Therapy of Fabry disease with pharmacological chaperones: from in silico predictions to in vitro tests

Abstract

BackgroundFabry 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.ResultsA 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.ConclusionsIn 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.

α-Galactosidase Aggregation Is a Determinant of Pharmacological Chaperone Efficacy on Fabry Disease Mutants

“…These MASS plots show that certain disease mutations are spread through the aggregation spectrum, suggesting that they might affect the intrinsic aggregation propensity of the protein as well as reduce its thermodynamic stability, which could lead to protein aggregation. tion, and using aggregation propensity as a classifier we can predict DGJ response with an accuracy that is equivalent to the most performing predictor algorithms using sequence information from ␣-Gal homologs (17,18). This demonstrates that the aggregation propensity of ␣-Gal mutants is an important determinant for DGJ response, although it is clear that other factors certainly also participate.…”

“…Structural analysis reveals that the most disruptive mutants are on average associated with the most severe forms of the disease, and they are also on average the least responsive to DGJ treatment (43,44). However, it remains very difficult to predict DGJ response from structure only, as only 40% of the nonresponsive mutants can be identified in this manner (17,18). Clearly, thermodynamic destabilization and thus severity of misfolding alone are not sufficient to characterize the therapeutic response (16).…”

“…Still, it remains very difficult to rationalize DGJ response solely from structural information (16) as only approximately 40% of nonresponsive mutants can be correctly predicted from structure alone (17). More robust prediction has been achieved recently by combining structural information with sequence conservation scoring (17,18). The addition of position specific scoring matrices derived from paralogs, but excluding far homologs, allows predicting DGJ response with an accuracy above 80%.…”

Identification of an Allosteric Binding Site on Human Lysosomal Alpha-Galactosidase Opens the Way to New Pharmacological Chaperones for Fabry Disease

“…The cells were transfected with individual plasmids containing mutant AGAL-encoding ORF using the LipofectAMINE2000 cationic lipid reagent in suspension [36]. …”

“…Tests on transfected mutants were carried out as described in [36]. Briefly: cell lysates (1–2 microliters) were added to 38 microliters of AGAL assay buffer (sodium citrate 27 mM-sodium phosphate dibasic 46 mM, 4-methylumbelliferyl-alpha-D-galactopyranoside 5 mM and N-acetyl-D-galactosamine 100 mM, pH 4.5) and incubated for 0.5–1 h at 37°C.…”