Interaction between the cytotoxic drug Clerocidin and DNA: evidence for covalent adduct formation

The diterpenoid Clerocidin causes DNA breaks by stimulating topoisomerase mediated DNA cleavage. The sequence analysis of the cleavage indicated the presence of two types of sites, one exhibtig easily reversible DNA cleavage, the other showing an irreversible cleavage, along with an abnormal migration during gel electrophoresis. This suggested an irreversible mcdification caused by the drug when interacting with the DNA-topoisomerase cleavable complex. Given the chemical structure of Clerocidin, it can be proposed that the dug is reactive towards DNA. To verify this hypothesis, the interaction of Clerocidin with single and double stranded DNA sequences, as well as with deoxyrihonucleatides, was investigated under various experimental conditions using gel electrophoresis, high-field NMR and high performance liquid chromatography coupled to mass spectrometry. Indeed, Clerocidin was able lo produce cleavage in the nucleic acid structure. In addition, the drug reacted with DNA bases giving several adducta, which were characterized and identified. In particular, our results suggest the formation of a covalent guanine-Clerocdin adduct to explain irreversible binding, and of a cytosine-Clerocidin Schiff base to account for reversible binding.