Glycine receptors; Tropeines; Ternary allosteric model; Glycine displacement

The synthesis and DNA binding characteristics of a polyamide–intercalator conjugate, designed to inhibit NF-Y binding to the ICB-2 site of the topoisomerase IIα promoter and up-regulate the expression of the enzyme in confluent cells, are reported. Thermal denaturation and CD titration studies demonstrated binding to the cognate sequence (5′-AAGCTA-3′). Formation of ligand-induced CD bands at ∼330 nm provided indication that the molecule interacts selectively in the minor groove of DNA. Intercalation was evidenced by a fivefold increase in emission of the intercalator moiety upon binding to the ICB-2 hairpin oligonucleotide. An increase in viscosity of a solution of calf-thymus DNA on addition of the conjugate provided further evidence. The binding affinity of the conjugate was ascertained using SPR (5.6 × 106 M−1), which according to a gel shift assay was capable of inhibiting the binding of NF-Y at a concentration of 50 μM. DNaseI footprinting, using the topoIIα promoter sequence, highlighted the specificity of the conjugate for the cognate site (5′-AAGCTA-3′). Finally, through Western blot analysis, confluent murine NIH 3T3 cells treated with conjugate were found to have enhanced expression of topoIIα. These results suggest that the conjugate can enter the nucleus, bind to its target site, presumably as a stacked dimer, and up-regulate the expression of topoIIα by blocking the binding of NF-Y.