A macrocyclic tweezers-shaped receptor for the biomimetic recognition of the Gal(α1-3)Gal disaccharide of the α-Gal antigen.

The Gal(α1-3)Gal is the terminal disaccharide unit of the α-Gal epitope [Gal(α1-3)Gal(ß1-4)GlcNAc], an exogenous antigenic determinant with several clinical implications, found in all non-primate mammals and in several dangerous pathogens, including certain protozoa and mycobacteria. Its absence in humans makes the α-Gal epitope an interesting target for several infectious diseases. Here we present the development of a macrocyclic tweezers-shaped receptor, resulting from the combination of the structural features of two predecessors belonging to the family of diaminocarbazole receptors, which exhibits binding properties in the low millimolar range toward the Gal(α1-3)Gal disaccharide of the α-Gal antigen.

Towards Biomimetic Recognition of Glycans by Synthetic Receptors.

Carbohydrates are abundant in Nature, where they are mostly assembled within glycans as free polysaccharides or conjugated to a variety of biological molecules such as proteins and lipids. Glycans exert several functions, including protein folding, stability, solubility, resistance to proteolysis, intracellular traffic, antigenicity, and recognition by carbohydrate-binding proteins. Interestingly, misregulation of their biosynthesis that leads to changes in glycan structures is frequently recognized as a mark of a disease state. Because of glycan ubiquity, carbohydrate binding agents (CBAs) targeting glycans can lead to a deeper understanding of their function and to the development of new diagnostic and prognostic strategies. Synthetic receptors selectively recognizing specific carbohydrates of biological interest have been developed over the past three decades. In addition to the success obtained in the effective recognition of monosaccharides, synthetic receptors recognizing more complex guests have also been developed, including di- and oligosaccharide fragments of glycans, shedding light on the structural and functional requirements necessary for an effective receptor. In this review, the most relevant achievements in molecular recognition of glycans and their fragments will be summarized, highlighting potentials and future perspectives of glycan-targeting synthetic receptors.

Niosomes Functionalized with a Synthetic Carbohydrate Binding Agent for Mannose-Targeted Doxorubicin Delivery.

Niosomes are a potential tool for the development of active targeted drug delivery systems (DDS) for cancer therapy because of their excellent behaviour in encapsulating antitumorals and the possibility to easily functionalise their surface with targeting agents. Recently, some of us developed a synthetic carbohydrate binding agent (CBA) able to target the mannosidic residues of high-mannose-type glycans overexpressed on the surface of several cancer cell lines, promoting their apoptosis. In this article, we modified the structure of this mannose receptor to obtain an amphiphilic analogue suitable for the functionalization of doxorubicin-based niosomes. Several niosomal formulations and preparation methods were investigated deeply to finally obtain functionalized niosomes suitable for parental administration, which were stable for over six months and able to encapsulate up to 85% of doxorubicin (DOXO). In vitro studies, carried out towards triple-negative cancer cells (MDA-MB231), overexpressing high-mannose-type glycans, showed a cytotoxic activity comparable to that of DOXO but with an appreciable increment in apoptosis given by the CBA. Moreover, niosomal formulation was observed to reduce doxorubicin-induced cytotoxicity towards normal cell lines of rat cardiomyocytes (H9C2). This study is propaedeutic to further in vivo investigations that can aim to shed light on the antitumoral activity and pharmacokinetics of the developed active targeted DDS.

Biomimetic Tweezers for N-Glycans: Selective Recognition of the Core GlcNAc2 Disaccharide of the Sialylglycopeptide SGP.

In recent years, glycomics have shown how pervasive the role of carbohydrates in biological systems is and how chemical tools are essential to investigate glycan function and modulate carbohydrate-mediated processes. Biomimetic receptors for carbohydrates can carry out this task but, although significant affinities and selectivities toward simple saccharides have been achieved, targeting complex glycoconjugates remains a goal yet unattained. In this work we report the unprecedented recognition of a complex biantennary sialylglycopeptide (SGP) by a tweezers-shaped biomimetic receptor, which selectively binds to the core GlcNAc2 disaccharide of the N-glycan with an affinity of 170 µM. Because of the simple structure and the remarkable binding ability, this biomimetic receptor can represent a versatile tool for glycoscience, opening the way to useful applications.

Lipoyl-Based Antagonists of Transient Receptor Potential Cation A (TRPA1) Downregulate Osteosarcoma Cell Migration and Expression of Pro-Inflammatory Cytokines.

Osteosarcoma is a heterogeneous tumor intimately linked to its microenvironment, which promotes its growth and spread. It is generally accompanied by cancer-induced bone pain (CIBP), whose main component is neuropathic pain. The TRPA1 ion channel plays a key role in metastasis and is increasingly expressed in bone cancer. Here, a novel TRPA1 inhibitor is described and tested together with two other known TRPA1 antagonists. The novel lipoyl derivative has been successfully assessed for its ability to reduce human osteosarcoma MG-63 cell viability, motility, and gene expression of the CIBP pro-inflammatory cytokines interleukin 6 (IL-6) and tumor necrosis factor α (TNF-α). A putative three-dimensional (3D) model of the inhibitor covalently bound to TRPA1 is also proposed. The in vitro data suggest that the novel inhibitor described here may be highly interesting and stimulating for new strategies to treat osteosarcomas.

Functionalized Hyaluronic Acid for "In Situ" Matrix Metalloproteinase Inhibition: A Bioactive Material to Treat the Dry Eye Sydrome.

Hyaluronic acid (HA) is a naturally occurring polysaccharide with many molecular functions, including maintaining the structure and physiology of the tissues, tissue remodeling, and inflammation. HA is found naturally in physiological tear fluid, possesses excellent mucus-layer-adhesive properties, and is successfully employed in the treatment of dry eye syndrome (DES). However, HA has as major drawback: its rapid in vivo degradation by hyaluronidase. We report on a unique material, namely, HA-3, obtained by the functionalization of HA with the metalloproteinase inhibitor 3 (MMPI). This material is characterized by an increased resistance to hyaluronidase degradation, associated with MMP inhibition properties. The ability of HA-3 to prevent dehydration of human corneal epithelial cells in vitro and in vivo may accelerate the development of more efficient DES treatment and broaden the application of HA in human diseases.

Synthetic carbohydrate-binding agents neutralize SARS-CoV-2 by inhibiting binding of the spike protein to ACE2.

Developing strategies against the SARS-CoV-2 is currently a main research subject. SARS-CoV-2 infects host cells by binding to human ACE2 receptors. Both, virus and ACE2, are highly glycosylated, and exploiting glycans of the SARS-CoV-2 envelope as binding sites for ACE2 represents a virus strategy for attacking the human host. We report here that a family of mannose-binding synthetic carbohydrate-binding agents (CBAs) inhibits SARS-CoV-2 infection, showing broad neutralizing activity vs. several variants of the spike protein. Preliminary tests indicated that the investigated CBAs interact with the spike protein rather than with ACE2. For a lead compound (IDS060), which has been selected among others for its lack of cytotoxicity, evidence of binding to the RBD of the spike protein has been found by NMR experiments, while competitive binding assays in the presence of IDS060 showed inhibition of binding of RBD to hACE2, although neutralizing activity was also observed with variants showing reduced or depleted binding.

Conformationally Constrained Sialyl Analogues as New Potential Binders of h-CD22.

Here, two conformationally constrained sialyl analogues were synthesized and characterized in their interaction with the inhibitory Siglec, human CD22 (h-CD22). An orthogonal approach, including biophysical assays (SPR and fluorescence), ligand-based NMR techniques, and molecular modelling, was employed to disentangle the interaction mechanisms at a molecular level. The results showed that the Sialyl-TnThr antigen analogue represents a promising scaffold for the design of novel h-CD22 inhibitors. Our findings also suggest that the introduction of a biphenyl moiety at position 9 of the sialic acid hampers canonical accommodation of the ligand in the protein binding pocket, even though the affinity with respect to the natural ligand is increased. Our results address the search for novel modifications of the Neu5Ac-α(2-6)-Gal epitope, outline new insights for the design and synthesis of high-affinity h-CD22 ligands, and offer novel prospects for therapeutic intervention to prevent autoimmune diseases and B-cell malignancies.

A Sulfonated Tweezer-Shaped Receptor Selectively Recognizes Caffeine in Water.

The selective recognition of caffeine in water among structurally related xanthines and purine or pyrimidine bases was achieved by a simple tweezer-shaped receptor featuring sulfonate hydrosolubilizing groups. The remarkable affinity for caffeine, among the highest reported thus far in the literature and larger than that shown by adenosine receptors of all subtypes, stems from a synergistic combination of hydrogen bonding, CH-π, and π-stacking interactions.

Characterization of Natural and Synthetic Sialoglycans Targeting the Hemagglutinin-Neuraminidase of Mumps Virus.

The inhibition of surface viral glycoproteins offers great potential to hamper the attachment of viruses to the host cells surface and the spreading of viral infection. Mumps virus (MuV) is the etiological agent of the mumps infectious disease and causes a wide spectrum of mild to severe symptoms due to the inflammation of the salivary glands. Here we focus our attention on the hemagglutinin-neuraminidase (HN) isolated from MuV SBL-1 strain. We describe the molecular features of host sialoglycans recognition by HN protein by means of NMR, fluorescence assays and computational studies. Furthermore, we also describe the synthesis of a N-acetylneuraminic acid-derived thiotrisaccharide targeting the viral protein, and the corresponding 3D-complex. Our results provide the basis to improve the design and synthesis of potent viral hemagglutinin-neuraminidase inhibitors.

Sodium hyaluronate-g-2-((N-(6-aminohexyl)-4-methoxyphenyl)sulfonamido)-N-hydroxyacetamide with enhanced affinity towards MMP12 catalytic domain to be used as visco-supplement with increased degradation resistance.

The present paper describes the functionalization of sodium hyaluronate (NaHA) with a small molecule (2-((N-(6-aminohexyl)-4-methoxyphenyl)sulfonamido)-N-hydroxyacetamide) (MMPI) having proven inhibitory activity against membrane metalloproteins involved in inflammatory processes (i.e. MMP12). The obtained derivative (HA-MMPI) demonstrated an increased resistance to the in-vitro degradation by hyaluronidase, viscoelastic properties close to those of healthy human synovial fluid, cytocompatibility towards human chondrocytes and nanomolar affinity towards MMP 12. Thus, HA-MMPI can be considered a good candidate as viscosupplement in the treatment of knee osteoarticular disease.

Molecular Recognition of Disaccharides in Water: Preorganized Macrocyclic or Adaptive Acyclic?

When facing the dilemma of following a preorganized or adaptive design approach in conceiving the architecture of new biomimetic receptors for carbohydrates, shape-persistent macrocyclic structures were most often chosen to achieve effective recognition of neutral saccharides in water. In contrast, acyclic architectures have seldom been explored, even though potentially simpler and more easily accessible. In this work, comparison of the binding properties of two structurally related diaminocarbazolic receptors, featuring a macrocyclic and an acyclic tweezer-shaped architecture, highlighted the advantages provided by the acyclic receptor in terms of selectivity in the recognition of 1,4-disaccharides of biological interest. Selective recognition of GlcNAc2 , the core fragment of N-glycans exposed on the surface of enveloped viruses, stands as an emblematic example. NMR spectroscopic data and molecular modeling calculations were used to ascertain the differences in binding mode and to shed light on the origin of recognition efficacy and selectivity.

A Simple Biomimetic Receptor Selectively Recognizing the GlcNAc2 Disaccharide in Water.

GlcNAc2 is the core disaccharide fragment present in N-glycans exposed on the surface of enveloped viruses of high health concern, such as coronaviruses. Because N-glycans are directly involved in the docking of viruses to host cells, recognition of GlcNAc2 by a biomimetic receptor may be a convenient alternative to the use of lectins to interfere with viral entry and infection. Herein, we describe a simple biomimetic receptor recognizing the methyl-ß-glycoside of GlcNAc2 in water with an unprecedented affinity of 160 µM, exceeding that of more structurally complex receptors reported in the literature. The tweezers-shaped acyclic structure exhibits marked selectivity among structurally related disaccharides, and complete discrimination between mono- and disaccharides. Molecular modelling calculations supported by NOE data provided a three-dimensional description of the binding mode, shedding light on the origin of the affinities and selectivities exhibited by the receptor.

A Class of Potent Inhibitors of the HIV-1 Nucleocapsid Protein Based on Aminopyrrolic Scaffolds.

The HIV-1 nucleocapsid protein 7 (NC) is a potential target for effective antiretroviral therapy due to its central role in virus replication, mainly linked to nucleic acid (NA) chaperone activity, and low susceptibility to drug resistance. By screening a compounds library, we identified the aminopyrrolic compound CN14_17, a known carbohydrate binding agent, that inhibits the NC chaperone activity in the low micromolar range. Different from most of available NC inhibitors, CN14_17 fully prevents the NC-induced annealing of complementary NA sequences. Using fluorescence assays and isothermal titration calorimetry, we found that CN14_17 competes with NC for the binding to NAs, preferentially targeting single-stranded sequences. Molecular dynamics simulations confirmed that binding to cTAR occurs preferably within the guanosine-rich single stranded sequence. Finally, CN14_17 exhibited antiretroviral activity in the low micromolar range, although with a moderate therapeutic index. Overall, CN14_17 might be the progenitor of a new promising class of NC inhibitors.

Effective Recognition of Caffeine by Diaminocarbazolic Receptors.

Caffeine is a competitive inhibitor of adenosine receptors and possesses wide pharmacological activity. Artificial receptors recognizing caffeine potentially have a wide range of biomedical and industrial applications. Herein, we describe two structurally related and readily available artificial receptors: 1) a macrocyclic receptor, which binds caffeine with the unprecedented affinity of 9.3 µM, though with poor selectivity; and 2) a tweezers-like structure, showing an affinity of 26 µM and a 4.5-fold and 6-fold selectivity compared to theophylline and theobromine, respectively. Binding affinities were measured by 1 H NMR titrations and were confirmed by isothermal titration calorimetry. The X-ray structure of the complex between caffeine and the acyclic receptor revealed the origin of the recognition, explained the selectivity, and shed light on the role of hydrogen bonding and CH-π/π-π interactions.

A Preorganized Hydrogen-Bonding Motif for the Molecular Recognition of Carbohydrates.

The choice between adaptive and preorganized architectures, or of the most effective hydrogen bonding groups to be selected, are dilemmas that supramolecular chemists must address in designing synthetic receptors for such a challenging guest as carbohydrates. In this paper, structurally related architectures featuring two alternative hydrogen bonding motifs were compared to ascertain the structural and functional origin of their binding differences and the advantages that can be expected in monosaccharide recognition. A set of structurally related macrocyclic receptors were prepared, and their binding properties were measured by NMR and ITC techniques in chloroform vs a common saccharidic target, namely, the ß-octyl glycoside of D-glucose. Results showed that the diaminocarbazolic motif, recently reported as the constituting unit of highly effective receptors for saccharides in water, is a superior hydrogen bonding motif compared to the previously described diaminopyrrolic motif, which was successfully employed in molecular recognition of carbohydrates in polar organic solvents, due to intrinsic structural and functional factors, rather than to hydrophobic contributions. In addition, the occurrence of a rare example of a thermodynamic template effect exerted by the beta-glucoside has been ascertained, enhancing the synthesis outcome of the otherwise low yielding preparation of the described macrocyclic receptors.

Niosomal Formulation of a Lipoyl-Carnosine Derivative Targeting TRPA1 Channels in Brain.

The transient receptor potential akyrin type-1 (TRPA1) is a non-selective cation channel playing a pivotal role in pain sensation and neurogenic inflammation. TRPA1 channels expressed in the central nervous system (CNS) have a critical role in the modulation of cortical spreading depression (CSD), which is a key pathophysiological basis of migraine pain. ADM_09 is a recently developed lipoic acid-based TRPA1 antagonist that is able to revert oxaliplatin-induced neuropathic pain and inflammatory trigeminal allodynia. In this context, aiming at developing drugs that are able to target TRPA1 channels in the CNS and promote an antioxidant effect, permeability across the blood-brain barrier (BBB) represents a central issue. Niosomes are nanovesicles that can be functionalized with specific ligands selectively recognized by transporters expressed on the BBB. In this work, the activity of ADM_09 on neocortex cultures was studied, and an efficient formulation to cross the BBB was developed with the aim of increasing the concentration of ADM_09 into the brain and selectively delivering it to the CNS rapidly after parenteral administration.

Biomimetic Carbohydrate-Binding Agents (CBAs): Binding Affinities and Biological Activities.

Mimicking nature in carbohydrate recognition-that is, by using noncovalent interactions exclusively-is a hot topic that has attracted the interest of many scientists in the last 30 years. Carbohydrates are challenging ligands of high biological relevance, playing central roles in several physiological and pathological processes. Carbohydrate-binding agents (CBAs) of proteic nature, such as lectins, have been extensively used in glycobiology to target carbohydrates, but intrinsic drawbacks conferred on them by their proteic nature limit their therapeutic development. Biomimetic CBAs, artificial small molecules designed for molecular recognition of carbohydrates through noncovalent interactions, have been shown to be effective alternatives in recognising carbohydrates in physiological media, opening the way to biological applications. Herein, we describe the recent achievements in this continually developing field, focusing on those biomimetic CBAs for which biological investigations have been carried out.

Interaction of Half Oxa-/Half cis-Platin Complex with Human Superoxide Dismutase and Induced Reduction of Neurotoxicity.

The formation of amorphous protein aggregates containing human superoxide dismutase (hSOD1) is thought to be involved in amyotrophic lateral sclerosis onset. cis-Platin inhibits the oligomerization of apo hSOD1, but its toxicity precludes any possible use in therapy. Herein, we propose a less toxic platinum complex, namely oxa/cis-platin, as hSOD1 antiaggregation lead compound. Oxa/cis-platin is able to interact with hSOD1 in the disulfide oxidized apo form by binding cysteine 111 (Cys111). The mild neurotoxic phenomena induced in vitro and in vivo by oxa/cis-platin can be successfully reverted by using lypoyl derivatives, which do not interfere with the antiaggregation properties of the platin derivative.

Antagonism of Transient Receptor Potential Ankyrin Type-1 Channels as a Potential Target for the Treatment of Trigeminal Neuropathic Pain: Study in an Animal Model.

Transient receptor potential ankyrin type-1 (TRPA1) channels are known to actively participate in different pain conditions, including trigeminal neuropathic pain, whose clinical treatment is still unsatisfactory. The aim of this study was to evaluate the involvement of TRPA1 channels by means of the antagonist ADM_12 in trigeminal neuropathic pain, in order to identify possible therapeutic targets. A single treatment of ADM_12 in rats 4 weeks after the chronic constriction injury of the infraorbital nerve (IoN-CCI) significantly reduced the mechanical allodynia induced in the IoN-CCI rats. Additionally, ADM_12 was able to abolish the increased levels of TRPA1, calcitonin gene-related peptide (CGRP), substance P (SP), and cytokines gene expression in trigeminal ganglia, cervical spinal cord, and medulla induced in the IoN-CCI rats. By contrast, no significant differences between groups were seen as regards CGRP and SP protein expression in the pars caudalis of the spinal nucleus of the trigeminal nerve. ADM_12 also reduced TRP vanilloid type-1 (TRPV1) gene expression in the same areas after IoN-CCI. Our findings show the involvement of both TRPA1 and TRPV1 channels in trigeminal neuropathic pain, and in particular, in trigeminal mechanical allodynia. Furthermore, they provide grounds for the use of ADM_12 in the treatment of trigeminal neuropathic pain.