Targeting osteoarthritis-associated galectins and an induced effector class by a ditopic bifunctional reagent: Impact of its glycan part on binding measured in the tissue context.

Pairing glycans with tissue lectins controls multiple effector pathways in (patho)physiology. A clinically relevant example is the prodegradative activity of galectins-1 and -3 (Gal-1 and -3) in the progression of osteoarthritis (OA) via matrix metalloproteinases (MMPs), especially MMP-13. The design of heterobifunctional inhibitors that can block galectin binding and MMPs both directly and by preventing their galectin-dependent induction selectively offers a perspective to dissect the roles of lectins and proteolytic enzymes. We describe the synthesis of such a reagent with a bivalent galectin ligand connected to an MMP inhibitor and of two tetravalent glycoclusters with a subtle change in headgroup presentation for further elucidation of influence on ligand binding. Testing was performed on clinical material with mixtures of galectins as occurring in vivo, using sections of fixed tissue. Two-colour fluorescence microscopy monitored binding to the cellular glycome after optimization of experimental parameters. In the presence of the inhibitor, galectin binding to OA specimens was significantly reduced. These results open the perspective to examine the inhibitory capacity of custom-made ditopic compounds on binding of lectins in mixtures using sections of clinical material with known impact of galectins and MMPs on disease progression.

Polynorepinephrine: state-of-the-art and perspective applications in biosensing and molecular recognition.

The polymerization of norepinephrine, and the properties of the related polymer polynorepinephrine, started to be investigated barely 9 years ago and only few works were produced so far, mainly in materials science and medicine. An unexpectedly low relevance, especially if compared with the interest toward dopamine and polydopamine, differing from norepinephrine only for a hydroxyl group and whose properties were deeply investigated and applied to an impressive number of subject areas. We show here that in some cases, norepinephrine and dopamine monomers can be exchanged without virtually affecting the experimental results. But even more interesting, the choice of norepinephrine can positively influence the properties of the final polymer. In particular, the smoother and more hydrophilic surface of polynorepinephrine may enhance cell adhesion and proliferation, increase the activity of conjugated biomolecules, and induce higher cellular uptake of nanodrugs. Moreover, polynorepinephrine presents an additional anchoring point that can be exploited for further functionalization. Nevertheless, despite its potential for bioconjugation and molecular recognition, polynorepinephrine has not yet been considered in biosensing. Here we report our feelings in terms of perspective use of polynorepinephrine as new functional monomer for biomimetic receptor development by molecular imprinting, with application in affinity biosensing. Graphical abstracts.

Characterization of troponin T binding aptamers for an innovative enzyme-linked oligonucleotide assay (ELONA).

Early diagnosis of acute myocardial infarction (AMI) is of outmost importance to reduce the mortality rate, and cardiac troponins are considered the gold standard biomarkers of myocardial necrosis. In this scenario, the characterization of two troponin T (TnT)-binding aptamers as viable alternative to antibodies employed on clinical immunoassays is here reported for the first time. Their recognition ability was first investigated through surface plasmon resonance (SPR). Subsequently, an enzyme-linked oligonucleotide assay (ELONA) was developed on common 96-well polystyrene plates, both by direct and sandwich detection strategies for comparison. In both cases, the assay exhibits a detection ability of TnT in the range of low nanomolar but a great advantage on serum interference was obtained by using both aptamers in a sandwich format, with excellent reproducibility and recovery values. Despite the sensitivity needing to be enhanced to the low picomolar range, these results are encouraging for the development of new, low-cost, and rapid antibody-free colorimetric assays for AMI studies based on aptamer-Troponin T recognition.

Multivalent Glycomimetics with Affinity and Selectivity toward Fucose-Binding Receptors from Emerging Pathogens.

Bacterial and fungal pathogens involved in lung infection in cystic fibrosis patients utilize a particular family of glycan-binding proteins, characterized by the presentation of six fucose-binding sites on a ring-shaped scaffold. These lectins are attractive targets for anti-infectious compounds that could interfere in the recognition of host tissues by pathogens. The design of a cyclopeptide-based hexavalent structure allowed for the presentation of six fucose residues. The synthetic hexavalent compound displays liable geometry resulting in high-avidity binding by lectins from Aspergillus fumigatus and Burkholderia ambifaria. Replacing the fucose residue with a conformationally constrained fucomimetic does not alter the affinity and provides fine specificity with no binding to other fucose-specific lectins.

Synthesis and binding monitoring of a new nanomolar PAMAM-based matrix metalloproteinases inhibitor (MMPIs).

Dendrimers are efficient drug delivery systems particularly useful in ocular diseases. In particular, low generation PAMAM dendrimers are non-toxic and non-immunogenic and they provide an enhancement of the residence time of drugs in the eyes. In this context, the synthesis of the PAMAM-based matrix metalloproteinases inhibitor 5, is reported. In particular, we demonstrated that 5 strongly binds (18.0nM±2.5nM) MMP-9, the most relevant MMP responsible of ocular surface damages in induced dry eyes syndrome (DES).

Sensitive 'two-steps' competitive assay for gonadotropin-releasing hormone detection via SPR biosensing and polynorepinephrine-based molecularly imprinted polymer.

The work reports an innovative bioassay for the detection of gonadorelin in urine, a gonadotropin-releasing hormone agonist widely used in fertility medicine and to treat hormonal dysfunctions. Gonadorelin is also a synthetic hormone listed by the World Anti-Doping Agency (WADA) and of interest in anti-doping controls. The main novelty relies on the development of a biocompatible, stable, and low-cost biomimetic receptor alternative to classic antibodies. Starting from norepinephrine monomer, a highly selective and sensitive molecularly imprinted polymer (MIP) was developed and optimized for optical real-time and label-free SPR biosensing. The selectivity has been addressed by testing a series of peptides, from high to low similarity, both in terms of molecular weight and primary sequence. Due to the very low molecular weight of gonadorelin (1182 Da), a 'two-steps' competitive assay was developed. Particular attention has been paid to the design of the competitor and its binding affinity constant towards the MIP, being a key step for the success of the competitive strategy. The SPR assay was first optimized in standard conditions and finally applied to untreated urine samples, achieving the sensitivity required by WADA guidelines. The MIP, tested in parallel with a monoclonal antibody, gave comparable results in terms of affinity constants and selectivity towards possible interfering analytes. However, the biomimetic receptor appears clearly superior in terms of sensitivity and reproducibility. This, together with its preparation simplicity, the extremely low-cost of the monomer and its reusability for hundreds of measurements, make polynorepinephrine-based MIPs powerful rivals to immune-based approaches in the near future for similar applications.

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.

A straightforward synthesis of phenyl boronic acid (PBA) containing BODIPY dyes: new functional and modular fluorescent tools for the tethering of the glycan domain of antibodies.

We report here on the efficient and straightforward synthesis of a series of modular and functional PBA-BODIPY dyes 1-4. They are an outstanding example of the efficient merge of the versatility of the 3,5-dichloro-BODIPY derivatives and the receptor-like ability of the PBA moiety. The potential bioanalytical applicability of these tools was assessed by measuring the binding to glycan chains of antibodies by a Quartz Crystal Microbalance (QCM).

Quantum Dot-Based Probes for Labeling and Imaging of Cells that Express Matrix Metalloproteinases.

The practical synthesis of novel multivalent fluorescent quantum-dot-based probes to target cellular matrix metalloproteinases (MMPs) (MT-MMPs) is reported. We show that these probes, which are decorated with a nanomolar water-soluble MMP inhibitor, can be used to label preferentially the surface of cancer cells that are known to express MMPs while no binding was observed on cells that do not.

Active-Site Targeting Paramagnetic Probe for Matrix Metalloproteinases.

The design and synthesis of the Ln3+ complexes of a DOTA-containing (DOTA=1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid) inhibitor of matrix metalloproteinases are reported. The tight binding of the sulfonamide scaffold to the catalytic domain of the investigated matrix metalloproteinase is not impaired by the presence of the Ln3+ -DOTA moiety. The paramagnetic properties of the Ln3+ complex are exploited to obtain insights into the structural features of the ligand-protein interactions and to evaluate the influence of the linker length on the quality of the paramagnetic restraints.

Structural Modifications of cis-Glycofused Benzopyran Compounds and Their Influence on the Binding to Amyloid-ß Peptide.

A small library of glycofused tricyclic compounds with a central pyran ring chemically modified in the position para to the ring oxygen has been synthesised. The influence of the chemical modification on the structural conformation of the compounds and on their ability to bind Aß peptide has been evaluated respectively using molecular mechanics (MM) and molecular dynamics (MD) simulations, and STD NMR spectroscopy. The introduction of particularly polar/charged groups leads to the loss of binding ability, without a significant change in the conformation, whilst other substitutions does not significantly affect either the structural conformation or the binding.