Abstract
We investigated the interaction with DNA of two synthetic derivatives of the antitumor antibiotic rebeccamycin: R-3, which is a potent topoisomerase I inhibitor and contains a methoxyglucose moiety appended to the indolocarbazole chromophore, and its aglycone, R-4. Spectroscopic measurements indicate that R-3intercalates into DNA and that its carbohydrate domain contributes significantly to reinforce the affinity for DNA. Two complementary ligation assays concur that R-3, but not its aglycone counterpart, exerts a significant effect on the curvature and/or the flexibility of DNA. The sugar moiety may be responsible for preferential binding of R-3 to circular (or bent) DNA molecules as opposed to linear DNA fragments. The sequence selectivity of binding to DNA has been studied thoroughly by footprinting with DNase I and two other nucleases. The glycosylated compound is highly selective for nucleotide sequences containing GpT (ApC) and TpG (CpA) steps. The derivative lacking the sugar moiety on the indolocarbazole chromophore binds at essentially identical sites but with considerably lower affinity, so it seems that the chromophore rather than the carbohydrate is responsible for the preferential binding to sequences surrounding GpT and TpG steps. The influence of the exocyclic substituents present on the bases at the recognition sites (i.e., the 2-amino group of guanine and the 5-methyl group of thymine) was evaluated using two series of modified DNA molecules prepared by polymerase chain reaction containing inosine and/or 2,6-diaminopurine and uridine and/or 5-methylcytosine residues. The introduction of the amino group onto purine residues or the addition of a methyl group to pyrimidine residues suffices to create new drug binding sites. Therefore, unlike most DNA-binding small molecules, the rebeccamycin analogue seems to be highly sensitive to any modification of the exocyclic substituents on the bases in both the major and minor grooves of the double helix. The footprinting profiles with the different DNA fragments bear a remarkable resemblance to those determined for nogalamycin and bisnaphthalimide compounds known to recognize their preferred GpT and TpG sites via intercalation from the major groove. The unique DNA binding characteristics of the rebeccamycin analogue correlate well with its inhibitory effects on topoisomerase I .
Footnotes
- Received July 23, 1997.
- Accepted September 18, 1997.
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Send reprint requests to: Dr. Christian Bailly, IRCL–INSERM U124, Place de Verdun, 59045 Lille cedex, France. E-mail:bailly{at}lille.inserm.fr
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This work was supported by research grants from the Association pour la Recherche sur le Cancer (Grant ARC6932) (C.B.) and the Fédération Nationale des Groupements des Entreprises Français dans la Lutte contre le Cancer; (M.J.W.) from the Cancer Research Campaign, the Wellcome Trust, Association for International Cancer Research, and the Sir Halley Stewart Trust. Support by the convention INSERM-CFB is acknowledged.
- The American Society for Pharmacology and Experimental Therapeutics
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