Ligand design and synthesis for Siglec-8 is important due to its roles in immune responses to allergens, chronic inflammatory disorders and malignancies. Here the synthesis, from readily accessible intermediates, of glycomimetics of Neu5Acα2-3 (6-O-sulfo-Gal), the minimal glycan ligand for Siglec-8, is reported. The mimetics retain Neu5Ac, while replacing the 6-O-sulfo-Gal component by a triazole containing linker presenting either carboxylate or sulfonate groups. Isothermal calorimetry (ITC) measurements showed that the sialyl triazole derivatives were ligands for Siglec-8, with a Kd of 45.5 μM established for the best ligand from the series. The ITC measurements showed that sulfonate was better than carboxylate as a replacement for sulfate, while the incorporation of the naphthylsulfonate group at the Neu5Ac C-9 of the ligands gave affinity improvement, consistent with earlier research. An NMR based binding study supports the conformational change to Siglec-8 to accommodate the 9-naphthylsulfonate group and indicates a similar mode of binding for the highest affinity triazole derivative to previously reported cyclohexane based glycomimetics. The perturbation of NMR signals, docking and binding pose metadynamics support binding of the ligand to the carbohydrate recognition domain. The research provides new Siglec-8 ligands from readily accessible intermediates as a basis for further development.

Anti-myelin associated glycoprotein (MAG) neuropathy patients exhibit high levels of monoclonal IgM autoantibodies against the carbohydrate epitope HNK-1 (human natural killer-1). This glycoepitope is abundantly presented on the adhesion molecule MAG as well as on other glycoconjugates of the peripheral nervous system. Binding of the autoantibodies results in demyelination of the peripheral nerves causing severe sensorimotor deficits in anti-MAG neuropathy patients, including paresthesias and sensory ataxia. We have previously reported the effective neutralization and removal of anti-HNK-1 IgM autoantibodies in an immunological mouse model with the glycopolymer PPSGG (poly-l-lysine400 loaded with phenyl disodium 3-O-sulfo-ß-d-glucopyranuronate)-(1 → 3)-ß-D-galactopyranoside). Here, we further explore its physicochemical characteristics and its mode of action. The linear glycopolymer PPSGG is highly negatively charged, with an approximate length of 100 nm, and is readily taken up by liver and spleen resident macrophages through scavenger receptors. No aggregate formation or immune complex deposition in these or other organs is observed and despite fast and extensive uptake of PPSGG into Kupffer cells of the liver, it does not exhibit hepatotoxic effects ex vivo. In the presence of the anti-HNK-1 IgM antibodies, it preferentially forms complexes in a 1:1 or 1:2 binding stoichiometry (PPSGG/anti-HNK-1 IgM), however, without leading to aggregate formation though crosslinking of the multivalent binding partners in vitro. The complexes are actively taken up by murine macrophages in vivo and in human macrophages in vitro. PPSGG is designed to selectively and quickly remove disease-causing anti-HNK-1 IgM antibodies from circulation. Here, we demonstrate the unique mode of action of PPSGG and its therapeutic potential as the first antigen-specific treatment for anti-MAG neuropathy.

The replacement of ionizable functional groups that are predominantly charged at physiological pH with neutral bioisosteres is a common strategy in medicinal chemistry; however, its impact on binding affinity is often context-dependent. Here, we investigated a series of amide derivatives of a glycomimetic E-selectin ligand, in which the carboxylate group of the lead compound is substituted with a range of amide and isosteric analogs. Despite the expected loss of the salt-bridge interaction with Arg97, several amides retained or even improved the binding affinity. Co-crystal structures revealed conserved binding poses across the series, with consistent interactions involving the carbonyl oxygen of the amide and the key residues Tyr48 and Arg97. High-level quantum chemical calculations ruled out a direct correlation between carbonyl partial charges and affinity. Instead, a moderate correlation was observed between ligand binding and the out-of-plane pyramidality of the amide nitrogen, suggesting a favorable steric adaptation within the binding site. Molecular dynamics (MD) simulations revealed that high-affinity ligands exhibit enhanced solution-phase pre-organization toward the bioactive conformation, likely reducing the entropic penalty upon binding. Further analysis of protein–ligand complexes using Molecular mechanics/Generalized born surface area (MM-GB/SA) decomposition suggested minor lipophilic contributions from amide substituents. Taken together, this work underscores the importance of geometric and conformational descriptors, beyond classical electrostatics, in driving affinity in glycomimetic ligand design and provides new insights into the nuanced role of amides as carboxylate isosteres in protein–ligand recognition.

The pharmacological modulation of disease-relevant carbohydrate-protein interactions represents an underexplored area of medicinal chemistry. One particular challenge in the design of glycomimetic compounds is the inherent instability of the glycosidic bond toward enzymatic cleavage. This problem has traditionally been approached by employing S-, N-, or C-glycosides with reduced susceptibility toward glycosidases. The application of ring-extended glycomimetics is an innovative approach to circumvent this issue. On the example of the bacterial adhesin FimH, it was explored how design principles from pyranose glycomimetics transfer to analogous septanose structures. A series of ring-extended FimH antagonists exhibiting the well-proven pharmacophore necessary for targeting the tyrosine-gate of FimH was synthesized. The resulting septanoses were evaluated for their affinity to the conformationally rigid isolated lectin domain of FimH (FimHLD), as well as a structurally flexible full-length FimH (FimHFL) construct. Some elements of potent mannoside-based FimH antagonists could be successfully transferred to septanose-based ligands, ultimately resulting in a 32-fold increase in binding affinity. Interestingly, the canonical ca. 100-fold loss of binding affinity between FimHLD and FimHFL is partly mitigated by the more flexible septanose antagonists, hinting at potentially differing interaction features of the flexible glycomimetics with intermediately populated states during the conformational transition of FimHFL.

The selectin family consisting of E-, P- and L-selectin plays dominant roles in atherosclerosis, ischemia-reperfusion injury, inflammatory diseases, and metastatic spreading of some cancers. An early goal in selectin-targeted drug discovery campaigns was to identify ligands binding to all three selectins, so-called pan-selectin antagonists. The physiological epitope, tetrasaccharide sialyl Lewisx (sLex, 1) binds to all selectins, albeit with very different affinities. Whereas P- and L-selectin require additional interactions contributed by sulfate groups for high binding affinity, E-selectin can functionally bind sLex-modified glycolipids and glycoproteins. Rivipansel (3) marked the first pan-selectin antagonist, which simultaneously interacted with both the sLex and the sulfate binding site. The aim of this contribution was to improve the pan-selectin affinity of rivipansel (3) by leveraging a new class of sLex mimetics in combination with an optimized linker length to the sulfate bearing group. As a result, the pan-selectin antagonist 11b exhibits an approximatively 5-fold improved affinity for E-, as well as P-selectin.

In this research article, we report on the strengthening of a non-classical hydrogen bond (C−H⋅⋅⋅O) by introducing electron withdrawing groups at the carbon atom. The approach is demonstrated on the example of derivatives of the physiological E-selectin ligand sialyl Lewis x ( 1 , sLe x ). Its affinity is mainly due to a beneficial entropy term, which is predominantly caused by the pre-organization of sLe x in its binding conformation. We have shown, that among the elements responsible for the pre-organization, the stabilization by a non-classical hydrogen bond between the H−C5 of l -fucose and the ring oxygen O5 of the neighboring d -galactose moiety is essential and yields 7.4 kJ mol −1 . This effect could be further strengthened by replacing l -fucose by 6,6,6-trifluoro- l -fucose leading to an improved non-classical H-bond of 14.9 kJ mol −1 , i.e., an improved pre-organization in the bioactive conformation. For a series of glycomimetics of sLe x ( 1 ), this outcome could be confirmed by high field NMR-shifts of the H−C5 Fuc , by X-ray diffraction analysis of glycomimetics co-crystallized with E-selectin as well as by isothermal titration calorimetry. Furthermore, the electron-withdrawing character of the CF 3 -group beneficially influences the pharmacokinetic properties of sLe x mimetics. Thus, acid-stability, a prerequisite for gastrointestinal stability, could be substantially improved.

Due to the shallow and hydrophilic binding sites of carbohydrate-binding proteins, the design of glycomimetics is often complicated by high desolvation costs as well as competition with solvent. Therefore, a careful optimization of interaction vectors and ligand properties is required in the design and optimization of glycomimetics. Here, we employ thermodynamics-guided design to optimize mannose-based glycomimetics targeting the human C-type lectin receptor dendritic cell-specific intercellular adhesion molecule 3 grabbing nonintegrin (DC-SIGN), a pathogenic host factor in viral infections. By exploring ligand rigidification and hydrogen bond engineering, a monovalent glycomimetic with an unprecedented affinity for DC-SIGN in the low μM range was discovered. A matched molecular pair analysis based on microcalorimetric data revealed a stereospecific hydrogen bond interaction with Glu358/Ser360 as the origin of this cooperative and enthalpically dominated interaction. This detailed insight into the binding mechanism paves the way for an improvement of monovalent glycomimetics targeting DC-SIGN.

Siglec-7 regulates immune cell activity and is a promising target for immunomodulation. Here, we report the discovery of novel sialic acid derivatives binding to Siglec-7. Synthesis and affinity measurements are complemented by high-quality models of sialoside-Siglec-7 complexes based on molecular dynamics (MD) simulations on the microsecond time scale. We provide details for the predicted binding modes for the new ligands, e.g., that an extension of the carbon backbone leads to a different molecular interaction pattern with the receptor and the nearby water structure than found for known Siglec-7 ligands. Further on, we uncover some shortcomings of the GLYCAM06 and GAFF2 force fields when used for the simulation of sialoside-based glycomimetics. Our results open new opportunities for the rational design of Siglec-7 inhibitors. In addition, we provide strategies on how to use and visualize MD simulations to describe and investigate sialoside-Siglec complexes in general.

Many viruses exploit the human C-type lectin receptor dendritic cell-specific ICAM-3 grabbing nonintegrin (DC-SIGN) for cell entry and virus dissemination. An inhibition of DC-SIGN-mediated virus attachment by glycan-derived ligands has, thus, emerged as a promising strategy toward broad-spectrum antiviral therapeutics. In this contribution, several cognate fragments of oligomannose- and complex-type glycans grafted onto a poly-l-lysine scaffold are evaluated as polyvalent DC-SIGN ligands. The range of selected carbohydrate epitopes encompasses linear (α- d-Man-(1→2)-α- d-Man, α- d-Man-(1→2)-α- d-Man-(1→2)-α- d-Man-(1→3)-α- d-Man) and branched (α- d-Man-(1→6)-[α- d-Man-(1→3)]-α- d-Man) oligomannosides, as well as α- l-Fuc. The thermodynamics of binding are investigated on a mono- and multivalent level to shed light on the molecular details of the interactions with the tetravalent receptor. Cellular models of virus attachment and DC-SIGN-mediated virus dissemination reveal a high potency of the presented glycopolymers in the low pico- and nanomolar ranges, respectively. The high activity of oligomannose epitopes in combination with the biocompatible properties of the poly- l-lysine scaffold highlights the potential for further preclinical development of polyvalent DC-SIGN ligands.

Mammarenaviruses are enveloped RNA viruses that can be associated with rodent-transmitted diseases in humans. Their virions are composed of a nucleocapsid surrounded by a lipid bilayer with glycoprotein (GP) spikes interacting with receptors on target cells. Both the GP and receptors are highly glycosylated, with glycosylation patterns being crucial for virus binding and cell entry, viral tropism, immune responses, or therapy strategies. These effects have been previously described for several different viruses. In case of arenaviruses, they remain insufficiently understood. Thus, it is important to determine the mechanisms of glycosylation of viral proteins and receptors responsible for infection, in order to fully understand the biology of arenaviruses. In this article, we have summarized and critically evaluated the available literature data on the glycosylation of mammarenavirus-associated proteins to facilitate further research in this field.

The d-GlcNAc moiety in sialyl Lewisx (sLex, 1) acts predominantly as a linker to position the d-Gal and the l-Fuc moieties in the bioactive spatial orientation. The hypothesis has been made that the NHAc group of GlcNAc pushes the fucose underneath the galactose and, thus, contributes to the stabilization of the bioactive conformation of the core of sLex (1). To test this hypothesis, GlcNAc mimetics consisting of (R,R)-1,2-cyclohexanediols substituted with alkyl and aryl substituents adjacent to the linking position of the fucose moiety were synthesized. To explore a broad range of extended and spatially demanding R-groups, an enzymatic approach for the synthesis of 3-alkyl/aryl-1,2-cyclohexanediols (3b-n) was applied. These cyclohexanediol derivatives were incorporated into the sLex mimetics 2b-n. For analyzing the relationship of affinity and core conformation, a 1H NMR structural-reporter-group concept was applied. Thus, the chemical shift of H-C5Fuc proved to be a sensitive indicator for the degree of pre-organization of the core of this class of sLex mimetics and therefore could be used to quantify the contribution of the R-groups.

Infectious diseases are still a major cause of death, disability, and social and economic disorder for millions of people throughout the world. Prevention and treatment strategies for infectious diseases are derived from a thorough understanding of the complex interactions between specific viral or bacterial pathogens and the human host. Glycans are found on the surfaces of all bacteria and viruses, as well as on their hosts. Thus, a majority of interactions between microbial pathogens and their hosts are based on the interaction of carbohydrate epitopes on the one hand and glycan-binding receptors on the other hand. This initially leads to the colonization of host epithelial surfaces, a prerequisite for spreading infection. In this chapter, the status of the development stage of Antiadhesive Carbohydrates and Glycomimetics is presented by means of some selected carbohydrate receptors, such as DC-SIGN, FimH PA-IL, and PA-IIL, and their carbohydrate/glycomimetic ligands.

The nature of alpha-D-mannose-natural aldohexose sugar, C-2 glucose epimer, whose intended use is for preventing urinary tract infections-in the interaction with E. coli is addressed in order to drive the issue of its regulatory classification as a medicinal product or medical device. PRISMA systematic review approach was applied; Delphi Panel method was used to target consensus on statements retrieved from evidence. Based on regulatory definitions and research evidence, the mechanism of D-mannose does not involve a metabolic or immunological action while there is uncertainty regarding the pharmacological action. Specific interaction between the product and the bacteria within the body occurs, but its nature is inert: it does not induce a direct response activating or inhibiting body processes. Moreover, the action of D-mannose takes place, even if inside the bladder, outside the epithelium on bacteria that have not yet invaded the urothelial tissue. Therefore, its mechanism of action is not directed to host structures but to structures (bacteria) external to the host's tissues. On the basis of current regulation, the uncertainty as regard a pharmacological action of alpha-D-mannose makes possible its medical device classification: new regulations and legal judgments can add further considerations. From a pharmacological perspective, research is driven versus synthetic mannosides: no further considerations are expected on alpha-D-mannose.

Carbohydrate-binding proteins are generally characterized by poor affinities for their natural glycan ligands, predominantly due to the shallow and solvent-exposed binding sites. To overcome this drawback, nature has exploited multivalency to strengthen the binding by establishing multiple interactions simultaneously. The development of oligovalent structures frequently proved to be successful, not only for proteins with multiple binding sites, but also for proteins that possess a single recognition domain. Herein we present the syntheses of a number of oligovalent ligands for Siglec-8, a monomeric I-type lectin found on eosinophils and mast cells, alongside the thermodynamic characterization of their binding. While the enthalpic contribution of each binding epitope was within a narrow range to that of the monomeric ligand, the entropy penalty increased steadily with growing valency. Additionally, we observed a successful agonistic binding of the tetra- and hexavalent and, to an even larger extent, multivalent ligands to Siglec-8 on immune cells and modulation of immune cell activation. Thus, triggering a biological effect is not restricted to multivalent ligands but could be induced by low oligovalent ligands as well, whereas a monovalent ligand, despite binding with similar affinity, showed an antagonistic effect.

Antibiotic resistance is a major worldwide concern, and new drugs with mechanistically novel modes of action are urgently needed. Here, we report the structure-based drug design, synthesis, and evaluation in vitro and in cellular systems of sialic acid derivatives able to inhibit the bacterial sialic acid symporter SiaT. We designed and synthesized 21 sialic acid derivatives and screened their affinity for SiaT by a thermal shift assay and elucidated the inhibitory mechanism through binding thermodynamics, computational methods, and inhibitory kinetic studies. The most potent compounds, which have a 180-fold higher affinity compared to the natural substrate, were tested in bacterial growth assays and indicate bacterial growth delay in methicillin-resistant Staphylococcus aureus. This study represents the first example and a promising lead in developing sialic acid uptake inhibitors as novel antibacterial agents.

Because of their large polar surface area, carbohydrates often exhibit insufficient pharmacokinetic properties. Specifically, the carboxylic acid function of the tetrasaccharide sialyl Lewisx , a pharmacophore crucial for the formation of a salt bridge with selectins, prevents oral availability. A common approach is the transfer of carboxylic acid into ester prodrugs. Once the prodrug is either actively or passively absorbed, the active principle is released by hydrolysis. In the present study, ester prodrugs of selectin antagonists with aliphatic promoieties were synthesized and their potential for oral availability was investigated in vitro and in vivo. The addition of lipophilic ester moieties to overcome insufficient lipophilicity improved passive permeation into enterocytes, however at the same time supported efflux back to the small intestines as well as oxidation into non-hydrolysable metabolites. In summary, our examples demonstrate that different modifications of carbohydrates can result in opposing effects and have to be studied in their entirety.

The recognition of oligomannose-type glycans in innate and adaptive immunity is elusive due to multiple closely related isomeric glycan structures. To explore the functions of oligomannoses, we developed a multifaceted approach combining mass spectrometry assignments of oligomannose substructures and the development of a comprehensive oligomannose microarray. This defined microarray encompasses both linear and branched glycans, varying in linkages, branching patterns, and phosphorylation status. With this resource, we identified unique recognition of oligomannose motifs by innate immune receptors, including DC-SIGN, L-SIGN, Dectin-2, and Langerin, broadly neutralizing antibodies against HIV gp120, N-acetylglucosamine-1-phosphotransferase, and the bacterial adhesin FimH. The results demonstrate that each protein exhibits a unique specificity to oligomannose motifs and suggest the potential to rationally design inhibitors to selectively block these protein-glycan interactions.

In their role as pattern-recognition receptors on cells of the innate immune system, myeloid C-type lectin receptors (CLRs) assume important biological functions related to immunity, homeostasis, and cancer. As such, this family of receptors represents an appealing target for therapeutic interventions for modulating the outcome of many pathological processes, in particular related to infectious diseases. This review summarizes the current state of research into glycomimetic or drug-like small molecule ligands for the CLRs Mincle, Langerin, and DC-SIGN, which have potential therapeutic applications in vaccine research and anti-infective therapy.

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.

Envelope glycoproteins of many viruses are heavily glycosylated. Among other functions, virusglycans can mediate interactions with host receptors and contribute to internalization and virusdissemination. The C-type lectin receptor DC-SIGN, which is expressed by cells of the innateimmune system, can act as an entry receptor for pathogens, including pandemic viruses such asSARS-CoV-2, ebola, and HIV. In the context of the recent SARS-CoV-2 pandemic, thismechanism has been linked to severe cases of COVID-19. Inhibition of the interaction betweenDC-SIGN and viral envelope glycoproteins has therefore the potential to generate broadspectrum antivirulent agents. Moreover, the important role of this mechanism in numerous viralinfections, as well as an interaction partner conserved in the host genome highlight the potentialof DC-SIGN-targeted therapeutics not only for the treatment of existing infections, but also forthe rapid response to future pandemics with newly emerging virus serotypes. Here, wedemonstrate that mannose-functionalized poly-L-lysine glycoconjugates efficiently inhibit theattachment of viral glycoproteins from SARS-CoV-2, ebola, and HIV to DC-SIGN-presentingcells with up to picomolar affinity. Treatment of susceptible cells leads to prolonged receptorinternalization and statistically significant inhibition of virus binding for up to 6 h. Furthermore,the polymers are fully biocompatible and readily cleared by the target cells. Finally, thethermodynamic analysis of these multivalent interactions revealed an entropy-driven affinityenhancement, opening promising perspectives for the future development of multivalenttherapeutics.