Molecular determinants of site-specific inhibition of human DNA topoisomerase I by fagaronine and ethoxidine - Relation to DNA binding

DNA topoisomerase (top) I inhibition activity of the natural alkaloid fagar onine (NSC157995) and its new synthetic derivative ethoxidine (12-ethoxy-be nzo[c] phenanthridine) has beers correlated with their molecular interactio ns and sequence specificity within the DNA complexes. Flow linear dichroism shows that ethoxidine exhibits the same inhibition of DNA relaxation as fa garonine at the 10-fold lower concentration. The patterns of DNA cleavage b y top I show linear enhancement of CPT-dependent sites at the 0.016-50 mu M concentrations of fagaronine, whereas ethoxidine suppress both top I-speci fic and CPT-dependent sites. Suppression of top I-mediated cleavage by etho xidine is found to be specific for the sites, including strand cut between A and T, Fagaronine and ethoxidine are DNA major groove intercalators. Etho xidine intercalates DNA in A-T sequences and its 12-ethoxy-moiety (absent i n fagaronine) extends into the DNA minor groove. These findings may explain specificity of suppression by ethoxidine of the strong top I cleavage site s with the A(+1), T(-l) immediately adjacent to the strand cut. Fagaronine does not show any sequence specificity of DNA intercalation, but its highly electronegative oxygen of hydroxy group (absent in ethoxidine) is shown to be an acceptor of the hydrogen bond with the NH, group of G base of DNA. A bility of fagaronine to stabilize top I-mediated ternary complex is propose d to be determined by interaction of its hydroxy group with the guanine at position (+1) of the DNA cleavage site and of quaternary nitrogen interacti on with top I, The model proposed provides a guidance for screening new top I-targeted drugs in terms of identification of molecular determinants resp onsible for their top I inhibition effects.