The emergence of antibiotic resistance has required that new approaches be applied in order to effectively fight a host of medically relevant bacterial infections. The currently used, imprecise antibiotics, need to be replaced with novel, rigorous, and safe treatments in order to combat the evolved bacterium of today. One way to destroy bacteria is to target their most essential, metabolic pathways. Riboswitches are RNA structural elements that bind cellular metabolites and control expression of essential metabolic genes providing a unique and distinct set of targets for development of artificial agonists to fight bacterial infections. Riboswitches are found in non-coding regions of mRNA molecules, and gene expression is modulated when metabolite binds directly to the RNA. Many riboswitches, once liganded, repress expression of associated or adjacent genes involved in the synthesis of the metabolite, providing an efficient feedback mechanism of genetic control. One particular riboswitch (the glmS riboswitch) binds to glucosamine-6-phosphate (GlcN6P), a building block of the cell wall in Gram-positive bacteria, and undergoes self-cleavage resulting in inactivation of the mRNA. We have shown that the ligand amine and phosphate functionalities are essential for binding of the metabolite to the riboswitch RNA and for catalysis by the catalytic RNA (ribozyme). These requirements for binding and catalysis of the GlcN6P-dependent riboswitch/ribozyme have been shared with our collaborator, Dr. David Berkowitz, to aid in design and organic syntheses of novel ligand analogs. We will test these analogs for their ability to induce glmS self-cleavage and inhibit bacterial growth. Already one ligand analog shows great promise in glmS self-cleavage assays. We also propose to continue our studies of the glmS self-cleavage reaction mechanism as further insight to acid-base catalysis may affect development of glmS ribozyme agonists that satisfy added chemical requirements for binding and activity. The aims of this renewal grant are focused on (1) ligand analog synthesis and characterization of glmS self-cleavage, (2) the structure, function and antibiotic properties of artificial agonists in regards to glmS riboswitch regulation of reporter gene expression and inhibition of bacterial growth, and (3) mechanistic studies of glmS-supported acid-base catalysis through coordinated proton transfer. Information gained from kinetic studies will further inform our continued design of ligand analogs that support glmS riboswitch/ribozyme catalysis and that act as novel antimicrobial agents against some of the hardest to treat human pathogens. PUBLIC HEALTH RELEVANCE: The threat of bacterial infections due to lack of effective antibiotics has come to the forefront as these pathogens become resistant to almost every antibiotic available to the public. The need is great for new classes of anti-microbial agents that target different, but specific and essential, metabolic pathways, such as those which utilize riboswitches to control gene expression. Structure-function and mechanistic studies of riboswitches have enabled detailed analyses of ligand recognition by RNA as well as rational design of non-natural agonists that ultimately could function as antibiotics.