Influence of compound structure on affinity, sequence selectivity, and mode of binding to DNA for unfused aromatic dications related to furamidine

In the course of a program aimed at developing sequence-specific gene-regulatory small organic molecules, we have investigated the DNA interactions of a new series of nine diphenylfuran dications related to the antiparasitic drug furamidine (DB75). Two types of structural modifications were tested: the terminal amidine groups of DB75 were shifted from the para to the meta position, and the amidines were replaced with imidazoline or dimethyl-imidazoline groups, to test the importance of both the position and nature of positively charged groups on DNA interactions. The interactions of these compounds with DNA and oligonucleotides were studied by a combination of biochemical and biophysical techniques. Absorption and CD measurements suggested that the drugs bind differently to AT and GC sequences in DNA. The para-para dications, like DB75, bind into the minor groove of poly(dAT)(2) and intercalate between the base pairs of poly(dGC)(2), as revealed by electric linear dichroism experiments. In contrast, the meta-meta compounds exhibit a high tendency to intercalate into DNA whatever the target sequence. The lack of sequence selectivity of the meta-meta compounds containing amidines or dimethyl-imidazoline groups was also evident from DNase I footprinting and surface plasmon resonance (SPR) experiments. Accurate binding measurements using the BIAcore SPR method revealed that all nine compounds bind with similar affinity to an immobilized GC sequence DNA hairpin but exhibit very distinct affinities for the corresponding AT hairpin oligonucleotide. The minor groove-binding para-para compounds have a high specificity for AT sequences. The biophysical data clearly indicate that shifting the cationic substituents from the para to the meta position results in a loss of specificity and change in binding mode. The strong AT selectivity of the para-para compounds was independently confirmed by DNase I footprinting experiments performed with a range of DNA restrictions fragments. In terms of AT selectivity, the compounds rank in the order para-para > para-meta > meta-meta. The para dications bind preferentially to sequences containing four contiguous AT base pairs. Additional footprinting experiments with substrates containing the 16 possible A.T](4) blocks indicated that the presence of a TpA step within an A.T] (4) block generally reduces the extent of binding. The diverse methods, from footprinting to SPR to dichroism, provide a consistent model for the interactions of the diphenylfuran dications with DNA of different sequences. Altogether, the results attest unequivocally that the binding mode for unfused aromatic cations can change completely depending on substituent position and DNA sequence. These data provide a rationale to explain the relationships between sequence selectivity and mode of binding to DNA for unfused aromatic dications related to furamidine.