Elucidating the role of context-specificity in oxazolidinone mechanism of action

PROJECT SUMMARY/ABSTRACT Antimicrobial resistance is an urgent and growing threat, with a projected toll of 10 million deaths annually by 2050. An improved understanding of the mechanisms of existing antibiotics, applied in the development of improved drugs capable of circumventing common resistance mechanisms, will be key towards combating this ongoing crisis. A class of fully synthetic ribosome-binding antibiotics, oxazolidinones are used clinically as a last defense against multidrug resistant gram-positive pathogens. Though these molecules have historically been considered global translation inhibitors, recent applications of ribosome profiling and cryo-EM have shown that clinical frontrunner linezolid in fact exhibits sequence-specific inhibition of translation. Although other classes of antibiotics have been shown to exhibit context-specific stalling, it remains unclear how and to what extent context-specificity contributes to antibiotic mechanism of action. The synthetic origin of oxazolidinone antibiotics has privileged them with status as a last-resort treatment for especially difficult infections, due to the decreased frequency of resistance compared to their natural product counterparts. As such, the recent discovery of multiple oxazolidinone resistance factors has been especially alarming. One of these proteins, Cfr, confers resistance to eight classes of antibiotic, including linezolid, through methylation of a single rRNA residue. The clinical oxazolidinones exhibit variable activity against this modification, however; while linezolid is highly susceptible to Cfr-mediated resistance, tedizolid maintains activity in Cfr-expressing strains. This proposal will explore 1) the mechanisms by which tedizolid avoids Cfr-mediated resistance and 2) the interconnectivity of context-specific stalling and overall antibiotic efficacy in the oxazolidinone class of antibiotics. In the first aim, I will use ribosome profiling and cryo-EM in concert with MIC assays for efficacy to explore the difference in context-selectivity between tedizolid and the parent compound, as well as in the Cfr- modified context. My 2nd aim will utilize an in vitro ribosome profiling assay in addition to cryo-EM to perform a detailed analysis of the structure-activity relationship between oxazolidinone context-specific stalling and antibiotic efficacy. Over the course of these two aims, I will investigate the model that context-specific stalling is integral to oxazolidinone function as an antibiotic and evaluate the effects of structural modification on said activity. I hypothesize that key structural differences induce a difference in context-specific stalling between the two clinical oxazolidinones which additionally contributes to tedizolid avoidance of Cfr-mediated resistance. This proposal is highly relevant to NIAID’s missions in understanding and treatment of infectious disease. The research will provide significant insight into the mechanism of action of a key class of antimicrobial compounds, including its mechanism of resistance avoidance, as well as the more general phenomenon of context- specificity in ribosome-binding antibiotics. The knowledge gained here will inform development of therapeutics with improved efficacy and ability to circumvent an emerging mechanism of resistance.