The current crisis in antibiotic resistance requires new therapeutic agents. The ribosome is the target for a class of antibiotics, the aminoglycosides, that bind directly to 16S rRNA in the region of the codon-anticodon interaction on the ribosome. A 27 nucleotide RNA, corresponding to the rRNA sequence, binds aminoglycoside antibiotics with similar affinity and specificity as to the ribosome, and the structure of a complex of this 27 nucleotide RNA complexes with paromomycin has been solved by NMR in our group. The current proposal builds on the structural results of the last funding period. The goal of this research is to understand the structural basis for aminoglycoside-ribosome interaction, such that novel antibiotics can be designed. In the paromomycin-RNA complex structure, potential hydrogen bonding and electrostatic contacts are observed between drug and RNA. Using organic synthesis methods, derivatives of paromomycin will be prepared with either methylated or acetylated amino groups, to test their role in complex formation. This analysis will pinpoint the essential chemical groups in the aminoglycoside structure. There are a large number of aminoglycosides, with distinctly different structures. In the paromomycin-RNA complex, rings I and II or paromomycin fit into a specific RNA binding pocket. However, several aminoglycoside have modified architectures, and the question is how they fit in the RNA binding site. Using NMR spectroscopy, the structures of several RNA- aminoglycoside complexes, including gentamicin C, G418, apramycin, sisomicin, butirosin and amikacin will be determined. Aminoglycosides distinguish among prokaryotic and eukaryotic ribosomal targets. The sequence differences between prokaryotic and eukaryotic rRNAs explains a 30-80-fold specificity of aminoglycoside action on prokaryotes. Certain human populations have an inherited predisposition to deafness on treatment with aminoglycosides, and this disorder arises from a mutation in human mitochondrial rRNA. The affinities of aminoglycoside antibiotics to ribosomes containing these sequences and to corresponding oligonucleotides will be determined biochemically. Structural studies on these eukaryotic sequences should reveal the detailed origins of aminoglycoside specificity. A final goal of this project is the design of aminoglycosides with increased discrimination between prokaryotic and eukaryotic sequences; addition of 2-deoxystreptamine may increase the number of specific contacts made prokaryotic rRNA.
Showing the most recent 10 out of 60 publications
