The inhibition of the enzymatic activity of DNMT1, DNMT3A, and DNMT3B was tested using the HotSpotSM platform for methyltransferase assays available at Reaction Biology Corporation. HotSpotSM is a low volume radioisotope-based assay that uses tritium-labeled AdoMet (3H-SAM) as a methyl donor. The three test compounds diluted in dimethyl sulfoxide were added using acoustic technology (Echo550, Labcyte) into an enzyme/substrate mixture in the nano-liter range. The corresponding reactions were commenced by adding 3H-SAM and incubated at 30°C. Total final methylations on the substrate (Poly dI-dC in DNMT1, 3A, and 3B assays) were identified by a filter binding method implemented in Reaction Biology. Data analysis was done with Graphed Prism software (La Jolla, CA, USA) for curve fits. The enzymatic inhibition assays were carried out at 1 μM of SAM. In all assays, SAH was used as a standard positive control. The three compounds were tested first with DNMT1 at one 100 μM concentration in duplicate. The most active molecules were tested as DNMT1, DNMT3A, and DNMT3B inhibitors in 10-dose IC 50 (effective concentration to inhibit enzymatic activity by 50%) with a threefold serial dilution starting at 100 μM. The research group has recently contracted the screening services of Reaction Biology Corporation to identify a novel DNMTi.