Galectins are a family of galactoside-recognizing proteins involved in different galectin-subtype-specific inflammatory and tumor-promoting processes, which motivates the development of inhibitors ...that are more selective galectin inhibitors than natural ligand fragments. Here, we describe the synthesis and evaluation of 3-
-methyl-gulopyranoside derivatives and their evaluation as galectin inhibitors. Methyl 3-deoxy-3-
-(hydroxymethyl)-β-d-gulopyranoside showed 7-fold better affinity for galectin-1 than the natural monosaccharide fragment analog methyl β-d-galactopyranoside, as well as a high selectivity over galectin-2, 3, 4, 7, 8, and 9. Derivatization of the 3-
-hydroxymethyl into amides gave gulosides with improved selectivities and affinities; methyl 3-deoxy-3-
-(methyl-2,3,4,5,6-pentafluorobenzamide)-β-d-gulopyranoside had K
700 µM for galectin-1, while not binding any other galectin.
Symmetrical and asymmetrical fluorinated phenyltriazolyl-thiodigalactoside derivatives have been synthesized and evaluated as inhibitors of galectin-1 and galectin-3. Systematic tuning of the ...phenyltriazolyl-thiodigalactosides’ fluoro-interactions with galectin-3 led to the discovery of inhibitors with exceptional affinities (K d down to 1–2 nM) in symmetrically substituted thiodigalactosides as well as unsurpassed combination of high affinity (K d 7.5 nM) and selectivity (46-fold) over galectin-1 for asymmetrical thiodigalactosides by carrying one trifluorphenyltriazole and one coumaryl moiety. Studies of the inhibitor–galectin complexes with isothermal titration calorimetry and X-ray crystallography revealed the importance of fluoro-amide interaction for affinity and for selectivity. Finally, the high affinity of the discovered inhibitors required two competitive titration assay tools to be developed: a new high affinity fluorescent probe for competitive fluorescent polarization and a competitive ligand optimal for analyzing high affinity galectin-3 inhibitors with competitive isothermal titration calorimetry.
A new series of orally available α-d-galactopyranosides with high affinity and specificity toward galectin-1 have been discovered. High affinity and specificity were achieved by changing six-membered ...aryl-triazolyl substituents in a series of recently published galectin-3-selective α-d-thiogalactosides (e.g., GB1107 K d galectin-1/3 3.7/0.037 μM) for five-membered heterocycles such as thiazoles. The in vitro pharmacokinetic properties were optimized, resulting in several galectin-1 inhibitors with favorable properties. One compound, GB1490 (K d galectin-1/3 0.4/2.7 μM), was selected for further characterization toward a panel of galectins showing a selectivity of 6- to 320-fold dependent on galectin. The X-ray structure of GB1490 bound to galectin-1 reveals the compound bound in a single conformation in the carbohydrate binding site. GB1490 was shown to reverse galectin-1-induced apoptosis of Jurkat cells at low μM concentrations. No cell cytotoxicity was observed for GB1490 up to 90 μM in the A549 cells. In pharmacokinetic studies in mice, GB1490 showed high oral bioavailability (F% > 99%).
The design of small and high‐affinity lectin inhibitors remains a major challenge because the natural ligand binding sites of lectin are often shallow and have polar character. Herein we report that ...derivatizing galactose with un‐natural structural elements that form multiple non‐natural lectin–ligand interactions (orthogonal multipolar fluorine–amide, phenyl–arginine, sulfur–π, and halogen bond) can provide inhibitors with extraordinary affinity (low nanomolar) for the model lectin, galectin‐3, which is more than five orders of magnitude higher than the parent galactose; moreover, is selective over other galectins.
Specific interactions working in concert! Derivatizing a low‐affinity monosaccharide with functionalities that form a combination of orthogonal multipolar fluorine–amide, phenyl–arginine, sulfur–π, and halogen bond interactions results in lectin ligands with affinities that far surpass those of common natural ligand fragments. These compounds are the smallest high‐affinity galectin‐3 inhibitors described and thus constitute a new class of promising drug lead structures.
Many pathogenic bacteria utilise sialic acids as an energy source or use them as an external coating to evade immune detection. As such, bacteria that colonise sialylated environments deploy specific ...transporters to mediate import of scavenged sialic acids. Here, we report a substrate-bound 1.95 Å resolution structure and subsequent characterisation of SiaT, a sialic acid transporter from Proteus mirabilis. SiaT is a secondary active transporter of the sodium solute symporter (SSS) family, which use Na
gradients to drive the uptake of extracellular substrates. SiaT adopts the LeuT-fold and is in an outward-open conformation in complex with the sialic acid N-acetylneuraminic acid and two Na
ions. One Na
binds to the conserved Na2 site, while the second Na
binds to a new position, termed Na3, which is conserved in many SSS family members. Functional and molecular dynamics studies validate the substrate-binding site and demonstrate that both Na
sites regulate N-acetylneuraminic acid transport.
Abstract
Among the responders to microbial invasion, neutrophils represent the earliest and perhaps the most important immune cells that contribute to host defense with the primary role to kill ...invading microbes using a plethora of stored anti-microbial molecules. One such process is the production of reactive oxygen species (ROS) by the neutrophil enzyme complex NADPH-oxidase, which can be assembled and active either extracellularly or intracellularly in phagosomes (during phagocytosis) and/or granules (in the absence of phagocytosis). One soluble factor modulating the interplay between immune cells and microbes is galectin-3 (gal-3), a carbohydrate-binding protein that regulates a wide variety of neutrophil functions. Gal-3 has been shown to potentiate neutrophil interaction with bacteria, including Staphylococcus aureus, and is also a potent activator of the neutrophil respiratory burst, inducing large amounts of granule-localized ROS in primed cells. Herein, the role of gal-3 in regulating S. aureus phagocytosis and S. aureus-induced intracellular ROS was analyzed by imaging flow cytometry and luminol-based chemiluminescence, respectively. Although gal-3 did not interfere with S. aureus phagocytosis per se, it potently inhibited phagocytosis-induced intracellular ROS production. Using the gal-3 inhibitor GB0139 (TD139) and carbohydrate recognition domain of gal-3 (gal-3C), we found that the gal-3-induced inhibitory effect on ROS production was dependent on the carbohydrate recognition domain of the lectin. In summary, this is the first report of an inhibitory role of gal-3 in regulating phagocytosis-induced ROS production.
Glycosylation is critical for the regulation of several cellular processes. One glycosylation pathway, the unusual O-linked β-N-acetylglucosamine glycosylation (O-GlcNAcylation) has been shown to be ...required for proper mitosis, likely through a subset of proteins that are O-GlcNAcylated during metaphase. As lectins bind glycosylated proteins, we asked if specific lectins interact with mitotic O-GlcNAcylated proteins during metaphase to ensure correct cell division. Galectin-3, a small soluble lectin of the Galectin family, is an excellent candidate, as it has been previously described as a transient centrosomal component in interphase and mitotic epithelial cells. In addition, it has recently been shown to associate with basal bodies in motile cilia, where it stabilizes the microtubule-organizing center (MTOC). Using an experimental mouse model of chronic kidney disease and human epithelial cell lines, we investigate the role of Galectin-3 in dividing epithelial cells. Here we find that Galectin-3 is essential for metaphase where it associates with NuMA in an O-GlcNAcylation-dependent manner. We provide evidence that the NuMA-Galectin-3 interaction is important for mitotic spindle cohesion and for stable NuMA localization to the spindle pole, thus revealing that Galectin-3 is a novel contributor to epithelial mitotic progress.
Inhibitors of galectin‐3, which has been implicated in cancer‐ and immunity‐related processes, can be synthesized from thiodigalactoside derivatives with aromatic amide substituents at both C3 ...positions (see structure). Dissociation constant (Kd) values as low as 33 nM have been obtained. Computer modeling suggests that such high affinity stems from double arginine–arene stacking interactions.
A chemical proteomics approach using Ca
/calmodulin-dependent protein kinase kinase (CaMKK) inhibitor-immobilized sepharose (TIM-063-Kinobeads) identified main targets such as CaMKKα/1 and β/2, and ...potential off-target kinases, including AP2-associated protein kinase 1 (AAK1), as TIM-063 interactants. Because TIM-063 interacted with the AAK1 catalytic domain and inhibited its enzymatic activity moderately (IC
= 8.51 µM), we attempted to identify potential AAK1 inhibitors from TIM-063-derivatives and found a novel AAK1 inhibitor, TIM-098a (11-amino-2-hydroxy-7H-benzodebenzo4,5imidazo2,1-aisoquinolin-7-one) which is more potent (IC
= 0.24 µM) than TIM-063 without any inhibitory activity against CaMKK isoforms and a relative AAK1-selectivity among the Numb-associated kinases family. TIM-098a could inhibit AAK1 activity in transfected cultured cells (IC
= 0.87 µM), indicating cell-membrane permeability of the compound. Overexpression of AAK1 in HeLa cells significantly reduced the number of early endosomes, which was blocked by treatment with 10 µM TIM-098a. These results indicate TIM-063-Kinobeads-based chemical proteomics is efficient for identifying off-target kinases and re-evaluating the kinase inhibitor (TIM-063), leading to the successful development of a novel inhibitory compound (TIM-098a) for AAK1, which could be a molecular probe for AAK1. TIM-098a may be a promising lead compound for a more potent, selective and therapeutically useful AAK1 inhibitor.
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