Journal: bioRxiv

Article Title: High-throughput screening of the ReFRAME, Pandemic Box, and COVID Box drug repurposing libraries against SARS-CoV2 nsp15 endoribonuclease to identify small-molecule inhibitors of viral activity

doi: 10.1101/2021.01.21.427657

Figure Lengend Snippet: Dose response curves of select nsp15 hits. Inhibitor structures and 3° FRET assay dose-response curves with calculated IC 50 values for Exebryl-1 ( A ), Piroxantrone ( C ), and MMV1580853 ( E ). Nsp15 (25 nM) was incubated with 2-fold or 3-fold serial concentrations of compound and incubated with the substrate 5’6- FAM/dArUdAdA/3’-TAMRA (0.5 μM) at ambient temperature for 1 h. Results of a polyacrylamide gel-based RNA cleavage assay for Exebryl-1 ( B ) and Piroxantrone ( D ). nsp15 (0.78 μM) and poly(rA) (500 ng) was incubated with 2-fold serial concentrations of each compound and incubated at ambient temperature for 2 h. Cleavage products were separated from uncleaved poly(rA) by electrophoresis on a 15% TBE-urea polyacrylamide gel and the % cleavage activity calculated by quantitating the relative amount of cleaved (20 nt lower band) vs uncleaved (31 nt top band) substrate in each sample relative to a DMSO control. Gel-based RNA cleavage inhibition of MMV1580853 was observed, but was variable from experiment to experiment, likely due to poor solubility in DMSO and assay buffer.

Article Snippet: The 5’6-FAM/dArUdAdA/3’-TAMRA oligonucleotide was purchased from IDT DNA Inc (Coralville, IA).

Techniques: Incubation, Cleavage Assay, Electrophoresis, Activity Assay, Inhibition, Solubility

Journal: bioRxiv

Article Title: High-throughput screening of the ReFRAME, Pandemic Box, and COVID Box drug repurposing libraries against SARS-CoV2 nsp15 endoribonuclease to identify small-molecule inhibitors of viral activity

doi: 10.1101/2021.01.21.427657

Figure Lengend Snippet: Primary SARS-CoV2 nsp15 endoribonuclease assay. A) Schematic illustrates the assay used to identify nsp15 inhibitors. Close proximity of the intact 5’-FAM and 3’-TAMRA fluorophores of the oligonucleotide substrate, 5’6-FAM/dArUdAdA/3’-TAMRA, results in quenching of fluorescence due to fluorescence resonance energy transfer (FRET). Enzymatic cleavage of the substrate by nsp15 leads to increased FAM fluorescence. B) The effects of Mn 2+ and Mg 2+ on SARS-CoV-2 nsp15 endoU activity in a 1 hr cleavage reaction at ambient temperature. The presence of Mn 2+ leads to a 2.7-fold increase in FAM fluorescence compared to reactions lacking metal supplementation while the addition of Mg 2+ appears to have minimal effect on activity (n=8, mean values ± standard deviations). ***, P

Article Snippet: The 5’6-FAM/dArUdAdA/3’-TAMRA oligonucleotide was purchased from IDT DNA Inc (Coralville, IA).

Techniques: Fluorescence, Förster Resonance Energy Transfer, Activity Assay