Biolayer interferometry
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Pulmonary hypertension is a cardiovascular disease with a low survival rate. The protein galectin-3 (Gal-3) binding β-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.
- MeSH
- arteria pulmonalis účinky léků metabolismus MeSH
- biologické markery MeSH
- fibroblasty účinky léků metabolismus MeSH
- galektin 3 * antagonisté a inhibitory metabolismus MeSH
- krysa rodu rattus MeSH
- kultivované buňky MeSH
- lidé MeSH
- plicní hypertenze * farmakoterapie metabolismus MeSH
- polymery chemie farmakologie MeSH
- tkáňová distribuce MeSH
- zvířata MeSH
- Check Tag
- krysa rodu rattus MeSH
- lidé MeSH
- mužské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
Galectins are lectins that bind β-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 β-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.
- MeSH
- galektiny * chemie MeSH
- glykokalyx * MeSH
- lidé MeSH
- ligandy MeSH
- molekulární konformace MeSH
- prospektivní studie MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Galectin-3 (Gal-3) participates in many cancer-related metabolic processes. The inhibition of overexpressed Gal-3 by, e.g., β-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.
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.
- MeSH
- galektiny * metabolismus MeSH
- glykokonjugáty * farmakologie chemie MeSH
- lidé MeSH
- ligandy MeSH
- polysacharidy metabolismus MeSH
- sacharidy chemie MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Monogenic diabetes is a gateway to precision medicine through molecular mechanistic insight. Hepatocyte nuclear factor 1A (HNF-1A) and HNF-4A are transcription factors that engage in crossregulatory gene transcription networks to maintain glucose-stimulated insulin secretion in pancreatic β cells. Variants in the HNF1A and HNF4A genes are associated with maturity-onset diabetes of the young (MODY). Here, we explored 4 variants in the P2-HNF4A promoter region: 3 in the HNF-1A binding site and 1 close to the site, which were identified in 63 individuals from 21 families of different MODY disease registries across Europe. Our goal was to study the disease causality for these variants and to investigate diabetes mechanisms on the molecular level. We solved a crystal structure of HNF-1A bound to the P2-HNF4A promoter and established a set of techniques to probe HNF-1A binding and transcriptional activity toward different promoter variants. We used isothermal titration calorimetry, biolayer interferometry, x-ray crystallography, and transactivation assays, which revealed changes in HNF-1A binding or transcriptional activities for all 4 P2-HNF4A variants. Our results suggest distinct disease mechanisms of the promoter variants, which can be correlated with clinical phenotype, such as age of diagnosis of diabetes, and be important tools for clinical utility in precision medicine.
- MeSH
- diabetes mellitus 2. typu * genetika metabolismus MeSH
- hepatocytární jaderný faktor 1-alfa * genetika metabolismus MeSH
- hepatocytární jaderný faktor 4 * genetika metabolismus MeSH
- krystalografie rentgenová MeSH
- lidé MeSH
- promotorové oblasti (genetika) * genetika MeSH
- regulace genové exprese MeSH
- vazba proteinů MeSH
- vazebná místa MeSH
- Check Tag
- lidé MeSH
- mužské pohlaví MeSH
- ženské pohlaví MeSH
- Publikační typ
- časopisecké články MeSH
