DNA binding by pixantrone

The binding of the anticancer drug pixantrone (6,9-bis[(2-aminoethyl)amino]benzo[g]isoquinoline-5,10-dione dimaleate) to the octanucleotide duplexes d(ACGATCGT)2 and the corresponding C-5 methylated cytosine (5MeC) analogue d(A5MeCGAT5MeCGT)2 has been studied by NMR spectroscopy and molecular modelling. The large upfield shifts observed for the resonances from the aromatic protons of pixantrone upon addition to either d(ACGATCGT)2 or the corresponding 5MeC analogue is consistent with the drug binding the octanucleotides by intercalation. The selective reduction in the sequential NOEs between the C2-G3 and C6-G7 nucleotides in NOESY spectra of either octanucleotide with added pixantrone confirms the intercalative binding mechanism. Strong NOEs from the side-chain ethylene protons of pixantrone to the H5 protons and the 5-CH3 protons of the C2 and C6 residues of d(ACGATCGT)2 and d(A5MeCGAT5MeCGT)2, respectively, indicate that pixantrone predominantly intercalates from the DNA major groove at the 5′-CG and 5′-5MeCG sites. Simple molecular models based on the conclusions from the NMR experiments indicated that the 5MeC groups do not represent a steric barrier to intercalation from the major groove. However, the observation of weak NOEs from the ethylene protons of pixantrone to a variety of minor groove protons from either octanucleotide suggests that the drug can also associate in the minor groove.