Abstract
As a class, minor-groove non-covalent binding small molecules generally show A/T selectivity. The lack of G/C specificity is thought to relate to the wider minor groove in G/C regions and the presence of exocyclic C2-NH2 groups of guanines which project into the groove and prevent close van der Waals contacts within the G/C-rich minor groove. Therefore, we have developed a new set of biaryl building blocks with the potential to alter the curvature of polyamides, thus increasing the number of van der Waals contacts within GC tracts of the DNA minor groove. These biaryl building blocks are phenyl-substituted heterocycles which are long enough to span two DNA base pairs. Initial fluorescent intercalator displacement (FID) assay results indicated that library members containing a 4-(1-methyl-1H-pyrrol-3-yl)benzenamine (MPB) motif switch binding preference from AT-rich to GC-rich sequences. To further confirm the sequence preference for MPB building blocks, a library of 50 biaryl-PBD conjugates has been synthesized using solution-phase parallel chemistry in which different five-membered heterocycles were used, and in which the MPB assembly is separated from the PBD moieties using a four carbon linker. An Ion Pair Reversed-Phase HPLC MS assay using AT-rich and GC-rich oligonucleotides was used to explore the sequence preference and rate of reaction of the MPB-PBD and dipyrrole-PBD conjugates with various oligonucleotides. The results showed that MPB units prefer GC sequences near to the PBD binding site (67% binding within 5 mins), while the dipyrrole-PBD showed poor tolerance for GC-rich sequences (12% within 5 mins) but had a strong preference for AT-rich ones (60-80% within 5 mins). A DNA melting study was used to evaluate the GC-targeting MPB building blocks on the stabilization of designed AT- and GC-rich oligonucleotides using a FRET-based assay. In this study, a hairpin-forming oligonucleotide containing only one PBD binding site but with modified flanking sequences was used. The MPB-PBD conjugates showed up to 13-fold greater stabilization of the GC-rich (3.9°C at 1µM) compared to the AT rich (0.3ºC at 1µm) oligonucleotides, whereas the opposite effect was observed for the dipyrrole-PBD which had a preference for AT-sequences. To evaluate the biological impact of this sequence-preference, we screened MPB-Py-MPB (66-KMR-77) and MPB-Phenyl-Thiazole (66-KMR-09) against the micro-organisms EMRSA-16 and Micrococcus luteus which have AT- and GC-rich genomes, respectively. While both compounds failed to show significant antibacterial activity against EMRSA-16, they both showed activity against Micrococcus luteus (MIC = ~8µg/ml) potentially due to their GC-preference. Finally, a series of the MPB-PBD conjugates were found to have sub-nanomolar IC50 values in the MCF7, A431, A2780, A549 and MDAMB231 tumour cell lines while being inactive in the non-tumour cell line WI38.
Citation Information: In: Proc Am Assoc Cancer Res; 2009 Apr 18-22; Denver, CO. Philadelphia (PA): AACR; 2009. Abstract nr 5357.
100th AACR Annual Meeting-- Apr 18-22, 2009; Denver, CO