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
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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 figure 1Optimization 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 (pmol.hr-1) 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 (pmol.hr-1) 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 (pmol.hr-1) 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 pmol.hr-1 and in the population consisting of 100% control cells 85.05 pmol.hr-1. 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)
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