No abstract

Because of the antimicrobial resistance crisis,lectins are considered novel drug targets.P seudomonas aeruginosa utilizes LecA and LecB in the infection process.I nhibition of both lectins with carbohydrate-derived molecules can reduce biofilm formation to restore antimicrobial susceptibility.Here, we focused on non-carbohydrate inhibitors for LecA to explore new avenues for lectin inhibition. From as creening cascade we obtained one experimentally confirmed hit, ac atechol, belonging to the well-known PAINS compounds. Rigorous analyses validated electron-deficient catechols as millimolar LecA inhibitors.T he first co-crystal structure of anon-carbohydrate inhibitor in complex with abacterial lectin clearly demonstrates the catechol mimicking the binding of natural glycosides with LecA. Importantly,c atechol 3 is the first non-carbohydrate lectin ligand that binds bacterial and mammalian calcium(II)-binding lectins,g iving rise to this fundamentally new class of glycomimetics.

Abstract The carbohydrate−binding protein LecA (PA-IL) from Pseudomonas aeruginosa plays an important role in the formation of biofilms in chronic infections. Development of inhibitors to disrupt LecA−mediated biofilms is desired, but limited to carbohydrate−based ligands. Moreover, discovery of drug−like ligands for LecA is challenging due to its weak affinities. Therefore, we established a protein−observed 19F (PrOF) NMR to probe ligand binding to LecA. LecA was labeled with 5 − fluoroindole to incorporate 5 − fluorotryptophanes and the resonances were assigned by site−directed mutagenesis. This incorporation did not disrupt LecA preference for natural ligands, Ca2+ and d − galactose. Following NMR resonance perturbation of W42, which is located in the carbohydrate−binding region of LecA, allowed to monitor binding of low affinity ligands such as N − acetyl d − galactosamine (d − GalNAc, Kd = 780 ± 97 μM). Moreover, PrOF NMR titration with glycomimetic of LecA p-nitrophenyl β-d-galactoside (pNPGal, Kd = 54 ± 6 μM) demonstrated a six-fold improved binding of d − Gal proving this approach to be valuable for ligand design in future drug discovery campaigns that aim to generate inhibitors of LecA.

Macrocyclic peptides have promising therapeutic potential but the scaling up of their chemical synthesis is challenging. The cyanobactin macrocyclase PatG is an efficient tool for production but is limited to substrates containing 6-11 amino acids and at least one thiazoline or proline. Here we report a new cyanobactin macrocyclase that can cyclize longer peptide substrates and those not containing proline/thiazoline and thus allows exploring a wider chemical diversity.

Pseudomonas aeruginosa is an opportunistic ESKAPE pathogen that produces two lectins, LecA and LecB, as part of its large arsenal of virulence factors. Both carbohydrate‐binding proteins are central to the initial and later persistent infection processes, i. e. bacterial adhesion and biofilm formation. The biofilm matrix is a major resistance determinant and protects the bacteria against external threats such as the host immune system or antibiotic treatment. Therefore, the development of drugs against the P. aeruginosa biofilm is of particular interest to restore efficacy of antimicrobials. Carbohydrate‐based inhibitors for LecA and LecB were previously shown to efficiently reduce biofilm formations. Here, we report a new approach for inhibiting LecA with synthetic molecules bridging the established carbohydrate‐binding site and a central cavity located between two LecA protomers of the lectin tetramer. Inspired by in silico design, we synthesized various galactosidic LecA inhibitors with aromatic moieties targeting this central pocket. These compounds reached low micromolar affinities, validated in different biophysical assays. Finally, X‐ray diffraction analysis revealed the interactions of this compound class with LecA. This new mode of action paves the way to a novel route towards inhibition of P. aeruginosa biofilms.

Carbohydrate-protein interactions are key for cell-cell and host-pathogen recognition and thus, emerged as viable therapeutic targets. However, their hydrophilic nature poses major limitations to the conventional development of drug-like inhibitors. To address this shortcoming, four fragment libraries were screened to identify metal-binding pharmacophores (MBPs) as novel scaffolds for inhibition of Ca2+-dependent carbohydrate-protein interactions. Here, we show the effect of MBPs on the clinically relevant lectins DC-SIGN, Langerin, LecA and LecB. Detailed structural and biochemical investigations revealed the specificity of MBPs for different Ca2+-dependent lectins. Exploring the structure-activity relationships of several fragments uncovered the functional groups in the MBPs suitable for modification to further improve lectin binding and selectivity. Selected inhibitors bound efficiently to DC-SIGN-expressing cells. Altogether, the discovery of MBPs as a promising class of Ca2+-dependent lectin inhibitors creates a foundation for fragment-based ligand design for future drug discovery campaigns.

Because of the antimicrobial resistance crisis,lectins are considered novel drug targets.P seudomonas aeruginosa utilizes LecA and LecB in the infection process.I nhibition of both lectins with carbohydrate-derived molecules can reduce biofilm formation to restore antimicrobial susceptibility.Here, we focused on non-carbohydrate inhibitors for LecA to explore new avenues for lectin inhibition. From as creening cascade we obtained one experimentally confirmed hit, ac atechol, belonging to the well-known PAINS compounds. Rigorous analyses validated electron-deficient catechols as millimolar LecA inhibitors.T he first co-crystal structure of anon-carbohydrate inhibitor in complex with abacterial lectin clearly demonstrates the catechol mimicking the binding of natural glycosides with LecA. Importantly,c atechol 3 is the first non-carbohydrate lectin ligand that binds bacterial and mammalian calcium(II)-binding lectins,g iving rise to this fundamentally new class of glycomimetics.

The Gram-negative pathogen Pseudomonas aeruginosa causes severe infections mainly in immunocompromised or cystic fibrosis patients and is able to resist antimicrobial treatments. The extracellular lectin LecB plays a key role in bacterial adhesion to the host and biofilm formation. For the inhibition of LecB, we designed and synthesized a set of fucosyl amides, sulfonamides, and thiourea derivatives. Then, we analyzed their binding to LecB in competitive and direct binding assays. We identified β-fucosyl amides as unprecedented high-affinity ligands in the two-digit nanomolar range. X-ray crystallography of one α- and one β-anomer of N-fucosyl amides in complex with LecB revealed the interactions responsible for the high affinity of the β-anomer at atomic level. Further, the molecules showed good stability in murine and human blood plasma and hepatic metabolism, providing a basis for future development into antibacterial drugs.