Structural and Functional Explorations of Novel Ribosome-Targeting Antibacterials

Protein synthesis is vital for cell survival. Ribosomes, the site of protein synthesis, are known to serve as targets for approx. half of the clinically useful antibiotics. The emergence of drug resistance in bacteria, however, creates dire need for new antibiotics. One approach to addressing this crisis is the discovery of novel chemical scaffolds. Alternatively, optimizing existing antibacterial drugs through chemical modifications can improve potency, enhance target affinity, or alter their mechanism of action for improved efficacy. In this work, we explored the mechanisms of under studied or “forgotten” ribosome targeting antibacterial compounds namely thermorubin, sparsomycin and bottromycin A2 which are naturally occurring secondary metabolites, and CAM BER, a semi-synthetic derivative of chloramphenicol. Varied in form yet unified in function, these compounds—despite their diverse sizes, chemical structures, and physical properties—converge on a common target i.e. protein synthesis. To establish each compound’s mode and mechanism of action, we used microbiological, biochemical, and structural methods. Using high-resolution X-ray crystallography, CryoEM, and primer extension inhibition (toe printing) assays, we demonstrated that thermorubin inhibits translation elongation, thereby disproving the prior hypothesis that it is a bona fide inhibitor of translation initiation. CAM-BER, a berberine analog of chloramphenicol, binds the ribosome 40X more strongly than its parent compound. Interestingly, our X-ray crystal structure demonstrated that CAM-BER binding causes a previously unseen rearrangement of 23S rRNA nucleotide A2059. Based on our structure, we predicted that CAM-BER should inhibit translation initiation, unlike elongation inhibitor chloramphenicol. Our toe-printing data supports this hypothesis. CAM-BER’s altered mechanism may help it bypass certain resistance pathways evolved against chloramphenicol. Sparsomycin's clinical use is limited by its broad activity, inhibiting translation not only in bacteria but also in archaea and eukarya. Structurally understanding sparsomycin’s mode of binding in a physiologically relevant complex is the key to designing effective derivatives. To this end, we first conducted toe-printing assays to identify the ideal stalling complex for crystallization. We report the first X-ray crystal structure of sparsomycin bound to the Thermus 70S ribosome with mRNA and full-length tRNA, resolving previous discrepancies and unambiguously establishing its binding mode in bacteria. Despite its discovery seventy years ago, the binding site and mechanism of bottromycin A2 remain unidentified. Significance of studying bottromycin A2 lies in the fact that it exhibits antibacterial activity against MRSA (Methicillin-resistant Staphylococcus aureus) and VRE (Vancomycin-resistant Enterococci), which pose serious threat to public health. Leveraging our toolkit of various in vivo and in vitro techniques, we demonstrated that bottromycin A2 inhibits translation elongation in a context-dependent manner. We also showed that the binding site of this drug likely resides near A2602 in the ribosome's catalytic center. Our efforts to determine its mode of binding in the ribosome using cryo EM are currently underway.