Gaucher disease (GD) is a rare genetic metabolic disorder characterized by a dysfunction of the lysosomal glycoside hydrolase glucocerebrosidase (GCase) due to mutations in the gene GBA1, leading to the cellular accumulation of glucosylceramide (GlcCer). While most of the current research focuses on the primary accumulated material, lesser attention has been paid to secondary storage materials and their reciprocal intertwining. By using a novel approach based on flow cytometry and fluorescent labelling, we monitored changes in storage materials directly in fibroblasts derived from GD patients carrying N370S/RecNcil and homozygous L444P or R131C mutations with respect to wild type. In L444P and R131C fibroblasts, we detected not only the primary accumulation of GlcCer accumulation but also a considerable secondary increase in GM1 storage, comparable with the one observed in infantile patients affected by GM1 gangliosidosis. In addition, the ability of a trivalent trihydroxypiperidine iminosugar compound (CV82), which previously showed good pharmacological chaperone activity on GCase enzyme, to reduce the levels of storage materials in L444P and R131C fibroblasts was tested. Interestingly, treatment with different concentrations of CV82 led to a significant reduction in GM1 accumulation only in L444P fibroblasts, without significantly affecting GlcCer levels. The compound CV82 was selective against the GCase enzyme with respect to the β-Galactosidase enzyme, which was responsible for the catabolism of GM1 ganglioside. The reduction in GM1-ganglioside level cannot be therefore ascribed to a direct action of CV82 on β-Galactosidase enzyme, suggesting that GM1 decrease is rather related to other unknown mechanisms that follow the direct action of CV82 on GCase. In conclusion, this work indicates that the tracking of secondary storages can represent a key step for a better understanding of the pathways involved in the severity of GD, also underlying the importance of developing drugs able to reduce both primary and secondary storage-material accumulations in GD.

Low‐valency multivalent iminosugars have recently emerged as promising inhibitors of the therapeutically relevant enzyme β‐glucocerebrosidase (GCase). A new synthetic strategy has been developed to simultaneously build more than one trihydroxypiperidine iminosugar unit onto a polyamine scaffold via double reductive amination (DRA) of a d ‐mannose derived dialdehyde. Five divalent derivatives, using both aliphatic and aromatic diamines and a trivalent compound based on an aromatic scaffold have been synthesized and evaluated as GCase inhibitors. Only oligomers with an aromatic core ( 26 , 31 , and 37 ) strongly inhibit GCase with IC ₅₀ values in the low micromolar range and activity enhancements, when compared to the monovalent counterpart, that confirm the occurrence of a positive multivalent effect. Kinetic analysis for divalent 31 and trivalent 37 revealed a mixed‐type inhibition. To rationalize the unexpected behavior of 37 , an integrated biophysical and computational approach based on STD‐NMR, docking, and MD simulations was employed, allowing to clarify the structural basis for its inhibitory profile and paving the way to the rational design of novel inhibitors able to bridge multiple interaction sites of the target enzyme.

Two selanyl compounds, 5-(phenylselanyl)pentanoic acid (1) and 5-(benzylselanyl)pentanoic acid (2), were investigated for their reactivity towards one-electron oxidants. Compound 2 was found to possess better antioxidant properties than compound 1.