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Molecular insights into microbial β-glucuronidase inhibition to abrogate CPT-11 toxicity
Adam B Roberts1, Bret D Wallace, Madhu Kumar Venkatesh
1Departments of Biochemistry, Chemistry and Microbiology, University of North Carolina at Chapel Hill, NC, USA.
Abstract:
Bacterial β-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 β-glucuronidases in the GI lumen can significantly damage the intestinal epithelium. Furthermore, selective disruption of bacterial β-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 β-glucuronidase inhibitors that inhibit Escherichia coli β-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 β-glucuronidase without inhibiting purified mammalian β-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 β-glucuronidase demonstrates that it contacts and orders only a portion of the "bacterial loop" present in microbial, but not mammalian, β-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 β-glucuronidase inhibition, which may improve human drug efficacy and toxicity.
Insights
Novel inhibitors selectively target bacterial β-glucuronidases, reducing drug toxicity in the gut. These compounds protect against chemotherapy side effects and may improve drug efficacy and safety.
Area of Science:
- Microbiology
- Pharmacology
- Structural Biology
Background:
- Bacterial β-glucuronidases in the gut microbiota contribute to drug-induced epithelial cell toxicity.
- Metabolites of drugs like CPT-11 (irinotecan) and diclofenac are detoxified by glucuronidation, but bacterial enzymes can reverse this process in the GI tract.
- This deconjugation can lead to severe intestinal damage and dose-limiting side effects for certain medications.
Purpose of the Study:
- To characterize novel small molecule inhibitors of microbial β-glucuronidases.
- To investigate the selectivity of these inhibitors against bacterial enzymes versus mammalian counterparts.
- To elucidate the structural basis for selective inhibition and assess in vivo efficacy.
Main Methods:
- In vitro enzyme inhibition assays using Escherichia coli β-glucuronidase and purified mammalian β-glucuronidase.
- Cell-based assays to assess the disruption of E. coli β-glucuronidase activity.
- X-ray crystallography to determine the binding mode of an inhibitor with E. coli β-glucuronidase at 2.8 Å resolution.
- In vivo studies in mice to evaluate protection against CPT-11-induced diarrhea.
Main Results:
- Novel inhibitors demonstrated potent inhibition of E. coli β-glucuronidase (Ki values from 180 nM to 2 μM) and cellular disruption (EC50 as low as 300 nM).
- Compounds were highly selective for bacterial E. coli β-glucuronidase, showing no inhibition of mammalian β-glucuronidase.
- The crystal structure revealed inhibitor interaction with a unique 'bacterial loop' present in microbial but not mammalian enzymes.
- The most potent inhibitor protected mice from CPT-11-induced diarrhea.
Conclusions:
- These findings provide a deeper understanding of the chemical and structural mechanisms underlying selective microbial β-glucuronidase inhibition.
- Targeting bacterial β-glucuronidases offers a promising strategy to mitigate drug-induced gastrointestinal toxicity.
- This approach holds potential for improving the efficacy and safety of various human therapeutics.
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