Exploring covalent strategies to target the bacterial lectin BC2L-C

The opportunistic Gram-negative pathogen Burkholderia cenocepacia is a globally spread multidrug-resistant bacterium that causes deadly lung infections in immunocompromised or cystic fibrosis patients.1 It employs lectins, i.e.carbohydrate-binding proteins, as virulence factors to target host tissues through recognition of and adhesion to the glycoconjugates on the host cells’ surface. The inhibition of these glycoconjugates-lectin interactions is being explored as an efficient approach to anti-adhesion therapy (AAT), which could be an alternative strategy to prevent and treat bacterial infections.2 Aims Among the B. cenocepacia’s lectins, the superlectin BC2L-C has been proposed as a major player in the adhesion process; in particular, its N-terminal fucose-binding domain (BC2L-C-Nt) represents an interesting target to design new antimicrobials for AAT. In this context, our aim consists of developing a new generation of glycomimetics targeting BC2L-C-Nt, to improve the sub-millimolar affinity with the target lectin displayed by the first-generation synthetic ligands.3,4 Methods The analysis of the fucose binding region in the BC2L-C-Nt domain identified the nucleophilic amino acid residues Cys72 and Lys108 as potential targets to develop covalent ligands. Hence, new glycomimetics were designed connecting a L-fucose scaffold through a linker to a spacer bearing an electrophilic warhead able to react with one of these residues. More than 100 compounds were screened in silico through non-covalent and covalent docking protocols. The most interesting candidates were synthetized and subjected to the first biophysical studies (TSA, SPR, MS, X-ray crystallography) showing promising results. Results The designed glycomimetics (Figure 1A) were analysed first by standard docking approaches to study the ligand-lectin interaction before the covalent bond formation. Then, a covalent docking protocol was performed, and the obtained poses were examined in detail, verifying the conformation of the ligands, the fucose binding mode and the interactions of the aglycone portion with the target lectin (Figure 1B). From this evaluation, the most promising ligands were selected and subjected to the synthesis.Preliminary studies with the new putative covalent glycomimetics were performed in collaboration with the CERMAV-CNRS Institute in Grenoble. Different biophysical techniques were exploited, such as thermal shift assays (TSA), surface plasmon resonance (SPR), mass spectrometry and X-ray crystallography. To evaluate the formation of a covalent ligand-protein adduct, mass spectrometry experiments were performed, using a MALDI-TOF instrument. For all compounds targeting Lys108, a shift in the protein peak was observed, corresponding to the mass of the adduct obtained after the condensation reaction with the lysine side chain. Mass experiments of glycomimetics targeting Cys72 were useful in assessing which compounds interact covalently with the protein, detecting in the mass spectra the peak corresponding to the protein-ligand covalent adduct. The ligand-lectin interaction was measured in a quantitative way through SPR competition assays, exploiting a fucosylated chip and injecting protein-ligand mixtures at different concentrations. Compounds targeting Lys108 provided IC50 values in the micro-molar range, displaying an improvement in the binding of about two orders of magnitude compared to non-covalent control ligands. Cys-targeting compounds yielded more variable IC50 values which however remain at most in the sub-millimolar range. A crystallization screening was performed with all the covalent compounds in order to obtain a crystal structure of the protein-ligand covalent adduct. Up to now, only one crystal of a Cys-targeting compound in complex with BC2L-C-Nt was obtained, in which the formation of the desired covalent bond with Cys72 was observed. The refinement of crystallography data is still ongoing. Conclusion With the aim to enhance the affinity and selectivity for the target bacterial lectin, new glycomimetics able to covalently bind BC2L-C-Nt’s nucleophilic residues Lys108 and Cys72 were designed and synthesized. The first biophysical studies (SPR competition assays and mass spectrometry) were performed showing promising results. In particular, compounds targeting Lys108 displayed a gain in lectin binding of about two orders of magnitude compared to non-covalent control compounds. Additional experiments to validate the interaction with the protein are ongoing.