We developed and evaluated a reporter assay for fast and efficient screening of inhibitors of the functionally conserved hCoV protease 3CLpro (Nsp5), which is among the most promising targets for treating coronavirus infections [32]. Our assay allows simultaneous quantification of a compound’s potency and cytotoxicity within 24 h, and can facilitate the discovery and evaluation of coronavirus protease inhibitors in the future. Whereas the currently most widely used COVID-19 treatment Paxlovid already targets 3CLpro, the development of further inhibitors is warranted due to the risk of drug resistance [8], [9], [10]. Our assay can be performed in a 96-well format, requires only a luminescence plate reader, and is compatible with the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) cytotoxicity assay, supporting high-throughput screening and rapid evaluation of potency and specificity.
hCoV 3CLpro proteases show 20–50% overall amino sequence diversity but share core structural homology and have similar substrate specificity [29]. Therefore, it is possible to develop broadly active hCoV protease inhibitors and a universal assay to study their activities [26]. We demonstrated that our reporter is compatible with 3CLpro homologs from all seven human coronaviruses as well as with most virus inactivation procedures. Furthermore, the assay has high sensitivity and a dynamic range of approximately 10-fold between the lowest and the highest tested dose, making it suitable for reliable quantification of potency. Using proof-of-concept assessments of IC50 values of nirmaltrevir, GC376, and lopinavir, we demonstrated the suitability of our system for evaluating SARS-CoV-2 3CLpro inhibitors.
There has been substantial interest in 3CLpro reporter assays since the start of the SARS-CoV-2 pandemic, and several versions of such assays exist, many of which rely on fluorescent Flip-GFP technology [26], [27], [28], [34], [35], [36]. Our luciferase-based assay overcomes several disadvantages of GFP-based reporters, such as their considerable confound by pH, the potential autofluorescence of tested compounds, and photobleaching. Compared to GFP, Gaussia luciferase offers higher stability and thus more reliable quantification [37], [38]. Furthermore, the secretion of the luciferase reporter into cell media allows non-invasive sampling and long-term sample storage.
While screening for potential 3CLpro inhibitors in a virus-free mode is certainly its most prominent application, our assay could also be further optimized towards higher sensitivity for the detection of endogenous 3CLpro activity resulting from actual hCoV infections. This is enabled by the fusion of the 3CLpro cleavage sequence to human ACE2, which serves as major entry receptor for SARS-CoV-1, SARS-CoV-2, and NL63. The resulting receptor-reporter fusion protein effectively promoted SARS-CoV-2 entry into the otherwise poorly permissive HEK293T cells[39]. Notably, the produced Gaussia luciferase signal withstood temperature and disinfectant-based virus inactivation procedures without compromising assay sensitivity.
The assay detected 3CLpro activity of all human coronaviruses (SARS-CoV-1, SARS-CoV-2, MERS, NL63, OC43, 229E, and HKU1), supporting rapid testing of inhibitors against both existing and potentially emerging pathogens. This finding also implies that it can be used to compare the drug sensitivity and protease function of different coronavirus lineages and to monitor the impact of 3CLpro and pp1ab mutations in emerging SARS-CoV-2 variants on nirmaltrevir sensitivity. While our assay is currently limited to the detection of inhibitors of 3CLpro, its design principle can be reiterated to develop reporter assays for other viral proteases, such as hCoV PLpro. Recent studies reported that SARS-CoV-2 3CLpro cleaves not only the viral polyprotein 1ab but also cellular factors such as NF-κB Essential Modulator (NEMO), septin and Caspase recruitment domain-containing protein 8 (CARD8) which play roles in immune sensing and cell homeostasis [40], [41], [42]. Our assay could be utilized for studying cellular 3CLpro substrates and to evaluate inhibitors that not only directly limit viral replication through blocking pp1ab processing but also prevent the 3CLpro associated cellular dysfunction.
Since our drug evaluations were performed using higher Nsp5 expression levels from overexpressed plasmid as compared to the endogenous levels observed during infection, the IC50 values assay are likely to be higher than those measured during experiments utilizing a genuine virus. Indeed, the reported IC50 values of nirmaltrevir obtained during SARS-CoV-2 infection assays in cell lines are 10 times lower than those measured in our study [8]. Therefore, it is highly advisable for any drug screening study to confirm the activity of newly discovered inhibitors using independent methods and in the context of genuine virus infection.
Overall, our findings highlight the application of the developed Gaussia luciferase-based reporter assay for studying coronavirus protease function and identifying/evaluating antiviral drug candidates. Other potential applications include monitoring the impact of emerging SARS-CoV-2 3CLpro mutations on proteolytic activity and drug sensitivity. Although already highly versatile in its current form, the assay could be easily modified to study the function other viral proteases, as well as the impact of mutations and polymorphisms at the 3CLpro recognition site. The sensitivity, compatibility, and rapid and broad applicability of this assay make it a valuable tool for drug discovery efforts aimed at combating current and future coronavirus outbreaks.