Identification and preclinical evaluation of MMV676558 as a promising therapeutic candidate against Clostridioides difficile

Clostridioides difficile, a gram-positive, toxin-producing, spore-forming anaerobe, is a major cause of antibioticassociated diarrhoea. The bacterium's intrinsic drug resistance limits current treatment options to fidaxomicin and vancomycin for initial episodes, with anti-toxin B monoclonal antibody or faecal microbiota transplantation recommended for complicated or recurrent cases. This underscores the urgent need for novel therapeutics. In this study, we screened the MMV Pathogen Box at a 10 mu M concentration against C. difficile R20291. Primary hits were evaluated for minimum inhibitory concentrations (MIC), killing kinetics, and biofilm inhibition. Bacterial cytological profiling (BCP) and transmission electron microscopy (TEM) were employed to study the mode of action. MMV676558 was further tested in a mouse model to assess survival, histopathology, and gut microbiota effects. We identified nineteen hits that inhibited over 50 % of C. difficile growth. MIC assays revealed three hits with MICs below 16 mu g/mL: MMV676558, MMV688755, and MMV690027. These hits were effective against various C. difficile ribotypes. Killing kinetics were comparable or superior to vancomycin and fidaxomicin, and biofilm assays showed inhibitory effects. BCP and TEM analyses suggested membrane function disruption as the mode of action. Furthermore, MMV676558 demonstrated a protective effect in mice, with favourable histopathology and gut microbiota profiles. Given the urgent threat posed by C. difficile antibiotic resistance, discovering new treatments is a top priority. Our study identified three promising hits from the MMV Pathogen Box, with MMV676558 showing significant in vivo potential for further evaluation.