| The object of this research was to identify drug structural determinants that contribute to the ability of DNA-binding drugs to interfere with the binding of protein transcription factors (TFs) to DNA. This was done with the expectation that these rules can be applied to develop drugs to interfere with the binding of specific TFs, and to modulate expression of the genes which these factors regulate.;To measure drug inhibition of protein binding to DNA, a quantitative mobility shift assay (MSA) system was developed. In this system, the $rm Cyssb2Hissb2$ zinc finger proteins EGR1, WT1 and NIL2A, the leucine zipper protein wbJun/wbFos, and the minor groove binding protein TBP were incubated with double-stranded oligodeoxyribonucleotides containing corresponding DNA recognition sites. Intercalating drugs, minor groove binding drugs (MGBDs), and covalently bonding drugs were added prior to protein or after the protein to examine the ability of drugs to interfere with TF association with DNA.;Submicromolar concentrations of the intercalating agents hedamycin and nogalamycin, and the MGBD chromomycin A$sb3$ inhibited complex formation for all transcription factors examined. Chromomycin A$sb3$ was able to disrupt preformed TF$spcdot$DNA complexes at drug concentrations two orders of magnitude lower than that required by the most potent intercalating agents.;Intercalating drugs and MGBDs were evaluated for their ability to inhibit DNA binding by the zinc finger proteins NIL2A and EGR1. These two proteins are similar in structure, but EGR1 binds a very G/C-rich DNA site, while NIL2A binds a site high in A/T content. Doxorubicin inhibited 50% of NIL2A$sp{cdot}$DNA complex formation at a drug concentration of 0.5 $mu$M; 100 times this amount of doxorubicin failed to inhibit EGR1$spcdot$DNA complex formation to the same degree. The covalently bonding minor groove drug CC-1065 inhibited NIL2A$spcdot$DNA complex formation by 50% at a drug concentration of 1 $mu$M, but had no effect on EGR1$spcdot$DNA complex formation. A/T-specific intercalating agents and MGBDs had no effect on either EGR1 or NIL2A binding to DNA. A/T-specific MGBDs inhibited DNA binding by TBP at submicromolar levels. |
