Abstract
| - Rebeccamycin derivatives represent a promising class of antitumor agents. In this series, twoglycosylated indolocarbazoles, NB-506 and NSC-655649, are currently undergoing clinical trials.Their anticancer activities are associated with their capacities to interact with DNA and toinhibit DNA topoisomerases. Previous studies revealed that the planar indolocarbazolechromophore can intercalate into DNA, locating the appended carbohydrate residue in one ofthe two helical grooves, probably the minor groove as is the case with the anthracyclines andother DNA-binding antibiotics. The sugar residue contributes significantly to the DNA bindingfree energy of NB-506. However, the exact positioning of the glycosyl residue of rebeccamycinderivatives in the drug−DNA complex remains poorly understood. To better understand howglycosylated indolocarbazoles interact with DNA, we investigated the interaction of arebeccamycin derivative (85) bearing a 2‘-amino group on the sugar residue. We show that thepresence of the 2‘-amino function permits the formation of covalent drug−DNA complexes inthe presence of formaldehyde. Complementary biochemical and spectroscopic measurementsattest that 85 reacts covalently with the 2-amino group of guanines exposed in the minor grooveof the double helix, as is the case with daunomycin. In contrast to daunomycin, 85 also formscross-links with an oligonucleotide containing only A·T base pairs. The covalent binding toA·T base pairs was detected using a gel mobility shift assay and was independently confirmedby thermal denaturation studies and by fluorescence measurements using a series of syntheticpolynucleotides. The HCHO-mediated alkylation reaction of the drug with A·T base pairsapparently involves the 6-amino group of adenines exposed in the major groove whereas thecovalent attachment to G·C base pairs implicates the 2-amino group of guanines situated inthe opposite minor groove. Therefore, the results suggest that either the drug is able to switchgrooves in response to sequence or it can simultaneously bind to both the minor and majorgrooves of the double helix. This study will help to guide the rational design of new DNA-binding antitumor indolocarbazole drugs and also provides a general experimental approachfor probing minor versus major groove interactions between small molecules and DNA.
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