Antibiotic Properties of Artificial Agonists for a Bacterial Riboswitch Notice Number NOT-OD-09-058 Notice Title: NIH Announces the Availability of Recovery Act Funds for Competitive Revision Applications 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 one of their most essential processes, metabolism. The discovery of RNA structural elements, termed riboswitches, that bind cellular metabolites and control expression of essential metabolic genes provides a unique and distinct target for development of artificial agonists to fight bacterial infections. Riboswitches are found in non-coding regions of messenger RNAs, and gene expression is modulated when metabolite binds directly to the RNA. Many riboswitches repress expression of nearby 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 inactivity of the mRNA. The amine functionality of GlcN6P seems to be directly involved in RNA catalysis, whereas the phosphate may play a role in ligand recognition. In order to develop effective artificial agonists/antibiotics that target the glmS riboswitch, an understanding of the structural and functional details of the riboswitch- metabolite complex is essential.
The aims of the original grant were focused on (1) investigating the structural and catalytic roles of metal ions in the glmS riboswitch, (2) deciphering ligand recognition by the glmS riboswitch, and (3) designing non-natural agonists with the ability to stimulate glmS riboswitch self-cleavage and control gene expression.
Aim 1 has been completed and Aim 2 is nearly finished. In regards to Aim 3, we have begun to test a small number of non-natural ligands, one of which does support glmS self-cleavage. This revision will significantly expand the scope of Aim 3 of the original grant in order to include additional ligand analog syntheses and bacterial growth experiments to test the antibiotic properties of the synthesized artificial agonists. Further expansion of the project will include an investigation of the mechanism of the self-cleavage reaction using kinetic isotope experiments. Both lines of experimentation will further aid in rational design of non-natural metabolite-like compounds that can function as agonists/antibiotics to halt bacterial growth through alteration of gene expression.
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 studies of ligand recognition by RNA as well as rational design of non-natural agonists that ultimately could function as antibiotics.
|Fei, Xiang; Holmes, Thomas; Diddle, Julianna et al. (2014) Phosphatase-inert glucosamine 6-phosphate mimics serve as actuators of the glmS riboswitch. ACS Chem Biol 9:2875-82|
|Klawuhn, Kevin; Jansen, Joshua A; Souchek, Joshua et al. (2010) Analysis of metal ion dependence in glmS ribozyme self-cleavage and coenzyme binding. Chembiochem 11:2567-71|