Analogues of the pan-selectin antagonist rivipansel (GMI-1070).

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.

Tetra- and Hexavalent Siglec-8 Ligands Modulate Immune Cell Activation.

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.

Simultaneous quantification of atorvastatin, erlotinib and OSI-420 in rat serum and liver microsomes using a novel liquid chromatography-mass spectrometry method.

Erlotinib is an epidermal growth factor receptor tyrosine kinase inhibitor used in the treatment of cancer. Atorvastatin is a statin commonly applied to treat hypercholesterolemia. In humans, both compounds are metabolized by CYP3A4 and are transported by OATP2B1, ABCB1 and ABCG2. We aimed to generate and validate a bioanalytical method for simultaneous determination of atorvastatin, erlotinib and its major metabolite OSI-420 applicable to biological samples. Quantification of erlotinib, OSI-420, and atorvastatin was achieved with an Agilent high-performance liquid chromatography system 1100/1200 coupled to a triple quadrupole G6410B. The method involved separation over the column Kinetex C8 (100 × 3 mm, 2.6 µm) using 2 mM ammonium acetate (pH 4.0) and acetonitrile as eluent. The method was assessed for selectivity, accuracy, recovery, matrix effect, and stability over a range from 1 to 4,000 ng/mL according to the respective guidelines. We applied the bioanalytical method to quantify the formation of OSI-420 in liver microsomes isolated from male and female Wistar rats. The optimized experiment revealed slower formation in microsomes of female compared to male rats, in which we observed lower amounts of CYP3A1 by Western blot analysis. Moreover, the presence of atorvastatin inhibited the CYP3A-mediated metabolism of erlotinib. Serum obtained from a drug-drug interaction study performed in male rats was also analyzed using the validated method. Non-compartmental pharmacokinetic analysis revealed a lower clearance of erlotinib when atorvastatin was co-administered. However, for atorvastatin we observed a lower systemic exposure in presence of erlotinib. In summary, we report a method to detect OSI-420, erlotinib and atorvastatin applicable to samples from ex vivo and in vivo studies.

A Structural-Reporter Group to Determine the Core Conformation of Sialyl Lewisx Mimetics.

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.

Sialic Acid Derivatives Inhibit SiaT Transporters and Delay Bacterial Growth.

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.

Selective Monovalent Galectin-8 Ligands Based on 3-Lactoylgalactoside.

Galectin-8 has gained attention as a potential new pharmacological target for the treatment of various diseases, including cancer, inflammation, and disorders associated with bone mass reduction. To that end, new molecular probes are needed in order to better understand its role and its functions. Herein we aimed to improve the affinity and target selectivity of a recently published galectin-8 ligand, 3-O-[1-carboxyethyl]-ß-d-galactopyranoside, by introducing modifications at positions 1 and 3 of the galactose. Affinity data measured by fluorescence polarization show that the most potent compound reached a KD of 12 µM. Furthermore, reasonable selectivity versus other galectins was achieved, making the highlighted compound a promising lead for the development of new selective and potent ligands for galectin-8 as molecular probes to examine the protein's role in cell-based and in vivo studies.

Prodrugs of E-selectin Antagonists with Enhanced Pharmacokinetic Properties.

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.

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.

Sweet Drugs for Bad Bugs: A Glycomimetic Strategy against the DC-SIGN-Mediated Dissemination of SARS-CoV-2.

The C-type lectin receptor DC-SIGN is a pattern recognition receptor expressed on macrophages and dendritic cells. It has been identified as a promiscuous entry receptor for many pathogens, including epidemic and pandemic viruses such as SARS-CoV-2, Ebola virus, and HIV-1. In the context of the recent SARS-CoV-2 pandemic, DC-SIGN-mediated virus dissemination and stimulation of innate immune responses has been implicated as a potential factor in the development of severe COVID-19. Inhibition of virus binding to DC-SIGN, thus, represents an attractive host-directed strategy to attenuate overshooting innate immune responses and prevent the progression of the disease. In this study, we report on the discovery of a new class of potent glycomimetic DC-SIGN antagonists from a focused library of triazole-based mannose analogues. Structure-based optimization of an initial screening hit yielded a glycomimetic ligand with a more than 100-fold improved binding affinity compared to methyl α-d-mannopyranoside. Analysis of binding thermodynamics revealed an enthalpy-driven improvement of binding affinity that was enabled by hydrophobic interactions with a loop region adjacent to the binding site and displacement of a conserved water molecule. The identified ligand was employed for the synthesis of multivalent glycopolymers that were able to inhibit SARS-CoV-2 spike glycoprotein binding to DC-SIGN-expressing cells, as well as DC-SIGN-mediated trans-infection of ACE2+ cells by SARS-CoV-2 spike protein-expressing viruses, in nanomolar concentrations. The identified glycomimetic ligands reported here open promising perspectives for the development of highly potent and fully selective DC-SIGN-targeted therapeutics for a broad spectrum of viral infections.

Therapeutic Peptides as Emerging Options to Restore Misguided Host Defence and Homeostasis: From Teaching to Concept to Clinic.

Among the many molecular entities suitable for therapeutic use, peptides have emerged as a particularly attractive option for academic drug discovery and development. Their modular structure and extendibility, the availability of powerful and affordable screening platforms, and the relative ease-of-synthesis render therapeutic peptides highly approachable for teaching and research alike. With a strong focus on the therapeutic modulation of host defence pathways, including the complement and renin-angiotensin systems, the Molecular Pharmacy group at the University of Basel strongly relies on peptides to introduce students to practical aspects of modern drug design, to discover novel therapeutics for immune and inflammatory diseases, and to expand on options for the preclinical development of a promising drug class. Current projects reach from student-driven iterative design of peptidic angiotensin-converting enzyme inhibitors and the use of phage display technology to discover novel immune modulators to the development of protective peptide coatings for biomaterials and transplants and the structure-activity-relationship-guided optimization of therapeutic peptide drug candidates in late-stage clinical trials. Even at the current stage, peptides allow for a perfect circle between pharmaceutical research and education, and the recent spark of clinical applications for peptide-based drugs may only increase the value and relevance of this versatile drug class.

A Potent Mimetic of the Siglec-8 Ligand 6'-Sulfo-Sialyl Lewisx.

Siglecs are members of the immunoglobulin gene family containing sialic acid binding N-terminal domains. Among them, Siglec-8 is expressed on various cell types of the immune system such as eosinophils, mast cells and weakly on basophils. Cross-linking of Siglec-8 with monoclonal antibodies triggers apoptosis in eosinophils and inhibits degranulation of mast cells, making Siglec-8 a promising target for the treatment of eosinophil- and mast cell-associated diseases such as asthma. The tetrasaccharide 6'-sulfo-sialyl Lewisx has been identified as a specific Siglec-8 ligand in glycan array screening. Here, we describe an extended study enlightening the pharmacophores of 6'-sulfo-sialyl Lewisx and the successful development of a high-affinity mimetic. Retaining the neuraminic acid core, the introduction of a carbocyclic mimetic of the Gal moiety and a sulfonamide substituent in the 9-position gave a 20-fold improved binding affinity. Finally, the residence time, which usually is the Achilles tendon of carbohydrate/lectin interactions, could be improved.

Improvement of Aglycone π-Stacking Yields Nanomolar to Sub-nanomolar FimH Antagonists.

Antimicrobial resistance has become a serious concern for the treatment of urinary tract infections. In this context, an anti-adhesive approach targeting FimH, a bacterial lectin enabling the attachment of E. coli to host cells, has attracted considerable interest. FimH can adopt a low/medium-affinity state in the absence and a high-affinity state in the presence of shear forces. Until recently, mostly the high-affinity state has been investigated, despite the fact that a therapeutic antagonist should bind predominantly to the low-affinity state. In this communication, we demonstrate that fluorination of biphenyl α-d-mannosides leads to compounds with perfect π-π stacking interactions with the tyrosine gate of FimH, yielding low nanomolar to sub-nanomolar KD values for the low- and high-affinity states, respectively. The face-to-face alignment of the perfluorinated biphenyl group of FimH ligands and Tyr48 was confirmed by crystal structures as well as 1 H,15 N-HSQC NMR analysis. Finally, fluorination improves pharmacokinetic parameters predictive for oral availability.

Target-directed Dynamic Combinatorial Chemistry: A Study on Potentials and Pitfalls as Exemplified on a Bacterial Target.

Target-directed dynamic combinatorial chemistry (DCC) is an emerging technique for the efficient identification of inhibitors of pharmacologically relevant targets. In this contribution, we present an application for a bacterial target, the lectin FimH, a crucial virulence factor of uropathogenic E. coli being the main cause of urinary tract infections. A small dynamic library of acylhydrazones was formed from aldehydes and hydrazides and equilibrated at neutral pH in presence of aniline as nucleophilic catalyst. The major success factors turned out to be an accordingly adjusted ratio of scaffolds and fragments, an adequate sample preparation prior to HPLC analysis, and the data processing. Only then did the ranking of the dynamic library constituents correlate well with affinity data. Furthermore, as a support of DCC applications especially to larger libraries, a new protocol for improved hit identification was established.

Urinary Tract Infection: Which Conformation of the Bacterial Lectin FimH Is Therapeutically Relevant?

Frequent antibiotic treatment of urinary tract infections has resulted in the emergence of antimicrobial resistance, necessitating alternative treatment options. One such approach centers around FimH antagonists that block the bacterial adhesin FimH, which would otherwise mediate binding of uropathogenic Escherichia coli to the host urothelium to trigger the infection. Although the FimH lectin can adopt three distinct conformations, the evaluation of FimH antagonists has mainly been performed with a truncated construct of FimH locked in one particular conformation. For a successful therapeutic application, however, FimH antagonists should be efficacious against all physiologically relevant conformations. Therefore, FimH constructs with the capacity to adopt various conformations were applied. By examining the binding properties of a series of FimH antagonists in terms of binding affinity and thermodynamics, we demonstrate that depending on the FimH construct, affinities may be overestimated by a constant factor of 2 orders of magnitude. In addition, we report several antagonists with excellent affinities for all FimH conformations.

FimH Antagonists: Phosphate Prodrugs Improve Oral Bioavailability.

The widespread occurrence of urinary tract infections has resulted in frequent antibiotic treatment, contributing to the emergence of antimicrobial resistance. Alternative approaches are therefore required. In the initial step of colonization, FimH, a lectin located at the tip of bacterial type 1 pili, interacts with mannosylated glycoproteins on the urothelial mucosa. This initial pathogen/host interaction is efficiently antagonized by biaryl α-d-mannopyranosides. However, their poor physicochemical properties, primarily resulting from low aqueous solubility, limit their suitability as oral treatment option. Herein, we report the syntheses and pharmacokinetic evaluation of phosphate prodrugs, which show an improved aqueous solubility of up to 140-fold. In a Caco-2 cell model, supersaturated solutions of the active principle were generated through hydrolysis of the phosphate esters by brush border-associated enzymes, leading to a high concentration gradient across the cell monolayer. As a result, the in vivo application of phosphate prodrugs led to a substantially increased Cmax and prolonged availability of FimH antagonists in urine.

The Conformational Variability of FimH: Which Conformation Represents the Therapeutic Target?

FimH is a bacterial lectin found at the tips of type 1 pili of uropathogenic Escherichia coli (UPEC). It mediates shear-enhanced adhesion to mannosylated surfaces. Binding of UPEC to urothelial cells initiates the infection cycle leading to urinary tract infections (UTIs). Antiadhesive glycomimetics based on α-d-mannopyranose offer an attractive alternative to the conventional antibiotic treatment because they do not induce a selection pressure and are therefore expected to have a reduced resistance potential. Genetic variation of the fimH gene in clinically isolated UPEC has been associated with distinct mannose binding phenotypes. For this reason, we investigated the mannose binding characteristics of four FimH variants with mannose-based ligands under static and hydrodynamic conditions. The selected FimH variants showed individually different binding behavior under both sets of conditions as a result of the conformational variability of FimH. Clinically relevant FimH variants typically exist in a dynamic conformational equilibrium. Additionally, we evaluated inhibitory potencies of four FimH antagonists representing different structural classes. Inhibitory potencies of three of the tested antagonists were dependent on the binding phenotype and hence on the conformational equilibrium of the FimH variant. However, the squarate derivative was the notable exception and inhibited FimH variants irrespective of their binding phenotype. Information on antagonist affinities towards various FimH variants has remained largely unconsidered despite being essential for successful antiadhesion therapy.

The tyrosine gate of the bacterial lectin FimH: a conformational analysis by NMR spectroscopy and X-ray crystallography.

Urinary tract infections caused by uropathogenic E. coli are among the most prevalent infectious diseases. The mannose-specific lectin FimH mediates the adhesion of the bacteria to the urothelium, thus enabling host cell invasion and recurrent infections. An attractive alternative to antibiotic treatment is the development of FimH antagonists that mimic the physiological ligand. A large variety of candidate drugs have been developed and characterized by means of in vitro studies and animal models. Here we present the X-ray co-crystal structures of FimH with members of four antagonist classes. In three of these cases no structural data had previously been available. We used NMR spectroscopy to characterize FimH-antagonist interactions further by chemical shift perturbation. The analysis allowed a clear determination of the conformation of the tyrosine gate motif that is crucial for the interaction with aglycone moieties and was not obvious from X-ray structural data alone. Finally, ITC experiments provided insight into the thermodynamics of antagonist binding. In conjunction with the structural information from X-ray and NMR experiments the results provide a mechanism for the often-observed enthalpy-entropy compensation of FimH antagonists that plays a role in fine-tuning of the interaction.

FimH antagonists: bioisosteres to improve the in vitro and in vivo PK/PD profile.

Urinary tract infections (UTIs), predominantly caused by uropathogenic Escherichia coli (UPEC), belong to the most prevalent infectious diseases worldwide. The attachment of UPEC to host cells is mediated by FimH, a mannose-binding adhesin at the tip of bacterial type 1 pili. To date, UTIs are mainly treated with antibiotics, leading to the ubiquitous problem of increasing resistance against most of the currently available antimicrobials. Therefore, new treatment strategies are urgently needed. Here, we describe the development of an orally available FimH antagonist. Starting from the carboxylate substituted biphenyl α-d-mannoside 9, affinity and the relevant pharmacokinetic parameters (solubility, permeability, renal excretion) were substantially improved by a bioisosteric approach. With 3'-chloro-4'-(α-d-mannopyranosyloxy)biphenyl-4-carbonitrile (10j) a FimH antagonist with an optimal in vitro PK/PD profile was identified. Orally applied, 10j was effective in a mouse model of UTI by reducing the bacterial load in the bladder by about 1000-fold.

SIGLEC-4 (MAG) Antagonists: From the Natural Carbohydrate Epitope to Glycomimetics.

Siglec-4, also known as myelin-associated glycoprotein (MAG), is a member of the siglec (sialic acid-binding immunoglobulin-like lectins) family. MAG binds with high preference to sialic acids α(2-3)-linked to D-galactose. Although the involvement and relevance of its sialic acid binding activity is still controversial, it could be demonstrated that interactions of MAG with sialylated gangliosides play an important role in axon stability and regeneration. In this article we describe in detail our current understanding of the biological role and the carbohydrate specificity of siglec-4. Furthermore, this review compiles the intensive research efforts leading from the identification of the minimal oligosaccharide binding epitope in gangliosides via micromolar oligosaccharide mimics to the development of small molecular weight and more drug-like sialic acid derivatives binding with low nanomolar affinities. Such compounds will be useful to elucidate MAG's biological functions, which are currently not fully understood.

Kinetic properties of carbohydrate-lectin interactions: FimH antagonists.

The lectin FimH is terminally expressed on type 1 pili of uropathogenic Escherichia coli (UPEC), which is the main cause of urinary tract infections (UTIs). FimH enables bacterial adhesion to urothelial cells, the initial step of infection. Various mannose derivatives have been shown to antagonize FimH and are therefore considered to be promising therapeutic agents for the treatment of UTIs. As part of the preclinical development process, when the kinetic properties of FimH antagonists were examined by surface plasmon resonance, extremely low dissociation rates (k(off)) were found, which is uncommon for carbohydrate-lectin interactions. As a consequence, the corresponding half-lives (t1/2) of the FimH antagonist complexes are above 3.6 h. For a therapeutic application, extended t1/2 values are a prerequisite for success, since the target occupancy time directly influences the in vivo drug efficacy. The long t1/2 value of the tested FimH antagonists further confirms their drug-like properties and their high therapeutic potential.