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Updated: May 4, 2026
In Vitro Enzyme Measurement to Test Pharmacological Chaperone Responsiveness in Fabry and Pompe Disease
Published on: December 20, 2017
A thermodynamic assay to test pharmacological chaperones for Fabry disease
Giuseppina Andreotti1, Valentina Citro2, Antonella Correra3
1Istituto di Chimica Biomolecolare, CNR, Pozzuoli, Italy.
Background:
The majority of the disease-causing mutations 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 excellent model system to 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 and Western blotting.
Results:
We measured 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.
Insights
This study shows urea-induced unfolding is a versatile method to test protein stability for rare diseases like Fabry disease. It helps assess drug responsiveness for pharmacological chaperones, aiding rare disease drug development.
Area of Science:
- Biochemistry
- Molecular Biology
- Pharmacology
Background:
- Many disease mutations impact protein stability, not function, making them targets for pharmacological chaperones.
- Pharmacological chaperones enhance target protein thermodynamic stability.
- Fabry disease, caused by alpha-galactosidase mutations, serves as a model for testing these drugs.
Purpose of the Study:
- To develop and validate experimental protocols for testing pharmacological chaperone efficiency.
- To assess the stability of lysosomal alpha-galactosidase under various conditions.
- To establish a method for evaluating drug responsiveness in rare disease mutations.
Main Methods:
- Urea-induced unfolding followed by limited proteolysis and Western blotting.
- Measurement of urea concentration for half-maximal unfolding as a stability indicator.
- Adaptation of the technique for cell extracts with low protein amounts.
Main Results:
- The study established a reliable method to quantify protein stability.
- The technique allows stability assessment across different pH levels and in the presence/absence of drugs.
- Results are independent of the protein expression method.
Conclusions:
- Urea-induced unfolding is a versatile in vitro method for assessing protein stability and drug responsiveness.
- This technique is crucial for developing clinical trials for rare diseases with limited patient populations.
- The protocol is applicable beyond Fabry disease for evaluating pharmacological chaperone efficacy on various mutations.
