Targeting a Multidrug-Resistant Pathogen: First Generation Antagonists of Burkholderia cenocepacia’s BC2L-C Lectin

Abstract

Multidrug-resistant pathogens such as Burkholderia cenocepacia have become a hazard in the context of healthcare-associated infections, especially for patients admitted with cystic fibrosis or immuno-compromising conditions. Like other opportunistic Gram-negative bacteria, this pathogen establishes virulence and biofilms through lectin-mediated adhesion. In particular, the superlectin BC2L-C is believed to cross-link human epithelial cells to B. cenocepacia during pulmonary infections. We aimed to obtain glycomimetic antagonists able to inhibit the interaction between the N-terminal domain of BC2L-C (BC2L-C-Nt) and its target fucosylated human oligosaccharides. In a previous study, we identified by fragment virtual screening and validated a small set of molecular fragments that bind BC2L-C-Nt in the vicinity of the fucose binding site. Here, we report the rational design and synthesis of bifunctional C- or N-fucosides, generated by connecting these fragments to a fucoside core using a panel of rationally selected linkers. A modular route starting from two key fucoside intermediates was implemented for the synthesis, followed by evaluation of the new compounds as BC2L-C-Nt ligands with a range of techniques (surface plasmon resonance, isothermal titration calorimetry, saturation transfer difference NMR, differential scanning calorimetry, and X-ray crystallography). This study resulted in a hit molecule with an order of magnitude gain over the starting methyl fucoside and in two crystal structures of antagonist/lectin complexes.

Identification of New L-Fucosyl and L-Galactosyl Amides as Glycomimetic Ligands of TNF Lectin Domain of BC2L-C from Burkholderia cenocepacia

“…In order to increase the water solubility of 2 , while preserving its ability to interact with BC2L-C-Nt, we tackled two structural features, one in the carbohydrate moiety and one in the aglycone ( Figure 1 ). On the aglycone side, we knew that the requirements for fitting the targeted crevice consist of a rather extended aromatic moiety, which generates a T-shaped interaction with Tyr58, and an amino group, targeting the side chain of Asp70 at the bottom end of the binding region [ 8 ]. We kept these features in the fucosylamide 3 , while transforming the aniline of 2 into an aminomethylene group.…”

“…On the monosaccharide side, we capitalized on the observation that L-galactose binds to BC2L-C-Nt with an affinity that is similar to that of L-fucose [ 12 ] to draw up also the L-galactosylamide 4 . The docking of 3 and 4 in BC2L-C-Nt, using Glide (version 7.8) [ 13 ] with the established protocol [ 8 ], supported their ability to fit within the expected region and to generate extensive interactions with the protein ( Figure 2 ). Thus, we set about to synthesize 3 and 4 and determine their activity as a BC2L-C ligand.…”

“…We have recently described the rational design and synthesis of a first group of antagonists of Burkholderia cenocepacia BC2L-C lectin, a candidate target for AAT against this pathogen, which is associated with fatal pulmonary infections of cystic fibrosis patients [ 7 , 8 ]. BC2L-C presents a double carbohydrate specificity, with a Man-specific carbohydrate recognition domain (CRD) in the C -terminal domain and a Fuc-specific CRD in the trimeric N- terminal domain [ 9 , 10 ].…”

“…These ligands bear an aglycone moiety derived from fragments, which were predicted in silico to engage a small crevice close to the fucose-binding area. An evaluation of the new compounds, including BC2L-C-Nt ligands, resulted in a hit molecule, the C-glycoside 1 ( Figure 1 ); this revealed an order of magnitude gain over the starting α-methyl fucoside, as judged by isothermal titration calorimetry [ 8 ]. X-ray crystallography strongly suggested that the fucosylamide 2 was also perfectly accommodated in the binding region and shared with 1 a number of stabilizing interactions with the protein surface.…”

“…However, the affinity of 2 for BC2L-C-Nt could not be determined by ITC, owing to its very low water solubility. A direct interaction SPR assay, performed by immobilizing BC2L-C-Nt on a CM5 chip, yielded an apparent Kd 2.4 mM for 2 , but affinities measured in this assay did not correlate with ITC results for a series of other molecules examined, and could not be relied upon [ 8 ]. In order to verify the potential of the chemotype represented by ligand 2 , we set about to generate water soluble versions of 2 ( Figure 1 ).…”