SARS-CoV-2 Viral Inhibition Assay

Compounds were dispensed
into 96-well plates using an ECHO 550 acoustic dispenser (Labcyte)
to give a final concentration range of 100–0.11 μM. Compounds
were preincubated with 50 μL of SARS-CoV-2 virus (Victoria strain–100
FFU) and 50 μL of VeroE6 cells (9 × 10⁵/mL)
in separate 96-well plates. Following incubation for 1 h at 37 °C
in a 5% CO₂ atmosphere, the virus was added to the well
containing compound-treated cells. The virus was allowed to infect
the cells for 2 h at 37 °C in a 5% CO₂ atmosphere,
followed by the addition of 100 μL of compound-adulterated carboxymethyl
cellulose (1.5%) to each well. Subsequently, the plates were incubated
for a further 20 h at 37 °C in a 5% CO₂ atmosphere.
All assays were carried out in technical duplicates.
Cells were
washed with 200 μL of DPBS and then fixed with paraformaldehyde
4%_v/v (100 μL/well) for 30 min at rt. Cells were
permeabilized with TritonX100 (1% in PBS) and then stained for SARS-CoV-2
nucleoprotein using a human monoclonal antibody (FB9B^{102 (link)}). Bound antibodies were detected following
incubation with a goat anti-human IgG HRP conjugate (Sigma, U.K.)
and following TrueBlue Peroxidase substrate (Insight Biotechnology,
U.K.) addition imaged using an ELISPOT reader. The half-maximal effective
concentration (EC₅₀) was defined as the concentration of
the compound that reduced the Foci forming unit (FFU) by 50% compared
to the control wells.