Molecular mechanisms of action of ribosome-targeting antibiotics.

SUMMARYRibosome-targeting antibiotics are indispensable both as therapeutic agents and as tools for basic research. Inspite of the importance of these inhibitors, there are significant gaps in our understanding of the mostfundamental principles of their action. Most of them interfere with protein synthesis by blocking the functionalcenters of the ribosome. Out of several functional centers, the catalytic peptidyl transferase center (PTC) andthe nascent peptide exit tunnel (NPET) are the sites targeted by the broadest array of inhibitors. In the proposedproject, we will explore the molecular mechanisms of action of the most basic PTC-targeting antibiotics andmacrolides ? chloramphenicol (CHL) and erythromycin (ERY). Recent studies yielded the unexpected conclusionthat, in contrast to the general view of CHL and ERY as global and indiscriminate inhibitors, these antibioticsinterfere with translation in a context-specific manner indicating that our understanding of their mechanism ofaction is incomplete and possibly even wrong. One way to obtain a clear explanation for the paradigm-shiftingphenomenon of context-specific activity of PTC-acting inhibitors and macrolides is to directly visualize themwithin the ribosome complexes conducive to their action. Previous crystal structures uncovered how CHL andERY bind to the PTC and NPET of the vacant bacterial ribosome and therefore provide information that isirrelevant for their context-specific activity. By determining the structures of CHL and ERY (as well as other PTC-acting drugs and macrolides) in functionally relevant ribosome complexes containing A-site aminoacyl-tRNA andP-site peptidyl-tRNA we will provide atomic-level view of their interactions not only with the ribosome (as before)but also with the growing peptide. Moreover, such structures could also reveal rearrangements that take placein the PTC of the ribosome upon drug binding and result in allosteric effects. Hence, in the Specific Aim 1, wewill focus on obtaining the structures of 70S complexes carrying various aminoacyl-tRNAs in the A site in thepresence and absence of CHL. Then, in the Specific Aim 2, we will obtain the first set of CHL-bound ribosomestructures featuring dipeptidyl-tRNAs in the P site containing alanine, serine, or threonine in the penultimateposition (the only sequence requirement for the efficient CHL-induced stalling). Finally, in the Specific Aim 3, wewill provide structural and mechanistic insights into the context-specific activity of ERY and other macrolides.Once our proposed methodology is established and refined, we will expand it onto the newest FDA-approvedclinically important drugs, such as linezolid, tedizolid, telithromycin, and solithromycin. The anticipated findingsshould significantly expand our understanding of the general mode of action of basic, as well as clinically-important, antibacterial drugs that act upon the catalytic center of the ribosome and may open new venues forrational development of protein synthesis inhibitors with superior antibiotic properties.