Antigen uptake and processing by innate immune cells is crucial to initiate the immune response. Therein, the endocytic C-type lectin receptors serve as pattern recognition receptors, detecting pathogens by their glycan structures. Herein, we studied the carbohydrate recognition domain of Langerin, a C-type lectin receptor involved in the host defense against viruses such as HIV and influenza as well as bacteria and fungi. Using a combination of nuclear magnetic resonance and molecular dynamics simulations, we unraveled the molecular determinants underlying cargo capture and release encoded in the receptor architecture. Our findings revealed receptor dynamics over several time scales associated with binding and release of the essential cofactor Ca(2+) controlled by the coupled motions of two loops. Applying mutual information theory and site-directed mutagenesis, we identified an allosteric intradomain network that modulates the Ca(2+) affinity depending on the pH, thereby promoting fast ligand release.

DC-SIGN is a cell-surface receptor for several pathogenic threats, such as HIV, Ebola virus, or Mycobacterium tuberculosis. Multiple attempts to develop inhibitors of the underlying carbohydrate-protein interactions have been undertaken in the past fifteen years. Still, drug-like DC-SIGN ligands are sparse, which is most likely due to its hydrophilic, solvent-exposed carbohydrate-binding site. Herein, we report on a parallel fragment screening against DC-SIGN applying SPR and a reporter displacement assay, which complements previous screenings using F NMR spectroscopy and chemical fragment microarrays. Hit validation by SPR and H- N HSQC NMR spectroscopy revealed that although no fragment bound in the primary carbohydrate site, five secondary sites are available to harbor drug-like molecules. Building on key interactions of the reported fragment hits, these pockets will be targeted in future approaches to accelerate the development of DC-SIGN inhibitors.

The replacement of hydroxyl groups by fluorine atoms on hexopyranoside scaffolds may allow access to invaluable tools for studying various biochemical processes. As part of ongoing activities toward the preparation of fluorinated carbohydrates, a systematic investigation involving the synthesis and biological evaluation of a series of mono‐ and polyfluorinated galactopyranosides is described. Various monofluorogalactopyranosides, a trifluorinated, and a tetrafluorinated galactopyranoside have been prepared using a Chiron approach. Given the scarcity of these compounds in the literature, in addition to their synthesis, their biological profiles were evaluated. Firstly, the fluorinated compounds were investigated as antiproliferative agents using normal human and mouse cells in comparison with cancerous cells. Most of the fluorinated compounds showed no antiproliferative activity. Secondly, these carbohydrate probes were used as potential inhibitors of galactophilic lectins. The first transverse relaxation‐optimized spectroscopy (TROSY) NMR experiments were performed on these interactions, examining chemical shift perturbations of the backbone resonances of LecA, a virulence factor from Pseudomonas aeruginosa. Moreover, taking advantage of the fluorine atom, the 19F NMR resonances of the monofluorogalactopyranosides were directly monitored in the presence and absence of LecA to assess ligand binding. Lastly, these results were corroborated with the binding potencies of the monofluorinated galactopyranoside derivatives by isothermal titration calorimetry experiments. Analogues with fluorine atoms at C‐3 and C‐4 showed weaker affinities with LecA as compared to those with the fluorine atom at C‐2 or C‐6. This research has focused on the chemical synthesis of “drug‐like” low‐molecular‐weight inhibitors that circumvent drawbacks typically associated with natural oligosaccharides.

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Protein-glycan interactions mediate important biological processes,i ncluding pathogen host invasion and cellular communication. Herein, we showcase an expedite approach that integrates automated glycan assembly (AGA) of 19 F-labeled probes and high-throughput NMR methods,e nabling the study of protein-glycan interactions.Synthetic Lewis type 2a ntigens were screened against seven glycan binding proteins (GBPs), including DC-SIGN and BambL, respectively involved in HIV-1 and lung infections in immunocompromised patients,c onfirming the preference for fucosylated glycans (Le x ,H type 2, Le y ). Previously unknown glycanlectin weak interactions were detected, and thermodynamic data were obtained. Enzymatic reactions were monitored in real-time,d elivering kinetic parameters.T hese results demonstrate the utility of AGAc ombined with 19 FNMR for the discovery and characterization of glycan-protein interactions, opening up new perspectives for 19 F-labeled complex glycans.

CD22, a sialic-acid binding immunoglobulin type-lectin (Siglec) family member, is an inhibitory co-receptor of the B-cell receptor (BCR) with established roles in health and disease. The restricted expression pattern of CD22 on B-cells and most B-cell lymphomas has made CD22 a therapeutic target for B-cell-mediated diseases. Models to better understand how in vivo targeting of CD22 translates to human disease are needed. Here, we report development of a transgenic mouse expressing human CD22 (hCD22) in B-cells and assess its ability to functionally substitute for murine CD22 (mCD22) for regulation of BCR signaling, antibody responses, homing, and tolerance. Expression of hCD22 on transgenic murine B-cells is comparable to expression on human primary B-cells, and co-localizes with mCD22 on the cell surface. Murine B-cells expressing only hCD22 have identical calcium (Ca2+) flux responses in response to anti-IgM as mCD22-expressing WT B-cells. Furthermore, hCD22 transgenic mice on a mCD22−/− background have restored levels of marginal zone B-cells and antibody responses compared to deficiencies observed in CD22−/− mice. Consistent with these observations, hCD22 transgenic mice develop normal humoral responses in a peanut allergy oral sensitization model. Homing of B-cells to Peyer’s patches (PP) was partially rescued by expression of hCD22 compared to CD22−/− B-cells, although not to WT levels. Notably, Siglec-engaging antigenic liposomes (STALs) formulated with a hCD22 ligand were shown to prevent B-cell activation, increase cell death, and induce tolerance in vivo. This hCD22 transgenic mouse will be a valuable model for investigating the function of hCD22 and pre-clinical studies targeting hCD22.

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.