Towards the development of new anti-infectives: Targeting bacterial RNAs and the ribosome with antibiotic leads.

摘要

Towards the development of new anti-infective compounds, two approaches were followed in this thesis work, with one being the development of a new class of compounds to an existing target, and the second one focusing on the interaction of an existing class of drugs to a new target.;The research first focused on interaction of the peptide HPVHHYQ with an A-site rRNA hairpin model. A single binding site was identified at the internal bulge of the RNA, with an average dissociation constant of 17 muM. The roles of the amino-acid side chains in the peptide•RNA interaction were studied by alanine scanning, and showed that enhancement of the affinity could be observed upon P2A and Y2A mutations; whereas H1A and Q7A were unfavorable for binding. Similar results were obtained for the A-site rRNA hairpin and the 30 S ribosome. The parent peptide HPVHHYQ retained flexibility in its binding, such that it was able to bind to three A-site rRNA mutant hairpins in a 1:1 fashion, at the asymmetric bulge. These results are significant with respect to the development os new ligands towards potentially resistant bacterial mutants.;The last part of the research focused on the interaction of aminoglycosides with a helix 69 rRNA hairpin model. The binding affinities and stochiometries of the interactions with the modified as well as the unmodified RNA were investigated, yielding Kds ranging from 0.5 to 14 muM. The preferred binding observed to the modified construct ober unmodified RNA was correlated to a long-range conformational change leading to base flipping of nucleotide A1913. Lower pH and lack of modifications lessen the affinity of the aminoglycosides for the target RNA, suggesting that a flipped-out conformation of 1913 is a driving force for the binding to helix 69.;Overall, this thesis work explored new ligand•RNA interactions, with the long-term objective to develop new antibiotic compounds. Studies with mutant RNAs will assist with identifying compounds that will have greater efficacy against drug-resistance bacterial strains.