Recent discoveries of novel classes of non-coding, regulatory RNAs have provided evidence for a variety of roles for RNA in disease, propelling interest in developing RNA-binding small molecules as both chemical probes and potential therapeutics. Riboswitches are naturally occurring, structured RNA elements often found in the 5’-untranstated regions of mRNAs in bacteria. Riboswitches regulate gene expression through molecular recognition of a specific cognate ligand, the binding of which induces an RNA conformational change that inhibits transcription or translation. With their unique biological and structural functions, riboswitches not only serve as important model systems for RNA-small molecule interactions, but also as potential antibacterial targets. In order to identify synthetic riboswitch-binding small molecules, we performed a small molecule microarray (SMM) screen on a preQ1 riboswitch found in B. subtilis. This riboswitch governs the expression of genes responsible for the biosynthesis of queuosine, a hypermodified nucleotide. One hit compound from this screen exhibited a binding affinity of approximately 500 nM to the preQ1 riboswitch. Further, in-line probing suggests that the synthetic ligand induces a riboswitch conformation that is different from that of the cognate ligand-bound form. The compound was also shown to bind a structurally similar, but functionally distinct preQ1 riboswitch from T. tengcongensis. Co-crystal structures with this riboswitch showed that the compound binds in the preQ1 binding site, is sandwiched by G11 and the G5/C16 pair and makes a hydrogen bonding interaction with A29. These key interactions are different from that of the cognate ligand but similarly occur at nucleotides that are conserved among the preQ1 riboswitch family. Importantly, initial in vitro transcription termination assays suggest that the small molecule is capable of regulating riboswitch function upon binding. The co-crystal structure will further be used for developing additional compounds that target the preQ1 riboswitch and the queuosine biosynthetic pathway through structure-guided design.

Chemical and structural insights into developing synthetic ligands for a PreQ1 riboswitch

256th National Meeting (2018)

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