DNA topoisomerase II-dependent cytotoxicity of alkylaminoanthraquinones and their N-oxides

Abstract

We studied the role of DNA topoisomerase II in the biological actions of a series of novel alkylaminoanthraquinones, including N-oxide derivatives designed as prodrugs liable to bioreductive activation in hypoxic tumour cells. Drug structures were based upon the DNA-binding anticancer topoisomerase II poison mitoxantrone with modifications to the alkylamino side chains. The agents included AQ4, 1,4-bis{[2-(dimethylamino)ethyl] amino}5,8-dihydroxy-anthracene-9,10-dione, and AQ6, 1{[2-dimethylamino)-ethyl]amino}4-{[2[(hydroxyethyl)amino]ethyl]- amino}5,8-dihydroxy-anthracene-9,10-dione, together with the corresponding mono-N-oxide (AQ6NO) and di-N-oxide (AQ4NO). The R3N(+)-O- modification renders the terminal nitrogen group electrically neutral and was found to reduce AQ6NO or effectively abolish AQ4NO-DNA binding. Comparative studies were carried out using two SV40-transformed fibroblast cell lines, MRC5-V1 and AT5BIVA, the latter being a relative overproducer of DNA topoisomerase II alpha. The inhibition of DNA topoisomerase II decatenation activity ranked according to DNA-binding capacity. A similar ranking was found for drug-induced DNA-protein cross-linking in intact cells, depending upon topoisomerase II availability. Inhibition of DNA synthesis in S-phase synchronized cultures ranked in the order of AQ6 > mitoxantrone > > AQ6NO and was independent of topoisomerase II availability. Cytotoxicity of acute 1-h exposures for all agents except the inactive AQ4NO was enhanced in the topoisomerase II-overproducing cell line. The results indicate an important role for enzyme targeting in anthraquinone action. However, DNA synthesis inhibition and cytotoxicity were greater than expected for AQ6, given its topoisomerase- and DNA-interaction properties, and parallel studies have provided evidence of an additional role for enhanced subcellular accumulation and nuclear targeting. The inactivity of AQ4NO and the retention of only partial activity of AQ6NO, allied with the effective topoisomerase II-targeting and high cytotoxic potential of their presumed metabolites, favour their use as prodrugs in tumour cells with enhanced bioreductive potential.

Spectral analysis of the DNA targeting bisalkylaminoanthraquinone DRAQ5 in intact living cells

“…The intercalation is stabilized by electrostatic interactions between the protonated tertiary amino group of the side chain and the phosphate backbone of the DNA. Previous molecular modeling studies have suggested that anthraquinone derivatives substituted at the 1,4-and 1,8-a-NH(CH 2 ) 2 NH(CH 2 CH 3 ) 2 1 side chain intercalate with DNA, with both substituents in the same groove (classical intercalation), while the similarly substituted 1,5-derivative intercalates, in a threading mode, with a side chain in each groove. The current study shows that binding appears to involve a preference for AT-containing sequences in living cells suggested by the results of Hoechst 33342 quenching studies, in which DRAQ5 appears to have the ability to colocate at a significant fraction of Hoechst 33342 binding sites, and to have the potential for minor groove interactions revealed by modeling studies.…”

“…tein targets for molecular therapeutics, as shown by the anthraquinone-mediated inhibition of DNA topoisomerase II function (2) and the anthracycline-mediated blocking of mismatch repair (3). Critically, both the development of novel drugs with DNA binding properties (i.e.…”

“…The visible absorption band of the parent molecule can be assigned to a single electronic state, with a reduced dipole moment in the excited state (8). Previous molecular modeling studies have suggested that anthraquinone derivatives, substituted at the 1,4-and 1,8-a-NH(CH 2 ) 2 NH(CH 2 CH 3 ) 2 1 side chain, intercalate with DNA, with both substituents in the same groove (classical intercalation), while the similarly substituted 1,5-derivative (DRAQ5) intercalates in a threading mode, with a side chain in each groove (9). DRAQ5 has high DNA binding affinity, while its anthraquinone chromophore is only weakly fluorescent (5,10).…”

AQ4N: a new approach to hypoxia-activated cancer chemotherapy

“…In other studies, where reduction was facilitated, potent cytotoxicity was observed in the nM range (Patterson, 1993;Wilson et al, 1996;Smith et al, 1997b). In vitro the differential activity of AQ4N, to its active metabolite AQ4, is considerably greater than any other N-oxide described including DACA-N-oxide and nitracrine N-oxide (Wilson et al, 1996).…”

Minor structural modifications to alchemix influence mechanism of action and pharmacological activity

“…The experiments was carried out as previously described using the anthraquinone-based clinical prodrug AQ4N and its metabolite AQ4 (topo IIα inhibitor) as negative and positive controls respectively [22]. All compounds were shown to completely inhibit topo IIα at 2.5 µM but at lower concentrations a dose-response was observed.…”