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. 2015 Oct 13;4(6):756-67.
doi: 10.1002/open.201500162. eCollection 2015 Dec.

Cinnamide Derivatives of d-Mannose as Inhibitors of the Bacterial Virulence Factor LecB from Pseudomonas aeruginosa

Roman Sommer  1 , Dirk Hauck  1 , Annabelle Varrot  2 , Stefanie Wagner  3 , Aymeric Audfray  2 , Andreas Prestel  4 , Heiko M Möller  4 , Anne Imberty  2 , Alexander Titz  1
Affiliations

Cinnamide Derivatives of d-Mannose as Inhibitors of the Bacterial Virulence Factor LecB from Pseudomonas aeruginosa

Roman Sommer et al. ChemistryOpen. .

Abstract

Pseudomonas aeruginosa is an opportunistic Gram-negative pathogen with high antibiotic resistance. Its lectin LecB was identified as a virulence factor and is relevant in bacterial adhesion and biofilm formation. Inhibition of LecB with carbohydrate-based ligands results in a decrease in toxicity and biofilm formation. We recently discovered two classes of potent drug-like glycomimetic inhibitors, that is, sulfonamides and cinnamides of d-mannose. Here, we describe the chemical synthesis and biochemical evaluation of more than 20 derivatives with increased potency compared to the unsubstituted cinnamide. The structure-activity relationship (SAR) obtained and the extended biophysical characterization allowed the experimental determination of the binding mode of these cinnamides with LecB. The established surface binding mode now allows future rational structure-based drug design. Importantly, all glycomimetics tested showed extended receptor residence times with half-lives in the 5-20 min range, a prerequisite for therapeutic application. Thus, the glycomimetics described here provide an excellent basis for future development of anti-infectives against this multidrug-resistant pathogen.

Keywords: LecB/PA-IIL; carbohydrates; glycoconjugates; glycomimetics; lectins.

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Figures

Figure 1
Figure 1
Reported natural and synthetically modified mannose based inhibitors 1, 2, and 3 and their thermodynamic dissociation constants (K d)with the lectin LecB.25, 26
Scheme 1
Scheme 1
Synthesis of compounds 5 a–r, 6, and 7 a,b. Reagents and conditions: a) carboxylic acids, EDC⋅HCl, Et3N, DMF, 0 °C → rt; b) FeSO4⋅7 H2O, NH4OH, H2O, rt; c) Pd/C, H2, MeOH, rt. Reaction times, yields, and purities are summarized in Table S1 in the Supporting Information.
Figure 2
Figure 2
Biochemical evaluation of LecB binding to ligands 5 a–r, 6, and 7 a,b. IC50 values were determined with a competitive fluorescence polarization assay. Means and standard deviations were determined from a minimum of three independent experiments.
Scheme 2
Scheme 2
Synthesis of glucose‐derivative 10. Reagents and conditions: a) Pd/C, H2, MeOH, rt; b) cinnamic acid, EDC⋅HCl, Et3N, DMF, 0 °C → rt.
Figure 3
Figure 3
Inhibition of LecB by manno‐ 3 is carbohydrate‐specific, and the control compound gluco‐ 10 has no effect up to a concentration of 3 mm on LecB functional binding to the fucosylated fluorescent probe of the assay system.
Figure 4
Figure 4
Isothermal titration microcalorimetry (ITC) of LecB with dimethoxycinnamide 7 b. Three independent titrations were performed; one representative example is shown here.
Figure 5
Figure 5
Single cycle kinetics analysis by surface plasmon resonance (SPR) of the direct binding of 1, 2, 3, or 7 b to immobilized LecB. Experimental data are shown in black; calculated fits using a 1:1 binding model with a global fitting on all injected concentrations are shown in red. Ligand concentrations injected were 120 nm, 600 nm, 3 μm, 15 μm, and 75 μm for 2, 3, and 7 b, and 3 μm, 15 μm, 75 μm, 375 μm, and 1.8 mm for 1.
Figure 6
Figure 6
A) CD spectroscopy of LecB (35 μm) in the presence or absence of compound 3 (100 μm) gave identical CD spectra indicative for beta‐sheet secondary structure. B) thermal unfolding followed by CD spectroscopy at 229 nm was not indicative of a destabilization of the protein in presence of 3.
Figure 7
Figure 7
Binding of dimethoxycinnamide 7 b to LecB was analyzed by 1H,15N‐TROSY‐HSQC NMR experiments in the absence (blue) and presence (red) of 7 b. A) Spectrum of 500 μm 15N‐labeled LecB without addition of ligand. B) Spectrum of 500 μm 15N‐labeled LecB in the absence (blue) and in presence (red) of 1 equivalent 7 b. Upon addition of 7 b, the intensities of about 15 peaks decreased, and a number of new crosspeaks appeared. This is indicative of chemical shift perturbations of a defined binding that interacts with 7 b in the slow‐exchange regime.
Figure 8
Figure 8
A) The crystal structure of the complex of 3 with LecB at 1.6 Å shows a surface binding of the cinnamide residue to the protein. The superposition of all four binding sites indicates coordination of the cinnamic acid residue to the protein in one monomer and no interaction with LecB in three of the four monomers. The latter results from extended interligand stacking interactions in neighboring binding sites due to the crystal packing. B) Surface for monomer 2 with electron density for 3: the cinnamide substituent forms lipophilic interactions with Gly97 and Thr98 and one water‐mediated hydrogen bond via the carbonyl oxygen with Ser23.
Figure 9
Figure 9
Aggregation assay of LecB (133 μm) with ligands (14 μm to 1.7 mm). Aggregation at OD600 observed for 3 at the two highest concentrations. No aggregation observed for 2, 7 b, and 10.
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