Unraveling A4GALT Mechanism and Its Modulation With Adamantyl‐Galactosylceramide Analogues: Advancing Fabry Disease Therapeutic Strategies

ABSTRACT Fabry disease (FD), one of the most prevalent lysosomal storage disorders in Europe, is caused by mutations in the GLA gene leading to deficient α‐galactosidase A activity with lysosomal accumulation of globotriaosylceramide (Gb3). Enzyme replacement therapy (ERT) and pharmacological chaperone therapy (PCT) are used in the clinic to treat FD but are limited in efficacy, underscoring the need for alternative therapeutic strategies. Inhibiting α‐1,4‐galactosyltransferase (A4GALT), the glycosyltransferase responsible for Gb3 biosynthesis, represents an attractive strategy. Here, we reveal the molecular mechanism of human A4GALT at atomic detail using QM/MM simulations. We reveal a conformational rearrangement involving a 3 10 ‐helix that stabilizes the donor substrate and promotes a front‐face S N i‐like catalytic mechanism, in which a short‐lived oxocarbenium‐ion intermediate forms. The simulations informed the synthesis of a panel of glycosylceramide substrate analogues. Among these, AdaGalCer (Ada = adamantyl) proved able to reduce Gb3 production in fibroblasts while simultaneously being converted by A4GALT into the galactosylated product AdaGb2. These results provide a clear path towards inhibiting A4GALT, paving the way for potential new and effective FD therapeutics.