title: Development and application of a high-throughput assay for glmS riboswitch activators.
abstract: Riboswitches are newly-discovered gene control elements that are promising targets for antibacterial drug development. To facilitate the rapid discovery and development of riboswitch-targeted compounds, modern drug discovery techniques such as structure-based design and high-throughput screening will need to be applied. One promising riboswitch drug target is the glmS riboswitch, which upon binding glucosamine-6-phosphate (GlcN6P) functions as a ribozyme and catalyzes self-cleavage. Herein we report the development of a high-throughput assay for glmS ribozyme cleavage that relies on fluorescence resonance energy transfer (FRET). This assay can be used to screen for compounds that bind to and activate glmS ribozyme cleavage. To validate the screen, we demonstrate that the assay can identify the active compounds from a library of GlcN6P analogs whose affinities for ribozyme were determined by commonly used electrophoretic methods with radiolabeled RNA. Furthermore, the primary screen of a library of 960 compounds previously approved for use in humans identified five active compounds, one of which is a GlcN6P analog known to stimulate ribozyme activity. These results demonstrate that modern high-throughput screening techniques can be applied to the discovery of riboswitch-targeted drug compounds.
authors.affiliation: Department of Molecular, Cellular and Developmental Biology, Yale University, New Haven, Connecticut 06520, USA.
authors.forename: Kenneth
authors.initials: K
authors.lastname: Blount
authors.forename: Izabela
authors.initials: I
authors.lastname: Puskarz
authors.forename: Robert
authors.initials: R
authors.lastname: Penchovsky
authors.forename: Ronald
authors.initials: R
authors.lastname: Breaker
mesh_terms: D001412:Bacillus subtilis / Q000378:metabolism; D001426:Bacterial Proteins / Q000737:chemistry*; D001483:Base Sequence; D002626:Chemistry, Pharmaceutical; D015195:Drug Design; D031541:Fluorescence Resonance Energy Transfer; D005944:Glucosamine / Q000031:analogs & derivatives / Q000737:chemistry; D019298:Glucose-6-Phosphate / Q000031:analogs & derivatives / Q000737:chemistry; D006801:Humans; D008956:Models, Chemical; D008957:Models, Genetic; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D009841:Oligonucleotides / Q000737:chemistry; D016337:RNA, Catalytic / Q000737:chemistry; D005945:Glutamine-Fructose-6-Phosphate Transaminase (Isomerizing)
chemical_list: D001426:Bacterial Proteins; D005944:Glucosamine; D019298:Glucose-6-Phosphate; D009841:Oligonucleotides; D016337:RNA, Catalytic; C001293:glucosamine 6-phosphate; C000731191:component S, glutamate mutase protein, Bacteria; D005945:Glutamine-Fructose-6-Phosphate Transaminase (Isomerizing)