RECOGNITION OF SPECIFIC SEQUENCES IN DNA BY A TOPOISOMERASE-I INHIBITOR DERIVED FROM THE ANTITUMOR DRUG REBECCAMYCIN

We investigated the interaction with DNA of two synthetic derivatives of the antitumor antibiotic rebeccamycin: R-3, which is a potent topoi somerase I inhibitor and contains a methoxyglucose moiety appended to the indolocarbazole chromophore, and its aglycone, R-4. Spectroscopic measurements indicate that R-3 intercalates into DNA and that its carb ohydrate domain contributes significantly to reinforce the affinity fo r 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 pr eferential binding of R-3 to circular (or bent) DNA molecules as oppos ed to linear DNA fragments. The sequence selectivity of binding to DNA has been studied thoroughly by footprinting with DNase I and two othe r nucleases. The glycosylated compound is highly selective for nucleot ide 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 i t seems that the chromophore rather than the carbohydrate is responsib le for the preferential binding to sequences surrounding GpT and TpG s teps. 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 inosin e and/or 2,6-diaminopurine and uridine and/or 5-methylcytosine residue s. The introduction of the amino group onto purine residues or the add ition 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 modifi cation of the exocyclic substituents on the bases in both the major an d minor grooves of the double helix. The footprinting profiles with th e different DNA fragments bear a remarkable resemblance to those deter mined for nogalamycin and bisnaphthalimide compounds known to recogniz e their preferred GpT and TpG sites via intercalation from the major g roove. The unique DNA binding characteristics of the rebeccamycin anal ogue correlate well with its inhibitory effects on topoisomerase I.