Insights Into The Mechanisms Of Action Of Ribosome-Targeting Antibiotics

Protein synthesis is a fundamental process that occurs at the ribosome in living organisms. Given its importance, the ribosome is a key target for antibiotics. The peptidyl transferase center (PTC) and nascent peptide exit tunnel (NPET) are the most targeted functional centers of the ribosome. Antibiotics that target these two centers exhibit various mechanisms of actions. Our understanding of these mechanisms have been greatly improved through the study of ribosome-antibiotic structure complexes. These structure studies has lead us to elucidate long-standing questions in the field and alter previously accepted insights. Antibiotic resistance is a rapidly-rising issue that has caused major financial and life-threatening repercussions all around the globe. This crisis emphasizes the absolute need to develop novel antibiotics. One of the most important aspects of antibiotic development is the understanding of antibiotic binding position, orientation and interactions within the ribosome. This is achieved by obtaining ribosome-antibiotic structure complexes at the highest resolution possible. Therefore, the main goal of my PhD study is to determine the structures of various ribosome-targeting antibiotics in complex with 70S ribosomes from Thermus thermophilus using high-resolution X-ray crystallography. In this thesis I present three projects that each provide significant impact to our understanding of antibiotics. In chapter 1, our high-resolution structure complexes of chloramphenicol and erythromycin unequivocally show that the competition the drugs is due to steric clash. These high resolution structures subsequently led us to formulate the following projects. In Chapter 2, through rational drug design we produced a novel chloramphenicol-triphenylphosphonium analog, CAM-C4-TPP. It exhibits a higher binding affinity to the ribosome and improved translational inhibition. Our structure analysis show that these improvements were caused by the additional interactions between the triphenylphosphonium moiety and the ribosome. In chapter 3, we discovered a novel synergistic pairing of antibiotics, the PTC-targeting HygA and NPET-targeting macrolides. They can cooperatively inhibit bacterial growth and exhibit higher bactericidal properties against pneumococcus. Furthermore, they can inhibit growth of the macrolide-resistant Erm-methyltransferase pneumococcus. Our structure complexes showcase the mechanisms of their cooperative binding, as well as the mechanism which allows it to be effective against erm strain resistant pathogens.