Dual-Targeting Small-Molecule Inhibitors of the Staphylococcus aureus FMN Riboswitch Disrupt Riboflavin Homeostasis in an Infectious Setting

Abstract

Riboswitches are bacterial-specific, broadly conserved, non-coding RNA structural elements that control gene expression of numerous metabolic pathways and transport functions essential for cell growth. As such, riboswitch inhibitors represent a new class of potential antibacterial agents. Recently, we identified ribocil-C, a highly selective inhibitor of the flavin mononucleotide (FMN) riboswitch that controls expression of de novo riboflavin (RF, vitamin B2) biosynthesis in Escherichia coli. Here, we provide a mechanistic characterization of the antibacterial effects of ribocil-C as well as of roseoflavin (RoF), an antimetabolite analog of RF, among medically significant Gram-positive bacteria, including methicillin-resistant Staphylococcus aureus (MRSA) and Enterococcus faecalis. We provide genetic, biophysical, computational, biochemical, and pharmacological evidence that ribocil-C and RoF specifically inhibit dual FMN riboswitches, separately controlling RF biosynthesis and uptake processes essential for MRSA growth and pathogenesis. Such a dual-targeting mechanism is specifically required to develop broad-spectrum Gram-positive antibacterial agents targeting RF metabolism.

Discovery of Small‐Molecule Antibiotics against a Unique tRNA‐Mediated Regulation of Transcription in Gram‐Positive Bacteria

“…Riboswitches are ideal targets because analogues of small‐molecule metabolites can be used to disrupt normal riboswitch regulation of the gene expression. Riboswitch inhibitors have been found . While inhibitors of metabolic riboswitches are promising sites for the design of new antibiotics, they are still prone to induce bacterial resistance because the drug binds to a single target molecule that could readily be mutated to decrease drug efficacy.…”

Thinking Outside the Box—Novel Antibacterials To Tackle the Resistance Crisis

“…[179] Confirmation that bacterial riboswitches can indeed be selectively targeted was obtained from the identification of two novel inhibitors of the FMN riboswitch in 2015:5FDQD and ribocil-C (Figure 13 b). [183] Although targeting riboswitches is avery promising approach which increases the number of new antibacterial targets,g ood in vivo inhibition without rapid development of resistance remain ac hallenge for future investigation. [180] Ribocil-C is a" drug-like" small molecule that is chemically unrelated to FMN.I tw as first identified by comparative phenotypic screening of as ynthetic smallmolecule library.…”

Superior cellular activities of azido- over amino-functionalized ligands for engineered preQ1riboswitches inE.coli

“…If the respective metabolite is essential for life, this will lead to a growth stop and/or death of the bacterial cell. Several studies have demonstrated that riboswitches are indeed druggable [6–8,16,47–49]. The most prominent investigation employed a phenotypic screen and identified ribocil that acts as a structurally distinct mimic of the natural ligand flavin mononucleotide to repress ribB gene expression and inhibit cell growth [7].…”

“…This makes the structure-based design of modified ligand analogs and ligand mimics rather challenging. Nevertheless, the identification of novel potent ligands is a topic of intense research because ever since their discovery, riboswitches have been viewed as promising targets for the development of novel antibiotic strategies [16]. Likewise, efforts to engineer riboswitches for imaging purposes [17–19] or as biotechnological tools for the detection of endogenous and non-endogenous small molecules are in the focus of synthetic biologists interested in understanding and reprogramming cellular behavior [20].…”