Gut Bacterial β-Glucuronidase Blockade by 1,3,4-Thiadiazole-Containing Thioester Inhibitors Mitigates Irinotecan-Induced Gastrointestinal Toxicity.

Gut microbial β-glucuronidase has attracted attention as a potential therapeutic target for mitigating irinotecan (CPT-11)-induced late-onset diarrhea and intestinal injury. Here, we designed and synthesized 60 thioester derivatives bearing a 1,3,4-thiadiazole moiety and evaluated their biological activities. In vitro, 3-5B (IC50 = 0.39 μM) exhibited potent inhibitory activity against Escherichia coliβ-glucuronidase (EcGUS). Kinetic studies showed that 3-5B acts as an uncompetitive EcGUS inhibitor (Ki = 0.60 μM). Furthermore, molecular docking analysis suggested that 3-5B binds EcGUS with high affinity through interactions with residues Asp163, Glu413, and Trp549. Notably, 3-5B showed no inhibitory effect on bovine liver β-glucuronidase, did not inhibit E. coli growth, and exhibited low cytotoxicity toward human Caco-2 cells. In vivo, oral administration of 3-5B (1 mg/kg/d) attenuated CPT-11-induced gastrointestinal toxicity in BALB/cJ mice, including weight loss, diarrhea, and colonic injury. Collectively, these findings identify 3-5B as a promising lead scaffold for the development of EcGUS inhibitors.