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

Irinotecan inhibits cell proliferation and thus is used for the primary treatment of colorectal cancer. Metabolism of irinotecan involves incorporation of ß-glucuronic acid to facilitate excretion. During transit of the glucuronidated product through the gastrointestinal tract, an induced upregulation of gut microbial ß-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.

Substituent Position of Iminocyclitols Determines the Potency and Selectivity for Gut Microbial Xenobiotic-Reactivating Enzymes.

Selective inhibitors of gut bacterial ß-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 (Ki ≥ 11 nM). C6-propyl analogue of 2 (6) displayed 700-fold selectivity for opportunistic bacterial GUSs (Ki = 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.

Characterization of protein serotonylation via bioorthogonal labeling and enrichment.

Protein serotonylation is a transglutaminase-mediated phenomenon whose biological mechanism of protein serotonylation is not yet fully understood, as the complete profiling of serotonylation targets in a proteome remains a critical challenge to date. Utilizing an alkyne-functionalized serotonin derivative bioorthogonally coupled to a cleavable linker, we developed a method to selectively enrich serotonylated proteins in a complex sample. With online nanoflow liquid chromatography and LTQ-Orbitrap Velos hybrid mass spectrometer detection, we identified 46 proteins with 50 serotonylation sites at their glutamine residues. Mass spectrometric analysis also generated direct residue-level evidence of various biological processes such as transglutaminase-chaperon interactions as well as actin assembly. An enrichment workflow utilizing click chemistry and on-bead digestion allowed us to achieve site-specific identification of protein serotonylation by mass spectrometry, and results obtained hereby also provided a great foundation in the elucidation of the true roles of protein serotonylation in biological systems.