Ceramide Is Involved in Temozolomide Resistance in Human Glioblastoma U87MG Overexpressing EGFR.

Glioblastoma multiforme (GBM) is the most frequent and deadly brain tumor. Many sphingolipids are crucial players in the regulation of glioma cell growth as well as in the response to different chemotherapeutic drugs. In particular, ceramide (Cer) is a tumor suppressor lipid, able to induce antiproliferative and apoptotic responses in different types of tumors including GBM, most of which overexpress the epidermal growth factor receptor variant III (EGFRvIII). In this paper, we investigated whether Cer metabolism is altered in the U87MG human glioma cell line overexpressing EGFRvIII (EGFR+ cells) to elucidate their possible interplay in the mechanisms regulating GBM survival properties and the response to the alkylating agent temozolomide (TMZ). Notably, we demonstrated that a low dose of TMZ significantly increases Cer levels in U87MG cells but slightly in EGFR+ cells (sensitive and resistant to TMZ, respectively). Moreover, the inhibition of the synthesis of complex sphingolipids made EGFR+ cells sensitive to TMZ, thus involving Cer accumulation/removal in TMZ resistance of GBM cells. This suggests that the enhanced resistance of EGFR+ cells to TMZ is dependent on Cer metabolism. Altogether, our results indicate that EGFRvIII expression confers a TMZ-resistance phenotype to U87MG glioma cells by counteracting Cer increase.

Unusual promoters and leaving groups in glycosylation reactions: The evolution of carbohydrate synthesis.

Glycosylation is the key reaction by which our body can produce and modify carbohydrates and their conjugates which are molecules essential for life. The study of the diversity of their functions is a current and ever-expanding topic that requires the ability to provide pure saccharides quickly, efficiently and in a controlled way which can be achieved by chemical synthesis. Although the influence of the donor and the promoter on the outcome of a glycosylation reaction is well documented, the search for new methodologies and new promoters/activators is constantly expanding. In this review, after an introduction dealing with well-known glycosylation strategies, we describe the most recent advances in terms of the use of innovative approaches, focalizing the study on new promoters and leaving groups exploited in the last ten years.

Recent advances on smart glycoconjugate vaccines in infections and cancer.

Vaccination is one of the greatest achievements in biomedical research preventing death and morbidity in many infectious diseases through the induction of pathogen-specific humoral and cellular immune responses. Currently, no effective vaccines are available for pathogens with a highly variable antigenic load, such as the human immunodeficiency virus or to induce cellular T-cell immunity in the fight against cancer. The recent SARS-CoV-2 outbreak has reinforced the relevance of designing smart therapeutic vaccine modalities to ensure public health. Indeed, academic and private companies have ongoing joint efforts to develop novel vaccine prototypes for this virus. Many pathogens are covered by a dense glycan-coat, which form an attractive target for vaccine development. Moreover, many tumor types are characterized by altered glycosylation profiles that are known as "tumor-associated carbohydrate antigens". Unfortunately, glycans do not provoke a vigorous immune response and generally serve as T-cell-independent antigens, not eliciting protective immunoglobulin G responses nor inducing immunological memory. A close and continuous crosstalk between glycochemists and glycoimmunologists is essential for the successful development of efficient immune modulators. It is clear that this is a key point for the discovery of novel approaches, which could significantly improve our understanding of the immune system. In this review, we discuss the latest advancements in development of vaccines against glycan epitopes to gain selective immune responses and to provide an overview on the role of different immunogenic constructs in improving glycovaccine efficacy.

Organotrifluoroborate Sugar Conjugates for a Guided Boron Neutron Capture Therapy: From Synthesis to Positron Emission Tomography.

Sugars are a versatile tool for targeting malignant cells and have been extensively used for drug delivery and imaging techniques. Their prototype, fluorodeoxyglucose ([18F]FDG), is currently used for positron emission tomography. Boron neutron capture therapy (BNCT) is a cancer treatment that relies on irradiation with thermal neutrons of cancer cells previously loaded with [10B]-containing compounds. The recent introduction of accelerators as a neutron source for clinical use prompts the planning of delivery compounds enriched with boron able to be traced in real time. This work describes the first synthesis of a new class of sugar derivatives conjugated to a trifluoroborate moiety as potential theranostic agents. Stability and cytotoxicity studies are reported for all compounds, together with [18F] radiolabeling optimization and in vivo preliminary positron emission tomography (PET) experiments on a selected compound.

Glycan Array Evaluation of Synthetic Epitopes between the Capsular Polysaccharides from Streptococcus pneumoniae 19F and 19A.

Vaccination represents the most effective way to prevent invasive pneumococcal diseases. The glycoconjugate vaccines licensed so far are obtained from capsular polysaccharides (CPSs) of the most virulent serotypes. Protection is largely limited to the specific vaccine serotypes, and the continuous need for broader coverage to control the outbreak of emerging serotypes is pushing the development of new vaccine candidates. Indeed, the development of efficacious vaccine formulation is complicated by the high number of bacterial serotypes with different CPSs. In this context, to simplify vaccine composition, we propose the design of new saccharide fragments containing chemical structures shared by different serotypes as cross-reactive and potentially cross-protective common antigens. In particular, we focused on Streptococcus pneumoniae (Sp) 19A and 19F. The CPS repeating units of Sp 19F and 19A are very similar and share a common structure, the disaccharide ManNAc-ß-(1→4)-Glc (A-B). Herein, we describe the synthesis of a small library of compounds containing different combinations of the common 19F/19A disaccharide. The six new compounds were tested with a glycan array to evaluate their recognition by antibodies in reference group 19 antisera and factor reference antisera (reacting against 19F or 19A). The disaccharide A-B, phosphorylated at the upstream end, emerged as a hit from the glycan array screening because it is strongly recognized by the group 19 antisera and by the 19F and 19A factor antisera, with similar intensity compared with the CPSs used as controls. Our data give a strong indication that the phosphorylated disaccharide A-B can be considered a common epitope among different Sp 19 serotypes.

2,3-Carbamate mannosamine glycosyl donors in glycosylation reactions of diacetone-d-glucose. An experimental and theoretical study.

The role of the cyclic 2,3-N,O-carbamate protecting group in directing the selectivity of mannosylation reactions of diacetone-d-glucose, promoted by BSP/Tf2O via α-triflate intermediates, has been investigated through a combined computational and experimental approach. DFT calculations were used to locate the transition states leading to the α or ß anomers. These data indicate the preferential formation of the ß-adduct with mannosyl donors either equipped with the 4,6-O-benzylidene protection or without it. The synthetic results confirmed this preference, showing in both cases an α/ß selectivity of 4:6. This highlights a role for the 2,3-N,O-carbamate in sharp contrast with what described in the case of 2,3-O-carbonate mannosyl donors.

Glyconanoparticles as tools to prevent antimicrobial resistance.

The increased phenomenon of antimicrobial resistance and the slow pace of development of new antibiotics are at the base of a global health concern regarding microbial infections. Antibiotic resistance kills an estimated 700,000 people each year worldwide, and this number is expected to increase dramatically if efforts are not made to develop new drugs or alternative containment strategies. Increased vaccination coverage, improved sanitation or sustained implementation of infection control measures are among the possible areas of action. Indeed, vaccination is one of the most effective tools of preventing infections. Starting from 1970s polysaccharide-based vaccines against Meningococcus, Pneumococcus and Haemophilus influenzae type b have been licensed, and provided effective protection for population. However, the development of safe and effective vaccines for infectious diseases with broad coverage remains a major challenge in global public health. In this scenario, nanosystems are receiving attention as alternative delivery systems to improve vaccine efficacy and immunogenicity. In this report, we provide an overview of current applications of glyconanomaterials as alternative platforms in the development of new vaccine candidates. In particular, we will focus on nanoparticle platforms, used to induce the activation of the immune system through the multivalent-displacement of saccharide antigens.

Emerging glyco-based strategies to steer immune responses.

Glycan structures are common posttranslational modifications of proteins, which serve multiple important structural roles (for instance in protein folding), but also are crucial participants in cell-cell communications and in the regulation of immune responses. Through the interaction with glycan-binding receptors, glycans are able to affect the activation status of antigen-presenting cells, leading either to induction of pro-inflammatory responses or to suppression of immunity and instigation of immune tolerance. This unique feature of glycans has attracted the interest and spurred collaborations of glyco-chemists and glyco-immunologists to develop glycan-based tools as potential therapeutic approaches in the fight against diseases such as cancer and autoimmune conditions. In this review, we highlight emerging advances in this field, and in particular, we discuss on how glycan-modified conjugates or glycoengineered cells can be employed as targeting devices to direct tumor antigens to lectin receptors on antigen-presenting cells, like dendritic cells. In addition, we address how glycan-based nanoparticles can act as delivery platforms to enhance immune responses. Finally, we discuss some of the latest developments in glycan-based therapies, including chimeric antigen receptor (CAR)-T cells to achieve targeting of tumor-associated glycan-specific epitopes, as well as the use of glycan moieties to suppress ongoing immune responses, especially in the context of autoimmunity.

Glycans Meet Sphingolipids: Structure-Based Design of Glycan Containing Analogues of a Sphingosine Kinase Inhibitor.

Sphingosine 1-phosphate (S1P) is a bioactive lipid mediator associated with diverse homeostatic and signaling roles. Enhanced biosynthesis of S1P, mediated by the sphingosine kinase isozymes (SK1 and SK2), is implicated in several pathophysiological conditions and diseases, including skeletal muscle fibrosis, inflammation, multiple sclerosis, and cancer. Therefore, therapeutic approaches that control S1P production have focused on the development of SK1/2 inhibitors. In this framework, we designed a series of natural monosaccharide-based compounds to enhance anchoring of the known SK1 inhibitor PF-543 at the polar head of the J-shaped substrate-binding channel. Herein, we describe the structure-based design and synthesis of new glycan-containing PF-543 analogues and we demonstrate their efficiency in a TGFß1-induced pro-fibrotic assay.

Exploring calixarene-based clusters for efficient functional presentation of Streptococcus pneumoniae saccharides.

Calixarenes are promising scaffolds for an efficient clustered exposition of multiple saccharide antigenic units. Herein we report the synthesis and biological evaluation of a calix[6]arene functionalized with six copies of the trisaccharide repeating unit of Streptococcus pneumoniae (SP) serotype 19F. This system has demonstrated its ability to efficiently inhibit the binding between the native 19F capsular polysaccharide and anti-19F antibodies, despite a low number of exposed saccharide antigens, well mimicking the epitope presentations in the polysaccharide. The calix[6]arene mobile scaffold has been selected for functionalization with SP 19F repeating unit after a preliminary screening of four model glycocalixarenes, functionalized with N-acetyl mannosamine, and differing in the valency and/or conformational properties. This work is a step forward towards the development of new fully synthetic calixarenes comprising small carbohydrate antigens as potential carbohydrate-based vaccine scaffolds.

Sperm can act as vectors for HIV-1 transmission into vaginal and cervical epithelial cells.

PROBLEM: Sperm are the major cells in semen. Human sperm possess a number of HIV-1 gp120 binding ligands including sulfogalactosylglycerolipid (SGG). However, the mechanisms of how sperm capture HIV-1 onto their surface are unclear. Furthermore, the ability of sperm to deliver HIV-1 to vaginal/cervical epithelial cells lining the lower female reproductive tract, as a first step in HIV-1 transmission, needs to be determined. METHOD OF STUDY: Sperm from healthy donors were incubated with dual-tropic HIV-1CS204 (clinical isolate), and virus capture was determined by p24 antigen ELISA. The involvement of SGG in HIV-1 capture was assessed by determining Kd values of HIV-1 gp120-SGG binding as well as computational docking of SGG to the gp120 V3 loop. The ability of sperm-associated HIV-1 to infect peripheral blood mononuclear cells (PBMCs) and TZM-bl indicator cells was determined. Lastly, infection of vaginal (Vk2/E6E7), ectocervical (Ect1/E6E7), and endocervical (End1/E6E7) epithelial cells mediated by HIV-1-associated sperm was evaluated. RESULTS: Sperm were able to capture HIV-1 in a dose-dependent manner, and the capture reached a maximum within 5 minutes. Captured HIV-1, however, could be removed from sperm by Percoll-gradient centrifugation. Affinity of gp120 for SGG was substantial, implicating sperm SGG in HIV-1 capture. Sperm-associated HIV-1 could productively infect PBMCs and TZM-bl cells, and was capable of being transmitted into vaginal/cervical epithelial cells. CONCLUSION: Sperm are able to capture HIV-1, which remains infectious and is able to be transmitted into vaginal/cervical epithelial cells, a result indicating the importance of sperm in HIV transmission.

A versatile synthesis of αGalCer and its analogues exploiting a cyclic carbonate as phytosphingosine 3,4-diol protecting group.

A convenient synthetic strategy to αGalCer and some relevant analogues by using a handily protected phytosphingosine is reported here. The conversion of the phytosphingosine amino group to azide and the protection of 3,4-diol as cyclic carbonate group, cleavable in mild basic conditions but resistant to acidic treatment, afforded quickly an excellent glycosyl acceptor. Its glycosylation with a proper galactosyl donor, gave a versatile intermediate in high yield and excellent stereoselectivity. To demonstrate the potentiality of the intermediate, three immunologically relevant compounds were chosen as model targets: αGalCer, dansyl alpha-galactosylceramide and 7DW8-5. These products were easily obtained in few steps and high yields to validate the synthetic route.

Allosteric Modulators of HSP90 and HSP70: Dynamics Meets Function through Structure-Based Drug Design.

Molecular chaperones HSP90 and HSP70 are essential regulators of the folding and activation of a disparate ensemble of client proteins. They function through ATP hydrolysis and the assembly of multiprotein complexes with cochaperones and clients. While their therapeutic relevance is recognized, important details underlying the links between ATP-dependent conformational dynamics and clients/cochaperones recruitment remain elusive. Allosteric modulators represent fundamental tools to obtain molecular insights into functional regulation. By selective perturbation of different aspects of HSP90/HSP70 activities, allosteric drugs can tune rather than completely inhibit signaling cascades, providing information on the relationships between structure-dynamics and function. Herein, we review advances in the design of HSP90 and HSP70 allosteric modulators. We consider inhibitors and activators in different biochemical and disease models. We discuss these compounds as probes to decipher the complexity of the chaperone machinery and that at the same time represent starting leads for the development of drugs against cancer and neurodegeneration.

Synthesis and biological evaluation of a trisaccharide repeating unit derivative of Streptococcus pneumoniae 19A capsular polysaccharide.

Streptococcus pneumoniae (SP) is a common human pathogen associated with a broad spectrum of diseases and it is still a leading cause of mortality and morbidity worldwide, especially in children. Moreover, SP is increasingly associated with drug resistance. Vaccination against the pathogen may thus represent an important strategy to overcome its threats to human health. In this context, revealing the molecular determinants of SP immunoreactivity may be relevant for the development of novel molecules with therapeutic perspectives as vaccine components. Serogroup 19 comprises the immune-cross reactive types 19F, 19A, 19B and 19C and it accounts for a high percentage of invasive pneumococcal diseases, mainly caused by serotypes 19F and 19A. Herein, we report the synthesis and biological evaluation of an aminopropyl derivative of the trisaccharide repeating unit of SP 19A. We compare two different synthetic strategies, based on different disconnections between the three monosaccharides which make up the final trisaccharide, to define the best approach for the preparation of the trisaccharide. Synthetic accessibility to the trisaccharide repeating unit lays the basis for the development of more complex biopolymer as well as saccharide conjugates. We also evaluate the binding affinity of the trisaccharide for anti-19A and anti-19F sera and discuss the relationship between the chemical properties of the trisaccharide unit and biological activity.

Properties, metabolism and roles of sulfogalactosylglycerolipid in male reproduction.

Sulfogalactosylglycerolipid (SGG, aka seminolipid) is selectively synthesized in high amounts in mammalian testicular germ cells (TGCs). SGG is an ordered lipid and directly involved in cell adhesion. SGG is indispensable for spermatogenesis, a process that greatly depends on interaction between Sertoli cells and TGCs. Spermatogenesis is disrupted in mice null for Cgt and Cst, encoding two enzymes essential for SGG biosynthesis. Sperm surface SGG also plays roles in fertilization. All of these results indicate the significance of SGG in male reproduction. SGG homeostasis is also important in male fertility. Approximately 50% of TGCs become apoptotic and phagocytosed by Sertoli cells. SGG in apoptotic remnants needs to be degraded by Sertoli lysosomal enzymes to the lipid backbone. Failure in this event leads to a lysosomal storage disorder and sub-functionality of Sertoli cells, including their support for TGC development, and consequently subfertility. Significantly, both biosynthesis and degradation pathways of the galactosylsulfate head group of SGG are the same as those of sulfogalactosylceramide (SGC), a structurally related sulfoglycolipid important for brain functions. If subfertility in males with gene mutations in SGG/SGC metabolism pathways manifests prior to neurological disorder, sperm SGG levels might be used as a reporting/predicting index of the neurological status.

Glyco-gold nanoparticles: synthesis and applications.

Glyco-gold nanoparticles combine in a single entity the peculiar properties of gold nanoparticles with the biological activity of carbohydrates. The result is an exciting nanosystem, able to mimic the natural multivalent presentation of saccharide moieties and to exploit the peculiar optical properties of the metallic core. In this review, we present recent advances on glyco-gold nanoparticle applications in different biological fields, highlighting the key parameters which inspire the glyco nanoparticle design.

Preparation and immunogenicity of gold glyco-nanoparticles as antipneumococcal vaccine model.

AIM: Nanotechnology-based fully synthetic carbohydrate vaccines are promising alternatives to classic polysaccharide/protein conjugate vaccines. We have prepared gold glyco-nanoparticles (GNP) bearing two synthetic carbohydrate antigens related to serotypes 19F and 14 of Streptococcus pneumoniae and evaluated their immunogenicity in vivo. RESULTS: A tetrasaccharide fragment of serotype 14 (Tetra-14), a trisaccharide fragment of serotype 19F (Tri-19F), a T-helper peptide and d -glucose were loaded onto GNP in different ratios. Mice immunization showed that the concomitant presence of Tri-19F and Tetra-14 on the same nanoparticle critically enhanced the titers of specific IgG antibodies toward type 14 polysaccharide compared with GNP exclusively displaying Tetra-14, while no IgG antibodies against type 19F polysaccharide were elicited. CONCLUSION: This work is a step forward toward synthetic nanosystems combining carbohydrate antigens and immunogenic peptides as potential carbohydrate-based vaccines.

Molecular Architecture and Symmetry Properties of 1,3-Alternate Calix[4]arenes with Orientable Groups at the Para Position of the Phenolic Rings.

Two glycoclusters constituted by four fully acetylated ß-acetylmannosamine residues linked through trimethylenethioureido spacers to a calix[4]arene core and differing for the presence of methoxy or propoxy groups at the lower rim were synthesized. One of the two compounds is fixed in the 1,3-alternate geometry by the presence of the propoxy groups, while the other is potentially free to assume one of the different geometries allowed in calix[4]arene. Their similar NMR spectra in chloroform clearly suggest the same 1,3-alternate geometry. Both compounds were submitted to a conformational investigation with the DFT approach at the standard B3LYP/6-31G(d) level. The two glycocalixarenes showed a large conformational preference for the same geometry that put the mannosamine moiety of one substituent close to the thioureido group of the opposite substituent. This allows the formation of intramolecular hydrogen bonds and originates a series of through-space close contacts. A comparison with the NOESY maps evidence an excellent correspondence between experimental and theoretical data, thus giving an experimental validation of the highly symmetrical conformation that the two glycocalixarenes assume in apolar solvents.

Cone Calix[4]arenes with Orientable Glycosylthioureido Groups at the Upper Rim: An In-Depth Analysis of Their Symmetry Properties.

The two glycoclusters α- and ß-d-mannosylthioureidocalix[4]arenes 1 and 2 in the cone geometry have been submitted to a conformational investigation with the DFT approach at the standard B3LYP/6-31G(d) level and using a water continuum solvent model. After a reasoned choice of the level of calculation and the evaluation of the properties of the monomeric components of 1 and 2, the intrinsic conformational properties of cone calix[4]arenes with orientable groups at the upper rim were thoroughly analyzed. From the possible combinations of the directions that the groups may assume, 10 different geometries derive, all chiral. These geometries are interchangeable through two different processes, named breathing equilibrium and arrow rotation, that allow a dense network connection among them. When the modeling of whole macrocycles 1 and 2 was performed, a huge difference in their conformational behavior that heavily influences the presentation mode of their saccharidic moieties was found.