A thermodynamic assay to test pharmacological chaperones for Fabry disease

Background: Themajority of the disease-causingmutations affect protein stability, but not functional sites and are
amenable, in principle, to be treated with pharmacological chaperones. These drugs enhance the thermodynamic
stability of their targets. Fabry disease, a disorder caused by mutations in the gene encoding lysosomal alpha-galactosidase,
represents an excellentmodel systemto develop experimental protocols to test the efficiency of such
drugs.
Methods: The stability of lysosomal alpha-galactosidase under different conditions was studied by urea-induced
unfolding followed by limited proteolysis andWestern blotting.
Results:Wemeasured the concentration of urea needed to obtain half-maximal unfolding because this parameter
represents an objective indicator of protein stability.
Conclusions: Urea-induced unfolding is a versatile technique that can be adapted to cell extracts containing tiny
amounts of wild-type or mutant proteins. It allows testing of protein stability as a function of pH, in the presence
or in the absence of drugs. Results are not influenced by the method used to express the protein in transfected
cells.
General significance: Scarce and dispersed populations pose a problem for the clinical trial of drugs for rare diseases.
This is particularly true for pharmacological chaperones that must be tested on each mutation associated
with a given disease. Diverse in vitro tests are needed.We used a method based on chemically induced unfolding
as a tool to assess whether a particular Fabry mutation is responsive to pharmacological chaperones, but, by no
means is our protocol limited to this disease.