Confirmation of the positive hits.
(A) Chemical structure of VM26 and three positive hits, betulinic acid, DDI and CET. (B) Anisotropy of enzyme/DNA/drug ternary complexes. The reaction was performed in 50 µl of reaction buffer with 50 nM Alexa Fluor 488-labeled DNA and 250 nM human Top2α. Negative control (filled) contained neither VM26 nor test compounds. Positive control (open) contained 300 µM VM26. Others (gray) contained 15 µM of test compounds. The reaction mixture was incubated at 37°C for 30 minutes. Before measuring the anisotropy, NaClO4 was added into the reaction mixture to give a final concentration of 200 mM. (C) Effects of positive hits on inhibition of DNA relaxation. The reaction contained 8.5 nM pUC19 and 0.125 nM human Top2α in 20 µl reaction buffer, and performed at 37°C for 5, 10 or 15 minutes. Solvent control contained 5% DMSO, and the other reactions contained 60 µM hit compound (DMSO was used as the solvent and its final concentration in each reaction was 5 %). (D) Effects of positive hits on DNA cleavage assay. The 20 µl reaction contained 8.5 nM pUC19 and 3.75 nM human Top2α with Top2α targeting compounds at the concentrations marked above each lane. Reaction products were analyzed by electrophoresis in a 1.2% agarose gel containing 0.15 µg/ml ethidium bromide. Positions of DNA were marked as NC (nicked circular), L (linear), NSC (negative supercoiled), and RC (relaxed).