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Access Type

WSU Access

Date of Award

January 2019

Degree Type

Dissertation

Degree Name

Ph.D.

Department

Chemistry

First Advisor

Christine S. Chow

Abstract

ABSTRACT

EXPLORING THE BINDING OF AMINOGLYCOSIDES AND EFFECT OF PEPTIDES

ON FUNCTIONALLY IMPORTANT REGIONS OF THE RIBOSOME

by

PRABUDDHA WADUGE

September 2019

Advisor: Prof. Christine S. Chow

Major: Chemistry

Degree: Doctor of Philosophy

Aminoglycosides (AGs) are potent antibiotics that target helix 44 (h44) of the functionally important aminoacyl-tRNA site (A site) of the bacterial ribosome. Despite potency in many cases, some limitations such as resistance and off-target binding restrict the use of AGs as antibiotics. Modification of currently available AGs is a major approach to overcome those limitations. Towards this end, it is of importance to examine the molecular interactions of modified AGs with their target ribosomal RNA (rRNA), h44. Therefore, we employed in vitro dimethyl sulfate (DMS) footprinting and a fluorescence assay to examine binding of AGs to bacterial ribosomes and minimal A-site RNA constructs, respectively. We utilized DMS’s ability to specifically methylate a h44 internal-loop residue, A1408, where binding of AGs has a negative effect on DMS reactivity. As observed in DMS footprinting patterns, chemical modifications on AGs altered h44 binding with bacterial ribosomes. The in vitro binding affinities determined from these probing experiments correlated well with the in vivo antibacterial activities of the tested compounds.

A fluorescence assay was employed to compare binding affinities of modified AGs to RNA constructs resembling the target bacterial A site and the human mitochondrial A site as an off target. Modified AGs showed higher specificity towards bacterial over mitochondrial A sites compared to the parent compound. The trends in binding affinities were consistent with previously reported in vitro translation inhibition of AGs, indicating the utility of the fluorescence assay with minimal RNA motifs in assessing the selectivity of modified AGs.

Helix 69 (H69) of 23S rRNA, which interacts with h44 during ribosome subunit assembly, was studied for its effects on AG antibacterial activity. H69 contains the modified nucleotide pseudouridine (Ψ), which is important for H69 structure and function. We determined minimum inhibitory concentration to evaluate the effects of Ψ modifications in H69 on modified AG activity. We observed that the loss of Ψ along with a defective ribosome release factor (RF2) rendered AG resistance. These results suggest the importance of H69 in AG activity through H69-RF2 interactions in translation termination.

In addition to A site and AGs, exploring novel antibiotic targets and antibiotic leads in the bacterial ribosome is important for overcoming antibiotic resistance. Towards this end, we employed an in vivo expression system to examine the in vivo effects and target binding of an in vitro selected peptide (TLWDLIP) targeting helix 31 (h31) of the bacterial ribosome. Our in vivo growth and in vivo DMS footprinting assays show that the expressed TLWDLIP moderately inhibits bacterial growth and induces conformational changes in the h31 loop region, respectively. Overall, the study of potential antibiotic leads binding to different functionally important regions of the ribosome using in vitro and in vivo systems yields a better understanding of rRNA interactions, which will be useful in developing both selective and potent antibiotics.

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