Determination of IC50

The concentration dependence of inhibition was examined for each validated direct DNMT1 inhibitor. IC₅₀ values were determined under identical assay conditions (10 nM hairpin DNA substrate and 10 μM SAM) using the DNA methylation assay buffer. Assays (0.1 mL) were conducted in triplicate and contained 1.5 nM RFTS(-) DNMT1, 0.6 U Gla I, 1% DMSO and varied inhibitor concentrations (0–100 μM). A matched control lacking DNMT1 was subtracted from each assay condition. Corrected fluorescence data was averaged and traces were fitted in Kaleidagraph to determine initial velocities. The percent activity at each inhibitor concentration was determined by comparing to a DMSO control assay. Each percent activity reported is an average of at least 3 independent experiments. IC₅₀ values were calculated by fitting the percent activity data using a unity Hill slope in KaleidaGraph.

To examine stability of the inhibitors, identical assay solutions were created that contained 100 μM of each compound or DMSO in the DNA methylation assay buffer. Substrates were added to one set of solutions and these solutions were assayed immediately by adding enzymes and observing the change in fluorescence over time. The other set was incubated at room temperature for 60 minutes and then substrates were added. Enzymes were added to initiate these assays and fluorescence changes were monitored. In both cases, assays contained 10 nM hairpin DNA substrate, 10 μM SAM, 1.5 nM RFTS(-) DNMT1, 0.6 U Gla I, and 1% DMSO. A matched control lacking DNMT1 was subtracted from each assay condition. Corrected fluorescence data was averaged and fitted in Kaleidagraph to determine initial velocities. Percent activity was calculated by comparing to a DMSO control assay.