Glycomimetic inhibitors of tandem-repeat galectins: Simple and efficient.

The binding of human galectins by glycomimetic inhibitors is a promising therapeutic approach. The structurally distinct group of tandem-repeat galectins has scarcely been studied so far, and there is hardly any knowledge on their ligand specificity or their inhibitory potential, particularly concerning non-natural carbohydrates. Here, we present the synthesis of a library of seven 3-O-disubstituted thiodigalactoside-derived glycomimetics and their affinity to two tandem-repeat galectins, Gal-8 and Gal-9. The straightforward synthesis of these glycomimetics involved dibutyltin oxide-catalyzed 3,3́-O-disubstitution of commercially available unprotected thiodigalactoside, and conjugation of various aryl substituents by copper-catalyzed Huisgen azide-alkyne cycloaddition (CuAAC). The inhibitory potential of the prepared glycomimetics for Gal-8 and Gal-9 was assessed, and compared with the established galectins Gal-1 and Gal-3. The introduction of C-3 substituents resulted in an over 40-fold increase in affinity compared with unmodified TDG. The structure-affinity relations within the studied series were discussed using molecular modeling. Furthermore, the prepared glycomimetics were shown to scavenge Gal-8 and Gal-9 from the surface of cancer cells. This pioneering study on the synthetic inhibitors especially of Gal-9 identified lead compounds that may be used in further biomedical research.

Synthesis and unexpected binding of monofluorinated N,N'-diacetylchitobiose and LacdiNAc to wheat germ agglutinin.

Fluorination of carbohydrate ligands of lectins is a useful approach to examine their binding profile, improve their metabolic stability and lipophilicity, and convert them into 19F NMR-active probes. However, monofluorination of monovalent carbohydrate ligands often leads to a decreased or completely lost affinity. By chemical glycosylation, we synthesized the full series of methyl ß-glycosides of N,N'-diacetylchitobiose (GlcNAcß(1-4)GlcNAcß1-OMe) and LacdiNAc (GalNAcß(1-4)GlcNAcß1-OMe) systematically monofluorinated at all hydroxyl positions. A competitive enzyme-linked lectin assay revealed that the fluorination at the 6'-position of chitobioside resulted in an unprecedented increase in affinity to wheat germ agglutinin (WGA) by one order of magnitude. For the first time, we have characterized the binding profile of a previously underexplored WGA ligand LacdiNAc. Surprisingly, 4'-fluoro-LacdiNAc bound WGA even stronger than unmodified LacdiNAc. These observations were interpreted using molecular dynamic calculations along with STD and transferred NOESY NMR techniques, which gave evidence for the strengthening of CH/π interactions after deoxyfluorination of the side chain of the non-reducing GlcNAc. These results highlight the potential of fluorinated glycomimetics as high-affinity ligands of lectins and 19F NMR-active probes.

Glycocalix[4]arenes and their affinity to a library of galectins: the linker matters.

Galectins are lectins that bind ß-galactosides. They are involved in important extra- and intracellular biological processes such as apoptosis, and regulation of the immune system or the cell cycle. High-affinity ligands of galectins may introduce new therapeutic approaches or become new tools for biomedical research. One way of increasing the low affinity of ß-galactoside ligands to galectins is their multivalent presentation, e.g., using calixarenes. We report on the synthesis of glycocalix[4]arenes in cone, partial cone, 1,2-alternate, and 1,3-alternate conformations carrying a lactosyl ligand on three different linkers. The affinity of the prepared compounds to a library of human galectins was determined using competitive ELISA assay and biolayer interferometry. Structure-affinity relationships regarding the influence of the linker and the core structure were formulated. Substantial differences were found between various linker lengths and the position of the triazole unit. The formation of supramolecular clusters was detected by atomic force microscopy. The present work gives a systematic insight into prospective galectin ligands based on the calix[4]arene core.

Selective referral or learning by doing? An analysis of hospital volume-outcome relationship of vascular procedures.

This paper analyzes the effects of hospital volume on outcomes of patients undergoing percutaneous transluminal angioplasty (PTA) with stent implant in Slovakia between 2014 and 2019. The volume-outcome relationship is estimated jointly using a discrete factor approach, where choice of hospital is correlated with durations until readmission or death, accounting for observed and unobserved characteristics. The results reveal the importance of controlling for between-hospital differences and selectivity in patient referral. Estimates without hospital fixed effects overstate the positive effect of volume on outcomes, but the results remain statistically significant. Once selectivity is accounted for in the joint correlated model, the positive volume-outcome relationship is not different from zero. Overall, the main driver of the volume-outcome relationship for PTA procedures appears to be related to selective referral and differences in quality of health care providers.

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.

Oligosaccharide Ligands of Galectin-4 and Its Subunits: Multivalency Scores Highly.

Galectins are carbohydrate-binding lectins that modulate the proliferation, apoptosis, adhesion, or migration of cells by cross-linking glycans on cell membranes or extracellular matrix components. Galectin-4 (Gal-4) is a tandem-repeat-type galectin expressed mainly in the epithelial cells of the gastrointestinal tract. It consists of an N- and a C-terminal carbohydrate-binding domain (CRD), each with distinct binding affinities, interconnected with a peptide linker. Compared to other more abundant galectins, the knowledge of the pathophysiology of Gal-4 is sparse. Its altered expression in tumor tissue is associated with, for example, colon, colorectal, and liver cancers, and it increases in tumor progression, and metastasis. There is also very limited information on the preferences of Gal-4 for its carbohydrate ligands, particularly with respect to Gal-4 subunits. Similarly, there is virtually no information on the interaction of Gal-4 with multivalent ligands. This work shows the expression and purification of Gal-4 and its subunits and presents a structure-affinity relationship study with a library of oligosaccharide ligands. Furthermore, the influence of multivalency is demonstrated in the interaction with a model lactosyl-decorated synthetic glycoconjugate. The present data may be used in biomedical research for the design of efficient ligands of Gal-4 with diagnostic or therapeutic potential.

Regioselective 3-O-Substitution of Unprotected Thiodigalactosides: Direct Route to Galectin Inhibitors.

The synthesis of tailored bioactive carbohydrates usually comprises challenging (de)protection steps, which lowers synthetic yields and increases time demands. We present here a regioselective single-step introduction of benzylic substituents at 3-hydroxy groups of ß-d-galactopyranosyl-(1→1)-thio-ß-d-galactopyranoside (TDG) employing dibutyltin oxide in good yields. These glycomimetics act as inhibitors of galectins-human lectins, which are biomedically attractive targets for therapeutic inhibition in, for example, cancerogenesis. The affinity of the prepared glycomimetics to galectin-1 and galectin-3 was studied in enzyme-linked immunosorbent (ELISA)-type assays and their potential to inhibit galectin binding on the cell surface was shown. We used our original in vivo biotinylated galectin constructs for easy detection by flow cytometry. The results of the biological experiments were compared with data from molecular modeling with both galectins. The present work reveals a facile and elegant synthetic route for the preparation of TDG-derived glycomimetics that exhibit differing selectivity and affinity to galectins depending on the choice of 3-O-substitution.

Long-term returns to local health-care spending.

This paper investigates the effects of health-care spending on mortality rates of patients who experienced a heart attack. We relate in-hospital deaths to in-hospital spending and post-discharge deaths to post-discharge health-care spending. In our analysis, we use detailed administrative data on individual personal characteristics including comorbidities, information about the type of medical treatment and information about health-care expenses at the regional level. To account for potential selectivity in the region of health-care treatment we compare local patients with visitors and stayers with recent movers from a different region. We find that in regions with higher health-care spending mortality after heart attacks is substantially lower. From this we conclude that there are long-term returns to local health-care spending.

Glycopolymer Inhibitors of Galectin-3 Suppress the Markers of Tissue Remodeling in Pulmonary Hypertension.

Pulmonary hypertension is a cardiovascular disease with a low survival rate. The protein galectin-3 (Gal-3) binding ß-galactosides of cellular glycoproteins plays an important role in the onset and development of this disease. Carbohydrate-based drugs that target Gal-3 represent a new therapeutic strategy in the treatment of pulmonary hypertension. Here, we present the synthesis of novel hydrophilic glycopolymer inhibitors of Gal-3 based on a polyoxazoline chain decorated with carbohydrate ligands. Biolayer interferometry revealed a high binding affinity of these glycopolymers to Gal-3 in the subnanomolar range. In the cell cultures of cardiac fibroblasts and pulmonary artery smooth muscle cells, the most potent glycopolymer 18 (Lac-high) caused a decrease in the expression of markers of tissue remodeling in pulmonary hypertension. The glycopolymers were shown to penetrate into the cells. In a biodistribution and pharmacokinetics study in rats, the glycopolymers accumulated in heart and lung tissues, which are most affected by pulmonary hypertension.

Galectin-targeting glycocalix[4]arenes can enter the cells.

Elevated levels of galectin-3 are associated with tumorigenesis. Its inhibition with high-affinity carbohydrate ligands opens new therapeutic routes. Targeting of intracellular galectin-3 is challenging for polar inhibitors like carbohydrates. We demonstrate the potential of novel biomedical research tools, glycocalix[4]arenes, to enter epithelial cells, which may allow their interaction with galectin-3.

Glycopolymers Decorated with 3-O-Substituted Thiodigalactosides as Potent Multivalent Inhibitors of Galectin-3.

Galectin-3 (Gal-3) participates in many cancer-related metabolic processes. The inhibition of overexpressed Gal-3 by, e.g., ß-galactoside-derived inhibitors is hence promising for cancer treatment. The multivalent presentation of such inhibitors on a suitable biocompatible carrier can enhance the overall affinity to Gal-3 and favorably modify the interaction with Gal-3-overexpressing cells. We synthesized a library of C-3 aryl-substituted thiodigalactoside inhibitors and their multivalent N-(2-hydroxypropyl)methacrylamide (HPMA)-based counterparts with two different glycomimetic contents. Glycopolymers with a higher content of glycomimetic exhibited a higher affinity to Gal-3 as assessed by ELISA and biolayer interferometry. Among them, four candidates (with 4-acetophenyl, 4-cyanophenyl, 4-fluorophenyl, and thiophen-3-yl substitution) were selected for further evaluation in cancer-related experiments in cell cultures. These glycopolymers inhibited Gal-3-induced processes in cancer cells. The cyanophenyl-substituted glycopolymer exhibited the strongest antiproliferative, antimigratory, antiangiogenic, and immunoprotective properties. The prepared glycopolymers appear to be prospective modulators of the tumor microenvironment applicable in the therapy of Gal-3-associated cancers.

Cannabis decriminalization and the age of onset of cannabis use.

BACKGROUND: In the Czech Republic in 2010 a law was introduced decriminalizing personal possession of small quantities of several illicit drugs, including cannabis. METHODS: We use 2012 survey data to examine the effect of a change in cannabis policy on the age of onset of cannabis use. We estimate the effect of the policy change using a mixed proportional hazards framework that models the transition to first cannabis use. RESULTS: The change in cannabis policy did not affect the transition to first cannabis use. CONCLUSION: We find no evidence of cannabis decriminalization affecting the age of onset of cannabis use.