CDBN-YGXZ, a Novel Small-Molecule Drug, Shows Efficacy against Clostridioides difficile Infection and Recurrence in Mouse and Hamster Infection Models

Abstract

Clostridioides difficile infection (CDI) causes severe diarrhea and colitis, leading to significant morbidity, mortality, and high medical costs worldwide. Oral vancomycin, a first-line treatment for CDI, is associated with a high risk of recurrence, necessitating novel therapies for primary and recurrent CDI. A novel small-molecule compound, CDBN-YGXZ, was synthesized by modifying the benzene ring of nitazoxanide with lauric acid. The mechanism of action of CDBN-YGXZ was validated using a pyruvate:ferredoxin/flavodoxin oxidoreductase (PFOR) inhibition assay. The efficacy of CDBN-YGXZ was evaluated using the MIC test and CDI infection model in mice and hamsters. Furthermore, metagenomics was used to reveal the underlying reasons for the effective reduction or prevention of CDI after CDBN-YGXZ treatment. The inhibitory activity against PFOR induced by CDBN-YGXZ. MIC tests showed that the in vitro activity of CDBN-YGXZ against C. difficile ranging from 0.1 to 1.5 μg/mL. In the mouse and hamster CDI models, CDBN-YGXZ provided protection during both treatment and relapse, while vancomycin treatment resulted in severe relapse and significant clinical scores. Compared with global effects on the indigenous gut microbiota induced by vancomycin, CDBN-YGXZ treatment had a mild influence on gut microbes, thus resulting in the disappearance or reduction of CDI recurrence. CDBN-YGXZ displayed potent activity against C. difficile in vitro and in vivo, reducing or preventing relapse in infected animals, which could merit further development as a potential drug candidate for treating CDI.

Keywords: CDBN-YGXZ; Clostridioides difficile infection; dysbiosis; hamster; microbiome; mouse; pyruvate:ferredoxin/flavodoxin oxidoreductases; relapse/recurrence.

FIG 1

Chemical structural formula of CDBN-YGXZ.

FIG 2

CDBN-YGXZ treatment protects mice from C. difficile infection. (A) Experimental design schematics: mice were administered an antibiotic cocktail for 5 days (day −7 to day −3). Two days later (day −1), they were administered clindamycin via intraperitoneal injection (10 mg/kg) and then challenged with C. difficile 1870 spores (10⁶ CFU/mouse) 1 day later (day 0), using uninfected animals as negative controls (NC). Treatment began 24 h after (day 1) infection and continued for 5 days. Two days later (day 8), recurrence was observed and samples were taken for analysis. (B) The percentage weight change, (C) diarrhea score, and (D) disease activity index of each group are shown. (E) Colon tissue samples were collected from each group for length measurement. (F) The histopathological changes in colon tissue in the different group (H&E staining, ×400, scale bar = 50 μm). NC (F-i), model (F-ii), vancomycin (F-iii), and CDBN-YGXZ groups (F-iv). (G) The data of colon length in each group. (H) Total histology score in each group. All data shown are mean ± standard error of the mean (n = 5 mice per group), and asterisks (*) show significant differences between group NC and group model. *, P < 0.05; ***, P < 0.001; octothorpe (^#) shows significant differences between the vancomycin and CDBN-YGXZ groups. ^#, P < 0.05; ^##, P < 0.01.

FIG 3

The proinflammatory cytokine expression in colon tissue. The mRNA expression levels of cytokines (TNF-α, IL-17, IL-1β, IL-6) were examined in colon tissue obtained from each group. The data shown are mean ± standard error of the mean (n = 5 mice per group). Octothorpes (^#) show significant differences between vancomycin and CDBN-YGXZ groups. ^#, P < 0.05; ^##, P < 0.01. NC, negative control.

FIG 4

Protective effect of CDBN-YGXZ on hamsters infected with C. difficile 630 spores. (A) Experimental design schematics: hamsters were treated orogastrically with 10 mg/kg clindamycin 1 day before challenge (day 2) with C. difficile 630 spores (5 × 10⁴ CFU/hamster). Hamsters were treated twice daily for a total of 20 days with saline (n = 5), vancomycin (10 mg/kg, n = 10), and CDBN-YGXZ (100 mg/kg, n = 10), respectively, after 24 h infection (day 3), using uninfected animals as negative controls (NC, n = 5). (B) Average weight. (C) Survival curve and (D) diarrhea score, and (E) disease activity index of each group are shown. (F) Total histology score in each group. (G) The histopathological changes in colon tissue in the different group (H&E staining, ×400, scale bar = 50 μm). NC (G-i), model (G-ii), vancomycin (G-iii), CDBN-YGXZ groups (G-iv). All data shown are mean ± standard error of the mean, and asterisks (*) show significant differences between group NC and group model. ****, P < 0.0001. Octothorpes (^#) show significant differences between vancomycin and CDBN-YGXZ groups. ^#, P < 0.05; ^##, P < 0.01; ^###, P < 0.001.

FIG 5

Effects of vancomycin and CDBN-YGXZ on the alterations of the gut bacterial taxonomic abundance at the endpoint of treatment (day 23). (A, C, and E) Linear discriminant analysis (LDA) effect size cladogram. (B, D, and F) Discriminative biomarkers with an LDA score > 4, (NC, n = 5; vancomycin, n = 8; CDBN-YGXZ, n = 9). NC, negative control.