Inhibition of Clostridium difficile TcdA and TcdB toxins with transition state analogues

Abstract

Clostridium difficile causes life-threatening diarrhea and is the leading cause of healthcare-associated bacterial infections in the United States. TcdA and TcdB bacterial toxins are primary determinants of disease pathogenesis and are attractive therapeutic targets. TcdA and TcdB contain domains that use UDP-glucose to glucosylate and inactivate host Rho GTPases, resulting in cytoskeletal changes causing cell rounding and loss of intestinal integrity. Transition state analysis revealed glucocationic character for the TcdA and TcdB transition states. We identified transition state analogue inhibitors and characterized them by kinetic, thermodynamic and structural analysis. Iminosugars, isofagomine and noeuromycin mimic the transition state and inhibit both TcdA and TcdB by forming ternary complexes with Tcd and UDP, a product of the TcdA- and TcdB-catalyzed reactions. Both iminosugars prevent TcdA- and TcdB-induced cytotoxicity in cultured mammalian cells by preventing glucosylation of Rho GTPases. Iminosugar transition state analogues of the Tcd toxins show potential as therapeutics for C. difficile pathology.

Fig. 1. Mechanism of action of C. difficile TcdA and TcdB.

a (top) Schematic of the Tcd toxin domain structure: N-terminal glucosyltransferase domain GTD (red), cysteine protease domain CPD (blue), delivery domain (orange) and receptor binding domain RBD (green). (Bottom) Schematic of mechanism of TcdA and TcdB-induced toxicity of mammalian cells (see “Introduction” for details), adapted from reference 25. b Schematic of TcdA-GTD and TcdB-GTD catalyzed reactions. Glucosyltransferase reaction catalyzed by TcdB-GTD and TcdA-GTD (left). Glucohydrolase reaction catalyzed by TcdB-GTD and TcdA-GTD with water acting as the nucleophile (right).

Fig. 2. KIEs measured for TcdA-GTD and TcdB-GTD glucohydrolase reaction.

Chemical structure of UDP-glucose. Atoms that were labeled for KIE measurements are colored and the type of KIE is indicated.

Fig. 3. Inhibition candidates for TcdA-GTD and TcdB-GTD catalytic activity.

Chemical structures of transition state analogues that were tested for inhibition of TcdA-GTD and TcdB-GTD in this study. Positions of relevant carbon atoms in isofagomine and noeuromycin are indicated.

Fig. 4. Isofagomine and noeuromycin binding to TcdB-GTD.

a Double reciprocal plots are shown for isofagomine (top) and noeuromycin (bottom). TcdB-GTD activity was measured in the presence of inhibitor and varying concentrations of UDP-glucose. Isofagomine or noeuromycin; 0 µM, circles; 2 µM isofagomine or 16 µM noeuromycin, triangles; 4 µM isofagomine or 32 µM noeuromycin, diamonds; 8 µM isofagomine or 64 µM noeuromycin Squares. Data points are the mean of experimental data from 3 independent experiments and lines represent the global fit to Eq. 12 for uncompetitive inhibition as described in Methods. b Isothermal titration calorimetry (ITC) analysis of isofagomine (top) or noeuromycin (bottom) binding to TcdB-GTD. c ITC analysis of isofagomine binding to TcdB-GTD in the presence of UDP. d ITC analysis of noeuromycin binding to TcdB-GTD in the presence of UDP. Conditions are provided in Methods. Top panels indicate raw heat data, and bottom panels show integrated heat injections which have been normalized per mole of injectant as a function of molar ratio. Results were best fit to a one-site binding model. Binding parameters represent mean ± SEM for three replicates. Source data are provided as a Source Data file.

Fig. 5. Binding interactions of TcdB-GTD in complex with inhibitors (stereo-views).

Inhibitor complexes of isofagomine (PDB ID: 7LOU, yellow) and noeuromycin (PDB ID: 7LOV, green) are shown in panels (a) and (b), respectively. Amino acid residues interacting with inhibitors and UDP are indicated. Selected hydrogen bond interactions are shown in black dotted lines. The π-π stacking interaction of uridine ring with Trp102 is also highlighted. c Superposition of the binding pocket of apo TcdB-GTD (PDB ID: 5UQT, yellow), isofagomine (PDB ID: 7LOU, cyan), and noeuromycin (PDB ID: 7LOV, gray) bound structures. Movement of the loop from residues Gln510 to Asp523 (10 Å) towards the binding pocket occurs on inhibitor binding and is highlighted with the black arrow.

Fig. 6. Isofagomine and noeuromycin prevent TcdA and TcdB-induced cytotoxicity in mammalian cells.

a Representative image from at least 3 experiments of Vero cells 2 h after treatment with TcdB and isofagomine (isof). Scale bar represents 10 µM. b Representative Western blot images for intracellular Rac1 glucosylation (n=3). Human IMR90 cells were treated with isofagomine (isof) or noeuromycin (noe) (doses indicated) for 30 min, followed by treatment with buffer control, 1 nM TcdA or 0.1 nM TcdB for 6 h. Cell lysates were prepared as described in Methods. Mab102 was used to detect unglucosylated Rac1 in cell lysates. Anti-Rac1 (23A8) was used to detect total Rac1 levels and anti-GAPDH served as the loading control. Uncropped Western blots are shown in Supplementary Figs. 9, 10. c Flow cytometry analysis of AnnexinV in HT-29 cells from 5 independent experiments (n=5). HT-29 cells were pre-treated with isofagomine (isof) or noeuromycin (noe) (250 µM or 500 µM) for 30 min before treatment with buffer control or 1 nM TcdA for 24 h. Cells were harvested and stained with AnnexinV as described in Methods. AnnexinV positivity (%) was normalized to untreated HT-29 cells. Untreated cells are shown as white circles, TcdA treated cells are shown in black circles, isofagomine and TcdA treated cells are shown as red circles and noeuromycin and TcdA treated cells are shown as blue circles. Ordinary one-way ANOVA with Tukey’s multiple comparison test, significance ****p < 0.0001, *** p < 0.001. Error bars show mean ± SEM. Source data are provided as a Source Data file.