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

Abstract

Burkholderia cenocepacia is an opportunistic Gramnegative 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.

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

“…The nature of these fragments is often defined by trial and error. The work we report here provides experimental validation to earlier work 26 describing the virtual screening of fragment libraries in the monosaccharide-lectin complex and thus shows that virtual screening of fragment libraries in the lectin complex of monosaccharides is an appropriate tool for fragment selection and rational design of glycomimetic structures. Finally, we have solved the first crystal structures of BC2L-C-Nt complexes with synthetic ligands, validating our computational and experimental work so far, as well as the choice of amide linkers.…”

“…This area is not occupied by the native oligosaccharide ligands, which extend from the α-face of the fucose ring, but is located at the interface of two protomers in the BC2L-C-Nt trimer. Virtual screening of a fragment library in the BC2L-C-Nt complex with α-methylselenyl-fucoside (PDB 2WQ4) resulted in the selection of molecular fragments predicted to bind the vicinal site, which were validated by biophysical techniques . Selected fragments are used, here, for the design of new bifunctional molecules, generated by connecting the fragment to a fucoside core.…”

“…Concerning the linker, a single “fit-all” function was not possible, as the structures, orientations, and distances to the anomeric position varied from fragment to fragment . Instead, a set of linkers were explored, with different characteristics in terms of bridging length, angle, flexibility, bulkiness, polarity, and metabolic stability.…”

“…The O -benzyl intermediate 3a was used to generate bifunctional molecules featuring a propargylic alcohol moiety. Indeed, some of the fragment screening hits bore a hydroxyl group directed toward the binding site and predicted to replace a crystallographic conserved water molecule . Entropically speaking, successfully replacing an ordered water molecule while maintaining its interactions can translate into a considerable affinity gain.…”

“…Thanks to the structural data obtained, computational work allowed mapping the protein surface for potential ligandable sites. A fragment screening campaign validated by X-ray data provided a small library of molecular entities predicted to bind near the fucose-occupied binding site . Based on these results, we now have rationally designed and synthesized a panel of first-generation BC2L-C-Nt glycomimetic ligands.…”