Minor groove binding compounds that jump a gc base pair and bind to adjacent AT base pair sites.

Many cationic-heterocyclic compounds selectively target A/T sequences by binding in the DNA minor groove. With the exception of polyamides, targeting GC and mixed AT/GC sequences has been difficult with this class of compounds. Minor groove binding diamidines are AT specific minor groove binding agents, such as DB75 and pentamidine (Figure 1), that have good uptake into many cells with relatively low toxicity (1–3). DB289, an orally active prodrug of DB75, which has reached phase III clinical trials against sleeping sickness, is active against other microbial parasites (1,4–6). Pentamidine has been available for many years and is widely used against trypanosomiasis, leishmaniasis and P. carinii pneumonia (4, 7–9). In a recent study of diamidine minor groove binders, it was shown that stacked minor groove dimer formation of DB293 in an ATGA binding site of the Pit-1 and Brn-3 promoter sequences inhibits binding of these two transcription factors to the major groove of DNA through structural changes in the minor groove (10). Cooperative stacked dimer formation in a single binding site is one of the methods to target DNA sequences with high specificity, but so far there is no general way to target mixed sequences with heterocyclic dications (11). Figure 1 Compound structures and DNA sequences. Kinetoplast minicircle mitochondrial DNAs (kDNA)1 in disease-causing kinetoplastid parasites have numerous A/T binding sites in thousands of repeated minicircles (5, 12, 13). Compounds that could bind cooperatively to such repeated sequences that are separated by one or more GC base pairs could have very high selectivity for targeting parasites. Cooperativity in adjacent binding sites in regions with A/T sequences, such as kDNA is, however, difficult to achieve with currently available compounds. Although some compounds show strong cooperativity in binding to two adjacent binding sites in short duplex DNAs (14), the cooperativity decreases in less dynamic, hairpin DNAs, which resemble longer sequences (15). Adjacent sites in kDNA might also be selectively targeted by compounds with linkers that could cross intervening GC base pairs in the minor groove. The kDNA minicircles have numerous sequences with three consecutive A/T bases separated by a single GC base pair (16). Compounds that could bind to such sequences, as well as to the classical binding sites with four or more AT base pairs, would have significantly enhanced selectivity for kDNA. Thus, the goal of this work is to design compounds that are able to selectively target sequences, such as AAAGTTT, and bind tightly to two A/T binding sites of 5–6 base pairs total while crossing 1–2 intervening GC base pairs. The AAAGTTT and AAAGCTTT sequences have been chosen as model systems (Figure 1) and the classical AATT site along with a single AAA site were used for comparison. To test which types of structures could bind to the target sequences, compounds that are similar to the biologically active anti-parasitic diamidines, DB75 and pentamidine (Figure 1) were initially designed and synthesized. This design concept includes two connected aromatic-amidine moieties that are specific for AT base pair sequences. Since the amino group of G in a GC base pair is in the minor groove, the AT specific groups were connected with oxy-alkyl linkers. The concept is to place oxygens in the linker to provide H-bond acceptors for interaction with the G-amino group. The interactions of the compounds with the different DNA model systems have been investigated by several different biophysical methods, and compounds that bind strongly to the AAAGTTT sequence have been identified.