doi: 10.1126/scitranslmed.aac9103. Epub 2015 Sep 23.
A small-molecule antivirulence agent for treating Clostridium difficile infection
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- PMID:26400909
- PMCID: PMC6025901
- DOI: 10.1126/scitranslmed.aac9103
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A small-molecule antivirulence agent for treating Clostridium difficile infection
Kristina Oresic Bender et al. Sci Transl Med..
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Abstract
Clostridium difficile infection (CDI) is a worldwide health threat that is typically triggered by the use of broad-spectrum antibiotics, which disrupt the natural gut microbiota and allow this Gram-positive anaerobic pathogen to thrive. The increased incidence and severity of disease coupled with decreased response, high recurrence rates, and emergence of multiple antibiotic-resistant strains have created an urgent need for new therapies. We describe pharmacological targeting of the cysteine protease domain (CPD) within the C. difficile major virulence factor toxin B (TcdB). Through a targeted screen with an activity-based probe for this protease domain, we identified a number of potent CPD inhibitors, including one bioactive compound, ebselen, which is currently in human clinical trials for a clinically unrelated indication. This drug showed activity against both major virulence factors, TcdA and TcdB, in biochemical and cell-based studies. Treatment in a mouse model of CDI that closely resembles the human infection confirmed a therapeutic benefit in the form of reduced disease pathology in host tissues that correlated with inhibition of the release of the toxic glucosyltransferase domain (GTD). Our results show that this non-antibiotic drug can modulate the pathology of disease and therefore could potentially be developed as a therapeutic for the treatment of CDI.
Copyright © 2015, American Association for the Advancement of Science.
Conflict of interest statement
Figures
(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.
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 (non-denaturing 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.
(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.
(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 associatedP value of <0.001.
Ebselen treatment blocks the pathology ofC. difficile infection in mice. Mice were orally challenged withC. difficile (strain 630) and treated daily with vehicle or ebselen (100 mg/kg). (A) Plot ofC. 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 indicate dmilligram 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 pairedt 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-WhitneyU 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).
Dose-dependent effects of ebselen on histopathology ofC. difficile infection in mice. Antibiotic-pretreated mice were left untreated (n = 5) or orally challenged withC. difficile (strain 630) and treated daily with vehicle (n = 5) or ebselen at three doses [100 mg/kg (n = 5), 10mg/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’st test. ****P < 0.0001, **P < 0.01, *P < 0.05.
Comment in
- Small Molecules Take A Big Step Against Clostridium difficile.Beilhartz GL, Tam J, Melnyk RA.Beilhartz GL, et al.Trends Microbiol. 2015 Dec;23(12):746-748. doi: 10.1016/j.tim.2015.10.009. Epub 2015 Nov 5.Trends Microbiol. 2015.PMID:26547239
- Comment on "A small-molecule antivirulence agent for treating Clostridium difficile infection".Beilhartz GL, Tam J, Zhang Z, Melnyk RA.Beilhartz GL, et al.Sci Transl Med. 2016 Dec 21;8(370):370tc2. doi: 10.1126/scitranslmed.aad8926.Sci Transl Med. 2016.PMID:28003550
References
- Surawicz CM, Brandt LJ, Binion DG, Ananthakrishnan AN, Curry SR, Gilligan PH, McFarland LV, Mellow M, Zuckerbraun BS. Guidelines for diagnosis, treatment, and prevention of Clostridium difficile infections. Am. J. Gastroenterol. 2013;108:478–498. - PubMed
- Center for Disease Control and Prevention. Antibiotic Resistance Threats in the United States 2013. CDC; Atlanta, GA: 2013.
- Douglas Scott R., II . The Direct Medical Costs of Healthcare-Associated Infections in U.S. Hospitals and the Benefits of Prevention, Division of Healthcare Quality Promotion National Center for Preparedness, Detection, and Control of Infectious Diseases. Coordinating Center for Infectious Diseases. CDC; Atlanta, GA: 2009.
- Tran M-C, Claros MC, Goldstein EJ. Therapy of Clostridium difficile infection: Perspectives on a changing paradigm. Expert Opin. Pharmacother. 2013;14:2375–2386. - PubMed
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