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High-Throughput Screen Fails to Identify Compounds That Enhance Residual Enzyme Activity of Mutant
O L M Meijer1,2, P van den Biggelaar2, R Ofman2
1Department of Pediatric Metabolic Diseases, Emma Children's Hospital and Amsterdam Lysosome Center "Sphinx", Academic Medical Center, Amsterdam, The Netherlands.
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.
Insights
This study screened 1,302 compounds to find treatments for mucopolysaccharidosis type IIIB (MPSIIIB), a neurodegenerative disorder. Unfortunately, no compounds effectively increased the activity of the deficient NAGLU enzyme in patient cells.
Area of Science:
- Biochemistry
- Genetics
- Neuroscience
Background:
- Mucopolysaccharidosis type IIIB (MPSIIIB) is a severe neurodegenerative disorder caused by deficient N-acetyl-α-glucosaminidase (NAGLU) activity, leading to heparan sulfate accumulation.
- Patients exhibit either a rapidly progressing (RP) or slowly progressing (SP) phenotype.
- Previous research showed that culturing SP patient fibroblasts at 30°C increased residual NAGLU activity, suggesting a role for improved protein folding and lysosomal targeting.
Purpose of the Study:
- To investigate the therapeutic potential of chaperone molecules in SP MPSIIIB patients.
- To screen a library of 1,302 compounds, including 1,280 approved drugs, for their ability to enhance residual NAGLU activity in SP MPSIIIB patient fibroblasts.
Main Methods:
- Utilized skin fibroblasts from healthy controls, an SP MPSIIIB patient (p.S612G mutation), and an RP MPSIIIB patient (p.R297* mutation).
- Developed and validated a high-throughput assay for measuring NAGLU activity.
- Screened 1,302 compounds for their effect on NAGLU activity in patient fibroblasts.
Main Results:
- None of the 1,302 tested compounds demonstrated an ability to enhance NAGLU activity in SP MPSIIIB patient fibroblasts.
Conclusions:
- The high-throughput screen did not identify any compounds capable of enhancing residual mutant NAGLU activity in fibroblasts from SP MPSIIIB patients with temperature-sensitive mutations.
- Further research is required to understand the mechanisms behind temperature-dependent increases in NAGLU activity before therapeutic strategies can be developed.
