Glucosylceramide synthase inhibition with lucerastat lowers globotriaosylceramide and lysosome staining in cultured fibroblasts from Fabry patients with different mutation types.

Fabry disease is an X-linked lysosomal storage disorder caused by mutations in the GLA gene coding for α-galactosidase A (α-GalA). The deleterious mutations lead to accumulation of α-GalA substrates, including globotriaosylceramide (Gb3) and globotriaosylsphingosine. Progressive glycolipid storage results in cellular dysfunction, leading to organ damage and clinical disease, i.e. neuropathic pain, impaired renal function and cardiomyopathy. Many Fabry patients are treated by bi-weekly intravenous infusions of replacement enzyme. While the only available oral therapy is an α-GalA chaperone, which is indicated for a limited number of patients with specific 'amenable' mutations. Lucerastat is an orally bioavailable inhibitor of glucosylceramide synthase (GCS) that is in late stage clinical development for Fabry disease. Here we investigated the ability of lucerastat to lower Gb3, globotriaosylsphingosine and lysosomal staining in cultured fibroblasts from 15 different Fabry patients. Patients' cells included 13 different pathogenic variants, with 13 cell lines harboring GLA mutations associated with the classic disease phenotype. Lucerastat dose dependently reduced Gb3 in all cell lines. For 13 cell lines the Gb3 data could be fit to an IC50 curve, giving a median IC50 [interquartile range (IQR)] = 11 µM (8.2-18); the median percent reduction (IQR) in Gb3 was 77% (70-83). Lucerastat treatment also dose dependently reduced LysoTracker Red staining of acidic compartments. Lucerastat's effects in the cell lines were compared to those with current treatments-agalsidase alfa and migalastat. Consequently, the GCS inhibitor lucerastat provides a viable mechanism to reduce Gb3 accumulation and lysosome volume, suitable for all Fabry patients regardless of genotype.

Oxidation by 2-oxoglutarate oxygenases: non-haem iron systems in catalysis and signalling.

The 2-oxoglutarate (2OG) and ferrous iron dependent oxygenases are a superfamily of enzymes that catalyse a wide range of reactions including hydroxylations, desaturations and oxidative ring closures. Recently, it has been discovered that they act as sensors in the hypoxic response in humans and other animals. Substrate oxidation is coupled to conversion of 2OG to succinate and carbon dioxide. Kinetic, spectroscopic and structural studies are consistent with a consensus mechanism in which ordered binding of (co)substrates enables control of reactive intermediates. Binding of the substrate to the active site triggers the enzyme for ligation of dioxygen to the metal. Oxidative decarboxylation of 2OG then generates the ferryl species thought to mediate substrate oxidation. Structural studies reveal a conserved double-stranded beta-helix core responsible for binding the iron, via a 2His-1carboxylate motif and the 2OG side chain. The rigidity of this core contrasts with the conformational flexibility of surrounding regions that are involved in binding the substrate. Here we discuss the roles of 2OG oxygenases in terms of the generic structural and mechanistic features that render the 2OG oxygenases suited for their functions.