Analytical and Bioanalytical Chemistry

, Volume 399, Issue 8, pp 2843–2853 | Cite as

Inhibitor screening of pharmacological chaperones for lysosomal β-glucocerebrosidase by capillary electrophoresis

Original Paper


Pharmacological chaperones (PCs) represent a promising therapeutic strategy for treatment of lysosomal storage disorders based on enhanced stabilization and trafficking of mutant protein upon orthosteric and/or allosteric binding. Herein, we introduce a simple yet reliable enzyme assay using capillary electrophoresis (CE) for inhibitor screening of PCs that target the lysosomal enzyme, β-glucocerebrosidase (GCase). The rate of GCase-catalyzed hydrolysis of the synthetic substrate, 4-methylumbelliferyl-β-d-glucopyranoside was performed using different classes of PCs by CE with UV detection under standardized conditions. The pH and surfactant dependence of inhibitor binding on recombinant GCase activity was also examined. Enzyme inhibition studies were investigated for five putative PCs including isofagomine (IFG), ambroxol, bromhexine, diltiazem, and fluphenazine. IFG was confirmed as a potent competitive inhibitor of recombinant GCase with half-maximal inhibitory concentration (IC50) of 47.5 ± 0.1 and 4.6 ± 1.4 nM at pH 5.2 and pH 7.2, respectively. In contrast, the four other non-carbohydrate amines were demonstrated to function as mixed-type inhibitors with high micromolar activity at neutral pH relative to acidic pH conditions reflective of the lysosome. CE offers a convenient platform for characterization of PCs as a way to accelerate the clinical translation of previously approved drugs for oral treatment of rare genetic disorders, such as Gaucher disease.


Inhibitor screening of previously approved drugs that function as pharmacological chaperones for glucocerebrosidase by capillary electrophoresis.


Enzyme kinetics Inhibitor screening Glucocerebrosidase Pharmacological chaperone Capillary electrophoresis Gaucher disease 



Modifying factor






Capillary electrophoresis




Endoplasmic reticulum


Enzyme-replacement therapy






Gaucher disease




Half-maximal inhibitory concentration




Inhibition constant


Michaelis–Menten binding constant


Lysosomal storage disorders






Pharmacological chaperone


Substrate-reduction therapy


Taurocholic acid


Maximum reaction velocity



Financial support was provided by the Natural Sciences and Engineering Research Council of Canada (NSERC). The authors wish to thank Dr. Michael Tropak and Dr. Don Mahuran from the Hospital for Sick Children in Toronto, Canada for their generous donation of Cerezyme.


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Copyright information

© Springer-Verlag 2011

Authors and Affiliations

  1. 1.Department of Chemistry and Chemical BiologyMcMaster UniversityHamiltonCanada

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