title: Novel riboswitch-binding flavin analog that protects mice against Clostridium difficile infection without inhibiting cecal flora.
abstract: Novel 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 against Clostridium difficile. In C57BL/6 mice, 5FDQD completely prevented the onset of lethal antibiotic-induced C. difficile infection (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 the Bacteroides, Lactobacillus, Bifidobacterium, Actinomyces, and Prevotella genera. 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.
authors.affiliation: Department of Molecular, Cellular and Developmental Biology, Yale University, New Haven, Connecticut, USA BioRelix Inc., Foxborough, Massachusetts, USA ken.blount@yale.edu ronald.breaker@yale.edu.
authors.forename: Kenneth F
authors.initials: KF
authors.lastname: Blount
authors.affiliation: Department of Molecular, Cellular and Developmental Biology, Yale University, New Haven, Connecticut, USA.
authors.forename: Cynthia
authors.initials: C
authors.lastname: Megyola
authors.affiliation: Yale Center for Molecular Discovery, Yale University, New Haven, Connecticut, USA.
authors.forename: Mark
authors.initials: M
authors.lastname: Plummer
authors.affiliation: BioRelix Inc., Foxborough, Massachusetts, USA.
authors.forename: David
authors.initials: D
authors.lastname: Osterman
authors.affiliation: BioRelix Inc., Foxborough, Massachusetts, USA.
authors.forename: Tim
authors.initials: T
authors.lastname: O'Connell
authors.affiliation: Aristoff Consulting LLC, Fort Collins, Colorado, USA.
authors.forename: Paul
authors.initials: P
authors.lastname: Aristoff
authors.affiliation: QnA Pharma Consulting, LLC, Minneapolis, Minnesota, USA.
authors.forename: Cheryl
authors.initials: C
authors.lastname: Quinn
authors.affiliation: Kalexsyn, Inc., Kalamazoo, Michigan, USA.
authors.forename: R Alan
authors.initials: RA
authors.lastname: Chrusciel
authors.affiliation: Kalexsyn, Inc., Kalamazoo, Michigan, USA.
authors.forename: Toni J
authors.initials: TJ
authors.lastname: Poel
authors.affiliation: Kalexsyn, Inc., Kalamazoo, Michigan, USA.
authors.forename: Heinrich J
authors.initials: HJ
authors.lastname: Schostarez
authors.affiliation: Kalexsyn, Inc., Kalamazoo, Michigan, USA.
authors.forename: Catherine A
authors.initials: CA
authors.lastname: Stewart
authors.affiliation: Kalexsyn, Inc., Kalamazoo, Michigan, USA.
authors.forename: Daniel P
authors.initials: DP
authors.lastname: Walker
authors.affiliation: Kalexsyn, Inc., Kalamazoo, Michigan, USA.
authors.forename: Peter G M
authors.initials: PG
authors.lastname: Wuts
authors.affiliation: Department of Molecular, Cellular and Developmental Biology, Yale University, New Haven, Connecticut, USA Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, Connecticut, USA Howard Hughes Medical Institute, Yale University, New Haven, Connecticut, USA ken.blount@yale.edu ronald.breaker@yale.edu.
authors.forename: Ronald R
authors.initials: RR
authors.lastname: Breaker
mesh_terms: D000818:Animals; D000900:Anti-Bacterial Agents / Q000627:therapeutic use*; D002432:Cecum / Q000382:microbiology*; D016360:Clostridioides difficile / Q000187:drug effects* / Q000472:pathogenicity; D004761:Enterocolitis, Pseudomembranous / Q000188:drug therapy*; D005260:Female; D005486:Flavin Mononucleotide / Q000627:therapeutic use*; D005415:Flavins / Q000627:therapeutic use*; D051379:Mice; D008810:Mice, Inbred C57BL; D058928:Riboswitch
chemical_list: D000900:Anti-Bacterial Agents; D005415:Flavins; D058928:Riboswitch; D005486:Flavin Mononucleotide