Abstract
Fluoride is a ubiquitous anion that inhibits a wide variety of metabolic processes. Here, we report the identification of a series of compounds that enhance fluoride toxicity in Escherichia coli and Streptococcus mutans. These molecules were isolated by using a high-throughput screen (HTS) for compounds that increase intracellular fluoride levels as determined via a fluoride riboswitch reporter fusion construct. A series of derivatives were synthesized to examine structure-activity relationships, leading to the identification of compounds with improved activity. Thus, we demonstrate that small molecule fluoride toxicity agonists can be identified by HTS from existing chemical libraries by exploiting a natural fluoride riboswitch. In addition, our findings suggest that some molecules might be further optimized to function as binary antibacterial agents when combined with fluoride.
INTRODUCTION
Although fluoride is commonly added to oral hygiene products to increase the strength of tooth enamel, it also has substantial anti-bacterial effects (Barbier et al., 2010; Li, 2003). For example, when in complex with a divalent metal ion and ADP, fluoride forms a nonfunctional mimic of ATP that can inhibit enolase (Curran et al., 1994; Qin et al., 2006), and similar complexes can inhibit a large diversity of other metabolic enzymes including phosphatases (Barbier et al., 2010; Nakai and Thomas, 1974). Due to these general mechanisms for enzyme inhibition, fluoride is toxic to organisms from all three domains of life.
Until recently, very little was known about how cells sense and respond to fluoride toxicity. New insights into the genes and mechanisms used by bacterial cells to overcome fluoride toxicity were revealed by the discovery of fluoride-responsive ribo-switches that control the expression of a number of genesconferring resistance to fluoride (Baker et al., 2012). Ribo-switches are structured RNAs that are typically found in the 50-UTRs of bacterial mRNAs where they regulate the expression of genes in response to binding a small molecule or ion (Breaker,2011; Peselis and Serganov, 2014; Serganov and Nudler, 2013). The most common gene associated with fluoride riboswitches termed crcB (also called fexorfluc), codes for a fluoride-specific channel protein (Baker et al., 2012; Li et al., 2013; Stockbridgeet al., 2013). Another commonly controlled gene, eriCF, encodes a fluoride-selective anti porter (Baker et al., 2012; Stockbridge et al., 2012). Numerous other genes presumably involved in mitigating fluoride toxicity are also associated with fluoride ribo-switches (Baker et al., 2012).
An Escherichia colistrain in which the fluc gene was deleted (?fluc) is 200-fold more sensitive to fluoride (Baker et al.,2012). Similarly, some eukaryotic species use homologous channel proteins to improve resistance to fluoride toxicity (Liet al., 2013), which underscores the importance of fluc to fluoride detoxification. Moreover, the deleterious phenotype resulting from fluc deletion in E. coli can be compensated by express in ganeri CF gene derived from another bacterium (Baker et al.,2012), indicating that the proteins expressed from eriCF appear to be biologically equivalent to those encoded by fluc. These findings demonstrate that a major strategy for overcoming toxicity is for cells to eject fluoride into the environment.We speculated that small molecules that specifically inhibit the protein products Fluc or EriCF, increase the membrane permeability of fluoride, or increase fluoride retention through some other mechanism could enhance fluoride toxicity. If such molecules could be found, then perhaps they could be optimized and made to function as novel antibacterial chemotherapeutics, particularly when used in combination with high fluoride concentrations. This speculation is supported by the fact that some previously characterized pore-forming antibacterial and antifungal compounds have been demonstrated to enhance the cytotoxic activity of fluoride (Li and Breaker, 2012; Nelson et al., 2014; Zasloff and Steinberg, 1993). However, these existing molecules only modestly increase fluoride toxicity, and no non-peptidic molecules have been identified that increase fluoride toxicity toward bacteria.
To identify novel fluoride toxicity agonists for bacteria, we developed an HTS strategy based on an E. colistrain in which a ß-galactosidase reporter gene controlled by a fluoride riboswitch provides a readout of intracellular fluoride concentration (Baker et al., 2012; Nelson et al., 2014). A number of studies have been conducted to identify compounds that directly target riboswitches and function as antibiotics (Blount and Breaker,2006; Deigan and Ferre ?-D’Amare ?, 2011; Kim et al., 2009; Leeet al., 2009; Lu ?nse et al., 2014a, 2014b; Mulhbacher et al.,2010; Ster et al., 2013). By contrast, our study is designed a riboswitch as a tool to identify compounds that target other aspects of bacterial physiology. Hit compounds from the HTS were validated via a variety of biochemical assays and were shown to sensitize bacteria to fluoride. A brief survey of analogs of the original hits identified more potent compounds with enhanced fluoride sensitization activity. Overall, this study illustrates the potential for optimization of fluoride toxicity agonists as antibacterial compounds.
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