Fabry Disease – Current Treatment and New Drug Development

Fabry disease is a rare inherited lysosomal storage disorder caused by a partial or complete deficiency of α-galactosidase A (GLA), resulting in the storage of excess cellular glycosphingolipids. Enzyme replacement therapy is available for the treatment of Fabry disease, but it is a costly, intravenous treatment. Alternative therapeutic approaches, including small molecule chaperone therapy, are currently being explored. High throughput screening (HTS) technologies can be utilized to discover other small molecule compounds, including non-inhibitory chaperones, enzyme activators, molecules that reduce GLA substrate, and molecules that activate GLA gene promoters. This review outlines the current therapeutic approaches, emerging treatment strategies, and the process of drug discovery and development for Fabry disease

High Throughput Screening for Inhibitors of Alpha-Galactosidase

Fabry disease is a rare X-linked lysosomal storage disorder caused by a deficiency in α-galactosidase A (GLA), which catalyzes the hydrolysis of terminal α-galactosyl groups from glycosphingolipids, such as globotriaosylceramide (Gb3). Many of the mutations in the GLA gene are missense alterations that cause misfolding, decreased stability, and/or mistrafficking of this protein. Small molecule compounds that correct the misfolding and mistrafficking, or activate the mutant enzyme, may be useful in the treatment of Fabry disease. We have screened a library of approximately 230,000 compounds using preparations of human recombinant protein and purified coffee bean enzyme in an effort to find activators and inhibitors of this enzyme. Lansoprazole was identified as a small molecule inhibitor of GLA derived from coffee beans (IC50 = 6.4 μM), but no inhibitors or activators were identified for the human enzyme. The screening results indicate that human GLA is a difficult target for small molecule inhibition or activation

Cloning and characterization of the mouse Mcoln1 gene reveals an alternatively spliced transcript not seen in humans

BACKGROUND: Mucolipidosis type IV (MLIV) is an autosomal recessive lysosomal storage disorder characterized by severe neurologic and ophthalmologic abnormalities. Recently the MLIV gene, MCOLN1, has been identified as a new member of the transient receptor potential (TRP) cation channel superfamily. Here we report the cloning and characterization of the mouse homologue, Mcoln1, and report a novel splice variant that is not seen in humans. RESULTS: The human and mouse genes display a high degree of synteny. Mcoln1 shows 91% amino acid and 86% nucleotide identity to MCOLN1. Also, Mcoln1 maps to chromosome 8 and contains an open reading frame of 580 amino acids, with a transcript length of approximately 2 kb encoded by 14 exons, similar to its human counterpart. The transcript that results from murine specific alternative splicing encodes a 611 amino acid protein that differs at the c-terminus. CONCLUSIONS: Mcoln1 is highly similar to MCOLN1, especially in the transmembrane domains and ion pore region. Also, the late endosomal/lysosomal targeting signal is conserved, supporting the hypothesis that the protein is localized to these vesicle membranes. To date, there are very few reports describing species-specific splice variants. While identification of Mcoln1 is crucial to the development of mouse models for MLIV, the fact that there are two transcripts in mice suggests an additional or alternate function of the gene that may complicate phenotypic assessment

High Throughput Screening for Small Molecule Therapy for Gaucher Disease Using Patient Tissue as the Source of Mutant Glucocerebrosidase

Gaucher disease (GD), the most common lysosomal storage disorder, results from the inherited deficiency of the lysosomal enzyme glucocerebrosidase (GCase). Previously, wildtype GCase was used for high throughput screening (HTS) of large collections of compounds to identify small molecule chaperones that could be developed as new therapies for GD. However, the compounds identified from HTS usually showed reduced potency later in confirmatory cell-based assays. An alternate strategy is to perform HTS on mutant enzyme to identify different lead compounds, including those enhancing mutant enzyme activities. We developed a new screening assay using enzyme extract prepared from the spleen of a patient with Gaucher disease with genotype N370S/N370S. In tissue extracts, GCase is in a more native physiological environment, and is present with the native activator saposin C and other potential cofactors. Using this assay, we screened a library of 250,000 compounds and identified novel modulators of mutant GCase including 14 new lead inhibitors and 30 lead activators. The activities of some of the primary hits were confirmed in subsequent cell-based assays using patient-derived fibroblasts. These results suggest that primary screening assays using enzyme extracted from tissues is an alternative approach to identify high quality, physiologically relevant lead compounds for drug development

Mucolipidosis type IV in a Turkish boy associated with a novel MCOLN1 mutation

Mucolipidosis type IV is a rare neurodegenerative lysosomal storage disorder that usually presents during the first year of life with severe mental retardation, delayed motor milestones and corneal opacities. Mucolipidosis IV is caused by mutations in MCOLN1, a gene encoding mucolipin-1 which is responsible for maintaining lysosomal function. The majority of known patients with this disorders are Ashkenazi Jews, and most have a splice IVS3-2 A>G, or a 6.4 kb deletion mutation in MCOLN1. Here, we present a Turkish patient who, in addition to the typical neurological and visceral characteristics of mucolipidosis type IV, also demonstrates defects in the posterior limb of internal capsule by MRI, micrognathia and clinodactyly of the fifth fingers. Direct sequencing of his DNA revealed a homozygous c. 1364C>T (S456L) mutation in MCOLN1, which was heterozygous in both consanguineous parents. This mutation, like several previously described, changes the protein sequence in the channel pore domain of the protein. Serine 456 is conserved in mucolipin proteins throughout evolution, therefore the mutation is considered as causative for the severe phenotype of this patient. (C) 2008 Elsevier B.V. All rights reserved