Abstract 665: Targeting the MluI cell cycle box (MCB) sequence 5’-ACGCGT-3’ in the human Dbf4 promoter using the rationally designed polyamide formamido-imidazole-pyrrole-imidazole (f-IPI)

Abstract

Abstract Dbf4 is the regulatory subunit of Cdc7 kinase, which is essential for the initiation of DNA replication throughout S-phase. The level of Dbf4 is critical for the activation of Cdc7. Human Cdc7/Dbf4 (HuCdc7/Dbf4) kinase activity is essential for cell proliferation by activating DNA replication. High levels of the Cdc7 kinase activity have been implicated with a large number of aggressive solid cancers. The MluI cell cycle box (MCB) sequence 5’-ACGCGT-3’ is the critical site for transcription factor binding that activates the expression of the HuCdc7/Dbf4 core gene in mammalian cells. HuDbf4 promoter activity has previously been shown to be significantly reduced by a point mutation within the MCB. We have previously shown that the rationally designed simple polyamide formamido-imidazole-pyrrole-imidazole (f-IPI) binds to its cognate sequence 5’-ACGCGT-3’ as a stacked dimer in an anti-parallel conformation (2:1 ligand/DNA complex) with exceptionally high affinity and selectivity, superior to the natural product distamycin A binding to its cognate sequence. DNAse I footprinting experiments indicated selective binding of f-IPI to the MCB sequence in the HuDbf4 promoter at 0.1µM. Eletrophoretic mobility shift assays using the MCB-containing sequence from the HuDbf4 promoter showed dose-dependent inhibition of protein binding by f-IPI which was complete at 0.5µM. RT-PCR showed decreased expression of HuDbf4 mRNA in response to incubation of exponentially growing MDA-MB231 cells with 10 μM f-IPI. Selective effects on protein expression, DNA replication and cell cycle are currently underway. These data demonstrate the potential of small molecule polyamides as modulators of gene expression. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 665. doi:10.1158/1538-7445.AM2011-665