A Modified ToxT Inhibitor Reduces Vibrio cholerae Virulence in Vivo

Abstract

We have previously designed and synthesized small-molecule inhibitors that reduce Vibrio cholerae virulence in vitro by targeting the transcription factor ToxT. Here we report the synthesis and biological activity of derivatives of our previous bicyclic, fatty acid-like inhibitors. All of the synthesized derivatives show antivirulence activity in vitro. For the most potent compounds, a concentration of 5 μM completely inhibited ToxT-mediated tcpA expression as measured in the β-galactosidase assay. One indole compound, 3-(1-butyl-1 H-indol-7-yl)propanoic acid (8), was also effective at inhibiting intestinal colonization in the infant mouse. These modified compounds may serve as good candidates for further anti-cholera drug development.

Figure 1.

General structures of the ‘8-methyl’ compounds, virstatin, and newly synthesized derivatives.

Figure 2.

A cross-section of the ToxT inhibitor-binding pocket containing an ‘8-methyl’ compound (from the crystal structure, PDB code 5SUW) (a) and an ‘8-alkyl’ derivative (predicted by AutoDock) (b). The surface of the pocket is colored by amino acid hydrophobicity: most hydrophilic (blue), neutral (white), most hydrophobic (orange red). Pocket calculated by CASTp.

Figure 3.

Effect of the ‘8-alkyl’ derivatives on tcpA-lacZ expression. Activity of the untreated wild-type strain (WT) is set to 100%.Relative β-gal activities of the negative control (△ToxT) and solvent control (DMSO) are calculated as a percentage of WT; samples containing inhibitor are calculated relative to DMSO. Final concentrations of inhibitor are 50 µM (white), 5 µM (dark blue), 0.5 µM (grey), and 0.05 µM (light blue). Error bars represent standard deviation where n is 3. *Compound 2 is a mixture of tetralins (see supplementary material).

Figure 4.

Effect of the indole derivatives on tcpA-lacZ expression. Relative β-gal activity is calculated as in Figure 3. Final concentrations of inhibitor are 50 µM (white), 5 µM (dark blue), 0.5 µM (grey), and 0.05 µM (light blue). Error bars represent standard deviation where n is 3–4.

Figure 5.

Effects of inhibitors on intestinal colonization. Mice were given the negative control strain (△ToxT), the wild-type strain containing a solvent control (DMSO), or the wild-type strain containing an inhibitor (0.1 mM virstatin, 0.1 mM compound 4, or 1 mM compound 8). A Box-Cox transformation of the colonization index raw data (output/input CFUs) was performed to normalize the data distribution (Figure S2). Pairwise means comparison using Tukey-Kramer HSD indicates statistically significant differences between 2 pairs: DMSO/△ToxT (p=0.0011) and DMSO/8 (p=0.0029). Note: A decrease in CI for compound 8 was also statistically significant when the untransformed (raw) data was analyzed using the nonparametric Kruskal-Wallis test (Figure S2).

Figure 6.

Influence of a toxT mutation on the expression of tcpA-lacZ in the presence or absence of inhibitors. β-gal activity was assayed in the wild-type toxT background (solid) or the toxT K230A background (striped). Samples were grown alone (WT and K230A), with a solvent control (DMSO), and in the presence of various inhibitors. Final concentrations of inhibitor are 708 µM (green), 50 µM (white/grey), and 5 µM (blue). Error bars represent standard deviation where n is 3. See Figure S1 for western blot.

Scheme 1.

Synthesis of 7-indole derivatives.

Scheme 2.

Synthesis of 4-indole derivatives.