Mode of binding of the cytotoxic alkaloid berberine with the double helix oligonucleotide d(AAGAATTCTT)(2).

Berberine, an isoquinoline plant alkaloid, belongs to the structural class of protoberberines. Recently, the ability of these compounds to act as Topoisomerase I or II poisons, was related to the antitumor activity. The binding of protoberberins to DNA has been studied and the partial intercalation into the double helix has been considered responsible for their activity. We have studied the interaction of berberine with the double helix oligonucleotides d(AAGAATTCTT)(2), d(GCGATCGC)(2), d(CGTATACG)(2), d(CGTACG)(2), 5'-d(ACCTTTTTGATGT)-3'/5(ACATCAAAAAGGT)-3' and with the single strand 5'-d(ACATCAAAAAGGT)-3', by 1H, 31P NMR and UV spectroscopy. Phosphorus resonance experiments were performed to detect small conformational changes of the phosphoribose backbone, in the case that an intercalation process occurs. Our data reveal that berberine does not intercalate into the duplexes studied, and binds preferentially to AT rich sequences. The structure of the complex with d(AAGAATTCTT)(2) was determined by using proton 2D NOESY spectra, which allowed to obtain several NOE contacts between the drug and the nucleotide. Structural models were built up by Molecular Mechanics (MM) and Molecular Dynamics (MD) calculations, by using the inter-proton distances derived from the NOE values. Berberine results to be located in the minor groove, lying with the convex side on the helix groove and presenting the positively charged nitrogen atom close to the negative ionic surface of the oligomer. The large 1H chemical shifts variation, observed for the drug when it is added to the above duplexes, as well as to the single strand oligomer, was interpreted with non-specific ionic interactions. The binding constants were measured by UV and NMR spectroscopy. They are strongly affected by the ionic strength and by the self-association process, which commonly occurs with this type of drugs. A dimerisation constant was measured and the value was included in the calculations of the binding constants. The results obtained show that the non-specific ionic interactions represent the major contribution to the values of the binding constants. These parameters, as well as the protons chemical shift variation of the ligand, are thus not diagnostic for the identification of a drug/DNA complex.