Gaucher disease (GD) is caused by mutations in the GBA1 gene that encodes the lysosomal enzyme acid β-glucosidase (GCase). Reduced GCase activity primarily leads to the accumulation of two substrates, glucosylceramide (GlcCer) and glucosylsphingosine (GlcSph). Current treatment options have not been shown to ameliorate the neurological pathology observed in the most severe forms of GD, clearly representing an unmet medical need. To better understand the relationship between GlcCer and GlcSph accumulation and ultimately their connection with the progression of neurological pathology, we developed LC-MS/MS methods to quantify GlcCer and GlcSph in mouse brain tissue. A significant challenge in developing these methods was the chromatographic separation of GlcCer and GlcSph from the far more abundant isobaric galactosyl epimers naturally occurring in white matter. After validation of both methods, we evaluated the levels of both substrates in five different GD mouse models, and found significant elevation of brain GlcSph in all five, while GlcCer was elevated in only one of the five models. In addition, we measured GlcCer and GlcSph levels in the brains of wild-type mice after administration of the GCase inhibitor conduritol β-epoxide (CBE), as well as the nonlysosomal β-glucosidase (GBA2) inhibitor N-butyldeoxygalactonojirimycin (NB-DGJ). Inhibition of GCase by CBE resulted in elevation of both sphingolipids; however, inhibition of GBA2 by NB-DGJ resulted in elevation of GlcCer only. Taken together, these data support the idea that GlcSph is a more selective and sensitive biomarker than GlcCer for neuronopathic GD in preclinical models.

Lysosomes are cellular organelles in which a variety of glycosphingolipids (GSLs), glycosaminoglycans, glycoproteins, and oligosaccharides are degraded into simpler substances that can be recycled or excreted from the cell. The current standard of care for several lysosomal storage disorders (LSDs) is enzyme replacement therapy (ERT) in which a manufactured enzyme is infused on a weekly or biweekly basis. Given the fact that the mutated enzymes associated with many LSDs are often catalytically competent but unable to traffic to lysosomes, an alternative therapeutic approach for LSDs involves the use of pharmacological chaperones (PCs). The clinical manifestations of Fabry disease (FD) span a broad spectrum of severity and can include progressive renal failure, cardiac disease, cerebrovascular disease, small-fiber peripheral neuropathy, and skin lesions, among others. Pompe disease shows a broad phenotypic spectrum that ranges from the severe infantile-onset form to more slowly progressing, later-onset forms.

Fabry disease is an X-linked lysosomal storage disorder caused by mutations in the gene that encodes a-galactosidase A and is characterized by pathological accumulation of globotriaosylceramide and globotriaosylsphingosine. Earlier, the authors demonstrated that oral coadministration of the pharmacological chaperone AT1001 (migalastat HCl; 1-deoxygalactonojirimycin HCl) prior to intravenous administration of enzyme replacement therapy improved the pharmacological properties of the enzyme. In this study, the authors investigated the effects of coformulating AT1001 with a proprietary recombinant human alpha-galactosidase A (ATB100) into a single intravenous formulation. AT1001 increased the physical stability and reduced aggregation of ATB100 at neutral pH in vitro, and increased the potency for ATB100-mediated globotriaosylceramide reduction in cultured Fabry fibroblasts. In Fabry mice, AT1001 coformulation increased the total exposure of active enzyme, and increased ATB100 levels in cardiomyocytes, cardiac vascular endothelial cells, renal distal tubular epithelial cells, and glomerular cells, cell types that do not show substantial uptake with enzyme replacement therapy alone. Notably, AT1001 coformulation also leads to greater tissue globotriaosylceramide reduction when compared with ATB100 alone, which was positively correlated with reductions in plasma globotriaosylsphingosine. Collectively, these data indicate that intravenous administration of ATB100 coformulated with AT1001 may provide an improved therapy for Fabry disease and thus warrants further investigation.

Visceral leishmaniasis is a fatal human disease caused by the intracellular protozoan parasite Leishmania chagasi that is captured by host cells in a process involving classics receptors mediated phagocytosis. The search for molecules involved in this process is important to design strategies to disease control. In this work, we verified the presence of heparin-binding protein (HBP) in L. chagasi promastigotes forms. HBP is a lectin of the group of ubiquitous proteins, whose main characteristic is to bind to carbohydrates present in glycoproteins or glycolipids, which is poorly studied in Leishmania species. L. chagasi HBP (HBPLc) was purified by affinity chromatography using heparin–agarose column in FPLC automated system. Its localization in the parasite was assessed by immunolabeling and electronic transmission microscopy tests using anti-HBPLc polyclonal antibodies, which showed HBP spread over the parasite outer surface and internally next to the kynetoplast. In addition, we verified that HBPLc participates in the process of parasite infection, since its blocking with heparin generated a partial reduction in the internalization of Leishmania by RAW macrophages “in vitro”. According to these results, it is believed that, in further “in vivo” studies, interference on this parasitic protein may provide us prophylactic and therapeutic alternatives against visceral leishmaniasis.

Objective: Genotype-phenotype correlation and the correlation between the genotype and the antibody formation against idursulfase were studied in the patients withmucopolysaccharidosis type II (MPS II) treated by enzyme replacement therapy (ERT) for more than 2 years. Patients and Methods: Fourteen patients with MPS II were divided into four groups by clinical phenotypes; four of type A (normal intelligence), one of B (normal-borderline intelligence), three of C (delay after 2 years of age), and six of D (delay before 2 years of age). Genotype and the titer of anti-idursulfase antibody in each patient were analyzed. Results: All the patients with type D developed anti-idursulfase antibody, while the patients with type A, B, and C did not. In the molecular analysis of idursulfase gene, missense mutation or normal exon sequence were detected in the patients with type A, B, and C, while nonsense mutation, nucleotides deletions, or pseudogene rearrangement were found in type D patients. Three out of six patients with type D had pseudogene rearrangement, whose titer of anti-idursulfase antibody plateaued at 6 months after ERT. However, the titer increased continuously in other three patients with type D, who showed poor efficacy of ERT with the large liver and high urinary glycosaminoglycan levels. Conclusion: Anti-idursurfase antibody, which reduced the efficacy of ERT, was developed in all the patients with type D, themost severe form of MPS II. The patients with extremely high titer of antibody had the mutations which may cause a severe deformation of enzyme protein or protein loss. Molecular diagnosis ofMPS IImay be useful to speculate the patient’s prognosis and the effectiveness of ERT.

Mutations in the gene that encodes the lysosomal enzyme acid β-glucosidase lead to reduced cellular activity and accumulation of glycosphingolipid substrates, biochemical hallmarks of the lysosomal storage disorder Gaucher disease (GD). Recently such mutations have been identified as risk factors for Parkinson’s disease (PD) and related disorders. Both gain-of-function (due to toxic cellular accumulation of mutant enzyme) and loss-of-function (due to accumulation of lipid substrates) hypotheses have been put forth to address the biochemical link between GD and PD. Similarly, links between Alzheimer’s disease and other lysosomal enzyme deficiencies have begun to emerge. The use of pharmacological chaperones to restore the cellular trafficking and activity of mutant lysosomal enzymes may offer a novel approach to treat these debilitating neurodegenerative diseases.

A variety of unwanted cellular waste materials and macromolecules (e.g. glycosphingolipids, oligosaccharides, glycoproteins, glycogen, peptides, cholesterol, etc.) are enzymatically degraded in a cellular organelle known as the lysosome, with the resulting products recycled into various biosynthetic pathways. Genetic defects that reduce the activity for any one of the enzymes that are responsible for these processes can result in accumulation of one or more substrates, compromised cellular function, and disease pathology. Over the last 10 to 20 years there have been significant advances in understanding the basic biochemistry and cell biology that underlie these diseases, which are known collectively as lysosomal storage diseases (LSDs). In turn, this has led to several treatment options, where virtually none had previously existed. A relatively new and promising therapeutic approach involves pharmacological chaperones (PCs), small molecules that are designed to bind and stabilise mutant lysosomal enzymes, and to improve trafficking to their proper cellular destination, the lysosome, where increased activity can aid in the clearance of storage material. This chapter will outline the mechanism of action of PCs, and describe how such molecules are identified and characterised pre-clinically using biochemical and cell-based assays, as well as animal models of LSDs.

Lysosomal enzymes are responsible for the degradation of a wide variety of glycolipids, oligosaccharides, proteins, and glycoproteins. Inherited mutations in the genes that encode these proteins can lead to reduced stability of newly synthesized lysosomal enzymes. While often catalytically competent, the mutated enzymes are unable to efficiently pass the quality control mechanisms of the endoplasmic reticulum, resulting in reduced lysosomal trafficking, substrate accumulation, and cellular dysfunction. Pharmacological chaperones (PCs) are small molecules that bind and stabilize mutant lysosomal enzymes, thereby allowing proper cellular translocation. Such compounds have been shown to increase enzyme activity and reduce substrate burden in a number of preclinical models and clinical studies. In this Perspective, we review several of the lysosomal diseases for which PCs have been studied and the SAR of the various classes of molecules.