Defining the Potential of Aglycone Modifications for Affinity/Selectivity Enhancement against Medically Relevant Lectins: Synthesis, Activity Screening, and HSQC‐Based NMR Analysis
Subhash Rao RauthuTze Chieh ShiaoSabine AndréMichelle C. MillerÉlodie MadejKevin H. MayoHans‐Joachim GabiusRené Roy
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Abstract The emerging significance of lectins for pathophysiological processes provides incentive for the design of potent inhibitors. To this end, systematic assessment of contributions to affinity and selectivity by distinct types of synthetic tailoring of glycosides is a salient step, here taken for the aglyconic modifications of two disaccharide core structures. Firstly we report the synthesis of seven N‐linked‐lactosides and of eight O‐linked N ‐acetyllactosamines, each substituted with a 1,2,3‐triazole unit, prepared by copper‐catalyzed azide–alkyne cycloaddition (CuAAC). The totally regioselective β‐ D ‐(1→4) galactosylation of a 6‐ O ‐TBDPSi‐protected N ‐acetylglucosamine acceptor provided efficient access to the N ‐acetyllactosamine precursor. The resulting compounds were then systematically tested for lectin reactivity in two binding assays of increasing biorelevance (inhibition of lectin binding to a surface‐presented glycoprotein and to cell surfaces). As well as a plant toxin, we also screened the relative inhibitory potential with adhesion/growth‐regulatory galectins (total of eight proteins). This type of modification yielded up to 2.5‐fold enhancement for prototype proteins, with further increases for galectins‐3 and ‐4. Moreover, the availability of 15 N‐labeled proteins and full assignments enabled 1 H, 15 N HSQC‐based measurements for hu‐ man galectins‐1, ‐3, and ‐7 against p ‐nitrophenyl lactopyranoside, a frequently tested standard inhibitor containing an aromatic aglycone. The measurements confirmed the highest affinity against galectin‐3 and detected chemical shift differences in its hydrophobic core upon ligand binding, besides common alterations around the canonical contact site for the lactoside residue. What can be accomplished in terms of affinity/selectivity by this type of core extension having been determined, the applied combined strategy should be instrumental for proceeding with defining structure–activity correlations at other bioinspired sites in glycans and beyond the tested lectin types.Keywords:
Galectin
Aglycone
Galactosides
Binding selectivity
Disaccharide
Carbohydrates are involved in many cellular processes, and most biomolecules are glycosylated. Thesemodifications are used in biological systems as information carriers, helping regulate organization on the cell surfaceand interactions between cells and the environment. Galectins are a family of carbohydrate binding proteins thatbind to polysaccharides containing a galactose. Galectins have the ability to crosslink glycosylated proteins –especially on the cell surface – giving galectins a role in modulating cell signalling and environmental interactions,influencing angiogenesis, immune regulation and cell adhesion. This implicates galectins in diseases like cancerand immune related disorders. Subsequently, many glycomimetics have been developed as galectin inhibitors,based on a variety of scaffolds, many with very high affinities, but selectivity between galectins remains a challenge.The galectin family of proteins has a very conserved binding motif, hence the differences in the binding pocket aresmall, making designing a selective inhibitor a challenge.We investigated C1-galactosides as possible galectin inhibitor scaffolds, exploiting one of the few differencesbetween galectin-1 and galectin-3 – histidine 52. We used C1-arylheterocycles to control the selectivity via theinteraction between the anomeric heterocycle and the histidine, an approach which turned out to be fruitful resultingin the inhibitors 1-naphthyloxazole galactose, a galectin-3 selective inhibitor with 90μM affinity and 2-fluorophenyltriazole galactose, a galectin-1 selective inhibitor with a 170 μM affinity with fivefold and eightfoldselectivity respectively. Extending the C1- system with a methylene linker resulted in the galectin-1 selective 4-fluorophenyltriazole 2-deoxygalactoheptulose, an inhibitor with 170 μM affinity and fourfold selectivity. In order topursue these 2-deoxyheptulose scaffolds we developed a diastereoselective hydroboration method for C1-exomethylene glycopyranosides. Combining C1-substitutions with substitution in position three on galactose with aphenyltriazole motif did not increase affinities in a straightforward way; instead of increasing affinity and perservingselectivity patterns set by the C1-substitutents, the disubstituted molecules emerged as galectin-4 selectiveinhibitors with affinities down to 2.3 μM and up to thirty-eightfold or better selectivity for galectin-4. This shows thatC1-galactosides can be selective galectin inhibitors with good affinities, but more work needs to be done tounderstand the interaction between substitution patterns. We also investigated aminpyrimidine substitutedgalactosides and identified compounds with a threehundred-fold selectivity for galectin-3 over galectin-1 andaffinities down to 1.7 μM. These results show that careful selection of heterocycles with an aim towards exploitingeven minute differences in the binding pocket can be effective in achieving selectivity. (Less)
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Multivalent protein-carbohydrate interactions that are mediated by sugar-binding proteins, i.e., lectins, have been implicated in a myriad of intercellular recognition processes associated with tumor progression such as galectin-mediated cancer cellular migration/metastatic processes. Here, using a modified ELISA, we show that glycodendrimers bearing mixtures of galactosides, lactosides, and N-acetylgalactosaminosides, galectin-3 ligands, multivalently affect galectin-3 functions. We further demonstrate that lactose functionalized glycodendrimers multivalently bind a different member of the galectin family, i.e., galectin-1. In a modified ELISA, galectin-3 recruitment by glycodendrimers was shown to directly depend on the ratio of low to high affinity ligands on the dendrimers, with lactose-functionalized dendrimers having the highest activity and also binding well to galectin-1. The results depicted here indicate that synthetic multivalent systems and upfront assay formats will improve the understanding of the multivalent function of galectins during multivalent protein carbohydrate recognition/interaction.
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Galectin-1
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Galactans are linear polysaccharides of β(1→4)-linked galactose residues. Although they can antagonize galectin function, the nature of their binding to galectins needs to be better defined to develop them as drugs. Here, we investigated interactions between galectin-3 (Gal-3) and a series of galactans ranging in weight average molecular weight from 670 to 7550 Da. 15N-1H HSQC NMR studies with 15N-labeled Gal-3 carbohydrate recognition domain (CRD) indicate that each of these galactans interacts primarily with residues in β-strands 4, 5 and 6 on the canonical, β-galactoside sugar binding S-face. Although these galactans also bind to full length Gal-3 (CRD plus N-terminal tail) to the same extent, it appears that binding to the S-face attenuates interactions between the CRD F-face and N-terminal tail, making interpretation of site-specific binding unclear. Following assignment of galactan 13C and 1H resonances using HSQC, HMBC and TOCSY experiments, we used 13C-1H HSQC data to demonstrate that the Gal-3 CRD binds to the terminal, non-reducing end of these galactans, regardless of their size, but with binding affinity increasing as the galactan chain length increases. Overall, our findings increase understanding as to how galactans interact with Gal-3 at the non-reducing, terminal end of galactose-containing polysaccharides as found on the cell surface.
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Galectin
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Galectin-3
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Galectins have a highly conserved carbohydrate-binding domain to which a variety of galactose-containing saccharides, both β- and α-galactosides, can interact with varying degrees of affinity. Recently, we demonstrated that the relatively large α(1 → 6)-d-galacto-β(1 → 4)-d-mannan (Davanat) binds galectin-1 (gal-1) primarily at an alternative carbohydrate-binding domain. Here, we used a series of α-galactomannans (GMs) that vary in their mannose-to-galactose ratios for insight into an optimal structural signature for GM binding to gal-1. Heteronuclear single-quantum coherence nuclear magnetic resonance spectroscopy with 15N-labeled gal-1 and statistical modeling suggest that the optimal signature consists of α-d-galactopyranosyl doublets surrounded by regions of about four or more "naked" mannose residues. These relatively large and complex GMs all appear to interact with varying degrees at essentially the same binding surface on gal-1 that includes the Davanat alternative binding site and elements of the canonical β-galactoside-binding region. The use of two small, well-defined GMs [61-α(1 → 6)-d-galactosyl-β-d-mannotriaose and 63,64-di-α(1 → 6)-d-galactosyl-β-d-mannopentaose] helped characterize how GMs, in general, interact in part with the canonical site. Overall, our findings contribute to better understanding interactions of gal-1 with larger, complex polysaccharides and to the development of GM-based therapeutics for clinical use.
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Heteronuclear molecule
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Synthetic introduction of aglyconic substitutions into carbohydrate ligands is an approach toward identifying potent inhibitors of medically relevant lectins. We tested a panel of 27 galactoside/lactoside derivatives harboring varying aglycone moieties together with some O-3/O-3′ functionality toward a biohazardous plant toxin and four human adhesion/growth-regulatory galectins. Differential sensitivity profiles of lectin binding with cases showing activity increase relative to galactose/lactose were revealed by systematic assessments using a solid-phase assay. Quantitative differences between the homologous human proteins could even be detected. Binding of substituted lactosides to galectins-1 and -3 was shown to be enthalpically driven. To determine the potential of substituted glycosides to protect cells from harmful lectin association, binding assays with human tumor cells were performed. Invariably, compounds were identified with increased potency relative to the unsubstituted parent sugars. However, aglyconic substitutions were shown to be able to convey cytotoxicity. This report directs further attention to examining additional 2′- and 3′-substitutions of the galactose core and the potential of ligand presentation in glycoclusters to enhance avidity and selectivity, continuing to use the herein applied strategic combination of a convenient biochemical test system with bioassays.
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In vitro toxicology
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Glycobiology of developing chicken kidney: Profiling the galectin family and selected β‐galactosides
The concept of the sugar code interprets the cellular glycophenotype as a rich source of information read by glycan-lectin recognition in situ. This study's aim is the comprehensive characterization of galectin expression by immunohistochemistry during chicken nephrogenesis along with mapping binding sites by (ga)lectin histochemistry. Light and two-color fluorescence microscopy were used. First, six plant/fungal lectins that are specific for galectin-binding parts of N- and O-glycans were applied. The spatiotemporally regulated distributions of these glycans in meso- and metanephros equip cells with potential binding partners for the galectins. Complete galectin profiling from HH Stage 20 (about 70-72 hr) onward revealed cell-, galectin-, and stage-dependent expression patterns. Representatives of all three types of modular architecture of the galectin family are detectable, and overlaps of signal distribution in light and two-color fluorescence microscopy illustrate a possibility for functional cooperation among them. Performing systematic galectin histochemistry facilitated comparisons between staining profiles of plant lectins and galectins. They revealed several cases for differences so that tissue lectins appear to be selective among the β-galactosides. Notably, selectivity is also disclosed in intrafamily comparison. Thus, combining experimental series with plant and tissue lectins is a means to characterize target populations of glycans presented by cellular glycoconjugates for individual galectins. Our results document the presence and sophisticated level of elaboration among β-galactosides and among the members of the family of galectins during organogenesis, using chicken galectins and kidney as model. Thus, they provide a clear guideline for functional assays using supramolecular tools, cells, and organ cultures.
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Galectins exhibit multiple roles through recognition of diverse structures of β-galactosides. However, this broad specificity often hinders their practical use as probes. In the present study we report a dramatic improvement in the carbohydrate specificity of a multi-specific fungal galectin from the mushroom Agrocybe cylindricea, which binds not only to simple β-galactosides, but also to their derivatives. Site-directed mutagenesis targeting five residues involved in β-galactose binding revealed that replacement of Asn46 with alanine (N46A) increased the binding to GalNAcα1-3Galβ-containing glycans, while eliminating binding to all other β-galactosides, as shown by glycoconjugate microarray analysis. Quantitative analysis by frontal affinity chromatography showed that the mutant N46A had enhanced affinity towards blood group A tetraose (type 2), A hexaose (type 1) and Forssman pentasaccharide with dissociation constants of 5.0 × 10⁻⁶ M, 3.8 × 10⁻⁶ M and 1.0 × 10⁻⁵ M respectively. Surprisingly, all the other mutants generated by saturation mutagenesis of Asn46 exhibited essentially the same specificity as N46A. Moreover, alanine substitution for Pro45, which forms the cis-conformation upon β-galactose binding, exhibited the same specificity as N46A. From a practical viewpoint, the derived N46A mutant proved to be unique as a specific probe to detect GalNAcα1-3Galβ-containing glycans by methods such as flow cytometry, cell staining and lectin microarray.
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Alanine
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Galectins are a galactoside specific subclass of carbohydrate binding proteins (lectins) involved in various cellular activities, certain cancers, infections, inflammations, and many other biological processes. The molecular basis for the selectivity of galectins is well-documented and revolves around appropriate interaction complementarity: an aromatic residue for C-H⋅⋅⋅π interactions and polar residues for (charge assisted) hydrogen bonds with the axial hydroxyl group of a galactoside. However, no synthetic mimics are currently available. We now report on the design and synthesis of the first galectin mimic (6), and show that it has a higher than 65-fold preference for n-octyl-β-galactoside (8) over n-octyl-β-glucoside (7) in CD2 Cl2 containing 5 % [D6 ]DMSO (with Ka ≥4500 M-1 for 6:8). Molecular modeling informed by nOe studies reveal a high degree of interaction complementarity between 6 and galactoside 8, which is very similar to the interaction complementarity found in natural galectins.
Galectin
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