Figure 1
Development of a biochemical assay for the identification of compounds that disrupt the SARS-CoV-2 N protein-RNA interaction. (A) Nucleotide sequences of the RNA molecules used as N-protein ligands. The blue, yellow and green regions represent the 3’ arm, the UUUUUU motif and the 5’ arm, respectively. (B) FP assay showing the binding affinity curves of the N protein with the FITC-labelled RNA probes 1 to 5 and the corresponding binding equilibrium constants (KD) determined by FP. High-affinity binding was observed with RNAs 1 and 3 derived from the putative SARS-CoV-2 PS sequence. The mutated RNA sequences 2 and 4, and RNA5, used as a negative control, show, in comparison, much lower binding affinities. Error bars represent standard deviation of the means from triplicate experiments. (C) Scatterplot of HTS results showing the binding percentage of each compound in the library calculated using polarization data from negative (RNA alone) and positive (RNA plus protein) controls. Seventy-eight compounds reduced the binding of the N protein to RNA1 to less than 30%, of which 45 were selected for concentration-dependent assays. Compounds that would promote RNA-N complex formation (activators, binding > 100%) were not further considered. (D) Z' values obtained for each assay plate confirming the robustness of the HTS trials.
