Macrolide antibiotics inhibit cell growth by interfering with protein synthesis. These drugs bind in the nascent peptide exit tunnel and, according to the widely accepted view, inhibit synthesis of all cellular proteins at the early rounds of translation In contrast to this conventional model of macrolide action, our preliminary studies showed that treatment of Gram-positive and Gram-negative bacteria with macrolides allows for continued translation of a defined subset of proteins. Further, the ability of the protein to evade inhibitio is determined by its N-terminal sequence which can bypass the antibiotic in the exit tunnel without displacing the drug from its binding site. After the initial bypass, translation of some proteins cn continue until their completion, whereas synthesis of some polypeptides can be arrested at later stages. Both of these effects depend on the structure of the antibiotic. In spite of the functional and medical significance of these phenomena, the molecular mechanisms underlying the ability of the protein to evade inhibition and the requirements for the drug-induced translation arrest are unknown and will be addressed in this project. Whole-cell proteomics will be used to comprehensively characterize proteins whose translation continues in the presence of the antibiotic. The highly-innovative technique of ribosome profiling will provide genome-wide information of the sites of drug-dependent 'late' translation arrest. The whole cell-studies will b followed by biochemical characterization of molecular mechanisms of bypass and arrest carried out in a cell-free translation system. Finally, the correlation between the structure of the antibiotic bound in the ribosomal exit tunnel and the spectrum of proteins synthesized in antibiotic-treated cells will be analyzed and physiological consequences of the variation in the composition of the resistome will be examined. The anticipated findings should significantly expand the understanding of the general mode of action of clinically-important macrolide antibacterials and open new venues for development of protein synthesis inhibitors with superior antibiotic properties.

Public Health Relevance

Understanding the molecular mechanisms of antibiotic action is required for rational, evidence-based, strategies for development of better drugs. The discovery that bacterial cells treated with protein synthesis inhibitors can synthesize specific proteins opened new directions for optimizing these clinically-important antibiotics. Unraveling the molecular mechanisms of protein escape from the inhibitory action of antibiotics will pave the way for novel strategies for developing superior drugs capable of curbing bacterial infections.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
Research Project (R01)
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Drug Discovery and Mechanisms of Antimicrobial Resistance Study Section (DDR)
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Fabian, Miles
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University of Illinois at Chicago
Schools of Pharmacy
United States
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