The inhibition of carbohydrate-lectin interactions is being explored as an efficient approach to anti adhesion therapy and biofilm destabilization, two alternative antimicrobial strategies that are being explored against resistant pathogens. BC2L-C is a new type of lectin from Burkholderia cenocepacia that binds (mammalian) fucosides at the N-terminal domain and (bacterial) mannosides at the C-terminal domain. This double carbohydrate specificity allows the lectin to crosslink host cells and bacterial cells. We have recently reported the design and generation of the first glycomimetic antagonists of BC2L-C, β-C- or β-N-fucosides that target the fucose-specific N-terminal domain (BC2L-C-Nt). The low water solubility of the designed N-fucosides prevented a full examination of this promising series of ligands. In this work, we describe the synthesis and biophysical evaluation of new L-fucosyl and L-galactosyl amides, designed to be water soluble and to interact with BC2L-C-Nt. The protein–ligand interaction was investigated by Saturation Transfer Difference NMR, Isothermal Titration Calorimetry and crystallographic studies. STD-NMR experiments showed that both fucosyl and galactosyl amides compete with α-methyl fucoside for lectin binding. A new hit compound was identified with good water solubility and an affinity for BC2L-C-Nt of 159 μM (ITC), which represents a one order of magnitude gain over α-methyl fucoside. The x-ray structure of its complex with BC2L-C-Nt was solved at 1.55 Å resolution.
Opportunistic infections from multidrug-resistant pathogens such as Burkholderia cenocepacia are a threatening risk for hospital-bound patients suffering from immunocompromised conditions or cystic fibrosis. B. cenocepacia BC2L-C lectin has been linked to bacterial adhesion and biofilm formation, thus hindering its activity is seen as a promising strategy to reduce the severity of the infection. We recently described the first bifunctional ligands of the trimeric N-terminal domain of BC2L-C (BC2L-C–Nt), capable of simultaneously engaging its fucose-specific sugar binding site and a vicinal region at the interface between two monomers. Here, we report a computational workflow for the study of these glycomimetic bifunctional ligands in complex with BC2L-C-Nt, aimed at investigating the molecular basis of ligand binding and the dynamics of glycomimetic/lectin interactions. In particular, we evaluated the use of molecular docking in the protein trimer, followed by refinement using MM-GBSA re-scoring and MD simulations in explicit water. Computational results were compared to experimental data derived from X-ray crystallography and isothermal titration calorimetry. The computational protocol proved suitable to provide a reliable description of the interactions between the ligands and BC2L-C-Nt, highlighting the contribution of MD simulations in explicit solvent for a good fit with the experimental observations. The information achieved in the study and the whole workflow appear promising for the structure-based design of improved BC2L-C-Nt ligands as novel antimicrobials with antiadhesive properties.
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