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

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 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.

Figure

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

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

IC50

Half-maximal inhibitory concentration

IFG

Isofagomine

Ki

Inhibition constant

Km

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

Vmax

Maximum reaction velocity

Notes

Acknowledgments

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.

References

  1. 1.
    Platt FM, Lachmann RH (2009) Treating lysosomal storage disorders: Current practice and future prospects. Biochim Biophys Acta Molecul Cell Res 1793:737–745CrossRefGoogle Scholar
  2. 2.
    Grabowski GA (1993) Gaucher Disease - Enzymology, genetics, and treatment. Adv Hum Genet 21:377–441Google Scholar
  3. 3.
    Beutler E, Nguyen NJ, Henneberger MW, Smolec JM, McPherson RA, West C, Gelbart T (1993) Gaucher Disease - Gene Frequencies in the Ashkenazi Jewish population Am. J Hum Genet 52:85–88Google Scholar
  4. 4.
    Sawkar AR, Adamski-Werner SL, Cheng WC, Wong CH, Beutler E, Zimmer KP, Kelly JW (2005) Gaucher disease-associated glucocerebrosidases show mutation-dependent chemical chaperoning profiles. Chem Biol 12:1235–1244CrossRefGoogle Scholar
  5. 5.
    Chang HH, Asano N, Ishii S, Ichikawa Y, Fan JQ (2006) Hydrophilic iminosugar active-site-specific chaperones increase residual glucocerebrosidase activity in fibroblasts from Gaucher patients. FEBS J 273:4082–4092CrossRefGoogle Scholar
  6. 6.
    Futerman AH, Sussman JL, Horowitz M, Silman I, Zimran A (2004) New directions in the treatment of Gaucher disease. Trends Pharmacol Sci 25:147–151CrossRefGoogle Scholar
  7. 7.
    Brooks DA (2007) Getting into the fold. Nature Chem Biol 3:84–85CrossRefGoogle Scholar
  8. 8.
    Arakawa T, Ejima D, Kita Y, Tsumoto K (2006) Small molecule pharmacological chaperones: From thermodynamic stabilization to pharmaceutical drugs. Biochim Biophys Acta Prot Proteomics 1764:1677–1687CrossRefGoogle Scholar
  9. 9.
    Bernier V, Lagace M, Bichet DG, Bouvier M (2004) Pharmacological chaperones: potential treatment for conformational diseases. Trends Endocrinol Metabol 15:222–228CrossRefGoogle Scholar
  10. 10.
    Fan JQ (2003) A contradictory treatment for lysosomal storage disorders: inhibitors enhance mutant enzyme activity. Trends Pharmacol Sci 24:355–360CrossRefGoogle Scholar
  11. 11.
    Lieberman RL, Wustman BA, Huertas P, Powe AC, Pine CW, Khanna R, Schlossmacher MG, Ringe D, Petsko GA (2007) Structure of acid beta-glucosidase with pharmacological chaperone provides insight into Gaucher disease. Nat Chem Biol 3:101–107CrossRefGoogle Scholar
  12. 12.
    Dvir H, Harel M, McCarthy AA, Toker L, Silman I, Futerman AH, Sussman JL (2003) X-ray structure of human acid-beta-glucosidase, the defective enzyme in Gaucher disease. EMBO Rep 4:704–709CrossRefGoogle Scholar
  13. 13.
    Walden CM, Sandhoff R, Chuang CC, Yildiz Y, Butters TD, Dwek RA, Platt FM, van der Spoel AC (2007) Accumulation of glucosylceramide in murine testis, caused by inhibition of beta-glucosidase 2 - Implications for spermatogenesis. J Biol Chem 282:32655–32664CrossRefGoogle Scholar
  14. 14.
    Zheng W, Padia J, Urban DJ, Jadhav A, Goker-Alpan O, Simeonov A, Goldin E, Auld D, LaMarca ME, Inglese J, Austin CP, Sidransky E (2007) Three classes of gluclocerebrosidase inhibitors identified by quantitative high-throughput screening are chaperone leads for Gaucher disease. Proc Nat Acad Sci U S A 104:13192–13197CrossRefGoogle Scholar
  15. 15.
    Lieberman RL, D’Aquino JA, Ringe D, Petsko GA (2009) Effects of pH and iminosugar pharmacological chaperones on lysosomal glycosidase structure and stability. Biochemistry 48:4816–4827CrossRefGoogle Scholar
  16. 16.
    Sawkar AR, D’Haeze W, Kelly JW (2006) Therapeutic strategies to ameliorate lysosomal storage disorders - a focus on Gaucher disease. Cell Molec Life Sci 63:1179–1192CrossRefGoogle Scholar
  17. 17.
    Wang GN, Reinkensmeier G, Zhang SW, Zhou J, Zhang LR, Zhang LH, Butters TD, Ye XS (2009)Rational design and synthesis of highly potent pharmacological chaperones for treatment of N370S mutant gaucher disease. J. Med. Chem. 52: 3146–3149.Google Scholar
  18. 18.
    Chalcraft KR, Britz-McKibbin P (2009) Newborn screening of inborn errors of metabolism by capillary electrophoresis − electrospray ionization-mass apectrometry: A second-tier method with improved specificity and sensitivity. Anal Chem 81:307–314CrossRefGoogle Scholar
  19. 19.
    Marsden D, Levy H (2009) Newborn screening of lysosomal storage disorders. Clin Chem 56:1071–1079CrossRefGoogle Scholar
  20. 20.
    Millington DS (2005) Newborn screening for lysosomal storage disorders. Clin Chem 51:808–809CrossRefGoogle Scholar
  21. 21.
    Inglese J, Auld DS, Jadhav A, Johnson RL, Simeonov A, Yasgar A, Zheng W, Austin CP (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 103:11473–11478CrossRefGoogle Scholar
  22. 22.
    Shen JS, Edwards NJ, Bin Hong Y, Murray GJ (2008) Isofagomine increases lysosomal delivery of exogenous glucocerebrosidase. Biochem Biophys Res Commun 369:1071–1075CrossRefGoogle Scholar
  23. 23.
    Sawkar AR, Cheng WC, Beutler E, Wong CH, Balch WE, Kelly JW (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.Google Scholar
  24. 24.
    Maegawa GHB, Tropak MB, Buttner JD, Rigat BA, Fuller M, Pandit D, Tang LI, Kornhaber GJ, Hamuro Y, Clarke JTR, Mahuran DJ (2009) Identification and characterization of ambroxol as an enzyme enhancement agent for Gaucher disease. J Biol Chem 284:23502–23516CrossRefGoogle Scholar
  25. 25.
    Bleicher KH, Bohm HJ, Muller K, Alanine AI (2003) Hit and lead generation: Beyond high-throughput screening. Nat Rev Drug Disc 2:369–378CrossRefGoogle Scholar
  26. 26.
    Zhang J, Hoogmartens J, Van Schepdael A (2008) Kinetic study of cytochrome P450 by capillary electrophoretically mediated microanalysis. Electrophoresis 29:3694–3700CrossRefGoogle Scholar
  27. 27.
    Gavina JMA, White CE, Finan TM, Britz-McKibbin P (2010) Determination of 4-hydroxyproline-2-epimerase activity by capillary electrophoresis: A stereoselective platform for inhibitor screening of amino acid isomerases. Electrophoresis 31:2831–2837CrossRefGoogle Scholar
  28. 28.
    Koval D, Kasicka V, Jiracek J, Collinsova M (2006) Determination of pKa values of diastereomers of phosphinic pseudopeptides by CZE. Electrophoresis 27:4648–4657CrossRefGoogle Scholar
  29. 29.
    Ma LJ, Gong XY, Yeung ES (2000) Combinatorial screening of enzyme activity by using multiplexed capillary electrophoresis. Anal Chem 72:3383–3387CrossRefGoogle Scholar
  30. 30.
    Pang HM, Kenseth J, Coldiron S (2004) High-throughput multiplexed capillary electrophoresis in drug discovery. Drug Discov Today 9:1072–1080CrossRefGoogle Scholar
  31. 31.
    Jambovane S, Duin EC, Kirn SK, Hong JW (2009) Determination of kinetic parameters, Km and kcat with a single experiment on a chip. Anal Chem 81:3239–3245CrossRefGoogle Scholar
  32. 32.
    Fan Y, Scriba GKE (2010) Advances in-capillary electrophoretic enzyme assays. J Pharma Biomed Anal 53:1076–1090CrossRefGoogle Scholar
  33. 33.
    Zhang J, Hoogmartens J, Van Schepdael A (2006) Advances in capillary electrophoretically mediated microanalysis: An update. Electrophoresis 27:35–43CrossRefGoogle Scholar
  34. 34.
    Zhang J, Hoogmartens J, Van Schepdael A (2009) Recent developments and applications of EMMA in enzymatic and derivatization reactions. Electrophoresis 31:65–73CrossRefGoogle Scholar
  35. 35.
    Telnarova M, Vytiskova S, Chaloupkova R, Glatz Z (2004) Study of enzymatic reaction by electrophoretically mediated microanalysis in a partially filled capillary with indirect or direct detection. Electrophoresis 25:290–296CrossRefGoogle Scholar
  36. 36.
    Iqbal J, Levesque SA, Sevigny J, Muller CE (2008) A highly sensitive CE-UV method with dynamic coating of silica-fused capillaries for monitoring of nucleotide pyrophosphatase/phosphodiesterase reactions. Electrophoresis 29:3685–3693CrossRefGoogle Scholar
  37. 37.
    Kanie Y, Kanie O (2003) Electrophoretically mediated reaction of glycosidases at a nanoliter scale. Electrophoresis 24:1111–1118CrossRefGoogle Scholar
  38. 38.
    Iqbal J, Jirovsky D, Lee SY, Zimmermann H, Muller CE (2008) Capillary electrophoresis-based nanoscale assays for monitoring ecto-5′-nucleotidase activity and inhibition in preparations of recombinant enzyme and melanoma cell membranes. Anal Biochem 373:129–140CrossRefGoogle Scholar
  39. 39.
    Van Dyck S, Van Schepdael A, Hoogmartens J (2002) Kinetic study of γ-glutamyltransferase activity by electrophoretically mediated microanalysis combined with micellar electrokinetic capillary chromatography. Electrophoresis 23:2854–2859CrossRefGoogle Scholar
  40. 40.
    Tang ZM, Wang ZY, Kang JW (2007) Screening of acetylcholinesterase inhibitors in natural extracts by CE with electrophoretically mediated microanalysis technique. Electrophoresis 28:360–365CrossRefGoogle Scholar
  41. 41.
    Martin-Biosca Y, Asensi-Bernardi L, Villanueva-Camanas RM, Sagrado S, Medina-Hernandez MJ (2009) Screening of acetylcholinesterase inhibitors by CE after enzymatic reaction at capillary inlet. J Sep Sci 32:1748–1756CrossRefGoogle Scholar
  42. 42.
    Roeber D, Achari A, Manavalan P, Edmunds T, Scott DL (2003) Cystallization and repliminary X-ray analysis of recombinant human acid beta-glucocerebrosidase, a treatment for Gaucher’s disease. Acta Crystallogr D59:343–344Google Scholar
  43. 43.
    Brumshtein B, Salinas P, Peterson B, Chan V, Silman I, Sussman JL, Savickas PJ, Robinson GS, Futerman AH (2010) Characterization of gene-activated human acid-beta-glucosidase: Crystal structure, glycan composition, and internalization into macrophages. Glycobiology 20:24–32CrossRefGoogle Scholar
  44. 44.
    Yoshino M, Murakami K (2009) A graphical method for determining inhibition constants. J Enzyme Inhibition Med Chem 24:1288–1290CrossRefGoogle Scholar
  45. 45.
    Kakkar T, Boxenbaum H, Mayersohn M (1999) Estimation of Ki in a competitive enzyme-inhibition model: Comparisons among three methods of data analysis. Drug Metab Dispos 27:756–762Google Scholar
  46. 46.
    Maurer T, Fung H-L (2000) Comparison of methods for analyzing kinetic data from mechanism-based enzyme inactivation: Application to nitric oxide synthase. AAPS PharmSci. 2: article 8.Google Scholar
  47. 47.
    Kemmer G, Keller S (2010) Nonlinear least-squares data fitting in Excel spreadsheets. Nat Protocol 5:267–281CrossRefGoogle Scholar
  48. 48.
    Chan WW-C (1995) Combination plots as graphical tools in the study of enzyme inhibition. Biochem J 311:981–985Google Scholar
  49. 49.
    Geng W, Ke J, Satoh H, Bush ED (2001) A numerical method for identification of inhibition mechanism and estimation of inhibition constant Ki0 in in-vitro enzyme inhibition study I : A computer simulation study AAPS PharmaSci 3: W4464.Google Scholar
  50. 50.
    Smid BE, Aerts JMFG, Boot RG, Linthorst FE, Hollak CEM (2010) Pharmacological small molecules for the treatment of lysosomal storage disorders. Expert Opin Investig Drugs 19:1367–1379CrossRefGoogle Scholar
  51. 51.
    Urban DJ, Zheng W, Goker-Alpan O, Jadhav A, LaMarca ME, Inglese J, Sidransky E, Austin CP (2008) Optimization and validation of two miniaturized glucocerebrosidase enzyme assays for high throughput screening. Combinat Chem High Throughput Screen 11:817–824CrossRefGoogle Scholar
  52. 52.
    Peters SP, Coyle P, Glew RH (1976) Differentiation of beta-glucocerebrosidase from beta-glucosidase in human tissues using sodium taurocholate. Arch Biochem Biophys 175:569–582CrossRefGoogle Scholar
  53. 53.
    Kaiser C, Segui-Lines G, D’Amaral JC, Ptolemy AS, Britz-McKibbin P (2008) Electrokinetic probes for single-step screening of polyol stereoisomers: the virtues of ternary boronate ester complex formation. Chem. Comm. 338–340.Google Scholar
  54. 54.
    Bulow A, Plesner IW, Bols M (2000) A large difference in the thermodynamics of binding of isofagomine and 1-deoxynojirimycin to beta-glucosidase. J Am Chem Soc 122:8567–8568CrossRefGoogle Scholar
  55. 55.
    Rigat B, Mahuran D (2009) Diltiazem, a L-type calcium channel blocker, also acts as a pharmacological chaperone in Gaucher patient cells. Molec Genet Metab 96:225–232CrossRefGoogle Scholar
  56. 56.
    Malerba M, Ragnoli B (2008) Ambroxol in the 21st century: Pharmacological and clinical update. Expert Opin Drug Metab Toxicol 4:1119–1129CrossRefGoogle Scholar
  57. 57.
    Gasiorowski K, Brokos B, Szyba K, Leszek J (2001) Antimutagenic activity of fluphenazine in short-term tests. Mutagenesis 16:31–38CrossRefGoogle Scholar
  58. 58.
    Weely SV, Aerts JMFG, Leeuwen MBV, Heikoop JC, Donker-Koopman WE, Barranger JA, Tager JM, Schram AW (1990) Function of oligosaccharide modification in glucocerebrosidase, a membrane-associated lysosomal hydrolase. Eur J Biochem 191:669–677CrossRefGoogle Scholar
  59. 59.
    Steet RA, Chung S, Wustman B, Powe A, Do H, Kornfeld SA (2006) The iminosugar isofagomine increases the activity of N370S mutant acid β-glucosidase in Gaucher fibroblasts by several mechanisms. Proc Nat Acad Sci U S A 103:13813–13818CrossRefGoogle Scholar
  60. 60.
    Kornhaber GJ, Tropak BB, Maegawa GH, Tuske SJ, Coales SJ, Mahuran DJ, Hamuro Y (2008) Isofagomine induced stabilization of glucocerebrosidase. Chembiochem 9:2643–2649CrossRefGoogle Scholar
  61. 61.
    Khanna R, Benjamin ER, Pellegrino L, Schilling A, Rigat BA, Soska R, Nafar H, Ranes BE, Feng J, Lun Y, Powe AC, Palling DJ, Wustman BA, Schiffmann R, Mahuran DJ, Lockhart DJ, Valenzano KJ (2010) The pharmacological chaperone isofagomine increases the activity of the Gaucher disease L444P mutant form of beta-glucosidase. FEBS J 277:1618–1638CrossRefGoogle Scholar
  62. 62.
    Gavina JMA, Mazhab-Jafari MT, Melacini G, Britz-McKibbin P (2009) Label-Free assay for thermodynamic analysis of protein-ligand interactions: A multivariate strategy for allosteric ligand screening. Biochemistry 48:223–225CrossRefGoogle Scholar
  63. 63.
    Christopoulos A (2002) Allosteric binding sites on cell-surface receptors: novel targets for drug discovery. Nat Rev Drug Discovery 1:198–210CrossRefGoogle Scholar
  64. 64.
    Tropak MB, Kornhaber GJ, Rigat BA, Maegawa GH, Buttner JD, Blanchard JE, Murphy C, Tuske SJ, Coales SJ, Hamuro Y, Brown ED, Mahuran DJ (2008) Identification of pharmacological chaperones for Gaucher disease and characterization of their effects on beta-glucocerebrosidase by hydrogen/deuterium exchange mass spectrometry. Chembiochem 9:2650–2662CrossRefGoogle Scholar
  65. 65.
    Maegawa GH, Tropak M, Buttner J, Stockley T, Kok F, Clarke JT, Mahuran DJ (2007) Pyrimethamine as a potential pharmacological chaperone for late-onset forms of GM2 gangliosidosis. J Biol Chem 282:9150–9161CrossRefGoogle Scholar
  66. 66.
    Cieslik-Boczula K, Szwed J, Jaszczyszyn A, Gasiorowski K, Koll A (2009) Interactions of dihydrochloride fluphenazine with DPPC liposomes: ATR-IR and 31P NMR Studies. J Phys Chem B 113:15495–15502CrossRefGoogle Scholar

Copyright information

© Springer-Verlag 2011

Authors and Affiliations

  1. 1.Department of Chemistry and Chemical BiologyMcMaster UniversityHamiltonCanada

Personalised recommendations