High-Mannose Oligosaccharide Hemimimetics that Recapitulate the Conformation and Binding Mode to Concanavalin A, DC-SIGN and Langerin.

The "carbohydrate chemical mimicry" exhibited by sp2 -iminosugars has been utilized to develop practical syntheses for analogs of the branched high-mannose-type oligosaccharides (HMOs) Man3 and Man5 . In these compounds, the terminal nonreducing Man residues have been substituted with 5,6-oxomethylidenemannonojirimycin (OMJ) motifs. The resulting oligomannoside hemimimetic accurately reproduce the structure, configuration, and conformational behavior of the original mannooligosaccharides, as confirmed by NMR and computational techniques. Binding studies with mannose binding lectins, including concanavalin A, DC-SIGN, and langerin, by enzyme-linked lectin assay and surface plasmon resonance revealed significant variations in their ability to accommodate the OMJ unit in the mannose binding site. Intriguingly, OMJMan segments demonstrated "in line" heteromultivalent effects during binding to the three lectins. Similar to the mannobiose (Man2 ) branches in HMOs, the binding modes involving the external or internal monosaccharide unit at the carbohydrate binding-domain exist in equilibrium, facilitating sliding and recapture processes. This equilibrium, which influences the multivalent binding of HMOs, can be finely modulated upon incorporation of the OMJ sp2 -iminosugar caps. As a proof of concept, the affinity and selectivity towards DC-SIGN and langerin were adjustable by presenting the OMJMan epitope in platforms with diverse architectures and valencies.

Processing of social and monetary rewards in autism spectrum disorders.

BACKGROUND: Reward processing has been proposed to underpin the atypical social feature of autism spectrum disorder (ASD). However, previous neuroimaging studies have yielded inconsistent results regarding the specificity of atypicalities for social reward processing in ASD. AIMS: Utilising a large sample, we aimed to assess reward processing in response to reward type (social, monetary) and reward phase (anticipation, delivery) in ASD. METHOD: Functional magnetic resonance imaging during social and monetary reward anticipation and delivery was performed in 212 individuals with ASD (7.6-30.6 years of age) and 181 typically developing participants (7.6-30.8 years of age). RESULTS: Across social and monetary reward anticipation, whole-brain analyses showed hypoactivation of the right ventral striatum in participants with ASD compared with typically developing participants. Further, region of interest analysis across both reward types yielded ASD-related hypoactivation in both the left and right ventral striatum. Across delivery of social and monetary reward, hyperactivation of the ventral striatum in individuals with ASD did not survive correction for multiple comparisons. Dimensional analyses of autism and attention-deficit hyperactivity disorder (ADHD) scores were not significant. In categorical analyses, post hoc comparisons showed that ASD effects were most pronounced in participants with ASD without co-occurring ADHD. CONCLUSIONS: Our results do not support current theories linking atypical social interaction in ASD to specific alterations in social reward processing. Instead, they point towards a generalised hypoactivity of ventral striatum in ASD during anticipation of both social and monetary rewards. We suggest this indicates attenuated reward seeking in ASD independent of social content and that elevated ADHD symptoms may attenuate altered reward seeking in ASD.

Advanced high-affinity glycoconjugate ligands of galectins.

Galectins are proteins of the family of human lectins. By binding terminal galactose units of cell surface glycans, they moderate biological and pathological processes such as cell signaling, cell adhesion, apoptosis, fibrosis, carcinogenesis, and metabolic disorders. The binding of monovalent glycans to galectins is usually relatively weak. Therefore, the presentation of carbohydrate ligands on multivalent scaffolds can efficiently increase and/or discriminate the affinity of the glycoconjugate to different galectins. A library of glycoclusters and glycodendrimers with various structural presentations of the common functionalized N-acetyllactosamine ligand was prepared to evaluate how the mode of presentation affects the affinity and selectivity to the two most abundant galectins, galectin-1 (Gal-1) and galectin-3 (Gal-3). In addition, the effect of a one- to two-unit carbohydrate spacer on the affinity of the glycoconjugates was determined. A new design of the biolayer interferometry (BLI) method with specific AVI-tagged constructs was used to determine the affinity to galectins, and compared with the gold-standard method of isothermal titration calorimetry (ITC). This study reveals new routes to low nanomolar glycoconjugate inhibitors of galectins of interest for biomedical research.

Multivalent glycocyclopeptides: conjugation methods and biological applications.

Click chemistry was extensively used to decorate synthetic multivalent scaffolds with glycans to mimic the cell surface glycocalyx and to develop applications in glycosciences. Conjugation methods such as oxime ligation, copper(I)-catalyzed alkyne-azide cycloaddition, thiol-ene coupling, squaramide coupling or Lansbury aspartylation proved particularly suitable to achieve this purpose. This review summarizes the synthetic strategies that can be used either in a stepwise manner or in an orthogonal one-pot approach, to conjugate multiple copies of identical or different glycans to cyclopeptide scaffolds (namely multivalent glycocyclopeptides) having different size, valency, geometry and molecular composition. The second part of this review will describe the potential of these structures to interact with various carbohydrate binding proteins or to stimulate immunity against tumor cells.

Correction to "Discovery of Novel α-Carboline Inhibitors of the Anaplastic Lymphoma Kinase".

[This corrects the article DOI: 10.1021/acsomega.2c00507.].

Characterization of the interaction of multivalent glycosylated ligands with bacterial lectins by biolayer interferometry.

The study of multivalent carbohydrate-protein interactions remains highly complicated and sometimes rendered impossible due to aggregation problems. Biolayer interferometry is emerging as a tool to monitor such complex interactions. In this study, various glycoclusters and dendrimers were prepared and evaluated as ligands for lectins produced by pathogenic bacteria Pseudomonas aeruginosa (LecA and Lec B) and Burkholderia ambifaria (BambL). Reliable kinetic and thermodynamic parameters could be measured, and immobilization of either lectin or ligands resulted in high quality data. The methods gave results in full agreement with previous isothermal titration calorimetry experiments, and presented strong advantages because they require less quantity and purity for the biomolecules.

Metabolic labelling of cancer cells with glycodendrimers stimulate immune-mediated cytotoxicity.

The recruitment of antibody naturally present in human blood stream at the surface of cancer cells have been proved a promising immunotherapeutic strategy to fight cancer. Antibody recruiting molecules (ARMs) combining tumor and antibody binding modules have been developed for this purpose, however the formation of the interacting complex with both antibody and cell is difficult to optimize to stimulate immune-mediated cytotoxicity. To circumvent this limitation, we report herein a more direct approach combining cell metabolism of azido-sugar and bio-orthogonal click chemistry to conjugate at the cell glycocalyx structurally well-defined glycodendrimers as antibody binding module (ABM). We demonstrate that this strategy allows not only the recruitment of natural antibody at the surface of isolated cells or solid tumor models but also activate a cytotoxic response with human serum as unique source of immune effectors.

Homo- and Heterovalent Neoglycoproteins as Ligands for Bacterial Lectins.

Click chemistry gives access to unlimited set of multivalent glycoconjugates to explore carbohydrate-protein interactions and discover high affinity ligands. In this study, we have created supramolecular systems based on a carrier protein that was grafted by Cu(I)-catalyzed azide-alkyne cycloaddition with tetravalent glycodendrons presenting αGal, ßGal and/or αFuc. Binding studies of the homo- (4 a-c) and heterovalent (5) neoglycoproteins (neoGPs) with the LecA and LecB lectins from P. aeruginosa has first confirmed the interest of the multivalent presentation of glycodendrons by the carrier protein (IC50 up to 2.8 nM). Moreover, these studies have shown that the heterovalent display of glycans (5) allows the interaction with both lectins (IC50 of 10 nM) despite the presence of unspecific moieties, and even with similar efficiency for LecB. These results demonstrate the potential of multivalent and multispecific neoGPs as a promising strategy to fight against resistant pathogens.

Structural influence of antibody recruiting glycodendrimers (ARGs) on antitumoral cytotoxicity.

The recruitment of endogenous antibodies against cancer cells has become a reliable antitumoral immunotherapeutic alternative over the last decade. The covalent attachment of antibody and tumor binding modules (ABM and TBM) within a single, well-defined synthetic molecule was indeed demonstrated to promote the formation of an interacting ternary complex between both the antibodies and the targeted cell, which usually results in the simultaneous immune-mediated cellular destruction. In a preliminary study, we have described the first Antibody Recruiting Glycodendrimers (ARGs), combining cRGD as ligands for the αVß3-expressing melanoma cell line M21 and Rha as ligand for natural IgM, and demonstrated that multivalency is an essential requirement to form this complex. In the present study, we synthesized a new series of ARGs composed of ABMs, i.e. self-condensed rhamnosylated cyclopeptide and polylysine dendrimer, which have been conjugated to the TBM with or without spacer. Flow cytometry and confocal microscopy experiments with human serum and different cell lines revealed that the ABM geometry significantly influences the ternary complex formation in M21, whereas no significant binding occurs in BT 549 having low integrin expression. In addition, we demonstrate with a cellular viability assay that ARGs induce high level of cytotoxicity against M21 which is also in close correlation with the ABM structure. In particular, we have shown that ARG combining cyclopeptide core and branches, with or without spacer, induce 40-57% of selective cytotoxicity against M21 cells in the presence of human serum as the unique source of immunity effectors. Finally, we also highlight that the spacer between ABM and TBM enables an increase of the immune-mediate cytotoxicity even with ABM of lower valency.

Multivalent Presentations of Glycomimetic Inhibitor of the Adhesion of Fungal Pathogen Candida albicans to Human Buccal Epithelial Cells.

Candida albicans causes some of the most prevalent hospital-acquired fungal infections, particularly threatening for immunocompromised patients. C. albicans strongly adheres to the surface of epithelial cells so that subsequent colonization and biofilm formation can take place. Divalent galactoside glycomimetic 1 was found to be a potent inhibitor of the adhesion of C. albicans to buccal epithelial cells. In this work, we explore the effect of multivalent presentations of glycomimetic 1 on its ability to inhibit yeast adhesion and biofilm formation. Tetra-, hexa-, and hexadecavalent displays of compound 1 were built on RAFT cyclopeptide- and polylysine-based scaffolds with a highly efficient and modular synthesis. Biological evaluation revealed that the scaffold choice significantly influences the activity of the lower valency conjugates, with compound 16, constructed on a tetravalent polylysine scaffold, found to inhibit the adhesion of C. albicans to human buccal epithelial cells more effectively than the glycomimetic 1; however, the latter performed better in the biofilm reduction assays. Interestingly, the higher valency glycoconjugates did not outperform the anti-adhesion activity of the original compound 1, and no significant effect of the core scaffold could be appreciated. SEM images of C. albicans cells treated with compounds 1, 14, and 16 revealed significant differences in the aggregation patterns of the yeast cells.

Social brain activation during mentalizing in a large autism cohort: the Longitudinal European Autism Project.

BACKGROUND: Autism spectrum disorder (ASD) is a neurodevelopmental condition with key deficits in social functioning. It is widely assumed that the biological underpinnings of social impairment are neurofunctional alterations in the "social brain," a neural circuitry involved in inferring the mental state of a social partner. However, previous evidence comes from small-scale studies and findings have been mixed. We therefore carried out the to-date largest study on neural correlates of mentalizing in ASD. METHODS: As part of the Longitudinal European Autism Project, we performed functional magnetic resonance imaging at six European sites in a large, well-powered, and deeply phenotyped sample of individuals with ASD (N = 205) and typically developing (TD) individuals (N = 189) aged 6 to 30 years. We presented an animated shapes task to assess and comprehensively characterize social brain activation during mentalizing. We tested for effects of age, diagnosis, and their association with symptom measures, including a continuous measure of autistic traits. RESULTS: We observed robust effects of task. Within the ASD sample, autistic traits were moderately associated with functional activation in one of the key regions of the social brain, the dorsomedial prefrontal cortex. However, there were no significant effects of diagnosis on task performance and no effects of age and diagnosis on social brain responses. Besides a lack of mean group differences, our data provide no evidence for meaningful differences in the distribution of brain response measures. Extensive control analyses suggest that the lack of case-control differences was not due to a variety of potential confounders. CONCLUSIONS: Contrary to prior reports, this large-scale study does not support the assumption that altered social brain activation during mentalizing forms a common neural marker of ASD, at least with the paradigm we employed. Yet, autistic individuals show socio-behavioral deficits. Our work therefore highlights the need to interrogate social brain function with other brain measures, such as connectivity and network-based approaches, using other paradigms, or applying complementary analysis approaches to assess individual differences in this heterogeneous condition.

Glucose-based spiro-oxathiazoles as in vivo anti-hyperglycemic agents through glycogen phosphorylase inhibition.

The design of glycogen phosphorylase (GP) inhibitors targeting the catalytic site of the enzyme is a promising strategy for a better control of hyperglycaemia in the context of type 2 diabetes. Glucopyranosylidene-spiro-heterocycles have been demonstrated as potent GP inhibitors, and more specifically spiro-oxathiazoles. A new synthetic route has now been elaborated through 1,3-dipolar cycloaddition of an aryl nitrile oxide to a glucono-thionolactone affording in one step the spiro-oxathiazole moiety. The thionolactone was obtained from the thermal rearrangement of a thiosulfinate precursor according to Fairbanks' protocols, although with a revisited outcome and also rationalised with DFT calculations. The 2-naphthyl substituted glucose-based spiro-oxathiazole 5h, identified as one of the most potent GP inhibitors (Ki = 160 nM against RMGPb) could be produced on the gram-scale from this strategy. Further evaluation in vitro using rat and human hepatocytes demonstrated that compound 5h is a anti-hyperglycaemic drug candidates performing slightly better than DAB used as a positive control. Investigation in Zucker fa/fa rat model in acute and subchronic assays further confirmed the potency of compound 5h since it lowered blood glucose levels by ∼36% at 30 mg kg-1 and ∼43% at 60 mg kg-1. The present study is one of the few in vivo investigations for glucose-based GP inhibitors and provides data in animal models for such drug candidates.

Chemical synthesis and immunological evaluation of new generation multivalent anticancer vaccines based on a Tn antigen analogue.

Tumor associated carbohydrate antigens (TACAs), such as the Tn antigen, have emerged as key targets for the development of synthetic anticancer vaccines. However, the induction of potent and functional immune responses has been challenging and, in most cases, unsuccessful. Herein, we report the design, synthesis and immunological evaluation in mice of Tn-based vaccine candidates with multivalent presentation of the Tn antigen (up to 16 copies), both in its native serine-linked display (Tn-Ser) and as an oxime-linked Tn analogue (Tn-oxime). The high valent vaccine prototypes were synthesized through a late-stage convergent assembly (Tn-Ser construct) and a versatile divergent strategy (Tn-oxime analogue), using chemoselective click-type chemistry. The hexadecavalent Tn-oxime construct induced robust, Tn-specific humoral and CD4+/CD8+ cellular responses, with antibodies able to bind the Tn antigen on the MCF7 cancer cell surface. The superior synthetic accessibility and immunological properties of this fully-synthetic vaccine prototype makes it a compelling candidate for further advancement towards safe and effective synthetic anticancer vaccines.

Multifunctional Glycoconjugates for Recruiting Natural Antibodies against Cancer Cells.

Invited for the cover of this issue is Olivier Renaudet and co-workers at the Université Grenoble Alpes and funded by the European Research Council (CoG "LEGO'" no. 647938). The image illustrates a synthetic chemist playing with supramolecular structures to kill cancer cells by using natural antibodies present in the blood stream. Read the full text of the article at 10.1002/chem.201903327.

Multifunctional Glycoconjugates for Recruiting Natural Antibodies against Cancer Cells.

We have developed a fully synthetic and multifunctional antibody-recruiting molecule (ARM) to guide natural antibodies already present in the blood stream against cancer cells without pre-immunization. Our ARM is composed of antibody and tumor binding modules (i.e., ABM and TBM) displaying clustered rhamnose and cyclo-RGD, respectively. By using a stepwise approach, we have first demonstrated the importance of multivalency for efficient recognition with naturel IgM and αv ß3 integrin expressing M21 tumor cell line. Once covalently conjugated by click chemistry, we confirmed by flow cytometry and confocal microscopy that the recognition properties of both the ABM and TBM are conserved, and more importantly, that the resulting ARM promotes the formation of a ternary complex between natural IgM and cancer cells, which is required for the stimulation of the cytotoxic immune response in vivo. Due to the efficiency of the synthetic process, a larger diversity of heterovalent ligands could be easily explored by using the same multivalent approach and could open new perspectives in this field.

Multivalent glycoligands with lectin/enzyme dual specificity: self-deliverable glycosidase regulators.

Multivalent mannosides with inherent macrophage recognition abilities, built on ß-cyclodextrin, RAFT cyclopeptide or peptide dendrimer cores, trigger selective inhibition of lysosomal ß-glucocerebrosidase or α-mannosidase depending on valency and topology, offering new opportunities in multitargeted drug design.

Heteroglycoclusters With Dual Nanomolar Affinities for the Lectins LecA and LecB From Pseudomonas aeruginosa.

Multivalent structures displaying different instead of similar sugar units, namely heteroglycoclusters (hGCs), are stimulating the efforts of glycochemists for developing compounds with new biological properties. Here we report a four-step strategy to synthesize hexadecavalent hGCs displaying eight copies of αFuc and ßGal. These compounds were tested for the binding to lectins LecA and LecB from Pseudomonas aeruginosa. While parent fucosylated (19) and galactosylated (20) homoclusters present nanomolar affinity with LecB and LecA, respectively, we observed that hGCs combining these sugars (11 and 13) maintain their binding potency with both lectins despite the presence of an unspecific sugar. The added multivalency is therefore not a barrier for efficient recognition by bacterial receptors and it opens the route for adding different sugars that can be selected for their immunomodulatory properties.

Selective high-resolution DNP-enhanced NMR of biomolecular binding sites.

Locating binding sites in biomolecular assemblies and solving their structures are of the utmost importance to unravel functional aspects of the system and provide experimental data that can be used for structure-based drug design. This often still remains a challenge, both in terms of selectivity and sensitivity for X-ray crystallography, cryo-electron microscopy and NMR. In this work, we introduce a novel method called Selective Dynamic Nuclear Polarization (Sel-DNP) that allows selective highlighting and identification of residues present in the binding site. This powerful site-directed approach relies on the use of localized paramagnetic relaxation enhancement induced by a ligand-functionalized paramagnetic construct combined with difference spectroscopy to recover high-resolution and high-sensitivity information from binding sites. The identification of residues involved in the binding is performed using spectral fingerprints obtained from a set of high-resolution multidimensional spectra with varying selectivities. The methodology is demonstrated on the galactophilic lectin LecA, for which we report well-resolved DNP-enhanced spectra with linewidths between 0.5 and 1 ppm, which enable the de novo assignment of the binding interface residues, without using previous knowledge of the binding site location. Since this approach produces clean and resolved difference spectra containing a limited number of residues, resonance assignment can be performed without any limitation with respect to the size of the biomolecular system and only requires the production of one protein sample (e.g. 13C,15N-labeled protein).

Dissecting the Heterogeneous Cortical Anatomy of Autism Spectrum Disorder Using Normative Models.

BACKGROUND: The neuroanatomical basis of autism spectrum disorder (ASD) has remained elusive, mostly owing to high biological and clinical heterogeneity among diagnosed individuals. Despite considerable effort toward understanding ASD using neuroimaging biomarkers, heterogeneity remains a barrier, partly because studies mostly employ case-control approaches, which assume that the clinical group is homogeneous. METHODS: Here, we used an innovative normative modeling approach to parse biological heterogeneity in ASD. We aimed to dissect the neuroanatomy of ASD by mapping the deviations from a typical pattern of neuroanatomical development at the level of the individual and to show the necessity to look beyond the case-control paradigm to understand the neurobiology of ASD. We first estimated a vertexwise normative model of cortical thickness development using Gaussian process regression, then mapped the deviation of each participant from the typical pattern. For this, we employed a heterogeneous cross-sectional sample of 206 typically developing individuals (127 males) and 321 individuals with ASD (232 males) (6-31 years of age). RESULTS: We found few case-control differences, but the ASD cohort showed highly individualized patterns of deviations in cortical thickness that were widespread across the brain. These deviations correlated with severity of repetitive behaviors and social communicative symptoms, although only repetitive behaviors survived corrections for multiple testing. CONCLUSIONS: Our results 1) reinforce the notion that individuals with ASD show distinct, highly individualized trajectories of brain development and 2) show that by focusing on common effects (i.e., the "average ASD participant"), the case-control approach disguises considerable interindividual variation crucial for precision medicine.

Tailor-made Janus lectin with dual avidity assembles glycoconjugate multilayers and crosslinks protocells.

We engineered the first chimeric, bispecific lectin, with two rationally oriented and distinct recognition surfaces. This lectin, coined Janus lectin in allusion to the two-faced roman god, is able to bind independently to both fucosylated and sialylated glycoconjugates. The multivalent presentation of binding sites on each face of the Janus lectin is very efficient, resulting in avidities in the low nanomolar range for both fucosylated and sialylated surfaces. Moreover, novel heterovalent, bifunctional glycoclusters were synthetized that match the topology of the Janus lectin. Based on these tools, we constructed organized and controlled supramolecular architectures by assembling Janus lectin and glycocompound layer-by-layer. Furthermore, the Janus lectin was employed as biomolecular linker to organize protocells made from giant unilamellar vesicles of different nature, to more complex prototissues. In summary, tailor-made Janus lectins open wide possibilities for creating biomimetic matrices or artificial tissues.