IKE1-IKE3 (C-1305 derivatives) inhibitory effect of the Yeast Topoisomerase II relaxation activity

Inhibition of Yeast Topoisomerase II were analyzed according to relaxation assay kit  from Inspiralis. Briefly, 250 ng of supercoiled pBR322 DNA, 1 mM ATP, 1-200 μM of analyzed compound were mixed with reaction buffer (1 mM Tris-HCl (pH 7.9), 10 mM KCl, 0.5 mM MgCl 2, 0.2 % (v/v) glycerol). The reaction was initiated by the addition of an enzyme, allowed to proceed at 30°C for 30 min and terminated by addition of 40% (w/v) sucrose, 100 mM Tris-HCl pH 8, 10 mM EDTA, 0.5 mg mL -1 Bromophenol Blue. Two step extraction with chloroform:isoamyl alcohol (24:1) and butanol water were made and mixtures were analyzed on the 1% agarose gel in 1x TAE buffer, 3h, 4.5 V cm -1. Gel was  stained in GelRed 3x staining solution for 30 min and photographed with Gel Doc XR+Gel Documentation System.