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

Abstract

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.

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

“…However, the nextstep reductive amination failed to reach a satisfying yield when compounds 8-10 were subjected to hydrogenation by following the condition from our previous study (in the presence of conc. HCl over 20% Pd(OH) 2 on charcoal at 50 psi H 2 ) 29 . Thin-layer chromatography (TLC) analysis indicated that the reaction bottleneck was the formation of the cyclic imine.…”

“…The final products 2-4 were obtained with total yield of 27-73% in three steps by N-Cbz protection, selective oxidation of the primary alcohol, and removal of Cbz by hydrogenation. Notably, the selective oxidation of the methyl hydroxyl group was the key step, avoiding our previous use of tedious protection and deprotection steps 29 . The selective oxidation of the primary alcohol could be achieved in moderate yield (38-78%) by bubbling oxygen through a mixture of 10% Pt/C and N-Cbz-protected isofagomines 11-13 under basic conditions in H 2 O/isopropanol/acetone ( Fig.…”

“…BdGUS (6.9 nM), EcGUS (2.5 nM), LgGUS (4.0 nM), CpGUS (3.8 nM), and HsGUS (7.5 nM) were used for the inhibition assays. The K i values for inhibitors 1-4 and HsGUS, LgGUS, CpGUS, and BdGUS were verified by Lineweaver-Burk plot 29 , and apparent K M values were calculated. Plotting the apparent K M values as a function of the inhibitor concentrations generated the secondary plot.…”

“…These structures displayed binding interactions similar to those with the UIFG moiety, which is equivalent to glucuronic acid (the reaction product). For BdGUS, the two catalytic residues Glu479 and Glu574 (equivalent to Glu413 and Glu504 in EcGUS that correspond to the acid/base and nucleophile, respectively) formed ionic interactions with the endocyclic amine of UIFG 29 . Asn636 and Lys638, characterized as a unique conserved N-K motif in loop 8 33 , provided hydrogen bonds (H-bonds) and electrostatic interactions, respectively, with the C5-carboxylate of inhibitors 1-4 ( Fig.…”

“…Uronic isofagomine (UIFG, 1) potently inhibits both mammalian and bacterial GUSs, owing to ionic interactions between the protonated ring nitrogen and the two catalytic glutamates 29 . Notably, we found that the incorporation of an alkyl substituent to UIFG could affect the binding affinity with different GUSs.…”