Design, synthesis and antibacterial activity of minor groove binders: the role of non-cationic tail groups

he design and synthesis of a new class of minor groove binder (MGBs) in which, the cationic tail group has been replaced by a neutral, polar variant including cyanoguanidine, nitroalkene, and trifluoroacetamide groups. Antibacterial activity (against Gram positive bacteria) was found for both the nitroalkene and trifluoroacetamide groups. For the case of the nitroalkene tail group, strong binding of a minor groove binder containing this tail group was demonstrated by both DNA footprinting and melting temperature measurements, showing a correlation between DNA binding and antibacterial activity. The compounds have also been evaluated for binding to the hERG ion channel to determine whether non-cationic but polar substituents might have an advantage compared with conventional cationic tail groups in avoiding hERG binding. In this series of compounds, it was found that whilst non-cationic compounds generally had lower affinity to the hERG ion channel, all of the compounds studied bound weakly to the hERG ion channel, probably associated with the hydrophobic head groups

The effects of DNA supercoiling and G-quadruplex formation

The self-association of guanine bases in a tetrameric square planar arrangement was first determined in the early 1960s. The tetramer, commonly termed the G-quartet, can stack upon other G-quartets to form four-stranded helices termed G-quadruplexes. Bioinformatics studies have revealed that guanine-rich sequences with the propensity to adopt these structures are found in telomeric DNA and throughout the human genome, particularly in gene promoter regions. It is thought that the location of these sequences is not a coincidence and that the folding potential of guanine-rich DNA in vivo may play an important role in biological events such as gene regulation. Repetitive guanine tracts of G-quadruplex-forming DNAs form highly polymorphic structures with parallel or antiparallel strand orientations, depending on the ionic condition and the length of the connecting loops, and can be assembled as inter- or intra-molecular complexes. While extensive research has demonstrated their formation in vitro, there is little direct evidence to support their formation in vivo. With the exception of the single-stranded telomeric DNA, all genomic guanine-rich sequences are always present in the duplex configuration. Therefore, these structures will need to compete with the duplex that is normally generated with the complementary cytosine-rich strand. In order for this to happen would first require the local dissociation of the strands. Negative supercoiling results from the unwinding of the DNA helix and is known to provide energy to facilitate the formation of a number of alternative DNA structures. The work described in this thesis therefore aims to investigate the formation of G-quadruplexes under negatively supercoiled conditions. This was examined by preparing plasmids that contained multiple copies of G-rich oligonucleotides, based on the sequences (G3T)n and (G3T4)n, cloned into the pUC19 vector. The formation of G-quadruplexes within these repeats has been assessed using the chemical probes dimethyl sulphate (DMS) and potassium permanganate, and the single-strand specific endonuclease S1. DMS probing revealed some evidence for G-quadruplex formation in (G3T)n sequences, though this was not affected by DNA supercoiling. However, probing with KMnO4 failed to detect exposed thymines in the loop regions, though there was some supercoil-dependent reactivity in the surrounding sequences, suggesting that this had been affected by the G-rich region. In contrast, the (G3T4)n sequences did not demonstrate protection from DMS, suggesting that G-quadruplex formation had not taken place. Surprisingly, the KMnO4 reactions identified structural alterations around, but not within, the inserted G-rich fragments. S1 nuclease digestions did not detect any structural perturbations in any of the sequences apart from a mutant plasmid containing an inverted quadruplex repeat at the 3’-end.Two-dimensional gel electrophoresis of DNA topoisomers was also conducted to detect any supercoil-dependent B-DNA to quadruplex transitions. Neither the (G3T)n nor (G3T4)n plasmids showed any such structural changes. However, the mutant plasmid did demonstrate some supercoil-dependent changes, though these may correspond to cruciform rather than G-quadruplex formation. These results do not support the suggestion that negative supercoiling can induce the formation of G-quadruplex structures

The effects of DNA supercoiling on G-quadruplex formation

Guanine-rich DNAs can fold into four-stranded structures that contain stacks of G-quartets. Bioinformatics studies have revealed that G-rich sequences with the potential to adopt these structures are unevenly distributed throughout genomes, and are especially found in gene promoter regions. With the exception of the single-stranded telomeric DNA, all genomic G-rich sequences will always be present along with their C-rich complements, and quadruplex formation will be in competition with the corresponding Watson-Crick duplex. Quadruplex formation must therefore first require local dissociation (melting) of the duplex strands. Since negative supercoiling is known to facilitate the formation of alternative DNA structures, we have investigated G-quadruplex formation within negatively supercoiled DNA plasmids. Plasmids containing multiple copies of (G3T)n and (G3T4)n repeats, were probed with dimethylsulphate, potassium permanganate, and S1 nuclease. While dimethylsulphate footprinting revealed some evidence for G-quadruplex formation in (G3T)n sequences, this was not affected by supercoiling, and permanganate failed to detect exposed thymines in the loop regions. (G3T4)n sequences were not protected from DMS and showed no reaction with permanganate. Similarly, both S1 nuclease and two-dimensional gel electrophoresis of DNA topoisomers did not detect any supercoil-dependent structural transitions. These results suggest that negative supercoiling alone is not sufficient to drive G-quadruplex formation