ORIGINAL PAPER Inhibitor screening of pharmacological chaperones for lysosomal β-glucocerebrosidase by capillary electrophoresis Meera Shanmuganathan & Philip Britz-McKibbin Received: 20 November 2010 / Revised: 4 January 2011 / Accepted: 5 January 2011 / Published online: 1 February 2011 # Springer-Verlag 2011 Abstract 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 concentra- tion (IC 50 ) 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 characteriza- tion 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. Keywords Enzyme kinetics . Inhibitor screening . Glucocerebrosidase . Pharmacological chaperone . Capillary electrophoresis . Gaucher disease Abbreviations α Modifying factor ABX Ambroxol BHX Bromhexine CE Capillary electrophoresis DTZ Diltiazem ER Endoplasmic reticulum ERT Enzyme-replacement therapy FLZ Fluphenazine GCase β-glucocerebrosidase GD Gaucher disease GlcCer Glucosylceramide IC 50 Half-maximal inhibitory concentration IFG Isofagomine K i Inhibition constant K m Michaelis–Menten binding constant LSDs Lysosomal storage disorders MU Methylumbelliferone MUG 4-methylumbelliferyl-β-D-glucopyranoside PC Pharmacological chaperone SRT Substrate-reduction therapy TC Taurocholic acid V max Maximum reaction velocity Introduction Over the past two decades, there has been remarkable progress in the development of therapies for treatment of lysosomal storage disorders (LSDs) [1]. LSDs are a diverse M. Shanmuganathan : P. Britz-McKibbin (*) Department of Chemistry and Chemical Biology, McMaster University, 1280 Main St. West, Hamilton, Ontario L8S 4M1, Canada e-mail: britz@mcmaster.ca Anal Bioanal Chem (2011) 399:2843–2853 DOI 10.1007/s00216-011-4671-6