Stimulation of site-specific topoisomerase II-mediated DNA cleavage by an N-methylpyrrolecarboxamide-anilinoacridine conjugate: relation to DNA binding.

Abstract

The DNA binding properties and effects on topoisomerase II of MePyGA, an anilinoacridine derivative bearing an N-methylpyrrolecarboxamide unit at position 1', have been compared with those of its precursor glycylanilinoacridine and the structurally related antileukaemic drug amsacrine. Electric linear dichroism spectroscopy reveals that MePyGA intercalates its acridine chromophore between DNA base pairs with a preference for GC-rich sequences, whereas both its structural analogue lacking the N-methylpyrrole unit and amsacrine intercalate into DNA without any strong sequence preference. The effects of the test drug on the catalytic activities of topoisomerase II were studied in vitro using purified calf thymus enzyme and 32P-labeled DNA. MePyGA stabilizes the topoisomerase II-DNA covalent complex and stimulates the cutting of DNA at a subset of preexisting topoisomerase II cleavage sites. The removal of the N-methylpyrrole unit abolishes both the GC-preferential binding to DNA and the topoisomerase II-mediated DNA cleavage. MePyGA and amsacrine stimulate the cleavage of DNA by topoisomerase II at different places: cleavage stimulated by amsacrine is consistent with the expected adenine requirement at position +1 whereas the predominant sites of DNA cleavage stimulated by MePyGA contain a cytosine at position +/- 1. This is the first instance where an anilinoacridine derivative differing only by the nature of the substituent at position 1' has been found to affect the catalytic activity of topoisomerase II differently. The spectroscopic and biochemical data lead to the conclusion that two functional domains can be identified in MePyGA: its anilino group can be regarded as a skeletal core to which are connected (i) the tricyclic acridine moiety which represents the DNA-binding domain and (ii) the N-methylpyrrole moiety which constitutes the topoisomerase II-targeted domain. The structure of the substituent at position 1' of the anilinoacridine chromophore evidently determines the location of the sites of DNA cleavage by topoisomerase II. These findings provide guidance for the synthesis and development of new topoisomerase II-targeted antitumor anilinoacridine derivatives.