Book of Abstracts: Albany 2011
June 14-18 2011
©Adenine Press (2010)
Minor Groove Ligands Based on Dimeric Bisbenzimidazoles as Inhibitors of DNA-Dependent Enzymes
Recently in this Journal there have been reports of both intercalators and groove binders interacting with DNA double helix (1-4). We have reported elsewhere that dimeric bisbenzimidazoles ( DB[n] ) differing in the length of methylene linkers can interact with dsDNA in the minor groove (5).
Figure 1: Structure of dimeric bisbenzimidazoles DB[n],where n = 1, 2, 3, 4, 5, 7, 11.
Compounds DB[n] were studied as inhibitors of two DNA-dependent enzymes, namely, calf thymus DNA topoisomerase I (topo-I) (6) and the catalytic domain Dnmt3a of murine DNA methyltransferase (MTase) (7). Ligands DB[3, 4, 5, 7, 11] inhibited activities of topo-I in vitro at 0.5-2.5 μM concentrations. Inhibitory activity of DB was 50-fold higher than that of camptothecin, a known topo-I inhibitor. DB[1, 2, 3, 11] reduced in vitro the MTase Dnmt3a activity by 50% at 5-12 μM concentrations. Bisbenzimidazoles DB[4, 5, 7] were shown to be less effective: 50% inhibition of MTase activity was observed only at a concentration of 20 μM and higher. Increased time of incubation of the tested DB[n] with DNA allowed for a substantial growth of inhibitory activity of the ligands toward both topo-I and MTase (6, 7). It should be noted that monomeric MB, a shortened DB[n] analogue, only negligibly inhibited both enzymes at 200 μM concentration. We assume that the found inhibitory activity of DB[n] is explained by their competition with DNA-dependent enzymes for binding sites on DNA. It was previously shown that the Hoechst 33258 dye containing the same bisbenzimidazole motive as DB[n] bound specifically to two consecutive AT-pairs (8, 9). Therefore, we suppose that compounds DB[n] would bind with the highest specificity to the -(A/T)n-(N)m-(A/T)n- DNA site, where N, a base pair, n = 2, m = 1-4, which correlates with computer modeling data. Indeed, both duplex [I] (a highly effective topo-I cleavage site) and duplex [II] sensitive to MTase Dnmt3a contain sequences optimal for DB[n] binding (fig. 2).
Figure 2: Oligonucleotide fragments used for studies of DB[n] inhibitory activities. Consecutive AT-pairs are in bold; potential binding sites of the corresponding ligands are underlined. For duplex I , the arrow shows the site cleaved by topo-I and for duplex II , cytosine residues to be methylated.
To summarize, DB[n] were proved to be inhibitors of at least two different DNA-dependent enzymes at low micromolar concentrations. Higher biological activity of DB[n] if compared with monomeric MB is due to the formation of dimeric molecules capable of binding to dsDNA in the form of bidentant ligands.
The work was supported by RFBR (grants 09-04-01126, 10-04-00809 and 11-04-00589) and by the Program of Presidium of RAS on Molecular and Cell Biology.
Alexander A. Ivanov1*
1Institute of Carcinogenesis