Novel Riboswitch-Binding Flavin Analog That Protects Mice against Clostridium difficile Infection without Inhibiting Cecal Flora

Abstract

ABSTRACTNovel mechanisms of action and new chemical scaffolds are needed to rejuvenate antibacterial drug discovery, and riboswitch regulators of bacterial gene expression are a promising class of targets for the discovery of new leads. Herein, we report the characterization of 5-(3-(4-fluorophenyl)butyl)-7,8-dimethylpyrido[3,4-b]quinoxaline-1,3(2H,5H)-dione (5FDQD)—an analog of riboflavin that was designed to bind riboswitches that naturally recognize the essential coenzyme flavin mononucleotide (FMN) and regulate FMN and riboflavin homeostasis.In vitro, 5FDQD and FMN bind to and trigger the function of an FMN riboswitch with equipotent activity. MIC and time-kill studies demonstrated that 5FDQD has potent and rapidly bactericidal activity againstClostridium difficile. In C57BL/6 mice, 5FDQD completely prevented the onset of lethal antibiotic-inducedC. difficileinfection (CDI). Against a panel of bacteria representative of healthy bowel flora, the antibacterial selectivity of 5FDQD was superior to currently marketed CDI therapeutics, with very little activity against representative strains from theBacteroides,Lactobacillus,Bifidobacterium,Actinomyces, andPrevotellagenera. Accordingly, a single oral dose of 5FDQD caused less alteration of culturable cecal flora in mice than the comparators. Collectively, these data suggest that 5FDQD or closely related analogs could potentially provide a high rate of CDI cure with a low likelihood of infection recurrence. Future studies will seek to assess the role of FMN riboswitch binding to the mechanism of 5FDQD antibacterial action. In aggregate, our results indicate that riboswitch-binding antibacterial compounds can be discovered and optimized to exhibit activity profiles that merit preclinical and clinical development as potential antibacterial therapeutic agents.