We have employed a broad range of spectroscopic, calorimetric, DNA cleavage, and
DNA winding/unwinding measurements to characterize the
DNA binding and
topoisomerase I (TOP1)
poisoning properties of three terbenzimidazole analogues,
5-phenylterbenzimidazole (5PTB), terbenzimidazole (TB), and 5-(naphthyl[2,3-d]imidazo-2-yl)bibenzimidazole (5NIBB), which differ with respect to the substitutions at their C5 and/or C6 positions. Our results reveal the following significant features. (i) The overall extent to which the three terbenzimidazole analogues
poison human TOP1 follows the hierarchy 5PTB > TB >> 5NIBB. (ii) The impact of the three terbenzimidazole analogues on the superhelical state of plasmid
DNA depends on the [total
ligand] to [base pair] ratio (rbp), having no effect on
DNA superhelicity at rbp ratios < or = 0.1, while weakly unwinding
DNA at rbp ratios > 0.1. This weak
DNA unwinding activity exhibited by the three terbenzimidazoles does not appear to be correlated with the abilities of these compounds to
poison TOP1. (iii) Upon complexation with both
poly(dA).poly(dT) and salmon testes
DNA, the three terbenzimidazole analogues exhibit flow linear dichroism properties characteristic of a minor groove-directed mode of binding to these host
DNA duplexes. (iv) The apparent minor groove binding affinities of the three terbenzimidazole analogues for the d(GA4T4C)2 duplex follow a qualitatively similar hierarchy to that noted above for
ligand-induced
poisoning of human TOP1-namely, 5PTB > TB > 5NIBB. In the aggregate, our results suggest that
DNA minor groove binding, but not
DNA unwinding, is important in the
poisoning of TOP1 by terbenzimidazoles.