Structure and Inhibition of Microbiome ß-Glucuronidases Essential to the Alleviation of Cancer Drug Toxicity.

The selective inhibition of bacterial ß-glucuronidases was recently shown to alleviate drug-induced gastrointestinal toxicity in mice, including the damage caused by the widely used anticancer drug irinotecan. Here, we report crystal structures of representative ß-glucuronidases from the Firmicutes Streptococcus agalactiae and Clostridium perfringens and the Proteobacterium Escherichia coli, and the characterization of a ß-glucuronidase from the Bacteroidetes Bacteroides fragilis. While largely similar in structure, these enzymes exhibit marked differences in catalytic properties and propensities for inhibition, indicating that the microbiome maintains functional diversity in orthologous enzymes. Small changes in the structure of designed inhibitors can induce significant conformational changes in the ß-glucuronidase active site. Finally, we establish that ß-glucuronidase inhibition does not alter the serum pharmacokinetics of irinotecan or its metabolites in mice. Together, the data presented advance our in vitro and in vivo understanding of the microbial ß-glucuronidases, a promising new set of targets for controlling drug-induced gastrointestinal toxicity.

Molecular insights into microbial ß-glucuronidase inhibition to abrogate CPT-11 toxicity.

Bacterial ß-glucuronidases expressed by the symbiotic intestinal microbiota appear to play important roles in drug-induced epithelial cell toxicity in the gastrointestinal (GI) tract. For the anticancer drug CPT-11 (irinotecan) and the nonsteroidal anti-inflammatory drug diclofenac, it has been shown that removal of the glucuronide moieties from drug metabolites by bacterial ß-glucuronidases in the GI lumen can significantly damage the intestinal epithelium. Furthermore, selective disruption of bacterial ß-glucuronidases by small molecule inhibitors alleviates these side effects, which, for CPT-11 {7-ethyl-10-[4-(1-piperidino)-1-piperidino]}, can be dose limiting. Here we characterize novel microbial ß-glucuronidase inhibitors that inhibit Escherichia coli ß-glucuronidase in vitro with Ki values between 180 nM and 2 µM, and disrupt the enzyme in E. coli cells, with EC50 values as low as 300 nM. All compounds are selective for E. coli ß-glucuronidase without inhibiting purified mammalian ß-glucuronidase, and they do not impact the survival of either bacterial or mammalian cells. The 2.8 Å resolution crystal structure of one inhibitor bound to E. coli ß-glucuronidase demonstrates that it contacts and orders only a portion of the "bacterial loop" present in microbial, but not mammalian, ß-glucuronidases. The most potent compound examined in this group was found to protect mice against CPT-11-induced diarrhea. Taken together, these data advance our understanding of the chemical and structural basis of selective microbial ß-glucuronidase inhibition, which may improve human drug efficacy and toxicity.