Multivalent Fucosides Targeting β-Propeller Lectins from Lung Pathogens with Promising Anti-Adhesive Properties

Abstract

Fungal and bacterial pathogens causing lung infections often use lectins to mediate adhesion to glycoconjugates at the surface of host tissues. Given the rapid emergence of resistance to the treatments in current use, β-propeller lectins such as FleA from Aspergillus fumigatus, SapL1 from Scedosporium apiospermum, and BambL from Burkholderia ambifaria have become appealing targets for the design of anti-adhesive agents. In search of novel and cheap anti-infectious agents, we synthesized multivalent compounds that can display up to 20 units of fucose, the natural ligand. We obtained nanomolar inhibitors that are several orders of magnitude stronger than their monovalent analogue according to several biophysical techniques (i.e., fluorescence polarization, isothermal titration calorimetry, and bio-layer interferometry). The reason for high affinity might be attributed to a strong aggregating mechanism, which was examined by analytical ultracentrifugation. Notably, the fucosylated inhibitors reduced the adhesion of A. fumigatus spores to lung epithelial cells when administered 1 h before or after the infection of human lung epithelial cells. For this reason, we propose them as promising anti-adhesive drugs for the prevention and treatment of aspergillosis and related microbial lung infections.

Figure 1

Overall structure of the targeted lectins: FleA, SapL1, and BambL (PDB codes 4AGI, 6TRV, and 3WZ2, respectively). (A) Side view. (B) Top view. Different monomers of the same protein are depicted in different shades of gray. The fucoside ligand is colored in blue and occupies each binding site. The longest distance between the opposite binding sites is indicated with a dashed line.

Scheme 1. Synthesis of the Elongated Ligands 10–12

Reagents and conditions: (a) 2-chloroethanol, Amberlite IR-120 H⁺, reflux; (b) Ac₂O, Py (68% over 2 steps); (c) NaN₃, TBAI, DMF, 90 °C (48% α); (d) SnCl₂, HCl aq., MeOH (99%); and (e) HATU, DIPEA, DCM (34–53%).

Scheme 2. Synthesis of the Tri-, Tetra-, and Hexa-Valent Cores

Reagents and conditions: (a) NaH, DMF; (b) THF/dioxane/NaOH 2 M 1:2:1 (41–87% over two steps).

Scheme 3. Final Steps in the Synthesis of Multivalent Fucosides

Reagents and conditions: (a) HATU, DIPEA, DMF; (b) MeONa/MeOH; (c) NaH, KI, propargyl bromide; and (d) 2a, CuSO₄, Na ascorbate, 100 °C, MW. Products were obtained in yields ranging from 24 to 66% over two steps.

Figure 2

Distribution of the sedimentation coefficients for mixtures of BambL with different concentrations of tri-4 and hexa-4.

Figure 3

WST-1 viability assay on A549 cells. The concentration of fucosides was 1 mM for αMeFuc, 0.1 mM for multivalent compounds. As control, 1% Triton X-100 was used. Values are represented as mean ± CI₉₅ from independent replicates, where ns: not significant, ****p < 0.0001, **p < 0.01 (Welch’s unpaired t-test).

Figure 4

Representative confocal images of spores of fungal strain Af293.1 (red) adhesion on alveolar A549 cells (blue), when cells were untreated (A) or treated with 100 μM tetra-4 (B). Spores were allowed to adhere for 4 h after infection, followed by washing and staining.

Figure 5

Adhesion of Af293.1 to A459. The black bars refer to the conditions where compounds and spores were pre-incubated 1 h prior infection. The white-striped bars refer to the conditions where compounds were added 1 h after infection. *Significant difference to the control (chi-square test, followed by Bonferroni’s multiple comparison test).