Hierarchy of Complex Glycomacromolecules: From Controlled Topologies to Biomedical Applications.

Carbohydrates bearing a distinct complexity use a special code (Glycocode) to communicate with carbohydrate-binding proteins at a high precision to manipulate biological activities in complex biological environments. The level of complexity in carbohydrate-containing macromolecules controls the amount and specificity of information that can be stored in biomacromolecules. Therefore, a better understanding of the glycocode is crucial to open new areas of biomedical applications by controlling or manipulating the interaction between immune cells and pathogens in terms of trafficking and signaling, which would become a powerful tool to prevent infectious diseases. Even though a certain level of progress has been achieved over the past decade, synthetic glycomacromolecules are still lagging far behind naturally existing glycans in terms of complexity and precision because of insufficient and inefficient synthetic techniques. Currently, specific targeting at a cellular level using synthetic glycomacromolecules is still challenging. It is obvious that multidisciplinary collaborations are essential between different specialized disciplines to enhance the carbohydrate receptor-targeting paradigm for new biomedical applications. In this Perspective, recent developments in the synthesis of sophisticated glycomacromolecules are highlighted, and their biological and biomedical applications are also discussed in detail.

Communication between cells: exosomes as a delivery system in prostate cancer.

Despite the considerable efforts in screening and diagnostic protocols, prostate cancer still represents the second leading cause of cancer-related death in men. Many patients with localized disease and low risk of recurrence have a favourable outcome. In a substantial proportion of patients, however, the disease progresses and becomes aggressive. The mechanisms that promote prostate cancer progression remain still debated. Many findings point to the role of cross-communication between prostate tumor cells and their surrounding microenvironment during the disease progression. Such a connection fosters survival, proliferation, angiogenesis, metastatic spreading and drug-resistance of prostate cancer. Recent years have seen a profound interest in understanding the way by which prostate cancer cells communicate with the surrounding cells in the microenvironment. In this regard, direct cell-to-cell contacts and soluble factors have been identified. Increasing evidence indicates that PC cells communicate with the surrounding cells through the release of extracellular vesicles, mainly the exosomes. By directly acting in stromal or prostate cancer epithelial cells, exosomes represent a critical intercellular communication system. By querying the public database ( https://pubmed.ncbi.nlm.nih.gov ) for the past 10 years, we have found more than four hundred papers. Among them, we have extrapolated the most relevant about the role of exosomes in prostate cancer malignancy and progression. Emerging data concerning the use of these vesicles in diagnostic management and therapeutic guidance of PC patients are also presented. Video Abstract.

Self-Assembled Multi- and Single-Chain Glyconanoparticles and Their Lectin Recognition.

In this work, we describe the physicochemical characterization of amphiphilic glycopolymers synthesized via copper(0)-mediated reversible-deactivation radical polymerization (Cu-RDRP). Depending on the chemical composition of the polymer, these glycopolymers are able to form multi-chain or single-chain polymeric nanoparticles. The folding of these polymers is first of all driven by the amphiphilicity of the glycopolymers and furthermore by the supramolecular formation of helical supramolecular stacks of benzene-1,3,5-tricarboxamides (BTAs) stabilized by threefold hydrogen bonding. The obtained polymeric nanoparticles were subsequently evaluated for their lectin-binding affinity toward a series of mannose- and galactose-binding lectins via surface plasmon resonance. We found that addition of 2-ethylhexyl acrylate to the polymer composition results in compact particles, which translates to a reduction in binding affinity, whereas with the addition of BTAs, the relation between the nature of the particle and the binding ability system becomes more unpredictable.

Multi-Arm Star-Shaped Glycopolymers with Precisely Controlled Core Size and Arm Length.

Star-shaped glycopolymers provide very high binding activities toward lectins. However, a straightforward synthesis method for the preparation of multi-arm glycopolymers in a one-pot approach has been challenging. Herein, we report a rapid synthesis of well-defined multi-arm glycopolymers via Cu(0)-mediated reversible deactivation radical polymerization in aqueous media. d-Mannose acrylamide has been homo- and copolymerized with NIPAM to provide linear arms and then core cross-linked with a bisacrylamide monomer. Thus, the arm length and core size of multi-arm glycopolymers were tuned. Moreover, the stability of multi-arm glycopolymers was investigated, and degradation reactions under acidic or basic conditions were observed. The binding activities of the obtained multi-arm glycopolymers with mannose-specific human lectins, DC-SIGN and MBL, were investigated via surface plasmon resonance spectroscopy. Finally, the encapsulation ability of multi-arm glycopolymers was examined using DHA and Saquinavir below and above the lower critical solution temperature (LCST) of P(NIPAM).

Effect of Arm Number and Length of Star-Shaped Glycopolymers on Binding to Dendritic and Langerhans Cell Lectins.

Many cell types in Nature are covered by glycans with a sugar shell on their surface. Synthetic glycopolymer-based materials can mimic these glycans in terms of their variety of biological processes, such as cell growth regulation, adhesion, inflammation by bacteria and viruses, and immune responses. However, the complexity of glycans is still very challenging to be mimicked completely to obtain specific and selective binding ability. Therefore, in this study we aimed to understand how the complexity in the sense of the effect of number of arms and lengths in star-shaped glycopolymers affect the binding activity with different lectins. The Cu-mediated reversible deactivation radical polymerization (Cu-RDRP) technique was employed for the synthesis of mannose containing star-shaped glycopolymers with varying arm number and length. Two sets of star-shaped glycopolymers with on average 1, 3, 7, 8, and 15 arms were successfully synthesized and characterized via 1H NMR, GPC, and DLS. The first set of glycopolymers (Set S1) encompasses 5 star-shaped glycopolymers with a different amount of arms per macromolecule but with equal arm length, whereas in the second set of 5 glycopolymers (Set S2), the amount of sugars per macromolecule was kept constant to obtain glycopolymers with similar glycovalency but in different configuration. Both glycopolymer sets were subsequently evaluated for their lectin-binding affinity toward a series of both newly and previously studied C-type mannose specific lectins present on dendritic and Langerhans cells. Briefly, while Set S1 glycopolymers with the same arm length and different molecular weight showed considerably different biological activities, Set S2 glycopolymers with different arm lengths and the same molecular weight displayed very similar binding abilities, which can indicate that multivalency can be more important than structure complexity to improve the binding behavior of glycopolymers.

Bottlebrush Glycopolymers from 2-Oxazolines and Acrylamides for Targeting Dendritic Cell-Specific Intercellular Adhesion Molecule-3-Grabbing Nonintegrin and Mannose-Binding Lectin.

Lectins are omnipresent carbohydrate binding proteins that are involved in a multitude of biological processes. Unearthing their binding properties is a powerful tool toward the understanding and modification of their functions in biological applications. Herein, we present the synthesis of glycopolymers with a brush architecture via a "grafting from" methodology. The use of a versatile 2-oxazoline inimer was demonstrated to open avenues for a wide range of 2-oxazoline/acrylamide bottle brush polymers utilizing aqueous Cu-mediated reversible deactivation radical polymerization (Cu-RDRP). The polymers in the obtained library were assessed for their thermal properties in aqueous solution and their binding toward the C-type animal lectins dendritic cell-specific intercellular adhesion molecule-3-grabbing nonintegrin (DC-SIGN) and mannose-binding lectin (MBL) via surface plasmon resonance spectrometry. The encapsulation properties of a hydrophobic drug-mimicking compound demonstrated the potential use of glyco brush copolymers in biological applications.

Synthesis of Brush-Like Glycopolymers with Monodisperse, Sequence-Defined Side Chains and Their Interactions with Plant and Animal Lectins.

The synthesis of brush glycopolymers mimicking the architecture of proteoglycans is achieved by grafting sequence-defined glycooligomers derived from solid-phase polymer synthesis onto a poly(active ester) scaffold. This approach gives access to a first library of brush glycopolymers with controlled variations in the degree of branching and number of carbohydrate ligands per branch. When studying lectin binding of linear and brush glycopolymers to lectins Concanavalin A (ConA), dendritic cell-specific intercellular adhesion molecule-3-grabbing non-integrin (DC-SIGN), and mannose-binding lectin (MBL), different preferences are observed with MBL showing higher binding to linear glycopolymer and ConA and DC-SIGN favoring brush glycopolymers. This finding suggests that the architecture of polymeric glycan mimetics affects binding to lectins not only in terms of creating higher avidity but potentially also selectivity ligands.

Short Total Synthesis of (±)-γ-Lycorane by a Sequential Intramolecular Acylal Cyclisation (IAC) and Intramolecular Heck Addition Reaction.

An intramolecular acylal cyclisation (IAC) approach to the synthesis of a range of bicyclic heterocycles is reported. As an example of the utility of the IAC reaction, the methodology was applied in a protecting-group-free five-step total synthesis of (±)-γ-lycorane, incorporating a new intramolecular Heck addition reaction to generate the pentacyclic core structure of the natural product in good yield.

Intramolecular Acylal Cyclisation (IAC) as an Efficient Synthetic Strategy towards the Total Synthesis of Erythrina Alkaloid Derivatives.

Compounds that comprise the erythrina alkaloid class of natural products are based on a tetracyclic spiroamine framework and exhibit a range of biological activities on the central nervous system. Herein, we report a new and efficient total synthesis of this multiple-ring system based on an intramolecular acylal cyclisation (IAC) approach. Using this methodology, the tetracyclic core was rapidly assembled over a two-step domino process catalysed by a Lewis acid. The effect of heteroatoms, substituents and ring size on the IAC has also been investigated, and the broad application of this procedure is demonstrated by the synthesis of a library of derivatives in good yields with excellent regioselectivity.

Synthesis of a non-peptidic PET tracer designed for α5ß1 integrin receptor.

Arginine-glycine-aspartic acid (RGD)-containing peptides have been traditionally used as PET probes to noninvasively image angiogenesis, but recently, small selective molecules for α5 ß1 integrin receptor have been developed with promising results. Sixty-one antagonists were screened, and tert-butyl (S)-3-(2-((3R,5S)-1-(3-(1-(2-fluoroethyl)-1H-1,2,3-triazol-4-yl)propanoyl)-5-((pyridin-2-ylamino)methyl)pyrrolidin-3-yloxy)acetamido)-2-(2,4,6-trimethylbenzamido)propanoate (FPMt) was selected for the development of a PET tracer to image the expression of α5 ß1 integrin receptors. An alkynyl precursor (PMt) was initially synthesized in six steps, and its radiolabeling was performed according to the azide-alkyne copper(II)-catalyzed Huisgen's cycloaddition by using 1-azido-2-[(18)F]fluoroethane ([(18)F]12). Different reaction conditions between PMt and [(18)F]12 were investigated, but all of them afforded [(18)F]FPMt in 15 min with similar radiochemical yields (80-83%, decay corrected). Overall, the final radiopharmaceutical ([(18)F]FPMt) was obtained after a synthesis time of 60-70 min in 42-44% decay-corrected radiochemical yield.

Synthesis and in vitro evaluation of a novel radioligand for αvß3 integrin receptor imaging: [18F]FPPA-c(RGDfK).

The development of RGD-based antagonist of αvß3 integrin receptor has enhanced the interest in PET probes to image this receptor for the early detection of cancer, to monitor the disease progression and the response to therapy. In this work, a novel prosthetic group (N-(4-fluorophenyl)pent-4-ynamide or FPPA) for the (18)F-labeling of an αvß3 selective RGD-peptide was successfully prepared. [(18)F]FPPA was obtained in three steps with a radiochemical yield of 44% (decay corrected). Conjugation to c(RGDfK(N3)) by the Cu(II) catalyzed Huisgen azido alkyne cycloaddition provided the [(18)F]FPPA-c(RGDfK) with a radiochemical yield of 29% (decay corrected), in an overall synthesis time of 140 min.

Levels of 24-hydroxycholesteryl esters in cerebrospinal fluid and plasma from patients with amyotrophic lateral sclerosis.

OBJECTIVE: In this context, our study aimed to ascertain whether the esterification of 24-hydroxycholesterol, a process heavily affected by oxidative stress, is altered in ALS. METHODS: The study examined the level of 24-hydroxycholesteryl esters in cerebrospinal fluid and plasma of 18 ALS patients by spectroscopic technique as Ultra-high performance liquid chromatography mass spectrometry (UPLC-MS). RESULTS: The level of 24-hydroxycholesteryl esters in cerebrospinal fluid was found to be lower as the brain-blood barrier was damaged. Such a level was positively correlated with the level of esters in plasma. Both cerebrospinal fluid (CSF) level and plasma level were lower in ALS patients (60.05 ± 4.24 % and 54.07 ± 20.37 % respectively) than in controls (79.51 ± 2.47 % and 80.07 ± 10.02 % respectively). CONCLUSIONS: The data suggest that the level 24-hydroxycholesteryl esters might be a new biomarker of ALS and can be measured for monitoring the disease progression.

New Insights and Emerging Therapeutic Approaches in Prostate Cancer.

Prostate cancer is the second most frequently diagnosed cancer in men and several therapeutic approaches are currently available for patient's care. Although the androgen receptor status represents a good predictor of response to androgen deprivation therapy, prostate cancer frequently becomes resistant to this approach and spreads. The molecular mechanisms that contribute to progression and drug-resistance of this cancer remain still debated. However, few therapeutic options are available for patient's management, at this stage. Recent years have seen a great expansion of the studies concerning the role of stromal-epithelial interactions and tumor microenvironment in prostate cancer progression. The findings so far collected have provided new insights into diagnostic and clinical management of prostate cancer patients. Further, new fascinating aspects concerning the intersection of the androgen receptor with survival factors as well as calcium channels have been reported in cultured prostate cancer cells and mouse models. The results of these researches have opened the way for a better understanding of the basic mechanisms involved in prostate cancer invasion and drug-resistance. They have also significantly expanded the list of new biomarkers and druggable targets in prostate cancer. The primary aim of this manuscript is to provide an update of these issues, together with their translational aspects. Exploiting the power of novel promising therapeutics would increase the success rate in the diagnostic path and clinical management of patients with advanced disease.

Glycopolymer-Functionalized MOF-808 Nanoparticles as a Cancer-Targeted Dual Drug Delivery System for Carboplatin and Floxuridine.

Codelivery of chemotherapeutics via nanomaterials has attracted much attention over the last decades due to improved drug delivery to tumor tissues, decreased systemic effects, and increased therapeutic efficacies. High porosities, large pore volumes and surface areas, and tunable structures have positioned metal-organic frameworks (MOFs) as promising drug delivery systems (DDSs). In particular, nanoscale Zr-linked MOFs such as MOF-808 offer notable advantages for biomedical applications such as high porosity, good stability, and biocompatibility. In this study, we report efficient dual drug delivery of floxuridine (FUDR) and carboplatin (CARB) loaded in MOF-808 nanoparticles to cancer cells. The nanoparticles were further functionalized by a poly(acrylic acid-mannose acrylamide) (PAAMAM) glycopolymer coating to obtain a highly selective DDS in cancer cells and enhance the therapeutic efficacy of chemotherapy. While MOF-808 was found to enhance the individual therapeutic effects of FUDR and CARB toward cancerous cells, combining FUDR and CARB was seen to cause a synergistic effect, further enhancing the cytotoxicity of the free drugs. Enhancement of CARB loading and therefore cytotoxicity of the CARB-loaded MOFs could be induced through a modified activation protocol, while coating of MOF-808 with the PAAMAM glycopolymer increased the uptake of the nanoparticles in cancer cells used in the study and offered a particularly significant selective drug delivery with high cytotoxicity in HepG2 human hepatocellular carcinoma cells. These results show how the enhancement of cytotoxicity is possible through both nanovector delivery and synergistic treatment, and that MOF-808 is a viable candidate for future drug delivery studies.

The Pandemic Response Box─Accelerating Drug Discovery Efforts after Disease Outbreaks.

The current Covid-19 pandemic has underlined the need for a more coordinated and forward-looking investment in the search for new medicines targeting emerging health care threats. Repositioning currently approved drugs is a popular approach to any new emerging disease, but it represents a first wave of response. Behind this would be a second wave of more specifically designed therapies based on activities against specific molecular targets or in phenotypic assays. Following the successful deployment and uptake of previous open access compound collections, we assembled the Pandemic Response Box, a collection of 400 compounds to facilitate drug discovery in emerging infectious disease. These are based on public domain information on chemotypes currently in discovery and early development which have been shown to have useful activities and were prioritized by medicinal chemistry experts. They are freely available to the community as a pharmacological test set with the understanding that data will be shared rapidly in the public domain.

Hyaluronan (HA)-inspired glycopolymers as molecular tools for studying HA functions.

Hyaluronic acid (HA), the only non-sulphated glycosaminoglycan, serves numerous structural and biological functions in the human body, from providing viscoelasticity in tissues to creating hydrated environments for cell migration and proliferation. HA is also involved in the regulation of morphogenesis, inflammation and tumorigenesis through interactions with specific HA-binding proteins. Whilst the physicochemical and biological properties of HA have been widely studied for decades, the exact mechanisms by which HA exerts its multiple functions are not completely understood. Glycopolymers offer a simple and precise synthetic platform for the preparation of glycan analogues, being an alternative to the demanding synthetic chemical glycosylation. A library of homo, statistical and alternating HA glycopolymers were synthesised by reversible addition-fragmentation chain transfer polymerisation and post-modification utilising copper alkyne-azide cycloaddition to graft orthogonal pendant HA monosaccharides (N-acetyl glucosamine: GlcNAc and glucuronic acid: GlcA) onto the polymer. Using surface plasmon resonance, the binding of the glycopolymers to known HA-binding peptides and proteins (CD44, hyaluronidase) was assessed and compared to carbohydrate-binding proteins (lectins). These studies revealed potential structure-binding relationships between HA monosaccharides and HA receptors and novel HA binders, such as Dectin-1 and DEC-205 lectins. The inhibitory effect of HA glycopolymers on hyaluronidase (HAase) activity was also investigated suggesting GlcNAc- and GlcA-based glycopolymers as potential HAase inhibitors.

Synthetic Glycomacromolecules of Defined Valency, Absolute Configuration, and Topology Distinguish between Human Lectins.

Carbohydrate-binding proteins (lectins) play vital roles in cell recognition and signaling, including pathogen binding and innate immunity. Thus, targeting lectins, especially those on the surface of immune cells, could advance immunology and drug discovery. Lectins are typically oligomeric; therefore, many of the most potent ligands are multivalent. An effective strategy for lectin targeting is to display multiple copies of a single glycan epitope on a polymer backbone; however, a drawback to such multivalent ligands is they cannot distinguish between lectins that share monosaccharide binding selectivity (e.g., mannose-binding lectins) as they often lack molecular precision. Here, we describe the development of an iterative exponential growth (IEG) synthetic strategy that enables facile access to synthetic glycomacromolecules with precisely defined and tunable sizes up to 22.5 kDa, compositions, topologies, and absolute configurations. Twelve discrete mannosylated "glyco-IEGmers" are synthesized and screened for binding to a panel of mannoside-binding immune lectins (DC-SIGN, DC-SIGNR, MBL, SP-D, langerin, dectin-2, mincle, and DEC-205). In many cases, the glyco-IEGmers had distinct length, stereochemistry, and topology-dependent lectin-binding preferences. To understand these differences, we used molecular dynamics and density functional theory simulations of octameric glyco-IEGmers, which revealed dramatic effects of glyco-IEGmer stereochemistry and topology on solution structure and reveal an interplay between conformational diversity and chiral recognition in selective lectin binding. Ligand function also could be controlled by chemical substitution: by tuning the side chains of glyco-IEGmers that bind DC-SIGN, we could alter their cellular trafficking through alteration of their aggregation state. These results highlight the power of precision synthetic oligomer/polymer synthesis for selective biological targeting, motivating the development of next-generation glycomacromolecules tailored for specific immunological or other therapeutic applications.

Selective Modulation of α5 GABAA Receptors Exacerbates Aberrant Inhibition at Key Hippocampal Neuronal Circuits in APP Mouse Model of Alzheimer's Disease.

Selective negative allosteric modulators (NAMs), targeting α5 subunit-containing GABAA receptors (GABAARs) as potential therapeutic targets for disorders associated with cognitive deficits, including Alzheimer's disease (AD), continually fail clinical trials. We investigated whether this was due to the change in the expression of α5 GABAARs, consequently altering synaptic function during AD pathogenesis. Using medicinal chemistry and computational modeling, we developed aqueous soluble hybrids of 6,6-dimethyl-3-(2-hydroxyethyl) thio-1-(thiazol-2-yl)-6,7-dihydro-2-benzothiophene-4(5H)-one, that demonstrated selective binding and high negative allosteric modulation, specifically for the α5 GABAAR subtypes in constructed HEK293 stable cell-lines. Using a knock-in mouse model of AD (APP NL-F/NL-F), which expresses a mutant form of human amyloid-ß (Aß), we performed immunofluorescence studies combined with electrophysiological whole-cell recordings to investigate the effects of our key molecule, α5-SOP002 in the hippocampal CA1 region. In aged APP NL-F/NL-F mice, selective preservation of α5 GABAARs was observed in, calretinin- (CR), cholecystokinin- (CCK), somatostatin- (SST) expressing interneurons, and pyramidal cells. Previously, we reported that CR dis-inhibitory interneurons, specialized in regulating other interneurons displayed abnormally high levels of synaptic inhibition in the APP NL-F/NL-F mouse model, here we show that this excessive inhibition was "normalized" to control values with bath-applied α5-SOP002 (1 µM). However, α5-SOP002, further impaired inhibition onto CCK and pyramidal cells that were already largely compromised by exhibiting a deficit of inhibition in the AD model. In summary, using a multi-disciplinary approach, we show that exposure to α5 GABAAR NAMs may further compromise aberrant synapses in AD. We, therefore, suggest that the α5 GABAAR is not a suitable therapeutic target for the treatment of AD or other cognitive deficits due to the widespread neuronal-networks that use α5 GABAARs.

Relevance of lactate level detection in migrane and fibromyalgia.

The aim of this study was to determine the blood lactate levels in healthy and pathological subjects, particularly with migraine and fibromyalgia. Moreover we investigated the possible correlation between lactate concentration, postural stability and balance disorders; the composition of the groups were: migraine (n = 25; age 49.7 ± 12.5), fibromyalgia (n = 10; age 43.7 ± 21.2), control group (n = 16 age 28.52 ± 2.4). The results showed that patients with fibromyalgia (FG) had higher lactate levels compared to migraine (MG) and control group (CG) (mean ± sd: FG = 1.78 ± 0.9 mmol/L; MG = 1.45±1 mmol/L; CG = 0,85 ± 0,07 mmol/L). The same situation was highlighted about the sway path length with eyes closed (FG = 518 ± 195 mm; MG = 465 ± 165 mm; CG = 405 ± 94,72 mm) and with eyes open (FG = 430 ± 220 mm; MG = 411 ± 143 mm; CG = 389 ± 107 mm). This can be explained by the fact that energy-intensive postural strategies must be used to optimize both static and dynamic coordination, in particular with repeated contractions of tonic oxidative muscle cells responsible for postural control.

Antioxidant and Anti-Inflammatory Activities of Buffalo Milk δ-Valerobetaine.

δ-Valerobetaine (δVB), a constitutive metabolite of ruminant milk, is produced in the rumen from free dietary Nε- trimethyllysine occurring ubiquitously in vegetable kingdom. The biological role of δVB is poorly known. Here, the antioxidant and anti-inflammatory potential of buffalo milk δVB was tested in vitro during high-glucose (HG)-induced endothelial damage. Results indicated that δVB (0.5 mM) ameliorated the HG cytotoxicity (0.57 ± 0.02 vs 0.41 ± 0.018 O.D. ( P < 0.01). Interestingly, buffalo milk extracts enriched with δVB showed improved significant efficacy in decreasing reactive oxygen species, lipid peroxidation, and cytokine release during HG treatment compared to milk extracts alone ( P < 0.05). It is noteworthy that δVB reduced the HG-activated inflammatory signal by modulating SIRT1 (0.96 ± 0.05 vs 0.85 ± 0.04 AU), SIRT6 (0.82 ± 0.04 vs 0.61 ± 0.03 AU), and NF-κB (0.85 ± 0.03 vs 1.23 ± 0.03 AU) ( P < 0.05). On the whole, our data show the first evidence of δVB efficacy in reducing endothelial oxidative stress and inflammation, suggesting a potential role of this betaine as a novel dietary compound with health-promoting properties.