Unveiling the potential of novel alkylated iminosugars as innovative pH-responsive pharmacological chaperones through endo-cavity complexation with water-soluble prismarene hosts

Gaucher disease (GD), the most prevalent lysosomal storage disorder, is caused by a genetic mutation in the GBA gene, leading to the misfolding of the acid-β-glucosidase (GCase) enzyme. This misfolding disrupts the enzyme proper trafficking to the lysosome, resulting in the accumulation of natural substrate, which, in turn, triggers a cascade of severe systemic symptoms. A promising therapeutic approach is Pharmacological Chaperone (PC) Therapy [1], which utilizes small molecules, such as iminosugars, to stabilize and restore proper folding of the GCase enzyme. These molecules act as chaperones, aiding enzyme folding and trafficking to the lysosome, thus enhancing activity. For optimal efficacy, the PC should have higher affinity for GCase in the endoplasmic reticulum (pH ~7) and lower affinity in the lysosome (pH ~4.5), allowing the natural substrate to access the catalytic site and enhancing the therapeutic potential. This study aims to develop innovative PC candidates for GD based on intelligent host-guest systems that are responsive to physiological pH fluctuations. In this context, we synthesized novel mono- and bis-alkylated iminosugars with piperidine and pyrrolidine scaffolds, starting from cost-effective carbohydrate derivatives. The complexation of these bioactive iminosugars with water-soluble prismarene hosts [2] was evaluated under different pH conditions using nuclear magnetic resonance (NMR), fluorescence titrations, and isothermal titration calorimetry (ITC) experiments. Finally, in vitro evaluations were conducted on cell lines from GD patients to assess the chaperone activity of the iminosugars, both in their free form and when complexed with the prismarene.