High-Throughput Screen Fails to Identify Compounds That Enhance Residual Enzyme Activity of Mutant N-Acetyl-α-Glucosaminidase in Mucopolysaccharidosis Type IIIB

  • O. L. M. Meijer
  • P. van den Biggelaar
  • R. Ofman
  • F. A. Wijburg
  • N. van Vlies
Research Report
Part of the JIMD Reports book series (JIMD, volume 39)


Background: In the severe neurodegenerative disorder mucopolysaccharidosis type IIIB (MPSIIIB or Sanfilippo disease type B), deficiency of the lysosomal enzyme N-acetyl-α-glucosaminidase (NAGLU) results in accumulation of heparan sulfate. Patients present with a severe, rapidly progressing phenotype (RP) or a more attenuated, slowly progressing phenotype (SP). In a previous study, residual NAGLU activity in fibroblasts of SP patients could be increased by culturing at 30°C, probably as a result of improved protein folding and lysosomal targeting under these conditions. Chaperones are molecules which influence protein folding and could therefore have therapeutic potential in SP MPSIIIB patients. Here we studied the effects of 1,302 different compounds on residual NAGLU activity in SP MPSIIIB patient fibroblasts including 1,280 approved compounds from the Prestwick Chemical Library.

Methods: Skin fibroblasts of healthy controls, an SP MPSIIIB patient (homozygous for the temperature sensitive mutation p.S612G) and an RP MPSIIIB patient (homozygous for the p.R297* mutation and non-temperature sensitive), were used. A high-throughput assay for measurement of NAGLU activity was developed and validated, after which 1,302 different molecules were tested for their potential to increase NAGLU activity.

Results: None of the compounds tested were able to enhance NAGLU activity.

Conclusions: This high-throughput screen failed to identify compounds that could enhance residual activity of mutant NAGLU in fibroblasts of SP MPSIIIB patients with temperature sensitive mutations. To therapeutically simulate the positive effect of lower temperatures on residual NAGLU activity, first more insight is needed into the mechanisms underlying this temperature dependent increase.


Chaperones Lysosomal storage disorder Mucopolysaccharidosis type IIIB N-acetyl-α-glucosaminidase Prestwick Chemical Library Sanfilippo disease type B 



The authors would like to thank Dr. S. F. van de Graaf of the Tytgat Institute for Liver and Intestinal Research/Department of Gastroenterology & Hepatology at the Academic Medical Center in Amsterdam, for being so kind to provide the Prestwick Chemical Library to us. This study was funded by grants from the private foundations “Stichting Stofwisselkracht,” “Zabawas,” “Zeldzame Ziekten Fonds,” and “Kinderen en Kansen,” the Netherlands.

Supplementary material (110 kb)
Supplementary figure 1 Optimization and validation of the 96-well NAGLU high-throughput assay. A. Time dependency of NAGLU activity (pmol) after incubation with 1.5 mg/mL 4MU-α-GlcNAc substrate at 37°C for the indicated time points, in control fibroblasts plated at a cell density of 20.000 cells/well grown for 24 hours. NAGLU activity was linear up to an incubation time of 24 hours B. NAGLU activity ( after incubation with various concentrations of 4MU-α-GlcNAc substrate at 37°C for 24 hours, in control fibroblasts plated at a cell density of 20.000 cells/well grown for 24 hours. Optimal enzyme activity was obtained at a substrate concentration of 1 mg/mL C. NAGLU activity ( in control fibroblasts plated at different cell densities and cultured for 5 days. A 4MU-α-GlcNAc substrate concentration of 1 mg/mL was used and plates were incubated at 37°C for 24 hours. The increase in NAGLU activity was linear with cell density up to 10.000 cells/well. D. Determination of the sensitivity of the assay using cell populations which would show small incremental increases in NAGLU activity. NAGLU activity ( is shown in populations of p.S612G MPSIIIB fibroblasts mixed with control fibroblasts in different ratios (total cell number 10.000 cells/well), after 5 days culturing. A 4MU-α-GlcNAc substrate concentration of 1 mg/mL was used and plates were incubated at 37°C for 24 hours. After 5 days culturing mean basal NAGLU activity in the population consisting of 100% p.S612G MPSIIIB fibroblasts was 0.26 and in the population consisting of 100% control cells 85.05 In the wells containing only 0.391% control cells and 99.609% MPSIIIB cells, a significant increase in NAGLU activity could already be detected accurately (* p < 0.001). In all cases mean ± SD is given. If error bars would be shorter than the height of the symbol, no error bars were drawn. Preliminary experiments showed that Triton X-100 at a final concentration of 0.1% had no adverse effect on NAGLU activity and could therefore be used for cell lysis (data not shown) (TIFF 13104 kb)


  1. Cortez L, Sim V (2014) The therapeutic potential of chemical chaperones in protein folding diseases. Prion 8:197–202CrossRefPubMedCentralGoogle Scholar
  2. Engin F, Hotamisligil GS (2010) Restoring endoplasmic reticulum function by chemical chaperones: an emerging therapeutic approach for metabolic diseases. Diabetes Obes Metab 12:108–115CrossRefPubMedGoogle Scholar
  3. Fan JQ (2003) A contradictory treatment for lysosomal storage disorders: inhibitors enhance mutant enzyme activity. Trends Pharmacol Sci 24:355–360CrossRefPubMedGoogle Scholar
  4. Feldhammer M, Durand S, Pshezhetsky AV (2009) Protein misfolding as an underlying molecular defect in mucopolysaccharidosis III type C. PLoS One 4:e7434CrossRefPubMedPubMedCentralGoogle Scholar
  5. Ficko-Blean E, Stubbs KA, Nemirovsky O et al (2008) Structural and mechanistic insight into the basis of mucopolysaccharidosis IIIB. Proc Natl Acad Sci U S A 105:6560–6565CrossRefPubMedPubMedCentralGoogle Scholar
  6. Germain DP, Hughes DA, Nicholls K et al (2016) Treatment of Fabry’s disease with the pharmacologic chaperone migalastat. N Engl J Med 375:545–555CrossRefPubMedGoogle Scholar
  7. Gootjes J, Schmohl F, Mooijer PA et al (2004) Identification of the molecular defect in patients with peroxisomal mosaicism using a novel method involving culturing of cells at 40°C: implications for other inborn errors of metabolism. Hum Mutat 24:130–139CrossRefPubMedGoogle Scholar
  8. Hartl FU, Bracher A, Hayer-Hartl M (2011) Molecular chaperones in protein folding and proteostasis. Nature 475:324–332CrossRefPubMedGoogle Scholar
  9. Hollak CE, Wijburg FA (2014) Treatment of lysosomal storage disorders: successes and challenges. J Inherit Metab Dis 37:587–598CrossRefPubMedGoogle Scholar
  10. Hughes DA, Nicholls K, Shankar SP et al (2017) Oral pharmacological chaperone migalastat compared with enzyme replacement therapy in Fabry disease: 18-month results from the randomised phase III ATTRACT study. J Med Genet 54:288–296CrossRefPubMedGoogle Scholar
  11. Lowry OH, Rosebrough NJ, Farr AL, Randall RJ (1951) Protein measurement with the Folin phenol reagent. J Biol Chem 193:265–275PubMedGoogle Scholar
  12. Macías-Vidal J, Girós M, Guerrero M et al (2014) The proteasome inhibitor bortezomib reduced cholesterol accumulation in fibroblasts from Niemann-Pick type C patients carrying missense mutations. FEBS J 281:4450–4466CrossRefPubMedGoogle Scholar
  13. Maegawa GH, Tropak MB, Buttner JD et al (2009) Identification and characterization of ambroxol as an enzyme enhancement agent for Gaucher disease. J Biol Chem 284:23502–23516CrossRefPubMedPubMedCentralGoogle Scholar
  14. Matos L, Canals I, Dridi L et al (2014) Therapeutic strategies based on modified U1 snRNAs and chaperones for Sanfilippo C splicing mutations. Orphanet J Rare Dis 9:180CrossRefPubMedPubMedCentralGoogle Scholar
  15. Mauri V, Lotfi P, Segatori L, Sardiello M (2013) A rapid and sensitive method for measuring N-acetylglucosaminidase activity in cultured cells. PLoS One 8:1–9CrossRefGoogle Scholar
  16. Meijer OL, Welling L, Valstar MJ et al (2016) Residual N-acetyl-α-glucosaminidase activity in fibroblasts correlates with disease severity in patients with mucopolysaccharidosis type IIIB. J Inherit Metab Dis 39:437–445CrossRefPubMedPubMedCentralGoogle Scholar
  17. Moog U, van Mierlo I, van Schrojenstein Lantman-de Valk HM et al (2007) Is Sanfilippo type B in your mind when you see adults with mental retardation and behavioral problems? Am J Med Genet C Semin Med Genet 145C:293–301CrossRefPubMedGoogle Scholar
  18. Muenzer J (2011) Overview of the mucopolysaccharidoses. Rheumatology 50(suppl 5):v4–v12CrossRefPubMedGoogle Scholar
  19. Parenti G (2009) Treating lysosomal storage diseases with pharmacological chaperones: from concept to clinics. EMBO Mol Med 1:268–279CrossRefPubMedPubMedCentralGoogle Scholar
  20. Parenti G, Andria G, Valenzano KJ (2015) Pharmacological chaperone therapy: preclinical development, clinical translation, and prospects for the treatment of lysosomal storage disorders. Mol Ther 23:1138–1148CrossRefPubMedPubMedCentralGoogle Scholar
  21. Pipalia NH, Cosner CC, Huang A et al (2011) Histone deacetylase inhibitor treatment dramatically reduces cholesterol accumulation in Niemann-Pick type C1 mutant human fibroblasts. Proc Natl Acad Sci U S A 108:5620–5625CrossRefPubMedPubMedCentralGoogle Scholar
  22. Shimada Y, Nishida H, Nishiyama Y et al (2011) Proteasome inhibitors improve the function of mutant lysosomal α-glucosidase in fibroblasts from Pompe disease patient carrying c.546G>T mutation. Biochem Biophys Res Commun 415:274–278CrossRefPubMedGoogle Scholar
  23. Valstar MJ, Bruggenwirth HT, Olmer R et al (2010) Mucopolysaccharidosis type IIIB may predominantly present with an attenuated clinical phenotype. J Inherit Metab Dis 33:759–767CrossRefPubMedPubMedCentralGoogle Scholar
  24. Zhang JH, Chung TD, Oldenburg KR (1999) A simple statistical parameter for use in evaluation and validation of high throughput screening assays. J Biomol Screen 4:67–73CrossRefPubMedGoogle Scholar
  25. Zhao KW, Neufeld EF (2000) Purification and characterization of recombinant human α-N-acetylglucosaminidase secreted by Chinese hamster ovary cells. Protein Expr Purif 19:202–211CrossRefPubMedGoogle Scholar

Copyright information

© Society for the Study of Inborn Errors of Metabolism (SSIEM) 2017

Authors and Affiliations

  • O. L. M. Meijer
    • 1
    • 2
  • P. van den Biggelaar
    • 2
  • R. Ofman
    • 2
  • F. A. Wijburg
    • 1
  • N. van Vlies
    • 1
    • 2
    • 3
  1. 1.Department of Pediatric Metabolic DiseasesEmma Children’s Hospital and Amsterdam Lysosome Center “Sphinx”, Academic Medical CenterAmsterdamThe Netherlands
  2. 2.Laboratory of Genetic Metabolic Diseases, Department of Clinical ChemistryAcademic Medical CenterAmsterdamThe Netherlands
  3. 3.Intravacc, Institute for Translational VaccinologyBilthovenThe Netherlands

Personalised recommendations