion protein separated by a 3CLpro recognition site. Cleavage by 3CLpro releases the Gal4 transcription factor, which then induces the expression of Gaussia luciferase. Our assay is compatible with 3CLpro proteases from all hCoVs, and allows simultaneous measurement of inhibitory and cytotoxic effects of the tested compounds. Proof-of-concept IC50 measurements confirmed that nirmaltrevir, GC376 and lopinavir inhibit SARS-CoV-2 3CLpro function without inducing cytotoxicity. Overall, the Gaussia luciferase-based reporter assay is suitable for evaluating viral protease function and screening of potential 3CLpro inhibitors.