The antitumor
anthracycline nemorubicin is converted by human liver microsomes to a major metabolite,
PNU-159682 (PNU), which was found to be much more potent than its parent
drug toward cultured tumor cells and in vivo
tumor models. The mechanism of action of
nemorubicin appears different from other
anthracyclines and until now is the object of studies. In fact PNU is deemed to play a dominant, but still unclear, role in the in vivo antitumor activity of
nemorubicin. The interaction of PNU with the
oligonucleotides d(CGTACG)(2), d(CGATCG)(2) and
d(CGCGCG)(2) was studied with a combined use of (1)H and (31)P NMR spectroscopy and by ESI-mass experiments. The NMR studies allowed to establish that the intercalation between the base pairs of the duplex leads to very stable complexes and at the same time to exclude the formation of covalent bonds. Melting experiments monitored by NMR, allowed to observe with high accuracy the behaviour of the
imine protons with temperature, and the results showed that the re-annealing occurs after melting. The formation of reversible complexes was confirmed by HPLC-tandem mass spectra, also combined with
endonuclease P1digestion. The MS/MS spectra showed the loss of neutral PNU before breaking the double helix, a behaviour typical of
intercalators. After digestion with the
enzyme, the spectra did not show any compound with PNU bound to the bases. The evidence of a reversible process appears from both
proton and
phosphorus NOESY spectra of PNU bound to d(CGTACG)(2) and to d(CGATCG)(2). The dissociation rate constants (k(off)) of the slow step of the intercalation process, measured by (31)P NMR NOE-exchange experiments, showed that the kinetics of the process is slower for PNU than for
doxorubicin and
nemorubicin, leading to
a 10- to 20-fold increase of the residence time of PNU into the intercalation sites, with respect to
doxorubicin. A relevant number of NOE interactions allowed to derive a model of the complexes in
solution from restrained MD calculations. The conformation of PNU bound to the
oligonucleotides was also derived from the coupling constant values.