Human topoisomerase I poisoning by protoberberines: Potential roles for both drug-DNA and drug-enzyme interactions

Protoberberines represent a structural class of organic cations that induce topoisomerase I-mediated DNA cleavage, a behavior termed topoisomerase I p oisoning. We have employed a broad range of biophysical, biochemical, and c omputer modeling techniques to characterize and cross-correlate the DNA-bin ding and topoisomerase poisoning properties of four protoberberine analogue s that differ with respect to the substituents on their A- and/or D-rings. Our data reveal the following significant features: (i) The binding of the four protoberberines unwinds duplex DNA by approximately 11 degrees, an obs ervation consistent with an intercalative mode of interaction. (ii) Enthalp ically favorable interactions, such as stacking interactions between the in tercalated ligand and the neighboring base pairs, provide <50% of the therm odynamic driving force for the complexation of the protoberberines to duple x DNA. Computer modeling studies on protoberberine-DNA complexes suggest th at only rings C and D intercalate into the host DNA helix, while rings A an d B protrude out of the helix interior into the minor groove. (iii) All fou r protoberberine analogues are topoisomerase I-specific poisons, exhibiting little or no topoisomerase II poisoning activity. (iv) Modifications of th e D-ring influence both DNA binding and topoisomerase I poisoning propertie s. Specifically, transference of a methoxy substituent from the 11- to the 9-position diminishes both DNA binding affinity and topoisomerase I poisoni ng activity, an observation suggesting that DNA binding is important in the poisoning of topoisomerase I by protoberberines, (v) Modifications of the A-ring have a negligible impact on DNA binding affinity, while exerting a p rofound influence on topoisomerase I poisoning activity. Specifically, prot oberberine analogues containing either 2,3-dimethoxy; 3,4-dimethoxy; or 3,4 -methylenedioxy substituents all bind DNA with a similar affinity. By contr ast, these analogues exhibit markedly different topoisomerase I poisoning a ctivities, with these activities following the hierarchy: 3,4-methylenediox y > 2,3-dimethoxy >> 3,4-dimethoxy. These differences in topoisomerase I po isoning activity may reflect the differing abilities of the analogues to in teract with specific functionalities on the enzyme, thereby stabilizing the enzyme in its cleavable state. In the aggregate, our results are consisten t with a mechanistic model in which both ligand-DNA and ligand-enzyme inter actions are important for the poisoning of topoisomerase I by protoberberin es, with the DNA-directed interactions involving ring D and the enzyme-dire cted interactions involving ring A. It is reasonable to suggest that the po isoning of topoisomerase I by a broad range of other naturally occurring an d synthetic ligands may entail a similar mechanism.