Characterization of Small-Molecule Scaffolds That Bind to the Shigella Type III Secretion System Protein IpaD

Abstract

Many pathogens such as Shigella and other bacteria assemble the type III secretion system (T3SS) nanoinjector to inject virulence proteins into their target cells to cause infectious diseases in humans. The rise of drug resistance among pathogens that rely on the T3SS for infectivity, plus the dearth of new antibiotics require alternative strategies in developing new antibiotics. The Shigella T3SS tip protein IpaD is an attractive target for developing anti-infectives because of its essential role in virulence and its exposure on the bacterial surface. Currently, the only known small molecules that bind to IpaD are bile salt sterols. In this study we identified four new small-molecule scaffolds that bind to IpaD, based on the methylquinoline, pyrrolidine-aniline, hydroxyindole, and morpholinoaniline scaffolds. NMR mapping revealed potential hotspots in IpaD for binding small molecules. These scaffolds can be used as building blocks in developing small-molecule inhibitors of IpaD that could lead to new anti-infectives.

Keywords: IpaD; NMR spectroscopy; small molecules; surface plasmon resonance; type III secretion system.

Figure 1

The four scaffolds that bind to IpaD (boxed) are 4-amino-2-methylquinoline (1), 4-[2-(pyrrolidin-1-yl) ethyl] aniline (2), 5-hydroxyindole (3), and 4-morpholinoaniline (4). Analogs of these four scaffolds that did not bind to IpaD identified which chemical moieties are important for binding to IpaD. The analogs are 4-hydroxy-2-methylquinoline (1a), 2-amino-3-methylquinoline (1b), 2-amino-6-bromoquinoline (1c), 5-bromoisoquinoline (1d), 3-(4-fluorobenzyl)-piperidine (2a), 4-(4-methyl-piperazin-1-ylmethyl)-phenylamine (2b), 3-indole acetic acid (3a), indole (3b), indole-3-carboxylic acid (3c), 3-morpholinophenol (4a) and 4-morpholin-4-ylmethyl-phenylamine (4b).

Figure 2

Surface plasmon resonance sensorgrams of IpaD with compounds A) 1, B) 2, C) 3 and D) 4.

Figure 3

STD NMR of IpaD with compounds A) 1, B) 2, C) 3 and D) 4. Top panel shows the off-resonance spectra and the assignment of protons for each scaffold. Lower panel shows the STD spectra, after the on-resonance spectra (not shown) were subtracted from their corresponding off-resonance spectra, indicating which protons of the scaffolds are in contact with the protein.

Figure 4

Selected regions from 2D ¹H ¹⁵N HSQC spectra of IpaD titrated with compounds A) 1, B) 2, C) 3 and D) 4. Only IpaD residues that showed changes in peak positions are shown. Arrows indicate the movement of peaks upon titration of IpaD with increasing molar ratios of each compound. Peaks are colored (black, red, blue, green, magenta) according to increasing molar ratios of IpaD:scaffold. The full HSQC spectra, including the molar ratios used for each titration, are in the Supplementary.

Figure 5

Plots of the weighted chemical shift deviations (Δδ) of IpaD with compounds A,B) 1, C,D) 2, E,F) 3 and G,H) 4. The results of the ¹⁵N titrations are on the left panels (A, C, E, G) and the results of the ILV-titrations are on the right panels (B, D, F, H).

Figure 6

The chemical shift deviation (Δδ) of compounds A) 1, B) 2, C) 3 and D) 4 is shown on the ribbon and surface structures of IpaD, and colored according to the value of (Δδ), with the least affected residues colored gray, to the highly affected residues colored red. The binding pockets x, y, and z are indicated. The different parts of IpaD are indicated as hairpin (hp), coiled-coil (cc), the mixed α/β domain (α/β) as well as the amino (N) and the carboxy (C) termini.