Modulation of Sequence Specificity of Duocarmycin-Dependent DNA Alkylation by Pyrrole−Imidazole Triamides

The ability of small molecules to modify nucleic acids irreversibly has generated considerable current interest. Many anticancer drugs interact with DNA, although most have little sequence specificity and often exhibit severe toxicity to normal tissues. Rational design of alkylating agents targeting specific sequences in the human genome may provide useful molecules for various applications, including better anticancer drugs. Anticancer antibiotic duocarmycin A (Duo) normally alkylates duplex DNA at the A-N3 site on the 3' side of 3-4 consecutive A times T base pairs. Previously, we found that the addition of distamycin A (Dist) markedly modulates the sequence specificity of Duo, causing the alkylation to shift primarily to the G residues in GC-rich sequences. Our 2D-NMR analysis revealed that the molecular mechanism of modulation of sequence specificity involves a cooperative heterodimer formation of Duo and Dist in the minor groove. In the ternary complex, Dist recognizes the DNA strand (complementary to the alkylated strand) using a binding mode similar to that of the dimers of N-methylimidazole-(Im)-N-methylpyrrole(Py) polyamides. These results suggest an intriguing possibility that the sequence specificity of Duo can be controlled in a predictable manner by pairing with Py/Im triamides. For example, DNA alkylation by Duo in the presence of ImImIm is expected to occur at 5'-CCCG-3' sequence according to the pairing rule for Im/Py polyamides. To test the above hypothesis, we synthesized six sets of Im/Py triamides and analyzed the site of DNA alkylation by Duo in the presence of these Im/Py triamides (Figure 1).