Exploring biomolecular interactions : : New ligands and novel assays

Many antibiotics bind the bacterial ribosome, the only validated RNA target. Derivatizing or mimicking these natural products is a potential way to create new RNA binders or antibacterials overcoming bacterial resistance. This motivated this work, which addressed the preparation of new RNA binders and the development of new assays. Semi -synthetic derivatives of clinically useful aminoglycosides, tobramycin and amikacin, were prepared by selectively modifying their 6'' positions. The binding to the rRNA A-site was probed by an in vitro Förster resonance energy transfer (FRET)-based assay, and antibacterial activity was quantified by determining minimum inhibitory concentrations (MICs). Most analogs displayed greater affinities for the bacterial A-site compared to the parent compounds. Several amikacin analogs showed potent and broad-spectrum antibacterial activity against resistant bacteria, suggesting they are overcoming resistance mechanisms. However, tobramycin analogs exhibited overall poor antibacterial activity. As an alternative approach to potentially new RNA binders, mimetics of the aminoglycoside pharmacophore, 2- deoxystreptamine, were synthesized. In noting structural similarities to aminoglycosides, polymyxin antibiotics, which target the cell wall, were examined as rRNA binders using a FRET assay. The polymyxins showed significant affinity for to the bacterial and eukaryotic A-sites. Additionally, in vitro translation assays showed all polymyxins interfered with eukaryotic translation, but not with bacterial, which could account for toxicity effects. Fluorescent molecules and labeled oligonucleotides are valuable tools to probe small molecule, RNA, and protein interactions. We have developed assays utilizing isomorphic fluorescent nucleosides to study RNA catalysis and small molecule-enzyme interactions. A fluorescent guanosine analog, thG triphosphate, was incorporated into oligonucleotides by T7 RNA polymerase. Additionally, modified transcripts were used to assemble a hammerhead ribozyme containing enzyme and substrate strands with thG replacing guanosine. The thG modified substrate was effectively cleaved by the natural enzyme. However, the thG modified enzyme showed no cleavage ability, suggesting the modifications likely disrupted the catalytic center. Toward exploring protein-nucleoside interactions with fluorescent nucleoside analogs, a fluorescent adenosine derivative, thA, was found to be deaminated by the enzyme adenosine deaminase (ADA) into the corresponding fluorescent inosine derivative, thI. A high-throughput assay utilizing the fluorescent properties of these molecules to discover ADA inhibitors was developed