Insights into the mechanism of antibiotic and toxin inhibition of ribosome function and antibiotic-resistant ribosomal subunits, using X-ray crystallography.
Final Report Abstract
The translational machinery represents one of the major targets within the cell for antibiotics. Many clinically used classes of antibiotics, such as the tetracyclines (tigecycline), phenylpropanoids (chloramphenicol), macrolides (erythromycin, tylosin), and oxazolidinones (linezolid) inhibit translation by binding to the ribosome. Despite the potency of many of these drug classes, antibiotic resistance among clinically relevant pathogens is an increasing problem and thus the need for new antibiotics is more urgent than ever before. The results of this proposal have shed light into the mechanism of action of many of these clinically used antibiotics. We have determined the first structure of the oxazolidinone linezolid on the large ribosomal subunit and reveal the interactions that this drug makes with the ribosomal RNA nucleotides that comprise its binding site. Based on this structure, we were able to predict regions of the drug that could be modified to develop new antibiotics that establish new interactions with the ribosome or have better pharmokinetic properties. In addition, we could present a model for the mechanism of action of this class of peptidyltransferase inhibitors. Likewise, we have determined the first and so far only structures of thiopeptide antibiotics bound to the ribosome. The surprising finding that micrococcin stabilizes an interaction between ribosomal proteins L7/L12 and L11 is consistent with the differential effects that this antibiotic has compared to thiostrepton on the GTPase activation of different translation factors. Additionally, we have developed high throughput translational machinery assays that has enabled us to identify thiopeptide precursor fragments that still interact with the ribosome and are thus potential lead compounds for future drug development. Our studies on the macrolide class of antibiotics demonstrated that these drugs do not inhibit translation of all polypeptides in the same manner. This surprising finding highlights the complex interplay that exists between the ribosome, macrolide and nascent chain being synthesized. Collectively, our studies therefore provide not only important information that aids the future development of improved antimicrobial agents, but also fundamental insight into the complicated process of protein synthesis – this latter point is important since large pharmaceutical companies are abandoning research and developed into antimicrobial agents and therefore it will be left to academics and small biotech companies to take up the challenge.
Publications
- (2008). The oxazolidinone antibiotics perturb the ribosomal peptidyl-transferase center and effect tRNA positioning. Proc Natl Acad Sci U S A 105, 13339-13344
Wilson, D. N., Schluenzen, F., Harms, J. M., Starosta, A. L., Connell, S. R., and Fucini, P.
- (2008). Translational regulation via L11: molecular switches on the ribosome turned on and off by thiostrepton and micrococcin. Mol Cell 30, 26-38
Harms, J. M., Wilson, D. N., Schluenzen, F., Connell, S. R., Stachelhaus, T., Zaborowska, Z., Spahn, C. M., and Fucini, P.
- (2009). Identification of distinct thiopeptide-antibiotic precursor lead compounds using translation machinery assays. Chem Biol 16, 1087-1096
Starosta, A. L., Qin, H., Mikolajka, A., Leung, G. Y., Schwinghammer, K., Nicolaou, K. C., Chen, D. Y., Cooperman, B. S., and Wilson, D. N.
- (2009). Non-hydrolyzable RNA- peptide conjugates: a powerful advance in the synthesis of mimics for 3'- peptidyl tRNA termini. Angew Chem Int Ed Engl 48, 4056-4060
Moroder, H., Steger, J., Graber, D., Fauster, K., Trappl, K., Marquez, V., Polacek, N., Wilson, D. N., and Micura, R.
- (2010). Interplay between the ribosomal tunnel, nascent chain, and macrolides influences drug inhibition. Chem. Biol. 17, 1-10
Starosta, A., Karpenko, V., Shishkina, A., Mikolajka, A., Sumbatyan, N., Schluenzen, F., Korshunova, G., Bogdanov, A., and Wilson, D.
- (2011). Differential effects of thiopeptide and orthosomycin antibiotics on translational GTPases. Chem. Biol., 18: 589- 600
Mikolajka, A., Liu, H., Chen, Y., Starosta, A. L., Márquez, V., Ivanova, M., Cooperman, B. S., and Wilson, D. N.
