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

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.

Processing of mutant N-acetyl-α-glucosaminidase in mucopolysaccharidosis type IIIB fibroblasts cultured at low temperature.

BACKGROUND: The autosomal recessive, neurodegenerative disorder mucopolysaccharidosis type IIIB (MPSIIIB) is caused by a deficiency of the lysosomal enzyme N-acetyl-α-glucosaminidase (NAGLU), resulting in accumulation of heparan sulfate. The disease spectrum comprises a severe, rapidly progressing (RP) phenotype and a more attenuated, slowly progressing (SP) phenotype. Previous studies showed significantly higher NAGLU activity in skin fibroblasts of SP patients when cultured at 30°C which may be relevant for development of novel therapeutic strategies. Here we report on the processes involved in this phenomenon. METHODS: Fibroblasts from controls, one RP patient (homozygous for the p.R297* mutation) and three SP MPSIIIB patients (homozygous for the mutation p.S612G or p.R643C, or compound heterozygous for the mutations p.A72_G79dup8 and p.R565Q) were cultured at temperatures ranging from 37°C to 27°C and harvested at different time points to assess NAGLU activity, mRNA and protein levels, and NAGLU glycosylation. Intracellular localization of wild-type and mutant mCherry-tagged NAGLU was analyzed by immunofluorescence. RESULTS: In control fibroblasts NAGLU was present as a 85kDa precursor and a 82kDa mature form. In SP patients' fibroblasts cultured at 37°C, only the 85kDa form was detected. Culturing at lower temperatures resulted in higher NAGLU mRNA levels, increased levels of both precursor and mature NAGLU protein and improved processing. The formation of mature NAGLU corresponded with higher NAGLU activity levels. CONCLUSION: We show that the NAGLU protein consists of a precursor and a mature form and that in SP MPSIIIB patients' fibroblasts only the precursor protein is present at 37°C. Culturing at lower temperatures resulted in the formation of the mature, enzymatically active form, due to higher mRNA levels and improved processing.

Residual N-acetyl-α-glucosaminidase activity in fibroblasts correlates with disease severity in patients with mucopolysaccharidosis type IIIB.

BACKGROUND: Mucopolysaccharidosis type IIIB (MPS IIIB) is a rare genetic disorder in which the deficiency of the lysosomal enzyme N-acetyl-α-glucosaminidase (NAGLU) results in the accumulation of heparan sulfate (HS), leading to progressive neurocognitive deterioration. In MPS IIIB a wide spectrum of disease severity is seen. Due to a large allelic heterogeneity, establishing genotype-phenotype correlations is difficult. However, reliable prediction of the natural course of the disease is needed, in particular for the assessment of the efficacy of potential therapies. METHODS: To identify markers that correlate with disease severity, all Dutch patients diagnosed with MPS IIIB were characterised as either rapid (RP; classical, severe phenotype) or slow progressors (SP; non-classical, less severe phenotype), based on clinical data. NAGLU activity and HS levels were measured in patients' fibroblasts after culturing at different temperatures. RESULTS: A small, though significant difference in NAGLU activity was measured between RP and SP patients after culturing at 37 °C (p < 0.01). Culturing at 30 °C resulted in more pronounced and significantly higher NAGLU activity levels in SP patients (p < 0.001) with a NAGLU activity of 0.58 nmol.mg-1.hr-1 calculated to be the optimal cut-off value to distinguish between the groups (sensitivity and specificity 100 %). A lower capacity of patients' fibroblasts to increase NAGLU activity at 30 °C could significantly predict for the loss of several disease specific functions. CONCLUSION: NAGLU activity in fibroblasts cultured at 30 °C can be used to discriminate between RP and SP MPS IIIB patients and the capacity of cells to increase NAGLU activity at lower temperatures correlates with disease symptoms.

Endoscopic ultrasonography in suspected pancreatic malignancy and indecisive CT.

BACKGROUND: In the assessment of patients with a clinical suspicion of malignant pancreatic disease, computed tomography (CT) findings are sometimes negative or inconclusive. AIMS: To determine whether endoscopic ultrasonography (EUS) with or without fine needle aspiration (EUS÷FNA) was conclusive in patients with a clinical suspicion of pancreatic malignancy, in whom CT scan was negative or inconclusive. METHODS: Retrospective case series in a tertiary referral centre. From February 2006 to December 2007, EUS÷FNA was performed in all patients suspected of having malignant pancreatic disease with negative or inconclusive CT findings. Main outcome measurement was the diagnostic yield of EUS in these patients. RESULTS: 34 patients had a negative (n=11) or inconclusive (n=23) CT scan. EUS÷FNA established a correct diagnosis in 30÷34 cases (88%). Malignancy was diagnosed in 19÷34 patients and nonmalignant disease in 8÷34 cases. In 3÷34 patients no lesions were found and no malignant disease developed during follow-up (mean=728 days). EUS÷FNA was inconclusive in 4÷34 patients. CONCLUSION: In patients with a clinical suspicion of pancreatic malignancy with negative or inconclusive CT findings, EUS÷FNA was able to establish a diagnosis in 88% of cases. EUS should therefore be considered a diagnostic modality in this complex group of patients.