Nature 526, 672–677 (2015)

Credit: NATURE

Antimicrobial strategies frequently target bacterial pathways that follow distinct routes from those in the human host or that humans lack entirely. Riboswitches—small noncoding RNA motifs that regulate gene expression in response to the specific binding of a small-molecule metabolite—are viewed as an attractive but underexplored class of antibacterial targets. Early efforts focused on the flavin mononucleotide (FMN) riboswitch, which regulates genes controlling the biosynthesis and transport of the essential vitamin riboflavin, have so far resulted in compounds with limited target selectivity. Howe et al. now report the identification of a specific inhibitor of the FMN riboswitch that displays potent antibacterial activity. The authors demonstrated that riboflavin biosynthesis genes such as ribA and ribB are conditionally essential in Escherichia coli, a property that facilitated the phenotypic screening of ∼57,000 growth-inhibitory compounds seeking agents with E. coli bioactivity that could also be suppressed by exogenous riboflavin. The screen identified ribocil, a synthetic compound with a chemical scaffold entirely distinct from that of riboflavin, which specifically inhibited the production of riboflavin and its metabolites in treated cells. To identify the potential target of ribocil, the authors selected compound-resistant E. coli strains and used whole-genome sequencing to show that all resistance mutations exclusively map to the FMN riboswitch that controls ribB expression. The FMN riboswitch was further established as ribocil's target through binding measurements, showing it to be a competitive inhibitor of FMN, and reporter gene assays, demonstrating that ribocil inhibited GFP expression under the control of wild-type, but not mutated, FMN riboswitch constructs. A crystal structure of (S)-ribocil in complex with the FMN riboswitch from Fusobacterium nucleatum confirmed that ribocil binds to the FMN pocket and suggested how resistance mutations may weaken ribocil binding. Finally, in a murine sepsis model, ribocil displays potent antibacterial activity that is dependent on its FMN-inhibitory properties. Beyond illustrating the utility of phenotypic screening and target identification in chemical biology, the study provides compelling evidence that bacterial riboswitches, and possibly other classes of noncoding RNA elements, may serve as new and as yet largely unexploited druggable targets.