A Fluorescent sp²‐Iminosugar With Pharmacological Chaperone Activity for Gaucher Disease: Synthesis and Intracellular Distribution Studies

Abstract

Gaucher disease (GD) is the most prevalent lysosomal-storage disorder, it is caused by mutations of acid β-glucosidase (β-glucocerebrosidase; β-Glu). Recently, we found that bicyclic nojirimycin (NJ) derivatives of the sp(2)-iminosugar type, including the 6-thio-N'-octyl-(5N,6S)-octyliminomethylidene derivative (6S-NOI-NJ), behaved as very selective competitive inhibitors of the lysosomal β-Glu and exhibited remarkable chaperone activities for several GD mutations. To obtain information about the cellular uptake pathway and intracellular distribution of this family of chaperones, we have synthesized a fluorescent analogue that maintains the fused piperidine-thiazolidine bicyclic skeleton and incorporates a dansyl group in the N'-substituent, namely 6-thio-(5N,6S)-[4-(N'-dansylamino)butyliminomethylidene]nojirimycin (6S-NDI-NJ). This structural modification does not significantly modify the biological activity of the glycomimetic as a chemical chaperone. Our study showed that 6S-NDI-NJ is mainly located in lysosome-related organelles in both normal and GD fibroblasts, and the fluorescent intensity of 6S-NDI-NJ in the lysosome is related to the β-Glu concentration level. 6S-NDI-NJ also can enter cultured neuronal cells and act as a chaperone. Competitive inhibition studies of 6S-NDI-NJ uptake in fibroblasts showed that high concentrations of D-glucose have no effect on chaperone internalization, suggesting that it enters the cells through glucose-transporter-independent mechanisms.

Candidate Molecules for Chemical Chaperone Therapy of G M1 -Gangliosidosis

“…These compounds are characterized by a glycone-type moiety that mimics the sugar substrate of the enzyme and a hydrophobic aglycone substituent that interacts with amino acids at the vicinity of the active site. The overall structure has amphiphilic character, imparting their ability to cross biological membranes [41]. Structure-activity relationship and x-ray studies suggested that the nature and orientation of the aglycon segment is critical for the enzyme selectivity [42,43].…”

Progress and potential of non-inhibitory small molecule chaperones for the treatment of Gaucher disease and its implications for Parkinson disease

“…Modification of iminosugars to sp 2 -iminosugars showed increased selectivity for β-glucosidases. However, enzyme activity assays and confocal microscopy-based imaging studies on patient fibroblasts and a neuronal mouse cell line treated with sp 2 -iminosugars revealed a high inhibitor to chaperone balance (71, 72). …”

Iminosugars: Therapeutic Applications and Synthetic Considerations

“…As expected all of these inhibitors present key attributes for glucose mimicry. As a direct glucose mimetic, analogues based on scaffold 5 have been evaluated extensively [174][175][176][177][178]. Although these compounds have demonstrated activity in vitro, historically, inhibitors based on this scaffold have been associated with signficant side effects resulting from limited selectivity.…”