Gut bacterial β-D-glucuronidases (GUSs) catalyze the removal of glucuronic acid from liver-produced β-D-glucuronides. These reactions can have deleterious consequences when they reverse xenobiotic metabolism. The human gut contains hundreds of GUSs of variable sequences and structures. To understand how any particular bacterial GUS(s) contributes to global GUS activity and affects human health, the individual substrate preference(s) must be known. Herein, we report that representative GUSs vary in their ability to produce various xenobiotics from their respective glucuronides. To attempt to explain the distinct substrate preference, we solved the structure of a bacterial GUS complexed with coumarin-3-β-D-glucuronide. Comparisons of this structure with other GUS structures identified differences in loop 3 (or the α2-helix loop) and loop 5 at the aglycone-binding site, where differences in their conformations, hydrophobicities and flexibilities appear to underlie the distinct substrate preference(s) of the GUSs. Additional sequence, structural and functional analysis indicated that several groups of functionally related gut bacterial GUSs exist. Our results pinpoint opportunistic gut bacterial GUSs as those that cause xenobiotic-induced toxicity. We propose a structure-activity relationship that should allow both the prediction of the functional roles of GUSs and the design of selective inhibitors.

Irinotecan inhibits cell proliferation and thus is used for the primary treatment of colorectal cancer. Metabolism of irinotecan involves incorporation of β-glucuronic acid to facilitate excretion. During transit of the glucuronidated product through the gastrointestinal tract, an induced upregulation of gut microbial β-glucuronidase (GUS) activity may cause severe diarrhea and thus force many patients to stop treatment. We herein report the development of uronic isofagomine (UIFG) derivatives that act as general, potent inhibitors of bacterial GUSs, especially those of Escherichia coli and Clostridium perfringens. The best inhibitor, C6-nonyl UIFG, is 23,300-fold more selective for E. coli GUS than for human GUS (Ki = 0.0045 and 105 μM, respectively). Structural evidence indicated that the loss of coordinated water molecules, with the consequent increase in entropy, contributes to the high affinity and selectivity for bacterial GUSs. The inhibitors also effectively reduced irinotecan-induced diarrhea in mice without damaging intestinal epithelial cells.

Selective inhibitors of gut bacterial β-glucuronidases (GUSs) are of particular interest in the prevention of xenobiotic-induced toxicities. This study reports the first structure–activity relationships on potency and selectivity of several iminocyclitols (2–7) for the GUSs. Complex structures of Ruminococcus gnavus GUS with 2–7 explained how charge, conformation, and substituent of iminocyclitols affect their potency and selectivity. N1 of uronic isofagomine (2) made strong electrostatic interactions with two catalytic glutamates of GUSs, resulting in the most potent inhibition (K i ≥ 11 nM). C6-propyl analogue of 2 (6) displayed 700-fold selectivity for opportunistic bacterial GUSs (K i = 74 nM for E. coli GUS and 51.8 μM for RgGUS). In comparison with 2, there was 200-fold enhancement in the selectivity, which was attributed to differential interactions between the propyl group and loop 5 residues of the GUSs. The results provide useful insights to develop potent and selective inhibitors for undesired GUSs.