Abstract
Clostridioides difficile infection (CDI) is a major public health issue, driven by antibiotic resistance and frequent recurrence. CD2068, an ABC protein in C. difficile, is associated with drug resistance, making it a potential target for novel therapies. This study explored FDA-approved non-antibiotic drugs for their ability to inhibit CD2068 through drug screening and experimental validation. Thioridazine exhibited moderate binding affinity to CD2068 and inhibited its ATP hydrolysis activity. It also suppressed the growth of multiple C. difficile ribotypes at 64-128 µg/mL, with rapid-killing effects. When combined with sub-MIC levels of standard antibiotics, thioridazine significantly reduced bacterial growth. In a mouse CDI model, thioridazine demonstrated potential in restoring gut microbial balance and improving survival, although it did not show superiority to vancomycin. These findings suggest that thioridazine has potential as a novel therapeutic for CDI, either as an adjunct to existing antibiotics or as part of a combination therapy to combat antibiotic resistance. Further research, including replication studies and dose optimization, is needed to fully evaluate thioridazine's therapeutic potential.