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Screening for pharmacological chaperones in Fabry disease.

ABSTRACT: As a prerequisite for clinical trials of pharmacological chaperone therapy (PCT) for Fabry disease, we developed a rapid screening assay for enhancement of endogenous alpha-galactosidase A (alpha-Gal A) in patient-derived cells. We used a T-cell based system to screen 11 mutations causing Fabry disease for enhanceability using 1-deoxygalactonojirimycin (DGJ). When patient-derived T-cells were grown in the presence of DGJ, alpha-Gal A activity increased to more than 50% of normal in several mutations but was unaffected in others. In addition to the mutation R301Q, reported previously, A97V, R112H, R112C, A143T, and L300P were enhanceable, but R356W, G132R, A143P, R220X, and 30delG were not. The level of alpha-Gal A activity achieved provides a basis for the therapeutic trial of DGJ in patients with similarly enhanceable enzyme. This assay method has general utility in other disorders in assessing the degree of enhancement of activity of mutated proteins by PCT.

SUBMITTER: Shin SH

PROVIDER: S-EPMC2729584 | biostudies-literature | 2007 Jul

REPOSITORIES: biostudies-literature

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Screening for pharmacological chaperones in Fabry disease.

Shin Sang-Hoon SH Murray Gary J GJ Kluepfel-Stahl Stefanie S Cooney Adele M AM Quirk Jane M JM Schiffmann Raphael R Brady Roscoe O RO Kaneski Christine R CR

Biochemical and biophysical research communications 20070522 1

As a prerequisite for clinical trials of pharmacological chaperone therapy (PCT) for Fabry disease, we developed a rapid screening assay for enhancement of endogenous alpha-galactosidase A (alpha-Gal A) in patient-derived cells. We used a T-cell based system to screen 11 mutations causing Fabry disease for enhanceability using 1-deoxygalactonojirimycin (DGJ). When patient-derived T-cells were grown in the presence of DGJ, alpha-Gal A activity increased to more than 50% of normal in several mutat ...[more]

PMID: 17532296

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Project description:Fabry disease is a rare disorder caused by a large variety of mutations in the gene encoding lysosomal alpha-galactosidase. Many of these mutations are unique to individual families. Fabry disease can be treated with enzyme replacement therapy, but a promising novel strategy relies on small molecules, so called "pharmacological chaperones", which can be administered orally. Unfortunately only 42% of genotypes respond to pharmacological chaperones.A procedure to predict which genotypes responsive to pharmacological chaperones in Fabry disease has been recently proposed. The method uses a position-specific substitution matrix to score the mutations. Using this method, we have screened public databases for predictable responsive cases and selected nine representative mutations as yet untested with pharmacological chaperones. Mutant lysosomal alpha galactosidases were produced by site directed mutagenesis and expressed in mammalian cells. Seven out of nine mutations responded to pharmacological chaperones. Nineteen other mutations that were tested with pharmacological chaperones, but were not included in the training of the predictive method, were gathered from literature and analyzed in silico. In this set all five mutations predicted to be positive were responsive to pharmacological chaperones, bringing the percentage of responsive mutations among those predicted to be positive and not used to train the classifier to 86% (12/14). This figure differs significantly from the percentage of responsive cases observed among all the Fabry mutants tested so far.In this paper we provide experimental support to an "in silico" method designed to predict missense mutations in the gene encoding lysosomal alpha galactosidase responsive to pharmacological chaperones. We demonstrated that responsive mutations can be predicted with a low percentage of false positive cases. Most of the mutations tested to validate the method were described in the literature as associated to classic or mild classic phenotype. The analysis can provide a guideline for the therapy with pharmacological chaperones supported by experimental results obtained in vitro. We are aware that our results were obtained in vitro and cannot be translated straightforwardly into benefit for patients, but need to be validated by clinical trials.

Dataset Information

Screening for pharmacological chaperones in Fabry disease.

Publications

Screening for pharmacological chaperones in Fabry disease.

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