The study addresses the dose-limiting side effect of the colon cancer chemotherapeutic CPT-11, which causes severe diarrhea due to the reactivation of the drug by symbiotic bacterial β-glucuronidases in the gut. The researchers identified potent and selective inhibitors of bacterial β-glucuronidases through high-throughput screening, which do not affect mammalian enzymes. Crystal structures revealed that selectivity is based on a unique "bacterial loop" in bacterial β-glucuronidases. These inhibitors were effective against the enzyme in both aerobic and anaerobic bacteria without harming mammalian cells. Oral administration of one inhibitor protected mice from CPT-11-induced toxicity, suggesting that inhibiting undesirable enzyme activities in essential microbial symbiotes can enhance chemotherapeutic efficacy. The findings highlight the potential for designing drugs to target specific bacterial enzymes without affecting commensal bacteria, which are crucial for human health.The study addresses the dose-limiting side effect of the colon cancer chemotherapeutic CPT-11, which causes severe diarrhea due to the reactivation of the drug by symbiotic bacterial β-glucuronidases in the gut. The researchers identified potent and selective inhibitors of bacterial β-glucuronidases through high-throughput screening, which do not affect mammalian enzymes. Crystal structures revealed that selectivity is based on a unique "bacterial loop" in bacterial β-glucuronidases. These inhibitors were effective against the enzyme in both aerobic and anaerobic bacteria without harming mammalian cells. Oral administration of one inhibitor protected mice from CPT-11-induced toxicity, suggesting that inhibiting undesirable enzyme activities in essential microbial symbiotes can enhance chemotherapeutic efficacy. The findings highlight the potential for designing drugs to target specific bacterial enzymes without affecting commensal bacteria, which are crucial for human health.