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

被引:0
作者
Meera Shanmuganathan
Philip Britz-McKibbin
机构
[1] McMaster University,Department of Chemistry and Chemical Biology
来源
Analytical and Bioanalytical Chemistry | 2011年 / 399卷
关键词
Enzyme kinetics; Inhibitor screening; Glucocerebrosidase; Pharmacological chaperone; Capillary electrophoresis; Gaucher disease;
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学科分类号
摘要
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.
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页码:2843 / 2853
页数:10
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  • [21] Ichikawa Y(2006)Quantitative high-throughput screening: A titration-based approach that efficiently identifies biological activities in large chemical libraries Proc Nat Acad Sci U S A 103 11473-11478
  • [22] Fan JQ(2008)Isofagomine increases lysosomal delivery of exogenous glucocerebrosidase Biochem Biophys Res Commun 369 1071-1075
  • [23] Futerman AH(2002)Chemical chaperones increase the cellular activity of N370S beta-glucosidase: A therapeutic strategy for Gaucher disease Proc. Nat. Acad. Sci. U.S.A. 99 15428-15433
  • [24] Sussman JL(2009)Identification and characterization of ambroxol as an enzyme enhancement agent for Gaucher disease J Biol Chem 284 23502-23516
  • [25] Horowitz M(2003)Hit and lead generation: Beyond high-throughput screening Nat Rev Drug Disc 2 369-378
  • [26] Silman I(2008)Kinetic study of cytochrome P450 by capillary electrophoretically mediated microanalysis Electrophoresis 29 3694-3700
  • [27] Zimran A(2010)Determination of 4-hydroxyproline-2-epimerase activity by capillary electrophoresis: A stereoselective platform for inhibitor screening of amino acid isomerases Electrophoresis 31 2831-2837
  • [28] Brooks DA(2006)Determination of pKa values of diastereomers of phosphinic pseudopeptides by CZE Electrophoresis 27 4648-4657
  • [29] Arakawa T(2000)Combinatorial screening of enzyme activity by using multiplexed capillary electrophoresis Anal Chem 72 3383-3387
  • [30] Ejima D(2004)High-throughput multiplexed capillary electrophoresis in drug discovery Drug Discov Today 9 1072-1080