TITLE: Development of multidisciplinary methods for identifying small molecules with FMN riboswitch binding capacity

ABSTRACT: Antibiotic resistance remains a pressing global concern, costing lives and taxing healthcare systems worldwide. One of the major factors of increasing rates of antibiotic resistance is that most antibiotics target the bacterial ribosome or a limited range of proteins. Due to the narrow range of targets and the relatively small number of antibiotics, resistance to commonly used treatments arises quickly and can spread across bacterial populations. While there have been efforts to identify new targets for antimicrobial compounds and develop antibiotics with different chemical structures and mechanisms of action, there continues to be a dearth of innovation in developing truly innovative antibiotic compounds.

In this thesis, I discuss efforts to develop a series of tools to aid in the identification of new molecular scaffolds, small molecules that can be altered to optimize antibacterial effects, for an underexplored antibiotic target. Specifically, these methods target bacterial riboswitches, structured RNA elements that regulate vital biosynthetic pathways by binding a specific small molecule. Riboswitches are present in many bacteria, including bacteria that pose the greatest risks to human health. Each riboswitch is defined by the specific molecule it binds to; this dissertation focuses on the flavin mononucleotide riboswitch (FRS) The FRS binds to flavin mononucleotide, an important cofactor for protein function and a key part of multiple metabolic cycles. Bacteria often use FRS to regulate riboflavin biosynthesis, which is crucial for cellular growth and energy production. Since the FRS is a regulator for a vital pathway, identifying molecules with distinct molecular structures that bind to the riboswitch could be optimized into novel antibiotics.

While FRS has a wealth of previous characterization and some previously identified binding partners; however, none of these candidates have advanced beyond preliminary mouse studies, far from actual implementation as a drug. We developed Fluorescent Ligand Equilibrium Displacement (FLED), a robust, rapid, and repeatable method, to identify molecules capable of interacting with the FRS. This method was applied to large libraries of compounds and their preliminary hits, investigated using classical biochemical techniques. We also pioneered methods for analyzing RNA using the analytical technology Ion Mobility Mass Spectrometry (IM-MS). We utilized it to study both transfer RNAs and FRS. While the previously mentioned methods are versatile tools for screening compound libraries, they do not account for the high cost of molecule libraries, nor the limitations inherent to screening vast molecular libraries. We employed two computational screening methods to reduce the number of compounds screened before identifying a new binding partner. These methods, coupled with FLED, were used to investigate the predictions and determine if the current setup does increase the rate of compound discovery.

The results described here support using FLED, IM-MS, and computational screening to identify small molecule candidates for development as FMN riboswitch-targeted antibiotics. It also provides preliminary work toward new a method to measure transcription termination of riboswitches. Finally, discusses the significance of such tools in the path toward antibiotic development.