The actions of a drug or a molecular target in vitro often do not translated in a cellular or in vivo context. Membrane permeability, off-target effects, and cytotoxicity are some of the factors that may contribute to this difference. This is a huge bottleneck in the effort to urgently identify novel antiviral therapies, especially in the wake of the coronavirus disease 2019 (COVID-19) pandemic.
The etiological agent of COVID-19 is severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). It has infected over 123.5 million lives, and caused over 2.7 million deaths. The only most promising antiviral agent against SARS-CoV-2 currently used in the clinic is the RNA-dependent RNA polymerase inhibitor remdesivir. However, it has presented mixed results and side-effects.
While effective vaccines against the SARS-CoV-2 are now being administered in many parts of the world, it is suspected that the virus may persist and continue to infect humans. Also, the emerging variants may contribute to the virus circulation and infection.
To fight this SAR-CoV-2 outbreak and any potential future zoonotic coronaviruses, it is essential to have efficient tools – direct biochemical assays – that provide more physiologically relevant information and allow the study of complex tertiary phenotypes such as viral replication.
Currently, the conventional plaque reduction neutralization tests (PRNT) are the gold standard for the quantification of viral replication. However, the low throughput and long turnaround time of these assays limit their utility for large-scale screening of drugs or sera.
To meet an urgent need for a simple, high-throughput cell-based assay, to quantitate authentic SARS-CoV-2 infection and new clinical variants, researchers exploited the presence of viral protease activity. The SARS-CoV-2 proteases are attractive therapeutic targets.
In a new study published recently on the bioRxiv* server, they generated protease-activatable fluorescent and luminescent biosensors for SARS-CoV-2 MPro (main protease) and PLPro (papain-like protease) activity, one based on “flip” GFP (FlipGFP), and the other based on circularly permuted firefly luciferase (FFluc). The team, from the University of Cambridge and the NHS Blood and Transplant, Cambridge, UK, developed a sensitive luminescent reporter cell line that accurately quantitates the infectious SARS-CoV-2 virus.
Compared with reverse-engineered fluorescent or luminescent reporter viruses, a key advantage of our luminescent reporter cell line is the potential to detect a range of clinical SARS-CoV-2 isolates, including emerging variants of concern.”
The researchers confirmed the specificity and utility of these reporters using recombinant viral proteases. They established that these reporters could detect and quantitate the infected cells.
Finally, they developed a stable, luminescent reporter cell line, in which the FFluc is activated by PLPro expression during the SARS-CoV-2 infection. They demonstrated the utility of these cells in assays of small molecule antivirals and neutralizing antibodies using a wild type SARS-CoV-2.
In this work, they demonstrated its utility for drug screening and titration of neutralizing antibodies.
After the researchers generated FlipGFP-based SARS-CoV-2 protease reporters, they tested if these biosensors could detect SARS-CoV-2 protease activity in cells. Of the three reporters studied here, they observed the strongest signal from the PLP2-FlipGFP reporter.
Further, they also confirmed that the selected SARS-CoV-2 biosensors are specific for their cognate proteases, and activated in a strictly sequence-dependent manner.
The data in this study supported the principle that cell-based assays for antiviral compounds correlate better with activity against viral replication than in vitro assays, the researchers stated.
Using the HEK293T cell line over-expressing ACE2 and furin (called the HEK293T-ACE2 cells), the researchers demonstrated that during a SARS-CoV-2 infection, the viral protease expression activated the FlipGFP-based reporters. Thus the protease-activatable biosensors may be exploited to signal SARS-CoV-2 infection. However, the study pointed out that it failed to demonstrate a usable window for high-throughput experiments.
They generated a luciferase-based SARS-CoV-2 protease reporter that can quantitate the SARS-CoV-2 infection, and also developed a facile luminescent assay for screening the inhibitors of SARS-CoV-2 replication.
Using this cell-based assay, the researchers differentiated, from a panel of inhibitors, between compounds that can inhibit viral replication (GC373 and GC376) and those which are not (carmofur, disulfiram, PX-12, tideglusib).
For a sensitive luminescent reporter cell line for drug, neutralization and general virological assays, the researchers then developed a stable cell line, HEK293T-ACE2-30F-PLP2 cells; used with a simple luminescent readout.
The first stable, luminescent reporter cell line for SARS-CoV-2 infection generated in this study is ideally suited to high-throughput screens of candidate antiviral compounds or therapeutic antibodies, and/or large-scale serological surveys for neutralizing activity against authentic virus.
In conclusion, the researchers in this study have developed a versatile toolkit of cell-based fluorescent and luminescent reporters activated by SARS-CoV-2 proteases.
Our data establish the feasibility of protease activatable biosensors for the detection of SARS-CoV-2 infection, and demonstrate the practical utility of a luciferase-based reporter cell line for quantification of infected cells, drug testing and serological assays.”
bioRxiv publishes preliminary scientific reports that are not peer-reviewed and, therefore, should not be regarded as conclusive, guide clinical practice/health-related behavior, or treated as established information.