Discovery of selective inhibitors of the Clostridium difficile dehydroquinate dehydratase.

Abstract

A vibrant and healthy gut flora is essential for preventing the proliferation of Clostridium difficile, a pathogenic bacterium that causes severe gastrointestinal symptoms. In fact, most C. difficile infections (CDIs) occur after broad-spectrum antibiotic treatment, which, by eradicating the commensal gut bacteria, allows its spores to proliferate. Hence, a C. difficile specific antibiotic that spares the gut flora would be highly beneficial in treating CDI. Towards this goal, we set out to discover small molecule inhibitors of the C. difficile enzyme dehydroquinate dehydratase (DHQD). DHQD is the 3(rd) of seven enzymes that compose the shikimate pathway, a metabolic pathway absent in humans, and is present in bacteria as two phylogenetically and mechanistically distinct types. Using a high-throughput screen we identified three compounds that inhibited the type I C. difficile DHQD but not the type II DHQD from Bacteroides thetaiotaomicron, a highly represented commensal gut bacterial species. Kinetic analysis revealed that the compounds inhibit the C. difficile enzyme with Ki values ranging from 10 to 20 µM. Unexpectedly, kinetic and biophysical studies demonstrate that inhibitors also exhibit selectivity between type I DHQDs, inhibiting the C. difficile but not the highly homologous Salmonella enterica DHQD. Therefore, the three identified compounds seem to be promising lead compounds for the development of C. difficile specific antibiotics.