The anthracenyl isoxazole amides (AIMs) have been demonstrated to possess anti-​tumor activity in the NCI-​60 cell line panel and inhibit growth of SNB-​19 glioblastoma cells at low micromolar and nanomolar concentrations. Our working hypothesis is that the mechanism of growth inhibition is through binding of quadruplex DNA. Various promoter sequences, such as the c-​MYC oncogene promoter, and repeating sequences formed at the end of telomeres adopt quadruplex structures. Ligands that stabilize this conformation have been reported to modulate expression of genes and inhibit telomerase activity. The objective of this research is to elucidate the underlying mechanism of the anti-​tumor activity of the AIMs. This work focused on utilizing circular dichroism (CD) spectroscopy thermal melting studies to establish whether interactions between the AIMs and various quadruplex-​forming sequences act to stabilize the quadruplex tertiary structure. Our results demonstrated the AIMs (2 equiv.) increase the melting temperature (Tm) of the c-​MYC promoter and telomeric sequences both by approx. 2°-3° C with strong statistical significance. This method allows the use of low micromolar concentrations of DNA and will be used in the future to rapidly develop structure-activity relationships between novel AIMs and quadruplex forming sequences. Our laboratory has also shown chemical shifts in the imino region upon treatment with the AIMs for both the c-MYC promotor and telomeric sequences by NMR. Additionally, fluorescence microscopy of SNB-​19 cells treated with AIMs show their localization is primarily in the mitochondria, and mitochondrial DNA contains several quadruplex sequences of interest. Mitochondrial-​dependent apoptosis has been suggested for other quadruplex ligands and therefore our future work will examine the potential stabilization of mitochondrial quadruplexes by these and other novel AIMs.

Anthracenyl isoxazole amides (AIMs) stabilize quadruplex DNA structures in telomeric and c-MYC promotor sequences

249th National Meeting (2015)

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