A TERBENZIMIDAZOLE THAT PREFERENTIALLY BINDS AND CONFORMATIONALLY ALTERS STRUCTURALLY DISTINCT DNA DUPLEX DOMAINS - A POTENTIAL MECHANISM FOR TOPOISOMERASE-I POISONING

The terbenzimidazoles are a class of synthetic ligands that poison the human topoisomerase 1 (TOP1) enzyme and promote cancer cell death. It has been proposed that drugs of this class act as TOP1 poisons by bin ding to the minor groove of the DNA substrate of TOP1 and altering its structure in a manner that results in enzyme-mediated DNA cleavage. T o test this hypothesis, we characterize and compare the binding proper ties of a 5-phenylterbenzimidazole derivative (5PTB) to the d(GA(4)T(4 )C)(2) and d(GT(4)A(4)C)(2) duplexes. The d(GA(4)T(4)C)(2) duplex cont ains an uninterrupted 8-bp A-T domain, which, on the basis of x-ray cr ystallographic data, should induce a highly hydrated ''A-tract'' confo rmation. This duplex also exhibits anomalously slow migration in a pol yacrylamide gel, a feature characteristic of a noncanonical global con formational state frequently described as ''bent.'' By contrast, the d (GT(4)A(4)C)(2) duplex contains two 4-bp A-T tracts separated by a TpA dinucleotide step, which should induce a less hydrated ''B-like'' con formation. This duplex also migrates normally in a polyacrylamide gel, a feature further characteristic of a global, canonical B-form duplex . Our data reveal that, at 20 degrees C, 5PTB exhibits an approximate to 2.3 kcal/mol greater affinity for the d(GA(4)T(4)C)(2) duplex than for the d(GT(4)A(4)C)(2) duplex. Significantly, we find this sequence/ conformational binding specificity of 5PTB to be entropic in origin, a n observation consistent with a greater degree of drug binding-induced dehydration of the more solvated d(GA(4)T(4)C)(2) duplex. By contrast with the differential duplex affinity exhibited by 5PTB, netropsin an d 4',6-diamidino-2-phenylindole (DAPI), two AT-specific minor groove b inding ligands that are inactive as human TOP1 poisons, bind to both d uplexes with similar affinities. The electrophoretic behaviors of the ligand-free and ligand-bound duplexes are consistent with 5PTB-induced bending and/or unwinding of both duplexes, which, for the d(GA(4)T(4) C)(2) duplex, is synergistic with the endogenous sequence-directed ele ctrophoretic properties of the ligand-free duplex state. By contrast, the binding to either duplex of netropsin or DAPI induces little or no change in the electrophoretic mobilities of the duplexes. Our results demonstrate that the TOP1 poison 5PTB binds differentially to and alt ers the structures of the two duplexes, in contrast to netropsin and D API, which bind with similar affinities to the two duplexes and do not significantly alter their structures. These results are consistent wi th a mechanism for TOP1 poisoning in which drugs such as 5PTB differen tially target conformationally distinct DNA sites and induce structura l changes that promote enzyme-mediated DNA cleavage.