Small Molecule Fluoride Toxicity Agonists

Removal of one such potential hydrogen-bond donor results in a dramatic decrease in compound activity.

“and the removal of one such potential hydrogen-bond donor results in a dramatic decrease in compound activity (Figure 3, 18).”

Compound 19 exhibits the greatest ability to increase fluoride toxicity when tested with S. mutans.

“This compound exhibits the greatest ability to increase fluoride toxicity when tested with S. mutans (Figure 5).”

Some similarities exist between our molecules and known inhibitors of bacterial cell wall biosynthesis and lipid biosynthesis.

“Some similarities exist between our molecules and known inhibitors of bacterial cell wall biosynthesis (Li et al., 2003; Francisco et al., 2004) and lipid biosynthesis (Russell, 2004).”

• cites Li et al., 2003Not matched

  Couldn’t be matched to a paper in Crossref, so it can’t be checked.

• cites Francisco et al., 2004 sourceNot checked — no full text of the source
• cites Russell, 2004 sourceNot checked — no full text of the source

Our chemical library was modest in compound number and diversity.

“Our chemical library was modest in compound number and diversity, and therefore it is not surprising that nearly all identified hits were based on the same chemical scaffold.”

Incorporation of similar substituents onto the aryl rings of our molecules enhances fluoride uptake and/or retention in E. coli.

“Since we observe that the incorporation of similar substituents onto the aryl rings of our molecules enhances fluoride uptake and/or retention E. coli (Figure 3 and Table S2)”

Hit validation resulted in five molecules that were chosen for future analysis.

“Hit validation consisted of repeating the screening assay and confirming that the signal increase was only observed in the presence of both cells and fluoride, resulting in five molecules that were chosen for future analysis (Figure 1 and Table S1).”

Significant bacterial toxicity was observed for certain compounds in the absence of any added fluoride.

“Additionally, we observed significant bacterial toxicity for certain compounds in the absence of any added fluoride (Figure 3 and Figure 5).”

The screen was successfully used to isolate compounds that increase intracellular fluoride concentrations.

“This screen was successfully used to isolate compounds that increase intracellular fluoride concentrations.”

Carboxamide linked compounds were generally less active than the corresponding urea- or thiourea-linked compounds.

“Notably, carboxamide linked compounds were generally less active than the corresponding urea- or thiourea-linked compounds (e.g., see 9 through 11).”

Based on SAR data, additional and more potent analogs were identified by the synthesis of analogs.

“Based on SAR data, additional and more potent analogs were identified by the synthesis of analogs.”

We have succeeded in identifying novel small molecules able to render both Gram-positive and Gram-negative bacteria susceptible to fluoride.

“Regardless, we have succeeded in identifying novel small molecules able to render both Gram-positive and Gram-negative bacteria susceptible to fluoride.”

Approximately 75,000 compounds from Chembridge, Maybridge, ChemDiv, and proprietary Yale small molecule libraries were tested.

“After optimizing screening conditions, approximately 75,000 compounds from Chembridge, Maybridge, ChemDiv, and proprietary Yale small molecule libraries were tested.”

These compounds do not increase the activity of the β-galactosidase enzyme in vitro.

“These compounds do not increase the activity of the β-galactosidase enzyme in vitro, and there was no increase in fluorescence upon treatment of cells containing a reporter construct carrying a fluoride riboswitch with a disruptive mutation (Figure S1).”

There was no increase in fluorescence upon treatment of cells containing a reporter construct carrying a fluoride riboswitch with a disruptive mutation.

“there was no increase in fluorescence upon treatment of cells containing a reporter construct carrying a fluoride riboswitch with a disruptive mutation (Figure S1).”

The most potent derivatives substantially increase the antibacterial potency of fluoride toward representative Gram-negative and Gram-positive bacteria.

“The most potent derivatives substantially increase the antibacterial potency of fluoride toward representative Gram-negative and Gram-positive bacteria.”

Addition of compound 1 alone at 3.2 μM causes no decrease in the viability of E. coli.

“Addition of 1 alone at 3.2 µM causes no decrease in the viability of E. coli but enhances the natural toxicity of fluoride.”

The antibacterial effects of these molecules are enhanced by the presence of fluoride.

“However, their antibacterial effects are enhanced by the presence of fluoride.”

All five selected hits isolated from the high-throughput screen contained an electron-deficient aryl group linked by either an amide or urea to another ring system.

“All five selected hits isolated from the HTS contained an electron-deficient aryl group linked by either an amide or urea to another ring system.”

The locations and identities of the electron-withdrawing groups do not appear to be of critical importance.

“The locations and identities of the electron-withdrawing groups, however, do not appear to be of critical importance (e.g., see 6, 8, and 9).”

No enhancement of fluoride toxicity was observed upon treatment with ciprofloxacin.

“No enhancement of fluoride toxicity was observed upon treatment with ciprofloxacin (Figure S2).”

Of the five hits, compound 1 was the most active, yielding an approximately 8-fold increase in reporter signal at 32 μM when 10 mM sodium fluoride was present relative to the addition of fluoride alone.

“Of these five hits, compound 1 was the most active, yielding an approximately 8-fold increase in reporter signal at 32 µM when 10 mM sodium fluoride was present relative to the addition of fluoride alone (Figure 2A).”

Nearly all identified hits were based on the same chemical scaffold.

“and therefore it is not surprising that nearly all identified hits were based on the same chemical scaffold.”

Compound 1 did not significantly change the minimal inhibitory concentration (MIC) of fluoride toward E. coli.

“Although 1 did not significantly change the minimal inhibitory concentration (MIC) of fluoride toward E. coli, it did reproducibly enhance the ability of 1 to slow E. coli growth (Figure 2B).”

A brief survey of analogs of the original hits identified more potent compounds with enhanced fluoride sensitization activity.

“A brief survey of analogs of the original hits identified more potent compounds with enhanced fluoride sensitization activity.”

An electron-deficient aromatic ring, such as those in compounds 6 and 7, is necessary for enhancing intracellular fluoride concentrations.

“The relative activities of various analogs (Figure 3 and Table S2) confirmed that an electron-deficient aromatic ring, such as those in compounds 6 and 7 (Figure 3B and C), were indeed necessary for enhancing intracellular fluoride concentrations.”

A subinhibitory concentration of compound 17 was less effective at increasing fluoride toxicity compared to compound 14.

“A subinhibitory concentration of 17 was less effective at increasing fluoride toxicity compared to 14.”

Reporter gene expression in the Δfluc E. coli strain was 15- to 20-fold higher compared to the WT strain.

“Reporter gene expression in the Δfluc E. coli strain was 15- to 20-fold higher compared to the WT strain.”

The SAR data derived from substitutions within the urea-linked series was not the same as with thiourea-linked.

“Interestingly, the SAR data derived from substitutions within the urea-linked series was not the same as with thiourea-linked (e.g., see 12 and 13).”

Compound 17 achieves its more modest effects at a lower concentration.

“However, 17 achieves its more modest effects at a lower concentration.”

Addition of compound 1 at 3.2 μM enhances the natural toxicity of fluoride.

“Addition of 1 alone at 3.2 µM causes no decrease in the viability of E. coli but enhances the natural toxicity of fluoride.”

New insights into the genes and mechanisms used by bacterial cells to overcome fluoride toxicity were revealed by the discovery of fluoride-responsive riboswitches that control the expression of genes conferring resistance to fluoride.

“New insights into the genes and mechanisms used by bacterial cells to overcome fluoride toxicity were revealed by the discovery of fluoride-responsive riboswitches that control the expression of a number of genes conferring resistance to fluoride (Baker et al., 2012).”

• cites Baker et al., 2012 sourceNot checked yet

Due to general mechanisms for enzyme inhibition, fluoride is toxic to organisms from all three domains of life.

“Due to these general mechanisms for enzyme inhibition, fluoride is toxic to organisms from all three domains of life.”

When in complex with a divalent metal ion and ADP, fluoride forms a non-functional mimic of ATP that can inhibit enolase.

“when in complex with a divalent metal ion and ADP, fluoride forms a non-functional mimic of ATP that can inhibit enolase (Curran et al., 1994; Qin et al., 2006)”

• cites Curran et al., 1994 sourceCheck pending
• cites Qin et al., 2006 sourceCheck pending

Similar fluoride complexes can inhibit a large diversity of other metabolic enzymes including phosphatases.

“similar complexes can inhibit a large diversity of other metabolic enzymes including phosphatases (Barbier et al., 2010; Nakai et al, 1974).”

• cites Barbier et al., 2010 sourceNot checked — no full text of the source
• cites Nakai et al, 1974 sourceNot checked — no full text of the source

Fluoride has substantial antibacterial effects.

“Although fluoride is commonly added to oral hygiene products to increase the strength of tooth enamel, it also has substantial antibacterial effects (Barbier et al., 2010; Li, 2003).”

• cites Barbier et al., 2010 sourceNot checked — no full text of the source
• cites Li, 2003Not matched

  Couldn’t be matched to a paper in Crossref, so it can’t be checked.

The fluoride riboswitch reporter system used in this study is sufficiently robust that it can be used to reveal hit compounds by HTS that only weakly increase the intracellular concentration of fluoride in bacteria.

“The fluoride riboswitch reporter system used in this study is sufficiently robust that it can be used to reveal hit compounds by HTS that only weakly increase the intracellular concentration of fluoride in bacteria.”

Any use of such compounds as potential antibacterial agents would likely require the concomitant application of additional fluoride.

“Thus, any use of such compounds as potential antibacterial agents would likely require the concomitant application of additional fluoride.”

It is likely that many other compounds could be identified by our HTS process that increase fluoride toxicity by different mechanisms.

“Although this collection of hits likely operate by a similar mechanism, it is likely that many other compounds could be identified by our HTS process that increase fluoride toxicity by different mechanisms.”

The deleterious phenotype resulting from fluc deletion in E. coli can be compensated by expressing an eriCF gene derived from another bacterium.

“Moreover, the deleterious phenotype resulting from fluc deletion in E. coli can be compensated by expressing an eriCF gene derived from another bacterium (Baker et al., 2012)”

• cites Baker et al., 2012 sourceNot checked yet

The specific nature of the hits obtained via our high-throughput screen would suggest these compounds might function by interacting with a specific target, rather than by non-specifically disrupting membranes.

“Furthermore, the specific nature of the hits obtained via our high-throughput screen would suggest these compounds might function by interacting with a specific target, rather than by non-specifically disrupting membranes.”

The results are consistent with the hypothesis that compound 1 increases intracellular fluoride concentrations.

“These results are consistent with our hypothesis that 1 increases intracellular fluoride concentrations.”

This study illustrates the potential for optimization of fluoride toxicity agonists as antibacterial compounds.

“Overall, this study illustrates the potential for optimization of fluoride toxicity agonists as antibacterial compounds.”

The Fluc protein channel is highly specific for fluoride, unlike compounds such as amphotericin B.

“One key difference between Fluc proteins and compounds such as amphotericin B is that the protein channel is highly specific for fluoride.”

Our findings indicate that small, non-peptidic molecules can be used to increase fluoride toxicity against bacteria.

“Our findings indicate that small, non-peptidic molecules can be used to increase fluoride toxicity against bacteria and that such molecules might be frequently identified as hit compounds via high-throughput screening.”

The proteins expressed from eriCF appear to be biologically equivalent to those encoded by fluc.

“indicating that the proteins expressed from eriCF appear to be biologically equivalent to those encoded by fluc.”

• cites Baker et al., 2012 sourceNot checked yet

A major strategy for overcoming fluoride toxicity is for cells to eject fluoride into the environment.

“These findings demonstrate that a major strategy for overcoming toxicity is for cells to eject fluoride into the environment.”

Small molecule fluoride toxicity agonists can be identified by high-throughput screening from existing chemical libraries by exploiting a natural fluoride riboswitch.

“Thus, we demonstrate that small molecule fluoride toxicity agonists can be identified by HTS from existing chemical libraries by exploiting a natural fluoride riboswitch.”

• AssumptionRests on an assumption: Until recently, very little was known about how cells sense and respond to fluoride toxicity.
• AssumptionRests on an assumption: No non-peptidic molecules have been identified that increase fluoride toxicity towards bacteria.

General membrane disruptors should be far more tolerant of changes to chemical substructures than our SAR data indicates.

“General membrane disruptors should be far more tolerant of changes to chemical substructures than our SAR data indicates.”

The ratio of the fluorescence signal of the positive control well to background was higher than 15:1.

“The ratio of the fluorescence signal of the positive control well (fluoride-treated Δfluc cells) to background (average fluorescence of all wells on a plate) was higher than 15:1.”

Some hits are structurally similar to known small molecule fluoride receptors.

“Intriguingly, some of our hits are also structurally similar to known small molecule fluoride receptors (Boiocchi et al., 2004).”

• cites Boiocchi et al., 2004 sourceNot checked — no full text of the source

Formation of non-selective ion pores, or general membrane disruption, will disrupt the membrane potential of the cell, which is critical for the productive use of Fluc channel proteins to expel fluoride from inside the cell.

“Formation of non-selective ion pores, or general membrane disruption will also disrupt the membrane potential of cell, which is critical for the activity of the productive use of Fluc channel proteins to expel fluoride from inside the cell.”

Fluc-type ion channels permit the selective and passive diffusion of fluoride through membranes.

“Curiously, evidence presented to date convincingly demonstrates that Fluc-type ion channels permit the selective and passive diffusion of fluoride through membranes (Stockbridge et al., 2013; Ji et al., 2014)”

• cites Stockbridge et al., 2013 sourceNot checked yet
• cites Ji et al., 2014Not matched

  Couldn’t be matched to a paper in Crossref, so it can’t be checked.

A wild-type E. coli strain was employed in which a fluoride-sensing riboswitch from Pseudomonas syringae controls the expression of a lacZ reporter gene in a fluoride concentration-dependent manner.

“Specifically, we employed a previously described wild-type (WT) E. coli strain in which a fluoride-sensing riboswitch from Pseudomonas syringae controls the expression of a lacZ reporter gene in a fluoride concentration-dependent manner (Baker et al., 2012; Nelson et al., 2014).”

• cites Baker et al., 2012 sourceCheck pending
• cites Nelson et al., 2014 sourceCheck pending

Some previously characterized pore-forming antibacterial and antifungal compounds have been demonstrated to enhance the cytotoxic activity of fluoride.

“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).”

• cites Li and Breaker, 2012 sourceCheck pending
• cites Nelson et al., 2014 sourceCheck pending
• cites Zasloff and Steinberg, 1993Not matched

  Couldn’t be matched to a paper in Crossref, so it can’t be checked.

The large negative membrane potential of bacterial cells should greatly favor fluoride efflux through the channel.

“However, the large negative membrane potential of bacterial cells should greatly favor fluoride efflux through the channel (Stockbridge et al., 2013).”

• cites Stockbridge et al., 2013 sourceNot checked yet

An E. coli strain in which the fluc gene was deleted (Δfluc) is approximately 200-fold more sensitive to fluoride.

“An E. coli strain in which the fluc gene was deleted (Δfluc) is ~200-fold more sensitive to fluoride (Baker et al., 2012).”

• cites Baker et al., 2012 sourceNot checked yet

The fluoride-sensing riboswitch is the most selective biosensor for fluoride currently known.

“Therefore, we designed a screen that utilized the most selective biosensor for fluoride currently known – a recently discovered fluoride sensing riboswitch (Baker et al., 2012).”

• cites Baker et al., 2012 sourceNot checked yet

Such molecules might be frequently identified as hit compounds via high-throughput screening.

“and that such molecules might be frequently identified as hit compounds via high-throughput screening.”

Bacteria that have genetic and biochemical responses to even rare exposures to high fluoride concentrations or to low concentrations of fluoride under slightly acidic conditions have a large selective advantage.

“Therefore, bacteria that have genetic and biochemical responses to even rare exposures to high fluoride concentrations or to low concentrations of fluoride under slightly acidic conditions have a large selective advantage.”

• AssumptionRests on an assumption: Fluoride in the form of HF will dominate under acidic conditions, and this acidic form is far more bioavailable.

Some eukaryotic species use homologous channel proteins to improve resistance to fluoride toxicity.

“Similarly, some eukaryotic species use homologous channel proteins to improve resistance to fluoride toxicity (Li et al., 2013), which underscores the importance of fluc to fluoride detoxification.”

• cites Li et al., 2013Not matched

  Couldn’t be matched to a paper in Crossref, so it can’t be checked.

Disruption of membranes in a non-specific fashion via externally added compounds will also disrupt membrane potential.

“The disruption of membranes in a non-specific fashion via various externally added compounds will also disrupt membrane potential.”

Compound 19 is known to promote the transport of chloride out of lipid vesicles.

“To evaluate this hypothesis we examined 19 (Figure 3), which is a compound known to promote the transport of chloride out of lipid vesicles (Busschaert et al., 2012).”

• cites Busschaert et al., 2012 sourceNot checked — no full text of the source

WT E. coli cultures can still grow at fluoride concentrations as high as 100 mM.

“which can still grow at concentrations as high as 100 mM (Baker et al., 2012).”

• cites Baker et al., 2012 sourceNot checked yet

The findings are consistent with the hypothesis that these molecules might enhance fluoride toxicity by targeting a cellular process that is critical for cell growth even in the absence of fluoride.

“Our findings are consistent with the hypothesis that these molecules might enhance fluoride toxicity by targeting a cellular process that is critical for cell growth even in the absence of fluoride.”

The determinants of activity for the urea-linked and thiourea-linked series might be different.

“This suggests that the determinants of activity for these two series might be different.”

The concentrations of fluoride used in the study are substantially higher than bacteria typically experience in a natural environment, except for those exposed to fluoride-containing oral hygiene products.

“Importantly, the concentrations of fluoride used in our study are substantially higher than bacteria typically experience in a natural environment, except for those exposed to fluoride-containing oral hygiene products.”

None of the existing methods for fluoride detection are sufficiently selective or sensitive to rapidly and easily detect intracellular fluoride concentrations in bacteria.

“However, none of these methods are sufficiently selective or sensitive to rapidly and easily detect intracellular fluoride concentrations in bacteria.”

• cites Cametti and Rissanen, 2009, 2013Not matched

  Couldn’t be matched to a paper in Crossref, so it can’t be checked.

• cites Drescher and Suttie, 1972 sourceNot checked — no full text of the source
• cites Jayaraman et al., 2000 sourceCheck pending

There is precedence for compounds that disrupt membrane integrity to enhance fluoride toxicity.

“There is precedence for compounds that disrupt membrane integrity to enhance fluoride toxicity (Li and Breaker, 2012; Nelson et al., 2014; Zasloff and Steinberg, 1993).”

• cites Li and Breaker, 2012 sourceCheck pending
• cites Nelson et al., 2014 sourceCheck pending
• cites Zasloff and Steinberg, 1993Not matched

  Couldn’t be matched to a paper in Crossref, so it can’t be checked.

High-throughput screening of a small molecule library with cells carrying a fluoride-dependent reporter gene can reveal compounds that exhibit antibacterial activity in the presence of fluoride.

“These results demonstrate that high-throughput screening of a small molecule library with cells carrying a fluoride-dependent reporter gene can reveal compounds that exhibit antibacterial activity in the presence of fluoride.”

This finding suggests that the compound series identified in our HTS might indeed function by directly facilitating the transport of fluoride into bacteria.

“This finding suggests that the compound series identified in our HTS might indeed function by directly facilitating the transport of fluoride into bacteria.”

Existing molecules only modestly increase fluoride toxicity.

“However, these existing molecules only modestly increase fluoride toxicity, and no non-peptidic molecules have been identified that increase fluoride toxicity towards bacteria.”

• cites Li and Breaker, 2012 sourceCheck pending
• cites Nelson et al., 2014 sourceCheck pending
• cites Zasloff and Steinberg, 1993Not matched

  Couldn’t be matched to a paper in Crossref, so it can’t be checked.