Methodological Development of a Multi-Readout Assay for the Assessment of Antiviral Drugs against SARS-CoV-2

Abstract

Currently, human infections with the severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2) are accelerating the ongoing spread of the pandemic. Several innovative types of vaccines have already been developed, whereas effective options of antiviral treatments still await a scientific implementation. The development of novel anti-SARS-CoV-2 drug candidates demands skillful strategies and analysis systems. Promising results have been achieved with first generation direct-acting antivirals targeting the viral polymerase RdRp or the protease 3CLpro. Such recently approved or investigational drugs like remdesivir and GC376 represent a basis for further development and optimization. Here, we establish a multi-readout assay (MRA) system that enables the antiviral assessment and mechanistic characterization of novel test compounds, drug repurposing and combination treatments. Our SARS-CoV-2-specific MRA combines the quantitative measurement of several parameters of virus infection, such as the intracellular production of proteins and genomes, enzymatic activities and virion release, as well as the use of reporter systems. In this regard, the antiviral efficacy of remdesivir and GC376 has been investigated in human Caco-2 cells. The readouts included the use of spike- and double-strand RNA-specific monoclonal antibodies for in-cell fluorescence imaging, a newly generated recombinant SARS-CoV-2 reporter virus d6YFP, the novel 3CLpro-based FRET CFP::YFP and the previously reported FlipGFP reporter assays, as well as viral genome-specific RT-qPCR. The data produced by our MRA confirm the high antiviral potency of these two drugs in vitro. Combined, this MRA approach may be applied for broader analyses of SARS-CoV-2-specific antivirals, including compound screenings and the characterization of selected drug candidates.

Anti‐SARS‐CoV‐2 Inhibitory Profile of New Quinoline Compounds in Cell Culture‐Based Infection Models

“…[a] The antiviral analysis was determined using the methodological protocols of a multi‐readout assay for SARS‐CoV‐2 replication in cultured cells as described recently [7b] . Cell viability was measured according to standard procedures using the Neutral Red assay.…”

“…Cell viability was measured according to standard procedures using the Neutral Red assay. The details of cell types and virus strains used, as well as the agents for detection and methodological readout systems, have been described before [7b] …”

“…Due to the published information that Vero cells lack some of the SARS‐CoV‐2‐supportive cellular signaling pathways that are maintained on Caco‐2 cells, the antiviral assessment was presented in a comparative manner. The additional use of various readouts, recently described as the SARS‐CoV‐2‐specific multi‐readout assay system (MRA), allows for an initial monitoring of the potential mechanistic mode of drug‐mediated interference with the regulatory levels of viral replication [7b] . Thereby, most pronounced antiviral activity was determined for compound 2 with 5.9±3.2 μM in Caco‐2 and compound 1 with 1.5±1.0 μM in Vero 76.…”

Genetically Encoded Fluorescent Sensors for SARS-CoV-2 Papain-like Protease PLpro

“…At the same time, we thought that for the end-point analysis, fluorescence lifetime imaging microscopy (FLIM) of the mScarlet donor was more appropriate as it ensured the absolute quantification of the FRET. In contrast to a previously published Mpro sensor based on a classical CFP–YFP (cyan–yellow) FRET pair [ 14 ], we used red and near-infrared fluorescent proteins. The red-shifted spectra of the sensor made it possible to reduce the phototoxicity of the observations, which is important for time-lapse imaging.…”

“…Hahn et al, developed a FRET-based 3CLpro based on CFP and YFP fluorescent proteins linked by a 3CLpro cleavage site [ 14 ]. In the presence of an active protease, the FRET signal was disrupted by protein separation after proteolysis whereas the addition of the protease inhibitor led to the restoration of the FRET signal.…”