Research ArticleInfectious Disease

A small-molecule antivirulence agent for treating Clostridium difficile infection

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Science Translational Medicine  23 Sep 2015:
Vol. 7, Issue 306, pp. 306ra148
DOI: 10.1126/scitranslmed.aac9103
  • Fig. 1. Screening for small-molecule activators and inhibitors of the CPD of TcdB.

    (A) Superposition of the crystal structures of TcdB (red) and TcdA (yellow) CPD domains with an IP6 allosteric activator bound. (B) Structure of the activity-based probe TAMRA–AWP-19 used for the high-throughput screen (HTS). The fluorophore is shaded in red, and the acyloxymethyl ketone electrophile specific for cysteine proteases is shown in blue. (C) Schematic of the fluorescence polarization (fluopol)–activity-based probe screen workflow. Compounds were added to the purified CPD domain from TcdB followed by addition of TAMRA–AWP-19 probe. The unbound probe produced low fluorescence polarization [millipolarization (mP)], which increased upon binding to the activated CPD. This change in millipolarization was measured on a plate reader and was used to assess the ability of a compound to either directly stimulate or inhibit (upon IP6 addition) CPD activation.

  • Fig. 2. Identification of ebselen as a potent inhibitor of CPD activation.

    (A) Chemical structure of the lead hit ebselen. (B) Assessment of CPD inhibition by labeling with TAMRA–AWP-19. The CPD was incubated with increasing amounts of ebselen, activated with IP6, and labeled with TAMRA–AWP-19. Samples were resolved by SDS–polyacrylamide gel electrophoresis (SDS-PAGE) and scanned for probe fluorescence (top gel) or stained by Coomassie (bottom gel). (C) Dose-response curve of ebselen inhibition of the CPD as measured by quantification of probe labeling shown in (B). Values are plotted for percent inhibition relative to dimethyl sulfoxide (DMSO) control (lane 0). (D) Deconvoluted mass spectra of intact CPD (nondenaturing conditions) incubated with IP6 (70 μM) with or without ebselen (100 nM) as indicated. The identified masses for the CPD plus ebselen addition are indicated.

  • Fig. 3. Inhibition of full-length TcdA and TcdB by ebselen.

    (A) TcdA was incubated with increasing amounts of ebselen, activated with IP6, and labeled with TAMRA–AWP-19. Samples were resolved by SDS-PAGE and scanned for probe fluorescence (top gel) or stained by Coomassie (bottom gel). Full-length TcdA and TcdA that has proteolytically released the GTD (TcdAΔ) are indicated by arrows. (B) TcdB was treated as in (A). Samples were resolved by SDS-PAGE and scanned for probe fluorescence (top gel) or stained by Coomassie (bottom gel). Full-length TcdB, TcdB that has proteolytically released the GTD (TcdBΔ), and the free GTD are indicated by arrows. The image shows a typical representation of three replicates. (C) Dose-response curve of ebselen inhibition of TcdB as measured by quantification of probe labeling as shown in (B). Values are plotted for percent inhibition relative to DMSO control (lane 0). (D) Uncoupling ebselen inhibition and IP6 activation. Full-length TcdB was incubated with ebselen before (third lane) or after (fourth lane) size exclusion chromatography. Toxin was then activated with IP6, labeled with TAMRA–AWP-19, and analyzed by SDS-PAGE and in-gel fluorescence analysis. Control lanes without ebselen treatment are marked in the red dashed box. All inhibition experiments were performed at least three times and, where applicable, represented as means ± SEM of triplicate analysis. (E) Proposed point of CPD inhibition mediated by ebselen (red dashed rectangle). Ebselen modifies the active-site cysteine upon IP6-mediated allosteric activation in the cytosol.

  • Fig. 4. Ebselen protects cells against TcdB-induced toxicity in vitro and in vivo.

    (A) HFF cells were challenged with DMSO, DMSO-treated TcdB, or TcdB treated with the indicated concentrations of ebselen. Images are representative fields for each sample. Scale bar, 10 μm. (B) EC50 values for cell rounding calculated by counting number of rounded cells and plotting as a percentage relative to the DMSO control. Results are means of three biological replicates ± SEM. (C) Western blot analysis of glucosyltransferase activity of TcdB in cell rounding assay. HFF cells were challenged as in (A), and cells were analyzed for total cellular Rac1 (mAb23A8, middle panel) and nonglucosylated Rac1 (mAb102, upper panel). β-Tubulin was used as a loading control (lower panel). Images are representative of three independent experiments. (D) Kinetic cell rounding assay of wild-type (WT) and noncleavable mutant toxin (L543A). HFF cells were challenged with 150 ng of full-length toxin (WT or L543A) that was preincubated with 100 nM ebselen or DMSO for 1 hour before coadministration to cells. Cell rounding was calculated as the percentage of rounded cells relative to DMSO-treated control cells at each time point. Results are means of three technical replicates ± SEM and representative of three independent experiments. (E) Mice (n = 5) were injected [intraperitoneally (i.p.)] with TcdB (1 μg/kg) or TcdB pretreated and coadministered with 100 nM ebselen. The plot shows clinical scores over time. Scores were assessed as follows: 0, healthy animal; 1, ruffled fur and BAR (bright, alert, and reactive); 2, ruffled fur, hunched, and QAR (quiet, alert, and reactive); 3, ruffled fur, hunched, inactive, and dehydrated; 4, moribund. Scores are plotted as the mean values for the five mice in each group ± SEM. (F) Survival plots showing the effect of ebselen treatment. Values were compiled using a Kaplan-Meier method using animals scored at 72 hours as the end point before humane termination. The χ2 statistic was 10.90 with associated P value of <0.001.

  • Fig. 5. Ebselen treatment blocks the pathology of C. difficile infection in mice.

    Mice were orally challenged with C. difficile (strain 630) and treated daily with vehicle or ebselen (100 mg/kg). (A) Plot of C. difficile CFU counts from fecal samples collected from ebselen- and vehicle-treated mice. (B) Plots showing the activity of TcdB in feces from vehicle- and ebselen-treated mice at day 5 after infection. The indicated milligram of feces equivalents collected from infected mice treated with ebselen (red, n = 8) or vehicle (blue, n = 7) were resuspended in 4× (w/v) phosphate-buffered saline (PBS) and applied to HFF cells. Cell rounding was calculated as the percentage of rounded cells relative to DMSO-treated control cells. Results are means ± SEM. Samples were statistically analyzed using paired t test. **P < 0.01. (C) Images [hematoxylin and eosin (H&E) stain] of colon tissue sections from infected mice treated with vehicle or ebselen. Black asterisks indicate neutrophilic infiltration in the lamina propria of the mucosa. Expansion/thickening of the submucosa with edema and neutrophilic infiltrates are highlighted with white bars. Magnification, ×200. Black scale bars, 100 μm. (D) Histopathological scores for vehicle-treated (circles) and ebselen-treated (squares) animals for inflammatory cell infiltration, submucosal edema, and mucosal hypertrophy. *P < 0.05 by Mann-Whitney U test. (E) Plot of the overall average histopathological scores for ebselen- and vehicle-treated animals. Statistical analysis was performed using the Mann-Whitney test. **P < 0.01. (F) Western blot analysis of the released GTD in the colon tissues from mice treated with ebselen or vehicle (upper panel). β-Tubulin was used as a loading control (lower panel).

  • Fig. 6. Dose-dependent effects of ebselen on histopathology of C. difficile infection in mice.

    Antibiotic-pretreated mice were left untreated (n = 5) or orally challenged with C. difficile (strain 630) and treated daily with vehicle (n = 5) or ebselen at three doses [100 mg/kg (n = 5), 10 mg/kg (n = 5), and 1 mg/kg (n = 5)] for 5 days. (A) Representative images (H&E stain) of colon tissue sections from uninfected mice, infected mice treated with vehicle, or infected mice treated with ebselen at indicated doses. Black scale bars, 100 μm. (B) Western blot analysis of the released GTD in colon tissues from the uninfected mice or infected mice treated with vehicle or indicated doses of ebselen. β-Tubulin was used as a loading control. (C) Overall histopathological scores for uninfected or infected animals treated with vehicle or ebselen at the indicated doses. Statistical analysis was performed using the Mann-Whitney test. **P < 0.01, *P < 0.05; NS, nonsignificant. (D) Scanning densitometry of the Western blot images (n = 5 for each group). Results represent mean for each group of animals (uninfected; infected treated with ebselen at 100, 10, and 1 mg/kg; or infected treated with vehicle) ± SEM normalized to the signal for uninfected animals. All signals were normalized to β-tubulin. Statistical analysis was performed using Student’s t test. ****P < 0.0001, **P < 0.01, *P < 0.05.

Supplementary Materials

  • www.sciencetranslationalmedicine.org/cgi/content/full/7/306/306ra148/DC1

    Materials and Methods

    Fig. S1. TcdB-induced cell rounding in HFF cells.

    Fig. S2. Efficacy of TcdB inhibitors in biochemical and cell-based assays.

    Fig. S3. Ebselen protects cells against TcdB-induced toxicity in vitro.

    Fig. S4. Ebselen inhibits toxin-induced cytopathic effect in a fecal sample from a C. difficile patient.

    Fig. S5. Effect of ebselen on the GTD domain of TcdB.

    Fig. S6. Ebselen protects against autoprocessing of full-length wild-type and L543A toxin over time.

    Fig. S7. Dose-response plots showing the activity of TcdB in feces of vehicle- and ebselen-treated mice from days 1 to 4 after C. difficile infection.

    Table S1. List of TcdB CPD inhibitors with corresponding IC50 values obtained from the fluorescence polarization high-throughput screen.

    Table S2. Primary data supporting Fig. 4E.

    Table S3. Primary data supporting Fig. 4F.

    Table S4. Primary data supporting Fig. 5 (D and E).

    Table S5. Primary data supporting Fig. 6C.

    References (6064)

  • Supplementary Material for:

    A small-molecule antivirulence agent for treating Clostridium difficile infection

    Kristina Oresic Bender, Megan Garland, Jessica A. Ferreyra, Andrew J. Hryckowian, Matthew A. Child, Aaron W. Puri, David E. Solow-Cordero, Steven K. Higginbottom, Ehud Segal, Niaz Banaei, Aimee Shen, Justin L. Sonnenburg, Matthew Bogyo*

    *Corresponding author. E-mail: mbogyo{at}stanford.edu

    Published 23 September 2015, Sci. Transl. Med. 7, 306ra148 (2015)
    DOI: 10.1126/scitranslmed.aac9103

    This PDF file includes:

    • Materials and Methods
    • Fig. S1. TcdB-induced cell rounding in HFF cells.
    • Fig. S2. Efficacy of TcdB inhibitors in biochemical and cell-based assays.
    • Fig. S3. Ebselen protects cells against TcdB-induced toxicity in vitro.
    • Fig. S4. Ebselen inhibits toxin-induced cytopathic effect in a fecal sample from a C. difficile patient.
    • Fig. S5. Effect of ebselen on the GTD domain of TcdB.
    • Fig. S6. Ebselen protects against autoprocessing of full-length wild-type and L543A toxin over time.
    • Fig. S7. Dose-response plots showing the activity of TcdB in feces of vehicle- and ebselen-treated mice from days 1 to 4 after C. difficile infection.
    • Table S1. List of TcdB CPD inhibitors with corresponding IC50 values obtained from the fluorescence polarization high-throughput screen.
    • Table S2. Primary data supporting Fig. 4E.
    • Table S3. Primary data supporting Fig. 4F.
    • Table S4. Primary data supporting Fig. 5 (D and E).
    • Table S5. Primary data supporting Fig. 6C.
    • References (6064)

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