Echo 550 acoustic

Curated compound collections from the University of Kansas High Throughput Screening laboratory which include Life Chemicals (15,040), ChemBridge (43,736), ChemDiv (56,232), Selleck Bioactives (1649), TimTec (5000), and FDA Repurposed Library (2,286) were used. Compound transfers from source (80 nL of 10 mM stocks) to assay plates were performed using an Echo 550 acoustic liquid handler (Labcyte, Sunnyvale, CA). For most libraries, a target final concentration of 10 μM of compound (in a final volume of 80 μL for the multiplexed reporter gene-based assay described below) was achieved; the FDA Repurposed Library compounds were plated to obtain final concentrations of 2.5 μM. Assay plates were hermetically sealed and stored at -80°C until used. AmpB and nystatin were purchased from Sigma-Aldrich (St. Louis, MO). Synthetic MPLA, lipoteichoic acid (LTA) from S. aureus, PAM₂CSK₄, Poly(I:C), ultrapure LPS from E. coli K12, flagellin from S. typhimurium, ODN-2006 (Vaccigrade), C12-iE-DAP, and Murabutide were procured from InvivoGen (San Diego, CA). The structures of small molecule PRR agonists synthesized by us are shown in S1 Fig. Responses to a variety of TLR and NLR agonists were first examined using THP1-Blue^™ NF-κB reporter cells (InvivoGen, San Diego, CA, Fig 1).

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.