Analogs of Tetrahydroisoquinoline Natural Products That Inhibit Cell Migration and Target Galectin-3 Outside of Its Carbohydrate-binding Site

Abstract

Cell migration is central to a number of normal and disease processes. Small organic molecules that inhibit cell migration have potential as both research probes and therapeutic agents. We have identified two tetrahydroisoquinoline natural product analogs with antimigratory activities on Madin-Darby canine kidney epithelial cells: a semisynthetic derivative of quinocarmycin (also known as quinocarcin), DX-52-1, and a more complex synthetic molecule, HUK-921, related to the naphthyridinomycin family. It has been assumed that the cellular effects of reactive tetrahydroisoquinolines result from the alkylation of DNA. We have reported previously that the primary target of DX-52-1 relevant to cell migration appears to be the membranecytoskeleton linker protein radixin. Here we extend the analysis of the protein targets of DX-52-1, reporting that the multifunctional carbohydrate-binding protein galectin-3 is a secondary target of DX-52-1 that may also be relevant to the antimigratory effects of both DX-52-1 and HUK-921. All known inhibitors of galectin-3 target its ␤-galactoside-binding site in the carbohydrate recognition domain. However, we found that DX-52-1 and HUK-921 bind galectin-3 outside of its ␤-galactoside-binding site. Intriguingly HUK-921, although a less potent inhibitor of cell migration than DX-52-1, had far greater selectivity for galectin-3 over radixin, exhibiting little binding to radixin, both in vitro and in cells. Overexpression of galectin-3 in cells led to a dramatic increase in cell adhesion on different extracellular matrix substrata as well as changes in cell-cell adhesion and cell motility. Galectin-3-overexpressing cells had greatly reduced sensitivity to DX-52-1 and HUK-921, and these compounds caused a change in localization of the overexpressed galectin-3 and reversion of the cells to a more normal morphology. The converse manipulation, RNA interference-based silencing of galectin-3 expression, resulted in reduced cell-matrix adhesion and cell migration. In aggregate, the data suggest that DX-52-1 and HUK-921 inhibit a carbohydrate binding-independent function of galectin-3 that is involved in cell migration.

Galectin-3 in Renal Pathology: More Than Just an Innocent Bystander?

“…Treatment of galectin-3-overexpressing MDCK cells with DX-52-1 or HUK-921 resulted in a change in localization of GFP-galectin-3 and reversion of the galectin-3-overexpressing cells from a highly spread state to a more normal epithelial morphology. The data suggest that DX-52-1 and HUK-921 inhibit a carbohydrate binding-independent function of galectin-3 that is involved in cell migration [90] .…”

Inhibition of Galectins with Small Molecules

“…[50] Although, comparatively little effort has been directed towards high-throughput screening of galectins against large compound libraries, a few examples have been published where non-carbohydrate synthetic inhibitors have been identified to act by allosteric inhibition of galectin-3. [51] Finally, synthetic, small-molecule inhibitors bind in competition with endogenous ligands to prevent normal galectin function. They can be made more hydrophobic than other inhibitors, allowing improved bioavailability.…”

Crystal structure of 4-(4-hydroxy-3-methoxy-phenyl)-2,7,7-trimethyl-5-oxo-1,4,5,6,7,8-hexahydro-quinoline-3-carboxylic acid methyl ester, C21H25NO5

“…The title compound was synthesized according to a reported procedure [5]. A mixture of 5,5-dimethyl-cyclohexane-1,3-dione (10 mmol), 3-methoxy-4-hydroxy-benzaldehyde (10 mmol), and 3-amino-2-butenoic acid methyl ester (10 mmol) in ethanol (100 mL) was refluxed for 2-3 h and then cooled to room temperature.…”