Novel therapeutic approaches targeting Clostridium difficile

Abstract

Clostridium difficile is a Gram-positive, obligately anaerobic, spore-forming bacillus that is the major cause of nosocomial antibiotic-associated diarrhea and pseudomembranous colitis (PMC).Current antibiotics for treating C. difficile infections (CDI), i.e. metronidazole, vancomycin, andfidaxomicin, are mostly effective. However, failure to cure CDI and relapse of the disease remain as significant clinical problems. The shortcomings of these agents are attributed to their low selectivity for C. difficile over normal gut microflora and their ineffectiveness against C. difficile spores. In order to develop novel therapeutics targeting C. difficile, firstly, we designed and implemented a comparative small molecule screen of approximately 75,000 compounds which produced a number of new classes of non-toxic and selective anti-Clostridium compounds. One of the hit compounds belonging to the diarylacylhydrazone class showed similar activity as vancomycin against several clinical C. difficile strains, as well as potent in vitro activity against non-growing cells. A structure-activity relationship (SAR) study suggested a protonophoric mechanism may play a role in the observed activity/selectivity of this diarylacylhydrazone class. Secondly, we found that the natural plant antimicrobial berberine was able to effectively kill both actively growing and stationary phase C. difficile cells when combined with Multidrug Resistance (MDR) pumps inhibitor INF271. We demonstrated that the berberine/INF271 combination was capable of rescuing Caenorhabditis elegans from Enterococcus faecalis infection. Thirdly, the hybrid molecules composed of berberine covalently linked to MDR pump inhibitors (INF55 and INF55 analogues) showed antibacterial properties. Interestingly, INF55-based MDR pump inhibitors that more strongly potentiate the antibacterial activity of berberine don't necessarily produce more potent berberine-INF55 hybrids.