A high-throughput response to the SARS-CoV-2 pandemic

Abstract

Four years after the beginning of the COVID-19 pandemic, it is important to reflect on the events that have occurred during that time and the knowledge that has been gained. The response to the pandemic was rapid and highly resourced; it was also built upon a foundation of decades of federally funded basic and applied research. Laboratories in government, pharmaceutical, academic, and non-profit institutions all played roles in advancing pre-2020 discoveries to produce clinical treatments. This perspective provides a summary of how the development of high-throughput screening methods in a biosafety level 3 (BSL-3) environment at Southern Research Institute (SR) contributed to pandemic response efforts. The challenges encountered are described, including those of a technical nature as well as those of working under the pressures of an unpredictable virus and pandemic.

Keywords: Antiviral; BSL-3; COVID; COVID-19; Coronavirus; Drug discovery; HTS; High-throughput; MERS; Pandemic; SARS; SARS-CoV; SARS-CoV-2; SARS2.

Fig. 1.

Illustration of the stacked plate concentration response experimental design. The format supports the creation of concentration response for 320 compounds when formatted in columns 3 – 22. Each plate contains a single concentration of each compound. This format provides a means to streamline performing a large number of concentration responses by formatting compounds into one plate and performing each dilution for all compounds simultaneously. Note: although the constraints of the fit model are 0 % - 100 % inhibition, experiments may produce readouts of 〈 0 % and 〉 100 % due to the ability of the biological signals to go outside the calculated average of the negative and positive controls.

Fig. 2.

Learning timeline with key method developments and screens that helped prepare for the SR HTS response to the COVID-19 pandemic.

Fig. 3.

Timeline showing milestones in the SR HTS’ SARS-CoV-2 screening efforts, therapeutic developments, and death tolls. Significant therapeutic developments are shown in red text. The introduction of variants into the population are shown in purple text.

Fig. 4.

Concentration response graphs for reference compounds run in the cytopathic effect assay (CPE) and corresponding cytotoxicity. Vero E6 cells with enhanced ACE-2 expression were infected with the WA1/2020 isolate of SARS-CoV-2. Inhibition of CPE is expressed as the 50 % inhibitory concentration (IC₅₀) and cytotoxicity is expressed as the 50 % cytotoxicity concentration (CC₅₀). EI = efflux inhibitor (CP-100356); Conc = Concentration.

Fig. 5.

Concentration response graphs for reference compounds run in the SARS-CoV-2 NanoLuc reporter virus assay in A549 cells that recombinantly express ACE-2. The left panel shows the antiviral assay results and the right panel the corresponding cytotoxicity assay results. Inhibition of reporter virus signal is expressed as the 50 % inhibitory concentration (IC₅₀) and cytotoxicity is expressed as the 50 % cytotoxicity concentration (CC₅₀). Note the exclusion of efflux inhibitor due to the lack of significant levels of MDR1 in A549 cells. Conc = Concentration.

Fig. 6.

Concentration response graphs for reference compounds run in the cytopathic effect (CPE) assay. Vero E6 cells with enhanced ACE-2 expression were infected with the Toronto-2 strain of SARS-CoV. Inhibition of CPE is expressed as the 50 % inhibitory concentration (IC₅₀). As illustrated in Fig. 4 (same cell line), none of the compounds tested exhibited measurable cytotoxicity. EI = efflux inhibitor (CP-100356); Conc = Concentration.

Fig. 7.

Concentration response graphs for reference compounds run in the cytopathic effect (CPE) assay in Vero CCL-81 cells infected with MERS-CoV and corresponding cytotoxicity. Inhibition of CPE is expressed as the 50 % inhibitory concentration (IC₅₀) and cytotoxicity is expressed as the 50 % cytotoxicity concentration (CC₅₀). EI = efflux inhibitor (CP-100356); Conc = Concentration.

Fig. 8.

Graph represents the number of compounds screened in single concentration (which includes assay ready plates) and the number of wells screened in concentration response in SARS-CoV-2 cell-based assays at Southern Research between May 2020 and January 2024. This represents a total of approximately 1.5 million antiviral data points. Note that the numbers do not include the parallel cytotoxicity assay data.

Fig. 9.

Concentration response of the MDR1 inhibitor CP-100356. A.) The measured effect in the SARS-CoV-2 CPE assay performed in Vero E6 cells. B.) The cytotoxic effect of CP-100356 observed in Vero E6 cells.