Prediction and Validation of a Druggable Site on Virulence Factor of Drug Resistant Burkholderia cenocepacia*.

Burkholderia cenocepacia is an opportunistic Gram-negative bacterium that causes infections in patients suffering from chronic granulomatous diseases and cystic fibrosis. It displays significant morbidity and mortality due to extreme resistance to almost all clinically useful antibiotics. The bacterial lectin BC2L-C expressed in B. cenocepacia is an interesting drug target involved in bacterial adhesion and subsequent deadly infection to the host. We solved the first high resolution crystal structure of the apo form of the lectin N-terminal domain (BC2L-C-nt) and compared it with the ones complexed with carbohydrate ligands. Virtual screening of a small fragment library identified potential hits predicted to bind in the vicinity of the fucose binding site. A series of biophysical techniques and X-ray crystallographic screening were employed to validate the interaction of the hits with the protein domain. The X-ray structure of BC2L-C-nt complexed with one of the identified active fragments confirmed the ability of the site computationally identified to host drug-like fragments. The fragment affinity could be determined by titration microcalorimetry. These structure-based strategies further provide an opportunity to elaborate the fragments into high affinity anti-adhesive glycomimetics, as therapeutic agents against B. cenocepacia.

BC2L-C N-Terminal Lectin Domain Complexed with Histo Blood Group Oligosaccharides Provides New Structural Information.

Lectins mediate adhesion of pathogens to host tissues, filling in a key role in the first steps of infection. Belonging to the opportunistic pathogen Burkholderia cenocepacia, BC2L-C is a superlectin with dual carbohydrate specificity, believed to mediate cross-linking between bacteria and host cells. Its C-terminal domain binds to bacterial mannosides while its N-terminal domain (BCL2-CN) recognizes fucosylated human epitopes. BC2L-CN presents a tumor necrosis factor alpha (TNF-) fold previously unseen in lectins with a novel fucose binding mode. We report, here, the production of a novel recombinant form of BC2L-CN (rBC2L-CN2), which allowed better protein stability and unprecedented co-crystallization with oligosaccharides. Isothermal calorimetry measurements showed no detrimental effect on ligand binding and data were obtained on the binding of Globo H hexasaccharide and l-galactose. Crystal structures of rBC2L-CN2 were solved in complex with two blood group antigens: H-type 1 and H-type 3 (Globo H) by X-ray crystallography. They provide new structural information on the binding site, of importance for the structural-based design of glycodrugs as new antimicrobials with antiadhesive properties.

Computational tools for drawing, building and displaying carbohydrates: a visual guide.

Drawing and visualisation of molecular structures are some of the most common tasks carried out in structural glycobiology, typically using various software. In this perspective article, we outline developments in the computational tools for the sketching, visualisation and modelling of glycans. The article also provides details on the standard representation of glycans, and glycoconjugates, which helps the communication of structure details within the scientific community. We highlight the comparative analysis of the available tools which could help researchers to perform various tasks related to structure representation and model building of glycans. These tools can be useful for glycobiologists or any researcher looking for a ready to use, simple program for the sketching or building of glycans.

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

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.