Structure-based design for the discovery of novel ligands targeting the FMN riboswitch
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Abstract
Abstract:
The FMN riboswitch is found in the 5'-UTR of bacterial mRNA and regulates gene expression of proteins involved in transport and biosynthesis of riboflavin via feedback control. This RNA structure is widespread among pathogenic bacteria and the ligand binding site is highly conserved among different species. Therefore, the FMN riboswitch represents a suitable drug target for novel antibacterial compounds.
For the identification of ligands with novel chemical scaffolds distinct from the chemical constitution of the cognate ligand FMN and its close analogues with known binding properties two successive virtual screening approaches were performed. Subsequently, an unbiased virtual screening with an in-house database of commercially available compounds and a privileged substructure search to facilitate mimicking the most important interactions between the target RNA and its cognate ligand were carried out.
The virtual screening hits were tested experimentally to confirm the in silico predictions. Three different methods were applied in order to determine the binding affinities of hit compounds. Whereas a method based on fluorescence quenching and ITC measurements indirectly determined the binding constants of the possible ligands, a SPR method was developed to directly sense the interactions between the FMN riboswitch RNA and possible small molecule ligands. One compound identified by the virtual screening methods was confirmed to bind experimentally. Close analogues of the hit substance are currently under investigation to initialize a small SAR study.