Extracellular and intracellular small-molecule galectin-3 inhibitors (original) (raw)

Galectin-3 is a carbohydrate binding protein which has important roles in cancer and immunity. Potent galectin-3 inhibitors have been synthesized, for experimental purposes and potential clinical use. As galectin-3 is implicated in both intra-and extracellular activities, permeability of galectin-3 inhibitors is an important parameter determining biological effects. We compared the cellular uptake of galectin-3 inhibitors and their potency in the intracellular or extracellular space. The inhibitors differed in their polar surface area (PSA), but had similar affinities for galectin-3. Using a well-established permeability assay, we confirmed that the uptake was significantly higher for the inhibitor with the lowest PSA, as expected. To analyze intracellular activity of the inhibitors, we developed a novel assay based on galectin-3 accumulation around damaged intracellular vesicles. The results show striking differences between the inhibitors intracellular potency, correlating with their PSAs. To test extracellular activity of the inhibitors, we analyzed their potency to block binding of galectin-3 to cell surfaces. All inhibitors were equally able to block galectin-3 binding to cells and this was proportional to their affinity for galectin-3. These inhibitors may serve as useful tools in exploring biological roles of galectin-3 and may further our understanding of intracellular versus extracellular roles of galectin-3. The galectin family of carbohydrate binding proteins have gained increasing interest as therapeutic targets in several diseases, such as chronic inflammation and cancer 1-4. Galectins are soluble proteins synthesized on free ribosomes in the cytosol. Even though they lack the classical characteristics of secreted proteins, they are rapidly translocated to the extracellular space through a yet unknown pathway 5. Once in the extracellular environment, the galectins are exposed to a large variety of glycan structures, where they recognize and bind specific β-galactosides. As some galectins are able to form multivalent structures or are multivalent in nature, they are able to cross-link glycoconjugates and form lattices. Formation of galectin/glycoconjugate lattices on the plasma membrane has been observed to influence the expression time, localization, and activity of several cell surface receptors, thus influencing numerous biological functions such as cell signaling, cell migration, and cell adherence 5,6. Furthermore, galectins can quickly (within minutes) be recycled back to the inside of cells trough the endocytic pathway, regulating sorting of both soluble and membrane bound glycoconjugates 5,7. Apart from the extracellular activities of the galectin family, mediated through glycan binding, galectins also play important roles in the intracellular compartments. Several studies have reported that galectins may influence cell signaling by interacting with signaling proteins in the cytosol, e.g. RAS proteins and β-catenin, and RNA splicing through binding of components of the spliceosome complex in the nucleus 8-12. As complex glycans are not found in the intracellular compartment, these activities are most likely mediated trough protein-protein interactions. Interestingly, however, several of these reported intracellular activates are inhibited by molecules interacting with the galectin carbohydrate binding site, such as lactose 8,10,13. Additionally, galectins play an important role at the interface between the cytosolic and intravesicular compartments by monitoring the integrity of vesicular membranes. It is now well established that several galectins (e.g. galectin-3 and-8 in particular) accumulate around disrupted vesicles by binding to exposed glycan structures and induce clearance of the damaged organelles by selective autophagy 14-20. The diverse roles of the galectins on a cellular level have consequences for several physiological