Drug-DNA adducts formed by formaldehyde activation of anthracyclines and related anti-cancer agents

The anthracyclines (doxorubicin, epirubicin, idarubicin and daunorubicin) are among the most widely-used anticancer chemotherapeutic agents. They are generally classified as topoisomerase II inhibitors but also have a variety of other targets in cells. It has been known for some years that the anthracyclines (particularly doxorubicin) are capable of forming DNA adducts, but the relevance and extent of these adducts in cells and their role in causing cell death has remained obscure. When the structure of adducts was solved, it became clear that formaldehyde was an absolute requirement for adduct formation. Adducts form primarily at GC sequences and involve an N-C-N aminal linkage between the drug and the N-2 of guanine, with the central carbon being derived from formaldehyde. The adducts have been extensively characterised in vitro by 2D-NMR, X-ray crystallography and mass spectrometry, and the aminal linkage has been shown to be reversible, with a half-life of up to 40 h. In cells, the DNA lesions produced appear to be dependent on two competing cellular pathways that can be switched from primarily topisomerase II lesions to primarily drug-DNA adducts by the use of formaldehyde-releasing prodrugs such as AN-9. The anthracyclines-DNA adducts are able to overcome the major forms of resistance to doxorubicin, are more cytotoxic than topoisomerase II lesions, and invoke conventional apoptotic pathways that are independent of topoisomerase II. Significant levels of adducts have now been detected in breast cancer cells (approximately 1,300 adducts per cell) following a 4 h treatment with clinically relevant levels of doxorubicin (25 nM) as a single agent, and this is elevated some 100-fold by co-treatment with formaldehyde-releasing prodrugs. The synthetic anthracendione anticancer agent, mitoxantrone, can also form adducts with DNA when activated by formaldehyde. These adducts exhibit specificity for CG and CA sequences but are rather unstable. More stable adducts have been detected with pixantrone, a second-generation mitoxantrone analogue that contains primary amino groups on the side-chain. However, the need to identify novel anthracenediones with enhanced anthracenedione-DNA adduct stability remains if these adducts ultimately are to be useful in the clinic.