Sugar-modified analogs of auranofin are potent inhibitors of

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Sugar-modified analogs of auranofin are potent inhibitors of the gastric pathogen Helicobacter pylori Tessa D. Epstein, Bin Wu, Karen D. Moulton, Mingdi Yan, and Danielle H. Dube ACS Infect. Dis., Just Accepted Manuscript • DOI: 10.1021/acsinfecdis.9b00251 • Publication Date (Web): 05 Sep 2019 Downloaded from pubs.acs.org on September 6, 2019

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ACS Infectious Diseases

Sugar-modified analogs of auranofin are potent inhibitors of the gastric pathogen Helicobacter pylori Tessa D. Epstein,1‡ Bin Wu,2‡ Karen D. Moulton,1 Mingdi Yan2* and Danielle H. Dube1* 1Department

2Department

of Chemistry & Biochemistry, Bowdoin College, 6600 College Station, Brunswick, ME 04011, USA. of Chemistry, University of Massachusetts Lowell, 1 University Ave., Lowell, MA 01854, USA.

*Corresponding authors: [email protected], [email protected]

Keywords: Helicobacter pylori, auranofin, thioredoxin, sugar-gold conjugate, antibiotic, inhibitor Helicobacter pylori infection poses a worldwide public health crisis, as chronic infection is rampant and can lead to gastric ulcers, gastritis, and gastric cancer. Unfortunately, frontline therapies cause harmful side effects and are often ineffective due to antibiotic resistance. The FDA-approved drug auranofin is a gold complex with a Au(I) core coordinated with triethylphosphine and peracetylated thioglucose as the ligands. Auranofin is used for the treatment of rheumatoid arthritis and also displays potent activity against H. pylori. One of auranofin’s modes of action involves cell death by disrupting cellular thiol-redox balance maintained by thioredoxin reductase (TrxR), but this disruption leads to unwanted side effects due to mammalian cell toxicity. Here we developed and tested sugar-modified analogs of auranofin as potential antibiotics against H. pylori, with the rationale that modulating the sugar moiety would bias uptake by targeting bacterial cells and mitigating mammalian cell toxicity. Sugar-modified auranofin analogs displayed micromolar minimum inhibitory concentrations against H. pylori, maintained nanomolar inhibitory activity against the target enzyme TrxR, and caused reduced toxicity to mammalian cells. Taken together, our results suggest that structurally modifying the sugar component of auranofin has the potential to yield superior antibiotics for the treatment of H. pylori infection. Broadly, glyco-tailoring is an attractive approach for repurposing approved drugs.

The infection of Helicobacter pylori is a global health crisis. H. pylori is a Gram-negative pathogenic bacterium that colonizes the stomach.1, 2 Infection of H. pylori is the major cause of duodenal and gastric ulcers, which are the leading cause of cancers throughout the gastric tract.1, 2 H. pylori infection has been identified to be an increased risk factor for the development of gastric cancer.3 In 1994, The World Health Organization International Agency for Research on Cancer classified H. pylori as a class I carcinogen, emphasizing the risks posed by H. pylori infection.4 More than 50% of the world population is infected with H. pylori, with a disproportionately high number of people from low socioeconomic background and developing countries among the infected populations.1, 2, 5, 6 Current treatments of H. pylori infection involve the potent “triple therapy,” which consists of the broadspectrum antibiotics amoxicillin and clarithromycin, and a proton pump inhibitor.1, 2, 7, 8 However, triple therapy causes harmful side effects and often fails to successfully eradicate H. pylori infection, even after multiple rounds of treatment.1, 2 Additionally, in recent years H. pylori antibiotic resistance has been drastically increasing, with confirmed reports of H. pylori strains resistant to the commonly utilized antibiotics clarithromycin and metronidazole.9 In 2017, the World Health Organization

listed clarithromycin resistant H. pylori as a high priority pathogen for which we need novel antibiotics.10 Thus, development of anti-H. pylori agents is a pressing need. Recent reports have demonstrated that auranofin, a sugargold conjugate approved for the treatment of rheumatoid arthritis by the Food and Drug Administration in 1985, displays potent activity against a range of Gram-positive bacteria.11-16 The antimicrobial properties of auranofin are attributed primarily to its ability to disrupt cellular thiol balance by inhibiting thioredoxin reductase (TrxR). In particular, auranofin’s inactivation of TrxR in bacteria that rely solely on the thioredoxin(Trx)–TrxR pathway for thiol balance causes cell death.11, 15 Auranofin was ineffective for bacteria that possess alternative antioxidant systems that can compensate for the loss of reducing capability of TrxTrxR, e.g., glutathione/glutathione reductase in most Gram-negatives. H. pylori, however, lacks such additional antioxidant systems.17, 18 As such, it relies on TrxR/Trx to combat oxidative stress, which makes auranofin effective in killing the bacterium. Auranofin displays a minimum inhibitory concentration (MIC) value against H. pylori G27 of 1.2 μM, suggesting that it is an intriguing lead for new antibiotics to treat H. pylori infection.19 Auranofin’s safety has been well documented, with no reported serious side effects or long-term safety issues.

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However, it has gradually fallen out of favor due to the development of new arthritis drugs and its minor but unpleasant side effects, which include diarrhea, stomatitis, conjunctivitis, proteinuria, and thrombocytopenia.20 An auranofin analog that retains toxicity against H. pylori but displays diminished toxicity against mammalian cells would be a superior antibiotic, as it could circumvent adverse side effects. Optimally, such an analog would maintain auranofin’s safety profile while enhancing its selectivity for target bacterial cells.

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are possible, indicating the potential of auranofin and its analogs to be active against antibiotic resistant H. pylori and to withstand the development of resistance. We reasoned that modification of the sugar ligand could tap into endogenous cellular uptake mechanisms and therefore modulate toxicity in a cell-specific manner, while leaving the pharmacophore intact. Motivated by the rationale that the identity of the sugar ligand could impact uptake and toxicity, we developed and tested sugar-modified analogs of auranofin as potential anti-H. pylori agents. Guided by previous work that established H. pylori robustly processes exogenously introduced peracetylated-N-azidoacetylglucosamine, an azide-containing analog of the common monosaccharide N-acetylglucosamine (GlcNAc) found within H. pylori’s glycans,23 we hypothesized that auranofin analogs bearing thio-GlcNAc analogs in place of thioglucose would be active against H. pylori. Here we report that thio-GlcNAccontaining variants of auranofin inhibit H. pylori cell growth and TrxR activity yet display reduced toxicity against mammalian cells relative to auranofin.

RESULTS AND DISCUSSION

Figure 1. Auranofin’s mode of action and the sugarmodified analogs used in this study. A) Auranofin is taken up by cells, followed by spontaneous release of the thiosugar to yield Au(I) or Au(I)-PEt3 that binds the thiol on TrxR to inactivate TrxR.21 Additional enzymes containing reactive cysteines may be inactivated by an analogous mechanism. B) A series of sugar-modified auranofin analogs based on a thioGlcNAc scaffold were explored in this study.

Development of auranofin analogs with desired properties requires an examination of auranofin’s structure. Auranofin is comprised of three key moieties, each with a critical role (Figure 1A). The central Au(I) atom is the presumed active component that binds and inactivates thiolates within TrxR’s active site.20 The Au(I) atom alone is sufficient to inhibit purified TrxR, yet this atom is relatively impermeable to cells.20 Though the precise molecular mechanism of auranofin is still unclear, it is generally believed that the triethylphosphine ligand aids with uptake of the Au(I) atom by cells by increasing its membrane permeability, whereas the thioglucose reduces the in vivo toxicity of its chloride precursor.20, 22 Following administration, the thioglucose is replaced by blood thiols, and the released gold species, Au(I) or [(PEt3)Au]+, then binds to redox-active cysteine residues in TrxR (Figure 1A).21 Emerging data indicates that TrxR is likely not the only target of auranofin, as the thiophilic nature of auranofin enables it to react with additional cysteinecontaining enzymes. Indeed, recent studies indicate that auranofin interferes with cell wall, DNA, protein and toxin synthesis in Staphylococcus aureus, which could be due to direct or indirect effects.16 Thus, multiple modes of action

To test the hypothesis that the identity of the sugar ligand on auranofin could impact uptake and toxicity, we designed a series of five novel auranofin derivatives (Figure 1B). All of these analogs are based on the GlcNAc scaffold (2) and thus bear an N-acyl moiety at the C-2 position of the thiosugar ligand. The panel of auranofin derivatives contains a range of structural modifications including (1) variation at the C-2 N-acyl position of the thiosugar ligand to make GlcNAc more stable (4) or more electron-deficient (5), (2) the presence of acetylated (-OAc) versus free (-OH) hydroxyls at the C-3, C-4, and C-5 positions on the sugars (3, 6), and (3) methyl (6) versus ethyl (3) substituents on the phosphine ligand (Figure 1B). Each of these structural changes has the potential to impact cellular uptake, compound stability, and bactericidal activity. The interplay of these factors is difficult to predict, so a structure-activity relationship analysis was undertaken.

Figure 2. Activity of auranofin and sugar-modified auranofin analogs against H. pylori. Minimum inhibitory concentration (MIC) was scored as the lowest concentration required to completely ablate H. pylori G27 growth on soft agar embedded with Au conjugates. All assays were performed

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in triplicate. Error bars represent standard deviation. * indicates significant difference from auranofin (1) based on an unpaired t test (p-value < 0.05).

Auranofin analogs retain potent anti-H. pylori activity. Compounds 1-6 were synthesized following literature and previously developed procedures.24 To assess whether auranofin derivatives bearing varied thiosugar substituents maintain anti-H. pylori activity, the minimum inhibitory concentrations (MICs) of the panel of auranofin derivatives against H. pylori strain G27 were measured. Briefly, MIC was scored as the lowest concentration that prevented H. pylori growth on solid media. The MIC value for auranofin against H. pylori was 0.44 M; this value is slightly lower than the previously reported MIC value (1.2 M),19 likely due to differences in experimental conditions (scoring growth on solid versus liquid media). Analogs 2-5 inhibited

H. pylori growth in the 0.30-0.65 M range, similar to the MIC of the parent compound auranofin (1) (Figure 2). In contrast, compound 6 exhibited an MIC value of 5 M, approximately ten-fold higher than the other compounds screened (Figure 2A). These results indicate that structural analogs of auranofin with altered thiosugar substituents retain potent H. pylori activity. A comparison of the activities of structurally similar compounds 3 and 6 — both of which have a thio-GlcNAc substituent but have triethyland trimethyl-phosphine ligands, respectively — suggests that the relative loss of activity in compound 6 appears to be due to modifying the phosphine rather than the sugar substituent. Indeed, analog 6, having a smaller trimethylphosphine and a deprotected GlcNAc ligand, is considerably less lipophilic than the other compounds, with a logP of -2.03 vs. 0.56, -0.26, -0.89, -0.36, 1.01 for 1-5, respectively.24 These logP values have the potential to influence cell uptake and ultimately delivery of the Au(I) pharmacophore within cells. Fortunately, structural changes to the thiosugar substituent in these compounds are well tolerated. Figure 3. In vitro inhibition of TrxR by auranofin and sugar-modified auranofin analogs. Activity of rat liver TrxR was monitored using a DTNB-based assay in the absence versus presence of compounds 1-6. Half maximal inhibitory concentration (IC50) was determined for each compound based on a comparison to the reaction in the absence of any inhibitor. All assays were performed in triplicate. Error bars

represent standard deviation. *indicates significant difference from auranofin (1) based on an unpaired t test (p-value < 0.05).

Auranofin analogs inhibit TrxR in the nanomolar range. Having confirmed that auranofin analogs retain potent activity against H. pylori, we next sought to assess the impacts of structural modification on the ability of these compounds to inhibit thioredoxin reductase (TrxR) in vitro. For these experiments we turned to commercially available rat TrxR, as inhibition of H. pylori TrxR and rat TrxR appear to correlate closely.19, 26 TrxR activity in the absence versus presence of auranofin analogs was measured using a 5,5’dithio-bis-[2-nitrobenzoic acid] (DTNB)-based colorimetric readout. Briefly, DTNB undergoes an NADPH-coupled reduction catalyzed by TrxR to produce the yellow compound 2-nitro-5-thiobenzoate (TNB); inhibition of TrxR was detected by suppression of TNB production. All five thio-GlcNAc analogs of auranofin, as well as auranofin, displayed a half maximal inhibitory concentration (IC50) of TrxR in the low nanomolar range (Figure 3). All analogs were at least as potent as auranofin and some displayed as much as ten-fold lower IC50 values. Surprisingly, the trimethylphosphine derivative 6, which displayed the least potent activity against H. pylori, inhibited TrxR most effectively. Given that these assays were conducted in vitro and that the Au(I) acts as a single covalent inhibitor of TrxR, the differences in IC50 values across compounds likely stems from ligand variation impacting release of the Au(I) atom. Irrespective of mechanistic details, we were encouraged to observe that all compounds inhibited TrxR at nanomolar concentrations and that their efficacy was maintained. Auranofin analogs deplete free thiols in H. pylori. Auranofin’s inhibition of TrxR alters the redox balance within cells and causes a depletion in detectable free thiols.15 In order to query whether the sugar-modified auranofin analogs used in this study cause similar effects in treated cells, we measured the concentration of free thiols in H. pylori samples treated with varying concentrations of each analog. Included in this assay were auranofin as a positive control and amoxicillin as a negative control, as this antibiotic has no effect on thiolredox balance.15 Samples treated with 5.0 μM compounds 1-6 displayed depleted thiol concentrations in comparison to untreated samples (Figure 4). Furthermore, a concentration-dependent trend was observed, with increased concentrations causing greater depletion in free thiols. By contrast, H. pylori samples treated with amoxicillin had no differences in free thiol concentrations in comparison to the untreated samples, even at a concentration of 5.0 μM amoxicillin (Figure 4). The observed decrease in free thiols measured in the presence of the auranofin analogs is consistent with these compounds impacting cellular free thiols rather than simply killing a portion of cells in the sample.



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peracetylated GlcNAc-analog 3 and dithiol benzene-analog 4 also had enhanced anti-H. pylori activity. Fortuitously, three of these compounds triggered considerably less cytotoxicity in mammalian cells than auranofin. These data suggest that modulating the sugar moiety can preferentially maintain or even enhance anti-H. pylori activity while diminishing unwanted mammalian cell toxicity.

Figure 4. Free thiol depletion after treatment with auranofin and auranofin analogs. The concentration of free thiols was measured in samples of H. pylori incubated with either 0.30 μM or 5.0 μM of compounds 1-6 or amoxicillin. Free thiol concentration for each sample are displayed as percent of free thiols detected in the sampled in comparison to free thiols detected in untreated H. pylori samples. Error bars represent standard deviation for two replicate measurements.

Auranofin analogs are less cytotoxic than auranofin. After establishing that sugar-modified analogs of auranofin retained potent activity against H. pylori and inhibited the target enzyme TrxR, we turned to reported mammalian cell cytotoxicity data to capture the effects of modifying the sugar scaffold on ameliorating auranofin’s toxicity.24 As previously described, the cytotoxicity of auranofin and analogs 1-6 was tested on A549 human lung carcinoma epithelial cells using an alamar blue (resazurin) assay.24 The IC50 value for auranofin was 11.3 μM after 20 h incubation, which is higher than a previously reported value (6.65 μM) obtained after 72 hours exposure.25 The difference could be that more resistant cells in the population succumb after a longer time exposure. Analogs 4 and 5 exhibited IC50 values of 18.7 μM and 10.1 μM, which are similar to auranofin (Supplemental Figure 1).24 By contrast, analogs 2, 3 and 6 were significantly less toxic than auranofin, exhibiting IC50 values around 4, 5 and 6 times higher than auranofin (Supplemental Figure 1).24 These results support the hypothesis that mammalian cell toxicity can be mitigated by modulating the sugar moiety on auranofin. Discussion This study sought to assess the inhibitory properties of a panel of sugar-modified auranofin analogs against H. pylori. A series of auranofin derivatives bearing variable thiosugar ligands was prepared and screened for MIC against H. pylori, IC50 against TrxR, and cytotoxicity against mammalian cells. As hypothesized, peracetylated GlcNAcanalog 2 bearing all other structural components as auranofin yielded increased inhibition of H. pylori and TrxR relative to auranofin. Two other compounds,

One striking observation was the dramatic differences in concentrations required to inhibit the enzymatic target TrxR in vitro versus the concentrations required to kill cells. The MIC and IC50 values differed by nearly three orders of magnitude (M versus nM, respectively), suggesting that the compounds were taken up by cells at relatively low levels but potently inhibited the target enzyme within cells. This observation, coupled to the enhanced survival of mammalian cells, supports the central hypothesis that glyco-tailoring of the thiosugar ligand serves as a means to bias uptake in target bacterial cells yet diminishes uptake in mammalian cells. Gaining further insight into auranofin analog and monosaccharide uptake will provide a platform to understand why and how thiosugar modifications modulate inhibitory properties. Future work will focus on the mechanism of cellular uptake of auranofin derivatives and other unnatural monosaccharides to gain insight into the role of the C-2 substituent and observed trends in toxicity. An exploration of sugar-free gold sulfide complexes undertaken in parallel will reveal the role of the sugar on uptake, physical properties (e.g. solubility), and interactions with the enzyme TrxR. Finally, the use of cell permeabilizing agents such as colistin will yield insight the role of cell uptake as a barrier to inhibitor activity. This information has the potential to guide rational design of antibiotics for the eradication of H. pylori infection with minimized physiological impacts.

METHODS Materials. All reagents and solvents were used as received from Sigma-Aldrich or Fisher Scientific. Compounds 1-6 were synthesized following the literature or our previously reported procedures.24 The purity of all compounds was determined by quantitative NMR to be ≥95%, except 94% for compound 2.24 H. pylori growth media and growth conditions. H. pylori strain G27 was used for all experiments and was received from Manuel Amieva (Stanford University). Horse blood agar (HBA) plates were prepared with Colombia agar base and 5% (w/v) horse blood, 1% (w/v) vancomycin, 0.5% (w/v) cefulosidan, 0.033% (w/v) polymyxin, 0.5% (w/v) trimethoprim, and 0.8% (w/v) amphotericin B. H. pylori was stored in freezer media (10% fetal bovine serum, 20% glycerol, 70% Brain Heart Infusion broth) at 80°C. H. pylori aliquots were spread onto warmed HBA plates via sterile Q-tip and allowed to grow for three days under incubation of 14% CO2 at 37°C. Liquid H. pylori cultures were prepared by suspending H. pylori grown on HBA plates in a liquid growth media composed of Brucella broth, 10% fetal bovine serum and 6 μg/mL vancomycin. Determination of MIC for auranofin and its derivatives. All auranofin analogs were prepared as 10 mg/mL solutions in


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DMSO and then diluted to 100 μg/mL in H2O for use in assays. The 100 μg/mL inhibitor solution was added to 20 mL of autoclaved HBA media solution prior to solidification to reach the desired concentrations ranging from 1250 ng/mL to 10 ng/mL. Inhibitor-supplemented media was vortexed to ensure an even distribution of the compound within the media, then added to petri dishes and allowed to solidify at room temperature overnight. HBA plates were stored at 4 °C and pre-warmed prior to inoculation with wildtype G27 H. pylori as described above. After three days of growth in 14% CO2 at 37°C, plates were visually analyzed for bacterial growth to determine the MIC. MIC was scored as the lowest concentration of compound at which no visible H. pylori growth was observed. TrxR inhibition assay. Inhibition of purified rat liver TrxR was measured in vitro for sugar-modified auranofin analogs 1-6. Inhibition of TrxR was determined through assays with 5,5’dithio-bis-[2-nitrobenzoic acid] (DTNB), which acts as a direct substrate in place of Trx by reacting with TrxR and NADPH to produce TNB, which has a signature absorbance at 412 nm. Inhibition assays were performed using an adapted protocol from the Thioredoxin Reductase Assay Kit (Sigma Aldrich CS0170), which includes rat liver TrxR. Manufacturer’s instructions were followed for all components except the TrxR, which was doubled in concentration. Auranofin analogs were diluted to 1 μg/mL in H2O and then serially diluted to concentrations ranging from 1000 nM to 62.5 nM before being added to the assay solutions to reach final concentrations ranging from 0.1825-50 nM. A412 was measured using a SPECTROstarNano plate reader. All assays were run in triplicate. Raw activity assay data are included in Supplemental Figure 2. Half maximal inhibitory concentration (IC50) was calculated based on TrxR inhibition assay results. The ΔA412 after 10 minutes was utilized for these calculations. Maximal inhibition (100%) was considered to be equivalent to the ΔA412 for highest concentration of inhibitor whereas the 0% inhibition was defined as ΔA412 for the sample with no inhibitor present. Compounds were considered to fully inhibit TrxR activity at concentrations in which the ΔA412 nm after 10 minutes was less than 0.05, mirroring the negative control. Total percent inhibition for each concentration measured was determined. Assuming a linear relationship around 50% inhibition, the ΔA412 for 50% inhibition was determined and the corresponding concentration of inhibitor was calculated as the IC50. Graphical analyses performed for IC50 calculations are included in Supplemental Figure 3. Free thiol depletion assay. H. pylori were grown overnight in liquid culture to an OD600 of 1.0, then incubated with either 0.30 μM or 5.0 μM compounds 1-6, amoxicillin, or no inhibitor for four hours. Bacteria were washed twice with PBS, resuspended in PBS, and lysed via sonication using a Branson Sonifier 450. Thiols were detected and quantified using the Thiol Detection Assay Kit (Cayman Chemicals) and GloMax Discover Plate Reader (Promega) using an excitation wavelength of 365 nm and an emission wavelength range of 500-550 nm. ASSOCIATED CONTENT

Supporting Information The Supporting Information is available free of charge on the ACS Publications website. Mammalian cell toxicity data, raw data from TrxR inhibition assay, graphical analyses performed for IC50 calculations (PDF)

AUTHOR INFORMATION Corresponding Authors * (MY) E-mail: [email protected] * (DHD) E-mail: [email protected]

Author Contributions The manuscript was written through contributions of all authors, and all authors have given approval to the final version of the manuscript. ‡These authors contributed equally.

Funding Sources National Institutes of Health grant numbers P20GM103423, R15GM109397 (to DD), R21AI140418 and R15GM128164 (to MY).

Notes

The authors declare no competing financial interest.

ACKNOWLEDGMENT We gratefully acknowledge insightful conversations with A. McBride and B. Kohorn, the technical support provided by C. Morin and R. Bernier, and members of our research laboratories for support and guidance. Research reported in this publication was supported by an Institutional Development Award (IDeA) from the National Institute of General Medical Sciences of the National Institutes of Health under grant number P20GM103423, by awards to T.E. from Kufe Family Research Fellowship and the Peter J. Grua and Mary G. O’Connell Research Award, as well as partial supports from NIH (R21AI140418 and R15GM128164 to MY and R15GM109397 to DD).

ABBREVIATIONS TrxR, thioredoxin reductase; Trx, thioredoxin; GlcNAc, Nacetylglucosamine; MIC, minimum inhibitory concentration; IC50, half maximal inhibitory concentration; DTNB, 5,5’dithio-bis-[2-nitrobenzoic acid]; TNB, 2-nitro-5-thiobenzoate; HBA, horse blood agar; DMSO, dimethyl sulfoxide; DMEM, Dulbecco's modified eagle medium;

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Auranofin’s mode of action and the sugar-modified analogs used in this study. A) Auranofin is taken up by cells, followed by spontaneous release of the thiosugar to yield Au(I) or Au(I)-PEt3 that binds the thiol on

TrxR to inactivate TrxR.21 Additional enzymes containing reactive cysteines may be in-activated by an analogous mechanism. B) A series of sugar-modified auranofin analogs based on a thio-GlcNAc scaffold were explored in this study. 228x177mm (300 x 300 DPI)


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Activity of auranofin and sugar-modified auranofin analogs against H. pylori. Minimum inhibitory concentration (MIC) was scored as the lowest concentration re-quired to completely ablate H. pylori G27 growth on soft agar embedded with Au conjugates. All assays were per-formed in triplicate. Error bars represent standard deviation. * indicates significant difference from auranofin (1) based on an unpaired t test (p-value < 0.05). 229x137mm (72 x 72 DPI)



In vitro inhibition of TrxR by auranofin and sugar-modified auranofin analogs. Activity of rat liver TrxR was monitored using a DTNB-based assay in the absence versus presence of compounds 1-6. Half maximal inhibitory concentration (IC50) was determined for each compound based on a comparison to the reaction in the absence of any inhibitor. All assays were performed in triplicate. Error bars represent stand-ard deviation. *indicates significant difference from auranofin (1) based on an unpaired t test (p-value < 0.05). 216x148mm (72 x 72 DPI)


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Free thiol depletion after treatment with auranofin and auranofin analogs. The concentration of free thiols was measured in samples of H. pylori incubated with either 0.30 μM or 5.0 μM of compounds 1-6 or amoxicillin. Free thiol concentration for each sample are displayed as percent of free thiols detected in the sampled in comparison to free thiols detected in untreated H. pylori samples. Error bars represent standard deviation for two replicate measurements. 253x164mm (72 x 72 DPI)




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Sugar-modified analogs of auranofin are potent inhibitors of

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