Small molecule fluoride toxicity agonists

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.

Figure 1. Structures of Hits from a High-throughput Screen for Compounds that Enhance Fluoride Uptake or Retention

Figure 2. Small Molecules Enhance Fluoride Uptake and Toxicity in E. coli

(A) Reporter activity in E. coli treated with 1 and NaF. Data points are the average of three replicates, and error bars represent standard deviations. Decreases in reporter expression at high sodium fluoride and 1 are due to cell death. See Figure S1 for additional control experiments. (B) Growth of bacteria cultured in the absence or presence of 1 and increasing concentrations of sodium fluoride following 16 hours of incubation at 37°C. DMSO (~1% [v/v] in the growth medium) was used to solublize and deliver test compounds. Data points are the average of three replicates, and error bars represent standard deviations.

Figure 3. Increase in Reporter Expression for Selected Hit Analogs

(A) Structures of selected compounds synthesized or purchased for determination of SAR. Analytical data is presented in Supplemental Information. (B) Reporter gene expression in E. coli treated with 6 and various concentrations of fluoride. When 6 was tested at or above 10 µM, bacterial growth was inhibited in 100 mM fluoride. Data points are the average of three replicates. Error bars are the standard deviation of the measurement. (C) Reporter gene expression in E. coli treated with 7 and various concentrations of fluoride. Details are as described for B. (D) Fold increase in reporter gene expression in E. coli treated with compound and 10 mM NaF. Values are normalized to that measured with cells treated with fluoride in the absence of any compound (value set to one, dashed line). White bars indicate the addition of 10 µM compound, while black bars indicate the addition of 100 µM compound, with the exceptions of 1, 14, and 15 (32 µM) and 16 and 17 (3.2 µM). The absence of a bar indicates no substantial bacterial growth was observed for that condition. See Tables S2 for data on additional compounds tested.

Figure 4. Summary of Structure Activity Relationships

Incorporation of electron-withdrawing groups (EWG) at the meta or para position of either aryl ring results in enhanced activity. Utilization of a thiourea typically has little effect on activity compared to a urea, and likewise the asymmetric incorporation of an additional ring system has little affect. Both amide protons are necessary for activity and might be important for hydrogen bonding (dashed lines).

Figure 5. Structural derivatives of hit compounds enhance fluoride toxicity

(A) MIC of hit compounds for E. coli and S. mutans in the absence of fluoride. Arrows indicate that the values for the analyses denoted by the symbols were not measurable at the highest concentrations tested. (B) Decrease in the MIC of sodium or potassium fluoride for E. coli in the presence of varying concentrations of hit compounds. For compounds whose MIC in the absence of fluoride was higher than 32 µg ml⁻¹, half and quarter MIC values were calculated as if the MIC were 64 µg ml⁻¹. Values are the average of three replicates. (C) Decrease in the MIC of sodium or potassium fluoride for S. mutans in the presence of varying concentrations of hit compounds. Values are the average of three replicates.

Scheme 1. Synthesis of analogs for structure-activity relationship analyses