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 I (TOP1) enzyme and promote cancer cell death. It has been proposed that drugs of this class act as TOP1 poisons by binding to the minor groove of the DNA substrate of TOP1 and altering its structure in a manner that results in enzyme-mediated DNA cleavage. To test this hypothesis, we characterize and compare the binding properties of a 5-phenylterbenzimidazole derivative (5PTB) to the d(GA4T4C)2 and d(GT4A4C)2 duplexes. The d(GA4T4C)2 duplex contains an uninterrupted 8-bp A.T domain, which, on the basis of x-ray crystallographic data, should induce a highly hydrated "A-tract" conformation. This duplex also exhibits anomalously slow migration in a polyacrylamide gel, a feature characteristic of a noncanonical global conformational state frequently described as "bent." By contrast, the d(GT4A4C)2 duplex contains two 4-bp A.T tracts separated by a TpA dinucleotide step, which should induce a less hydrated "B-like" conformation. 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 approximately 2. 3 kcal/mol greater affinity for the d(GA4T4C)2 duplex than for the d(GT4A4C)2 duplex. Significantly, we find this sequence/conformational binding specificity of 5PTB to be entropic in origin, an observation consistent with a greater degree of drug binding-induced dehydration of the more solvated d(GA4T4C)2 duplex. By contrast with the differential duplex affinity exhibited by 5PTB, netropsin and 4',6-diamidino-2-phenylindole (DAPI), two AT-specific minor groove binding ligands that are inactive as human TOP1 poisons, bind to both duplexes 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(GA4T4C)2 duplex, is synergistic with the endogenous sequence-directed electrophoretic 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 alters the structures of the two duplexes, in contrast to netropsin and DAPI, which bind with similar affinities to the two duplexes and do not significantly alter their structures. These results are consistent with a mechanism for TOP1 poisoning in which drugs such as 5PTB differentially target conformationally distinct DNA sites and induce structural changes that promote enzyme-mediated DNA cleavage.
Pilch
D S
DS
Department of Pharmacology, University of Medicine and Dentistry of New Jersey, Robert Wood Johnson Medical School, Piscataway, NJ 08854, USA.
Xu
Z
Z
Sun
Q
Q
LaVoie
E J
EJ
Liu
L F
LF
Breslauer
K J
KJ
D001483:Base Sequence; D001562:Benzimidazoles / Q000737:chemistry / Q000378:metabolism* / Q000494:pharmacology*; D001665:Binding Sites; D002942:Circular Dichroism; D004247:DNA / Q000737:chemistry / Q000187:drug effects* / Q000378:metabolism*; D004264:DNA Topoisomerases, Type I / Q000378:metabolism*; D006801:Humans; D066298:In Vitro Techniques; D007211:Indoles / Q000378:metabolism; D008024:Ligands; D009429:Netropsin / Q000378:metabolism; D009690:Nucleic Acid Conformation / Q000187:drug effects; D013379:Substrate Specificity; D013816:Thermodynamics
C103388:5-phenyl-2-(2'-(benzimidazol-5''-yl)benzimidazol-5'-yl)benzimidazole; D001562:Benzimidazoles; D007211:Indoles; D008024:Ligands; C007293:DAPI; D009429:Netropsin; D004247:DNA; D004264:DNA Topoisomerases, Type I