Poster abstracts
Poster number 79 submitted by Ingrid Kilde
Discovery of novel FMN Riboswitch Binding Partners using an Interdisciplinary Approach
Ingrid R. Kilde (Program in Chemical Biology, University of Michigan - Ann Arbor), Dr. Elizabeth D. Tidwell (Life Sciences Institute, University of Michigan - Ann Arbor), Anna Anders, Dr. Brandon T. Ruotolo (Department of Chemistry, University of Michigan - Ann Arbor), Dr. Aaron T. Frank (Arrakis Therapeutics), Zhijian Hu, Dr. Kevin Wood (Department of Physics and Department of Biophysics, University of Michigan - Ann Arbor), Dr. Markos Koutmos (Department of Chemistry and Department of Biophysics, University of Michigan - Ann Arbor)
Abstract:
Prokaryotic flavin homeostasis (import and biosynthesis) is regulated via flavin mononucleotide riboswitches (FMN-RS) in most bacterial species groups, including 41 of the 49 highest priority pathogens identified by the World Health Organization. Riboswitches are structured RNA elements capable of altering gene expression at the transcriptional or translational level in response to specific binding of an essential metabolite ligand such as FMN. While dysregulation of flavin metabolism can be deleterious for bacterial robustness, investigation into antibacterials targeting the FMN riboswitch has been complicated by the off-target effects inherent to flavin analogues, and the lack of whole-cell potency in current lead compounds. To overcome these challenges, we aim to identify potent small molecule (SM) inhibitors of the FMN-RS using an interdisciplinary pipeline incorporating computational, analytical, biochemical, and biophysical techniques. We have developed a robust label-free and cell-free high throughput fluorescent equilibrium displacement (FLED) assay for monitoring the impact of SM on the FMN:FMNRS complex. In collaboration with UM Center for Chemical Genomics, we utilized this assay to screen over 20K compounds from diverse libraries including traditional unbiased libraries, commercial libraries curated for RNA targets, and libraries we assembled through computational prescreening using either docking (rDock) or ligand-based chemical and structural overlay (ROCS). For rigorous hit characterization, we are using ion mobility-mass spectrometry (IM-MS) to quantify binding capacity and collision-induced unfolding (CIU) to determine the structural impact (stabilization or destabilization) of SM binding. Furthermore, we have optimized a FMNRS-specific transcription termination assay and are in the process of implementing a high throughput in vivo growth inhibition assays for functional validation. Results from the SM characterization will inform future in silico prescreening for RNA:SM interactions, with the goal of iterative improvement of virtual screening for RNA bioactive compounds and further development of RNA drug discovery pipelines.
Keywords: FMN Riboswitch, Drug discovery, High throughput