Fragment screening against the thiamine pyrophosphate riboswitchthiM

Abstract

Riboswitches are regions of mRNA that bind selectively certain metabolites, thereby regulating gene expression. We have developed a method, which uses a combination of biophysical techniques (equilibrium dialysis, waterLOGSY and T₂ relaxation-edited NMR spectroscopy and isothermal titration calorimetry) that allows screening and identification of novel ligands for riboswitches. By this method a library of 1300 fragments was screened on the E. colithiamine pyrophosphate (TPP) riboswitchthiM, resulting in the identification of 17 hits. The compounds showed K_D values from 22 to 670 μM, with four compounds having K_D values <100 μM. The fragments are structurally diverse and their ligand efficiency indicates good prospects for structure-guided elaboration. In addition, a riboswitch functional assay based on in vitrotranscription translation (IVTT) of the reporter gene luciferase was developed. This assay system is an alternative to in vivo assays and constitutes a valuable tool to determine the effect of small molecules on riboswitch-regulated gene expression.