Secondary Sites of the C-type Lectin-Like Fold.

C-type lectins are a large superfamily of proteins involved in a multitude of biological processes. In particular, their involvement in immunity and homeostasis has rendered them attractive targets for diverse therapeutic interventions. They share a characteristic C-type lectin-like domain whose adaptability enables them to bind a broad spectrum of ligands beyond the originally defined canonical Ca2+-dependent carbohydrate binding. Together with variable domain architecture and high-level conformational plasticity, this enables C-type lectins to meet diverse functional demands. Secondary sites provide another layer of regulation and are often intricately linked to functional diversity. Located remote from the canonical primary binding site, secondary sites can accommodate ligands with other physicochemical properties and alter protein dynamics, thus enhancing selectivity and enabling fine-tuning of the biological response. In this review, we outline the structural determinants allowing C-type lectins to perform a large variety of tasks and to accommodate the ligands associated with it. Using the six well-characterized Ca2+-dependent and Ca2+-independent C-type lectin receptors DC-SIGN, langerin, MGL, dectin-1, CLEC-2 and NKG2D as examples, we focus on the characteristics of non-canonical interactions and secondary sites and their potential use in drug discovery endeavors.

Glycomimetics for the inhibition and modulation of lectins.

Carbohydrates are essential mediators of many processes in health and disease. They regulate self-/non-self- discrimination, are key elements of cellular communication, cancer, infection and inflammation, and determine protein folding, function and life-times. Moreover, they are integral to the cellular envelope for microorganisms and participate in biofilm formation. These diverse functions of carbohydrates are mediated by carbohydrate-binding proteins, lectins, and the more the knowledge about the biology of these proteins is advancing, the more interfering with carbohydrate recognition becomes a viable option for the development of novel therapeutics. In this respect, small molecules mimicking this recognition process become more and more available either as tools for fostering our basic understanding of glycobiology or as therapeutics. In this review, we outline the general design principles of glycomimetic inhibitors (Section 2). This section is then followed by highlighting three approaches to interfere with lectin function, i.e. with carbohydrate-derived glycomimetics (Section 3.1), novel glycomimetic scaffolds (Section 3.2) and allosteric modulators (Section 3.3). We summarize recent advances in design and application of glycomimetics for various classes of lectins of mammalian, viral and bacterial origin. Besides highlighting design principles in general, we showcase defined cases in which glycomimetics have been advanced to clinical trials or marketed. Additionally, emerging applications of glycomimetics for targeted protein degradation and targeted delivery purposes are reviewed in Section 4.

Antigen targeting to dendritic cells: Still a place in future immunotherapy?

The hallmark of DCs is their potent and outstanding capacity to activate naive resting T cells. As such, DCs are the sentinels of the immune system and instrumental for the induction of immune responses. This is one of the reasons, why DCs became the focus of immunotherapeutical strategies to fight infections, cancer, and autoimmunity. Besides the exploration of adoptive DC-therapy for which DCs are generated from monocytes or purified in large numbers from the blood, alternative approaches were developed such as antigen targeting of DCs. The idea behind this strategy is that DCs resident in patients' lymphoid organs or peripheral tissues can be directly loaded with antigens in situ. The proof of principle came from mouse models; subsequent translational studies confirmed the potential of this therapy. The first clinical trials demonstrated feasibility and the induction of T-cell immunity in patients. This review will cover: (i) the historical aspects of antigen targeting, (ii) briefly summarize the biology of DCs and the immunological functions upon which this concept rests, (iii) give an overview on attempts to target DC receptors with antibodies or (glycosylated) ligands, and finally, (iv) discuss the translation of antigen targeting into clinical therapy.

The molecular basis for the pH-dependent calcium affinity of the pattern recognition receptor langerin.

The C-type lectin receptor langerin plays a vital role in the mammalian defense against invading pathogens. Langerin requires a Ca2+ cofactor, the binding affinity of which is regulated by pH. Thus, Ca2+ is bound when langerin is on the membrane but released when langerin and its pathogen substrate traffic to the acidic endosome, allowing the substrate to be degraded. The change in pH is sensed by protonation of the allosteric pH sensor histidine H294. However, the mechanism by which Ca2+ is released from the buried binding site is not clear. We studied the structural consequences of protonating H294 by molecular dynamics simulations (total simulation time: about 120 µs) and Markov models. We discovered a relay mechanism in which a proton is moved into the vicinity of the Ca2+-binding site without transferring the initial proton from H294. Protonation of H294 unlocks a conformation in which a protonated lysine side chain forms a hydrogen bond with a Ca2+-coordinating aspartic acid. This destabilizes Ca2+ in the binding pocket, which we probed by steered molecular dynamics. After Ca2+ release, the proton is likely transferred to the aspartic acid and stabilized by a dyad with a nearby glutamic acid, triggering a conformational transition and thus preventing Ca2+ rebinding. These results show how pH regulation of a buried orthosteric binding site from a solvent-exposed allosteric pH sensor can be realized by information transfer through a specific chain of conformational arrangements.

Targeting the Central Pocket of the Pseudomonas aeruginosa Lectin LecA.

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.

Microbe-focused glycan array screening platform.

Interactions between glycans and glycan binding proteins are essential for numerous processes in all kingdoms of life. Glycan microarrays are an excellent tool to examine protein-glycan interactions. Here, we present a microbe-focused glycan microarray platform based on oligosaccharides obtained by chemical synthesis. Glycans were generated by combining different carbohydrate synthesis approaches including automated glycan assembly, solution-phase synthesis, and chemoenzymatic methods. The current library of more than 300 glycans is as diverse as the mammalian glycan array from the Consortium for Functional Glycomics and, due to its microbial focus, highly complementary. This glycan platform is essential for the characterization of various classes of glycan binding proteins. Applications of this glycan array platform are highlighted by the characterization of innate immune receptors and bacterial virulence factors as well as the analysis of human humoral immunity to pathogenic glycans.

Druggable Allosteric Sites in ß-Propeller Lectins.

Carbohydrate-binding proteins (lectins) are auspicious targets in drug discovery to combat antimicrobial resistance; however, their non-carbohydrate drug-like inhibitors are still unavailable. Here, we present a druggable pocket in a ß-propeller lectin BambL from Burkholderia ambifaria as a potential target for allosteric inhibitors. This site was identified employing 19 F NMR fragment screening and a computational pocket prediction algorithm SiteMap. The structure-activity relationship study revealed the most promising fragment with a dissociation constant of 0.3±0.1 mM and a ligand efficiency of 0.3 kcal mol-1 HA-1 that affected the orthosteric site. This effect was substantiated by site-directed mutagenesis in the orthosteric and secondary pockets. Future drug-discovery campaigns that aim to develop small molecule inhibitors can benefit from allosteric sites in lectins as a new therapeutic approach against antibiotic-resistant pathogens.

Protein-observed 19F NMR of LecA from Pseudomonas aeruginosa.

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 it is limited to carbohydrate-based ligands. Moreover, discovery of drug-like ligands for LecA is challenging because of its weak affinities. Therefore, we established a protein-observed 19F (PrOF) nuclear magnetic resonance (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 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 ß-d-galactoside (pNPGal, Kd = 54 ± 6 µM) demonstrated a 6-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.

A Remote Secondary Binding Pocket Promotes Heteromultivalent Targeting of DC-SIGN.

Dendritic cells (DC) are antigen-presenting cells coordinating the interplay of the innate and the adaptive immune response. The endocytic C-type lectin receptors DC-SIGN and Langerin display expression profiles restricted to distinct DC subtypes and have emerged as prime targets for next-generation immunotherapies and anti-infectives. Using heteromultivalent liposomes copresenting mannosides bearing aromatic aglycones with natural glycan ligands, we serendipitously discovered striking cooperativity effects for DC-SIGN+ but not for Langerin+ cell lines. Mechanistic investigations combining NMR spectroscopy with molecular docking and molecular dynamics simulations led to the identification of a secondary binding pocket for the glycomimetics. This pocket, located remotely of DC-SIGN's carbohydrate bindings site, can be leveraged by heteromultivalent avidity enhancement. We further present preliminary evidence that the aglycone allosterically activates glycan recognition and thereby contributes to DC-SIGN-specific cell targeting. Our findings have important implications for both translational and basic glycoscience, showcasing heteromultivalent targeting of DCs to improve specificity and supporting potential allosteric regulation of DC-SIGN and CLRs in general.

Automated Glycan Assembly of 19 F-labeled Glycan Probes Enables High-Throughput NMR Studies of Protein-Glycan Interactions.

Protein-glycan interactions mediate important biological processes, including 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, enabling the study of protein-glycan interactions. Synthetic Lewis type 2 antigens were screened against seven glycan binding proteins (GBPs), including DC-SIGN and BambL, respectively involved in HIV-1 and lung infections in immunocompromised patients, confirming the preference for fucosylated glycans (Lex , H type 2, Ley ). Previously unknown glycan-lectin weak interactions were detected, and thermodynamic data were obtained. Enzymatic reactions were monitored in real-time, delivering kinetic parameters. These results demonstrate the utility of AGA combined with 19 F NMR for the discovery and characterization of glycan-protein interactions, opening up new perspectives for 19 F-labeled complex glycans.

Non-Carbohydrate Glycomimetics as Inhibitors of Calcium(II)-Binding Lectins.

Because of the antimicrobial resistance crisis, lectins are considered novel drug targets. Pseudomonas aeruginosa utilizes LecA and LecB in the infection process. Inhibition 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 a screening cascade we obtained one experimentally confirmed hit, a catechol, belonging to the well-known PAINS compounds. Rigorous analyses validated electron-deficient catechols as millimolar LecA inhibitors. The first co-crystal structure of a non-carbohydrate inhibitor in complex with a bacterial lectin clearly demonstrates the catechol mimicking the binding of natural glycosides with LecA. Importantly, catechol 3 is the first non-carbohydrate lectin ligand that binds bacterial and mammalian calcium(II)-binding lectins, giving rise to this fundamentally new class of glycomimetics.

On-Chip Neo-Glycopeptide Synthesis for Multivalent Glycan Presentation.

Single glycan-protein interactions are often weak, such that glycan binding partners commonly utilize multiple, spatially defined binding sites to enhance binding avidity and specificity. Current array technologies usually neglect defined multivalent display. Laser-based array synthesis technology allows for flexible and rapid on-surface synthesis of different peptides. By combining this technique with click chemistry, neo-glycopeptides were produced directly on a functionalized glass slide in the microarray format. Density and spatial distribution of carbohydrates can be tuned, resulting in well-defined glycan structures for multivalent display. The two lectins concanavalin A and langerin were probed with different glycans on multivalent scaffolds, revealing strong spacing-, density-, and ligand-dependent binding. In addition, we could also measure the surface dissociation constant. This approach allows for a rapid generation, screening, and optimization of a multitude of multivalent scaffolds for glycan binding.

Rational Design of a DNA-Scaffolded High-Affinity Binder for Langerin.

Binders of langerin could target vaccines to Langerhans cells for improved therapeutic effect. Since langerin has low affinity for monovalent glycan ligands, highly multivalent presentation has previously been key for targeting. Aiming to reduce the amount of ligand required, we rationally designed molecularly defined high-affinity binders based on the precise display of glycomimetic ligands (Glc2NTs) on DNA-PNA scaffolds. Rather than mimicking langerin's homotrimeric structure with a C3-symmetric scaffold, we developed readily accessible, easy-to-design bivalent binders. The method considers the requirements for bridging sugar binding sites and statistical rebinding as a means to both strengthen the interactions at single binding sites and amplify the avidity enhancement provided by chelation. This gave a 1150-fold net improvement over the affinity of the free ligand and provided a nanomolar binder (IC50 =300 nM) for specific internalization by langerin-expressing cells.

The 3F Library: Fluorinated Fsp3 -Rich Fragments for Expeditious 19 F†NMR Based Screening.

Fragment-based drug discovery (FBDD) is a popular method in academia and the pharmaceutical industry for the discovery of early lead candidates. Despite its wide-spread use, the approach still suffers from laborious screening workflows and a limited diversity in the fragments applied. Presented here is the design, synthesis, and biological evaluation of the first fragment library specifically tailored to tackle both these challenges. The 3F library of 115 fluorinated, Fsp3 -rich fragments is shape diverse and natural-product-like with desirable physicochemical properties. The library is perfectly suited for rapid and efficient screening by NMR spectroscopy in a two-stage workflow of 19 F NMR and subsequent 1 H NMR methods. Hits against four diverse protein targets are widely distributed among the fragment scaffolds in the 3F library and a 67 % validation rate was achieved using secondary assays. This collection is the first synthetic fragment library tailor-made for 19 F NMR screening and the results demonstrate that the approach should find broad application in the FBDD community.

A Liposomal Platform for Delivery of a Protein Antigen to Langerin-Expressing Cells.

The skin is an attractive site for vaccination and harbors a dense network of Langerhans cells that are the prime target for antigen delivery approaches in the epidermis. While specific targeting of Langerhans cells has been shown to elicit the necessary T-cell response using antibody-based delivery approaches, the targeted administration of particulate antigens in the form of nanoparticle-based vaccine formulations has been challenging. We previously reported on a specific targeting ligand for human Langerin, a C-type lectin expressed on Langerhans cells. This ligand is presented on liposomes and renders them highly specific for the uptake by Langerhans cells. Here we show a detailed study of the uptake and intracellular routing of the particles in model cell lines by confocal and live cell imaging as well as flow cytometric assays. Liposomes are internalized into early endosomal compartments and accumulate in late endosomes and lysosomes, shortly followed by a release of the cargo. Furthermore, we show the encapsulation of protein antigens and their delivery to cell lines and primary human Langerhans cells. These data further support the applicability of the targeted liposomal particles for protein vaccine applications.

Stereoselective Synthesis of Fluorinated Galactopyranosides as Potential Molecular Probes for Galactophilic Proteins: Assessment of Monofluorogalactoside-LecA Interactions.

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 19 F 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.

Asymmetrically Branched Precision Glycooligomers Targeting Langerin.

Asymmetrically branched precision glycooligomers are synthesized by solid-phase polymer synthesis for studying multivalent carbohydrate-protein interactions. Through the stepwise assembly of Fmoc-protected oligo(amidoamine) building blocks and Fmoc/Dde-protected lysine, straightforward variation of structural parameters such as the number and length of arms, as well as the number and position of carbohydrate ligands, is achieved. Binding of 1-arm and 3-arm glycooligomers toward lectin receptors langerin and concanavalin A (ConA) was evaluated where the smallest 3-arm glycooligomer shows the highest binding toward langerin, and stepwise elongation of one, two, or all three arms leads to decreased binding. When directly comparing binding toward langerin and ConA, we find that structural variation of the scaffold affects glycomimetic ligand binding differently for the different targets, indicating the potential to tune such ligands not only for their avidity but also for their selectivity toward different lectins.

Bacterial Polysaccharide Specificity of the Pattern Recognition Receptor Langerin Is Highly Species-dependent.

The recognition of pathogen surface polysaccharides by glycan-binding proteins is a cornerstone of innate host defense. Many members of the C-type lectin receptor family serve as pattern recognition receptors facilitating pathogen uptake, antigen processing, and immunomodulation. Despite the high evolutionary pressure in host-pathogen interactions, it is still widely assumed that genetic homology conveys similar specificities. Here, we investigate the ligand specificities of the human and murine forms of the myeloid C-type lectin receptor langerin for simple and complex ligands augmented by structural insight into murine langerin. Although the two homologs share the same three-dimensional structure and recognize simple ligands identically, a screening of more than 300 bacterial polysaccharides revealed highly diverging avidity and selectivity for larger and more complex glycans. Structural and evolutionary conservation analysis identified a highly variable surface adjacent to the canonic binding site, potentially forming a secondary site of interaction for large glycans.

Allosteric Inhibition of a Mammalian Lectin.

Glycan-binding proteins are key components of central physiological and cellular processes such as self-/non-self-recognition, cellular tissue homing, and protein homeostasis. Herein, C-type lectins are a diverse protein family that play important roles in the immune system, rendering them attractive drug targets. To evaluate C-type lectin receptors as target proteins for small-molecule effectors, chemical probes are required, which are, however, still lacking. To overcome the supposedly poor druggability of C-type lectin receptors and to identify starting points for chemical probe development, we screened murine langerin using 1H and 19F NMR against a library of 871 drug-like fragments. Subsequently, hits were validated by surface plasmon resonance and enzyme-linked lectin assay. Using structure-activity relationship studies and chemical synthesis, we identified thiazolopyrimidine derivatives with double-digit micromolar activity that displayed langerin selectivity. Based on 1H-15N HSQC NMR and competitive binding and inhibition experiments, we demonstrate that thiazolopyrimidines allosterically inhibit langerin. To the best of our knowledge, this is the first report of drug-like allosteric inhibitors of a mammalian lectin.

Glycomimetic, Orally Bioavailable LecB Inhibitors Block Biofilm Formation of Pseudomonas aeruginosa.

The opportunistic Gram-negative bacterium Pseudomonas aeruginosa is a leading pathogen for infections of immuno-compromised patients and those suffering from cystic fibrosis. Its ability to switch from planktonic life to aggregates, forming the so-called biofilms, is a front-line mechanism of antimicrobial resistance. The bacterial carbohydrate-binding protein LecB is an integral component and necessary for biofilm formation. Here, we report a new class of drug-like low molecular weight inhibitors of the lectin LecB with nanomolar affinities and excellent receptor binding kinetics and thermodynamics. This class of glycomimetic inhibitors efficiently blocked biofilm formation of P. aeruginosa in vitro while the natural monovalent carbohydrate ligands failed. Furthermore, excellent selectivity and pharmacokinetic properties were achieved. Notably, two compounds showed good oral bioavailability, and high compound concentrations in plasma and urine were achieved in vivo.