title: Crystallographic analysis of TPP riboswitch binding by small-molecule ligands discovered through fragment-based drug discovery approaches.
abstract: Riboswitches are structured mRNA elements that regulate gene expression in response to metabolite or second-messenger binding and are promising targets for drug discovery. Fragment-based drug discovery methods have identified weakly binding small molecule "fragments" that bind a thiamine pyrophosphate (TPP) riboswitch. However, these fragments require substantial chemical elaboration into more potent, drug-like molecules. Structure determination of the fragments bound to the riboswitch is the necessary next step. In this chapter, we describe the methods for co-crystallization and structure determination of fragment-bound TPP riboswitch structures. We focus on considerations for screening crystallization conditions across multiple crystal forms and provide guidance for building the fragment into the refined crystallographic model. These methods are broadly applicable for crystallographic analyses of any small molecules that bind structured RNAs.
authors.affiliation: National Heart, Lung and Blood Institute, Bethesda, Maryland, USA; Department of Chemistry, University of Cambridge, Cambridge, United Kingdom.
authors.forename: Katherine Deigan
authors.initials: KD
authors.lastname: Warner
authors.affiliation: National Heart, Lung and Blood Institute, Bethesda, Maryland, USA. Electronic address: adrian.ferre@nih.gov.
authors.forename: Adrian R
authors.initials: AR
authors.lastname: Ferré-D'Amaré
mesh_terms: D001665:Binding Sites; D018360:Crystallography, X-Ray / Q000379:methods*; D055808:Drug Discovery / Q000379:methods*; D008024:Ligands; D008958:Models, Molecular; D058928:Riboswitch; D054852:Small Molecule Libraries / Q000737:chemistry* / Q000494:pharmacology; D013835:Thiamine Pyrophosphate / Q000378:metabolism*
chemical_list: D008024:Ligands; D058928:Riboswitch; D054852:Small Molecule Libraries; D013835:Thiamine Pyrophosphate
keywords: Fragment-based drug discovery; Riboswitch; X-ray crystallography