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. Author manuscript; available in PMC: 2022 Jan 3.
Published in final edited form as: Prog Biophys Mol Biol. 2021 Mar 3;163:143–159. doi: 10.1016/j.pbiomolbio.2021.02.004

Fig. 1.

Fig. 1.

High-throughput small-molecule screening for human UDG inhibitors.

Panel A: A 60 min reaction time-course experiment was performed to measure human UDG enzymatic activity in the presence of 10 nM UDG (red line) or absence of UDG (black line) uracil DNA hairpin (25 nM) only control. Each data point is the average of 12 reaction replicates from a 384-well plate. Error bars represent standard deviation from the mean.

Panel B: Two independent screens using a plate of 320 compounds were plotted on the same graph with the trend line showing a positive correlation between the two runs with an R2 value of 0.711 using y = mx + b.

Panel C: High-throughput screening with 3115 bioactive compounds. Normalized percent activity of UDG-mediated unquenching of fluorescence DNA hairpin is shown. See methods for further detail regarding the equation that was used to normalize the percent activity of UDG. Control condition denoted as RFU+UDG AVG (DMSO controls without compound); the average RFU value was computed from a set of 32 wells per screening plate. Control condition denotes RFU−UDG AVG (DNA alone, no protein); the average relative fluorescence unit (RFU) value was computed using a set of 32 replicate wells per screening plate.

Panel D: A dose-response curve showing the normalized average % inhibition of UDG-mediated un-quenching of fluorescence from the DNA hairpin over nine different concentrations of ATA, starting with the highest dose of 12.5 μM, then serially diluted to 3.96 μM, 1.25 μM, 0.396 μM, 0.125 μM, 0.0396 μM, 0.01255 μM, 0.003972 μM, 0.001257 μM, and 0.0003978 μM. Each point at each concentration represents a normalized average percent inhibition from three replicate wells from three different 384-well plates. IC50 was generated using Prism software version 8.4.3, non-linear fit, [inhibitor] vs. response (three parameters, R2 = 0.99). Y error bars represent standard deviation from the mean. The average RFU+UDG AVG (UDG + DMSO control without compound) value was computed from a set of 18 wells per screening plate. The average RFU.UDG AVG (uracil DNA hairpin alone) was computed using a set of 9 wells per screening plate.