Bisanthracycline WP631 inhibits basal and Sp1-activated transcription initiation in vitro

An in vitro transcription assay was used to compare the capacity of the bis intercalating anthracycline WP631 (which displays a remarkably high DNA-bin ding affinity) and the monointercalating anthracycline daunomycin to inhibi t transcription initiation of the adenovirus major late promoter linked to a G-less transcribed DNA template. Both drugs inhibit basal RNA synthesis i n a concentration-dependent way, and the drug concentrations required to in hibit transcription initiation are similar. However, in this study WP631 wa s around 15 times more efficient at inhibiting transcription initiation whe n used with an adenovirus promoter containing an upstream Spl-protein bindi ng site under experimental conditions in which the Spl protein acted as a t ransactivator in vitro. The differences in the ability of each drug to inhi bit transcription initiation were related to the competition between Spl an d the drugs for the same binding site. Concentrations of WP631 as low as 60 nM could inhibit the Spl-activated transcription initiation in vitro, In c ontrast, the concentration of daunomycin required to inhibit Spl-activated transcription by 50% was almost the same as the concentration required to i nhibit basal transcription. The efficiency of WP631 at displacing Spl from its putative binding site was confirmed using gel retardation and footprint ing assays, These results are the first unequivocal example of a direct eff ect of an intercalator on activated transcription initiation.