Abstract
Late-onset GM2 gangliosidosis is composed of two related, autosomal recessive, neurodegenerative diseases, both resulting from deficiency of lysosomal, heterodimeric beta-hexosaminidase A (Hex A, alphabeta). Pharmacological chaperones (PC) are small molecules that can stabilize the conformation of a mutant protein, allowing it to pass the quality control system of the endoplasmic reticulum. To date all successful PCs have also been competitive inhibitors. Screening for Hex A inhibitors in a library of 1040 Food Drug Administration-approved compounds identified pyrimethamine (PYR (2,4-diamino 5-(4-chlorophenyl)-6-ethylpyrimidine)) as the most potent inhibitor. Cell lines from 10 late-onset Tay-Sachs (11 alpha-mutations, 2 novel) and 7 Sandhoff (9 beta-mutations, 4 novel) disease patients, were cultured with PYR at concentrations corresponding to therapeutic doses. Cells carrying the most common late-onset mutation, alphaG269S, showed significant increases in residual Hex A activity, as did all 7 of the beta-mutants tested. Cells responding to PC treatment included those carrying mutants resulting in reduced Hex heat stability and partial splice junction mutations of the inherently less stable alpha-subunit. PYR, which binds to the active site in domain II, was able to function as PC even to domain I beta-mutants. We concluded that PYR functions as a mutation-specific PC, variably enhancing residual lysosomal Hex A levels in late-onset GM2 gangliosidosis patient cells.
Highlights
Terized by progressive deterioration of motor, cerebral, and spinocerebellar function caused by deficiency of lysosomal -hexosaminidase A
We report here the application of a small molecule library screening approach to search for novel inhibitors of lysosomal Hex A with the potential to treat late-onset variants of GM2 gangliosidosis by functioning as Pharmacological chaperones (PC)
Ten of the 11 different HEXA mutations, and 5 of the 9 different HEXB mutations that we identified in patients were reported previously
Summary
Study Subject—Fibroblast cell lines were obtained from 17 patients with late-onset forms of GM2 gangliosidosis. Rabbit polyclonal antibodies against human Hex A (Western blot) and sheep polyclonal IgG against human -subunit (used for immunoprecipitation and cell immunofluorescence) were prepared as previously described [22]. Real-time Hex assays were performed in final volumes of 100 l, containing 1 mM of each drug, 12 ng of purified Hex B diluted in citrate phosphate buffer (CP) (pH 4.1) containing 0.025% human serum albumin, and 25 l of MUG (0.4 mM). 10-cm culture plates of each cell line were treated with PYR and NGT diluted in Me2SO at final concentrations of 20, 10, and 5 g/ml. To concentrate Hex A, as well as other soluble lysosomal enzymes, 3.5–3.8 mg of total cell lysate protein was incubated with concanavalin A beads (30 l of drain solution) overnight at 4 °C. 18 h, as well as further procedures for stopping the reaction and preparation for liquid scintillation counting analysis, were performed as previously described [4]
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