Download files
Abstract
The binding of the aza-anthracenedione anti-neoplastic drug pixantrone (6,9-bis[92- aminoethyl)amino]benzo[g]isoquinoline-5,10-dione fumarate) to the tridecanucleotide d(CCGAGAATTCCGG)2 that contains a single bulged adenine on each strand and the oligonucleotide duplex d(TACGATGAGTA) : d(TACCATCGTA), that contains a single bulge and a single 5’-CpG site, have been studied by low temperature NMR spectroscopy and molecular modelling. Large upfield shifts of the pixantrone aromatic protons have been observed, consistent with pixantrone binding d(CCGAGAATTCCGG)2 by intercalation. Strong NOEs between the pixantrone H7/H8 and H4 with the tridecanucleotide A4H1’ and G3H1’ protons, respectively, were observed suggesting that pixantrone binds d(CCGAGAATTCCGG)2 from minor groove at the bulge 5’-G3pA4 site and from major groove at the C2pG3 site.
The oligonucleotide d(TACGATGAGTA) : d(TACCATCGTA) was chosen to determine the pixantrone binding preference between isolated 5’-CpG and bulge sites. The selective reduction in the sequential NOE between the G7-A8 and A8-G9 nucleotides suggested that pixantrone binds at the bulge site by intercalation. Additionally, only NOEs from the pixantrone H7/H8 to A8H1’ were observed, indicating pixantrone preferentially binds from minor groove at the bulge site. Furthermore, strong NOEs from pixantrone Ha to G9H1’ were found while only weak NOEs were observed between Ha and G7H1’, consistent with the energy minimized models that showed the A8pG9 is the more favoured site than the G7pA8 site. Large upfield shifts for the G9 imino resonance, compared to the G7 resonance, upon pixantrone binding were also observed, consistent with pixantrone binding at the A8-G9 bulge site. The results of this study highlight a new favourable binding site for pixantrone, and suggest that pixantrone may be a particularly effective anti-cancer agent for cell lines that have a significant number of unrepaired bulge sites, i.e. those with a deficient mismatch repair pathway.