Entropy-driven binding of gut bacterial β-glucuronidase inhibitors ameliorates irinotecan-induced toxicity

Abstract

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 (K_i = 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.

Fig. 1. Molecular structures of inhibitors and synthetic scheme for UIFG derivatives.

a Molecular structures of UIFG (1) and its C6-alkylated derivatives 2–4 and ASN03273363. b Synthesis of 2–4 and substituted isofagomines 11–13.

Fig. 2. Crystal structures of inhibitors bound to BdGUS.

A water-mediated H-bond network within the substrate binding site of a EcGUS in complex with glucaro-δ-lactam and b–e BdGUS in complex with inhibitors 1–4. The interactions were similar between the sugar moiety and BdGUS. The 2F_o–F_c density maps of water molecules are colored blue. The alkyl substituents of 2–4 appear to expel some of the water molecules. f Superimposition of inhibitors 1–4 in the crystal structures. C6 of 2–4 deviates 11.4° from that of 1.

Fig. 3. Entropy-driven binding of inhibitors 1–4 with EcGUS and BdGUS.

a Graphical presentation to show the thermodynamic parameters of the binding interactions of EcGUS and BdGUS in complex with inhibitors 1–4 at 298 K. The detailed analysis is shown in Supplementary Table 1. ^a 250 mM NaCl, 20 mM KH₂PO₄, 100 mM Na₂HPO₄, pH 8.0. ^b 250 mM NaCl, 20 mM Tris, pH 8.0. b Cartoon presentation to demonstrate that several water molecules (represented by red circles) are orderly located at the active site of gut bacterial GUSs, as shown in EcGUS (the left model), and can be displaced by binding with UIFGs. The best inhibitor, C6-nonyl UIFG, displays tight binding with EcGUS (K_i = 4.5 nM) and 23,300-fold higher selectivity for EcGUS than for human GUS (the bottom right). GUSs are mainly different in the aglycone-binding site, which can be leveraged to develop selective inhibitors. If UIFGs have no substitution (the top right), selective inhibition no longer exists.

Fig. 4. Treatment with inhibitor 3 demonstrates inhibition of GUS and protection against diarrhea caused by CPT-11 damage in mice.

a Inhibition of GUS in vivo. Mice were given 3 via oral gavage. After 1 h, fluorescein-di-β-d-glucuronide (FDGiCu; 500 µg in 100 µL) was injected intravenously. The hydrolysis product (fluorescein) generated in the gut was quantified over a 2 h period by in vivo imaging (excitation 465 nm, emission 520 nm). The maximum fluorescein fluorescence was observed 60 min after injection of vehicle control. At 2 h post injection, most of the FDGiCu has been excreted and thus fluorescence is reduced. The region of interest (ROI) was analyzed with Living Image Software. b Effect of inhibitor 3 to protect against diarrhea caused by CPT-11. Diarrhea severity was scored as described in methods. Mice receiving CPT-11 (blue squares) experienced severe diarrhea from days 7–11, whereas mice receiving inhibitor 3 with CPT-11 (red triangles) displayed significantly reduced diarrhea (Welch’s unpaired t-test, day 9, p = 0.3632; day 10, *p = 0.0284; day 11, ***p = 0.0005; day 12, **p = 0.0057).