Hepatocyte Targeting and Intracellular Copper Chelation by a Thiol-Containing Glycocyclopeptide

Abstract

Metal overload plays an important role in several diseases or intoxications, like in Wilson's disease, a major genetic disorder of copper metabolism in humans. To efficiently and selectively decrease copper concentration in the liver that is highly damaged, chelators should be targeted at the hepatocytes. In the present work, we synthesized a molecule able to both lower intracellular copper, namely Cu(I), and target hepatocytes, combining within the same structure a chelating unit and a carbohydrate recognition element. A cyclodecapeptide scaffold displaying a controlled conformation with two independent faces was chosen to introduce both units. One face displays a cluster of carbohydrates to ensure an efficient recognition of the asialoglycoprotein receptors, expressed on the surface of hepatocytes. The second face is devoted to metal ion complexation thanks to the thiolate functions of two cysteine side-chains. To obtain a chelator that is active only once inside the cells, the two thiol functions were oxidized in a disulfide bridge to afford the glycopeptide P(3). Two simple cyclodecapeptides modeling the reduced and complexing form of P(3) in cells proved a high affinity for Cu(I) and a high selectivity with respect to Zn(II). As expected, P(3) becomes an efficient Cu(I) chelator in the presence of glutathione that mimics the intracellular reducing environment. Finally, cellular uptake and ability to lower intracellular copper were demonstrated in hepatic cell lines, in particular in WIF-B9, making P(3) a good candidate to fight copper overload in the liver.

A Series of Tripodal Cysteine Derivatives as Water‐Soluble Chelators that are Highly Selective for Copper(I)

“…[12] We have shown here that the introduction of three cysteine residues in a tripodal architecture provides even more efficient Cu I chelators with large selectivities with respect to Zn II . Three tripodal molecules derived from nitrilotriacetic acid were synthesised and differ by the nature of the carbonyl function adjacent to the thiol group.…”

“…In particular, intracellular copper chelation would be an efficient tool to remove metal ions from organs where it is accumulated. [12] As the cytoplasm of most eukaryotic cells is a reducing environment, the predominant oxidation state of copper in cells is Cu I , [7] which has a soft character and thus a high affinity for soft donors like thiolates. This preference for soft sulfur ligands is exemplified in proteins involved in copper homeostasis, [3] which mainly bind these ions with several thiolates of cysteine side chains.…”

“…To design efficient and selective soft metal ion chelators, we take advantage of the high affinity of cysteine sulfur donors for Cu I , evidenced in proteins trafficking or sequestering this metal in cells. [12,14,15,19] As trithiolato coordination environments afford very stable Cu I complexes in metallothioneins, ligands with three cysteine residues that promote a MS 3 geometry are very attractive. Therefore, three cysteine moieties can be attached with peptide bonds to nonbiological scaffolds to get pseudopeptide ligands.…”

Crucial role of copper in detection of metal-coordinating odorants

“…, presumably the form in which it reduces S-S bonds and forms odorant complexes (13 ) did not affect receptor activation ( Fig. 2A).…”

Methanobactin reverses acute liver failure in a rat model of Wilson disease

“…Intriguingly, this positive effect was not due to a reduction of cellular copper but rather to effects on sphingolipid homeostasis (14). A third study reported hepatocyte targeting and intracellular copper chelation by specifically designed glycocyclopeptides (15). Despite this recent progress involving new therapeutic routes in WD, these studies also emphasize the unmet need for new medications that especially counteract acute liver failure in WD.…”