l-NBDNJ allosteric chaperone for multiple defective enzymes involved in lysosomal storage disorders (LSDs)

Protein misfolding underlies a wide range of conformational diseases, including lysosomal storage disorders (LSDs) such as Gaucher, Pompe, and Fabry diseases. Pharmacological chaperones (PCs) have emerged as promising small molecules capable of rescuing mutant enzymes from endoplasmic reticulum (ER)-associated degradation by stabilizing their correct folding. While first-generation PCs act as competitive inhibitors at the active site, their therapeutic window is narrow due to concomitant enzyme inhibition. Second-generation PCs, or allosteric chaperones, aim to stabilize mutant enzymes without interfering with catalysis. In this study, the unnatural iminosugar l-NBDNJ (l-N-butyldeoxynojirimycin), enantiomer of the drug Miglustat, was synthesized and assessed for its ability to act as a non-inhibitory chaperone. Enzyme inhibition assays demonstrated no activity against a broad glycosidase panel, supporting its potential as an allosteric ligand. In vitro assays on patient-derived fibroblasts showed that l-NBDNJ alone failed to restore activity of GCase, GAA, or α-Gal A mutants. However, co-administration with active-site chaperones (Isofagomine, DNJ, or DGJ) yielded synergistic improvements, increasing residual enzyme activities by 16-30%. In silico docking and molecular dynamics confirmed stable binding at predicted allosteric sites, forming tertiary complexes with active-site chaperones and enhancing binding energies. Collectively, these findings identify l-NBDNJ as a second-generation allosteric enhancer of active-site chaperones, able to potentiate their efficacy across multiple LSDs.