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Research Paper
Development and Application of a High-Throughput Assay for glmS Riboswitch Activators
Kenneth Blount, Izabela Puskarz, Robert Penchovsky and Ronald Breaker
volume 3 | issue 2
april/may/june 2006Pages: 77 - 81
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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) 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 identifies the compounds known to be active from a focused library of GlcN6P analogs whose affinities for the ribozyme had been determined by commonly used electrophoretic methods using 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.
We now provide open access to journal articles published online for one year or more. This article may be downloaded at the following link:
Download PDF If the document does not open, please right-click on the link (control-click on a Macintosh) and select the option to save the file to disk.