Gaucher disease is caused by an inherited deficiency of glucocerebrosidase that manifests with storage of glycolipids in lysosomes, particularly in macrophages. Available cell lines modeling Gaucher ...disease do not demonstrate lysosomal storage of glycolipids; therefore, we set out to develop two macrophage models of Gaucher disease that exhibit appropriate substrate accumulation. We used these cellular models both to investigate altered macrophage biology in Gaucher disease and to evaluate candidate drugs for its treatment. We generated and characterized monocyte-derived macrophages from 20 patients carrying different Gaucher disease mutations. In addition, we created induced pluripotent stem cell (iPSC)-derived macrophages from five fibroblast lines taken from patients with type 1 or type 2 Gaucher disease. Macrophages derived from patient monocytes or iPSCs showed reduced glucocerebrosidase activity and increased storage of glucocerebroside and glucosylsphingosine in lysosomes. These macrophages showed efficient phagocytosis of bacteria but reduced production of intracellular reactive oxygen species and impaired chemotaxis. The disease phenotype was reversed with a noninhibitory small-molecule chaperone drug that enhanced glucocerebrosidase activity in the macrophages, reduced glycolipid storage, and normalized chemotaxis and production of reactive oxygen species. Macrophages differentiated from patient monocytes or patient-derived iPSCs provide cellular models that can be used to investigate disease pathogenesis and facilitate drug development.
A major challenge in the field of Gaucher disease has been the development of new therapeutic strategies including molecular chaperones. All previously described chaperones of glucocerebrosidase are ...enzyme inhibitors, which complicates their clinical development because their chaperone activity must be balanced against the functional inhibition of the enzyme. Using a novel high throughput screening methodology, we identified a chemical series that does not inhibit the enzyme but can still facilitate its translocation to the lysosome as measured by immunostaining of glucocerebrosidase in patient fibroblasts. These compounds provide the basis for the development of a novel approach toward small molecule treatment for patients with Gaucher disease.
Gaucher disease (GD) is an autosomal recessive disorder caused by mutations in the acid β-glucocerebrosidase gene. To model GD, we generated human induced pluripotent stem cells (hiPSC), by ...reprogramming skin fibroblasts from patients with type 1 (N370S/N370S), type 2 (L444P/Rec NciI), and type 3 (L444P/L444P) GD. Pluripotency was demonstrated by the ability of GD hiPSC to differentiate to all three germ layers and to form teratomas in vivo. GD hiPSC differentiated efficiently to the cell types most affected in GD, i.e., macrophages and neuronal cells. GD hiPSC-macrophages expressed macrophage-specific markers, were phagocytic, and were capable of releasing inflammatory mediators in response to LPS. Moreover, GD hiPSC-macrophages recapitulated the phenotypic hallmarks of the disease. They exhibited low glucocerebrosidase (GC) enzymatic activity and accumulated sphingolipids, and their lysosomal functions were severely compromised. GD hiPSC-macrophages had a defect in their ability to clear phagocytosed RBC, a phenotype of tissue-infiltrating GD macrophages. The kinetics of RBC clearance by types 1, 2, and 3 GD hiPSC-macrophages correlated with the severity of the mutations. Incubation with recombinant GC completely reversed the delay in RBC clearance from all three types of GD hiPSC-macrophages, indicating that their functional defects were indeed caused by GC deficiency. However, treatment of induced macrophages with the chaperone isofagomine restored phagocytosed RBC clearance only partially, regardless of genotype. These findings are consistent with the known clinical efficacies of recombinant GC and isofagomine. We conclude that cell types derived from GD hiPSC can effectively recapitulate pathologic hallmarks of the disease.
Gaucher disease (GD), the most common lysosomal storage disorder, results from the inherited deficiency of the lysosomal enzyme glucocerebrosidase (GCase). Previously, wildtype GCase was used for ...high throughput screening (HTS) of large collections of compounds to identify small molecule chaperones that could be developed as new therapies for GD. However, the compounds identified from HTS usually showed reduced potency later in confirmatory cell-based assays. An alternate strategy is to perform HTS on mutant enzyme to identify different lead compounds, including those enhancing mutant enzyme activities. We developed a new screening assay using enzyme extract prepared from the spleen of a patient with Gaucher disease with genotype N370S/N370S. In tissue extracts, GCase is in a more native physiological environment, and is present with the native activator saposin C and other potential cofactors. Using this assay, we screened a library of 250,000 compounds and identified novel modulators of mutant GCase including 14 new lead inhibitors and 30 lead activators. The activities of some of the primary hits were confirmed in subsequent cell-based assays using patient-derived fibroblasts. These results suggest that primary screening assays using enzyme extracted from tissues is an alternative approach to identify high quality, physiologically relevant lead compounds for drug development.
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Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Gaucher disease is an autosomal recessive lysosomal storage disorder caused by mutations in the glucocerebrosidase gene. Missense mutations result in reduced enzyme activity that may be due to ...misfolding, raising the possibility of small-molecule chaperone correction of the defect. Screening large compound libraries by quantitative high-throughput screening (qHTS) provides comprehensive information on the potency, efficacy, and structure-activity relationships (SAR) of active compounds directly from the primary screen, facilitating identification of leads for medicinal chemistry optimization. We used qHTS to rapidly identify three structural series of potent, selective, nonsugar glucocerebrosidase inhibitors. The three structural classes had excellent potencies and efficacies and, importantly, high selectivity against closely related hydrolases. Preliminary SAR data were used to select compounds with high activity in both enzyme and cell-based assays. Compounds from two of these structural series increased N370S mutant glucocerebrosidase activity by 40-90% in patient cell lines and enhanced lysosomal colocalization, indicating chaperone activity. These small molecules have potential as leads for chaperone therapy for Gaucher disease, and this paradigm promises to accelerate the development of leads for other rare genetic disorders.
Mucolipidosis type IV (MLIV) is an autosomal recessive lysosomal storage disease characterized by severe psychomotor retardation, achlorhydria, and ophthalmological abnormalities. Cells from several ...tissues in MLIV patients accumulate large vacuoles that are presumed to be lysosomes, but whose exact nature remains to be determined. Other defects include the deterioration of neuronal integrity in the retina and the cerebellum. MCOLN1, the gene mutated in MLIV patients, encodes a protein called h-mucolipin-1 that has six predicted transmembrane domains and functions as a Ca2+-permeable channel that is modulated by changes in Ca2+ concentration. CUP-5 is the Caenorhabditis elegans functional orthologue of h-mucolipin-1. Mutations in cup-5 result in the accumulation of large vacuoles in several cells, in increased cell death, and in embryonic lethality. We demonstrate here that CUP-5 functions in the biogenesis of lysosomes originating from hybrid organelles. We also show that at least two h-mucolipin family members rescue cup-5 mutant endocytic defects, indicating that there may be functional redundancy among the human proteins. Finally, we propose a model that relates the lysosome biogenesis defect in the absence of CUP-5/h-mucolipin-1 to cellular phenotypes in worms and in humans.
A previously unrecognized nonmuscle myosin II heavy chain (NMHC II), which constitutes a distinct branch of the nonmuscle/smooth muscle myosin II family, has recently been revealed in genome data ...bases. We characterized the biochemical properties and expression patterns of this myosin. Using nucleotide probes and affinity-purified antibodies, we found that the distribution of NMHC II-C mRNA and protein (MYH14) is widespread in human and mouse organs but is quantitatively and qualitatively distinct from NMHC II-A and II-B. In contrast to NMHC II-A and II-B, the mRNA level in human fetal tissues is substantially lower than in adult tissues. Immunofluorescence microscopy showed distinct patterns of expression for all three NMHC isoforms. NMHC II-C contains an alternatively spliced exon of 24 nucleotides in loop I at a location analogous to where a spliced exon appears in NMHC II-B and in the smooth muscle myosin heavy chain. However, unlike neuron-specific expression of the NMHC II-B insert, the NMHC II-C inserted isoform has widespread tissue distribution. Baculovirus expression of noninserted and inserted NMHC II-C heavy meromyosin (HMM II-C/HMM II-C1) resulted in significant quantities of expressed protein (mg of protein) for HMM II-C1 but not for HMM II-C. Functional characterization of HMM II-C1 by actin-activated MgATPase activity demonstrated a Vmax of 0.55 + 0.18 s–1, which was half-maximally activated at an actin concentration of 16.5 + 7.2 μm. HMM II-C1 translocated actin filaments at a rate of 0.05 + 0.011 μm/s in the absence of tropomyosin and at 0.072 + 0.019 μm/s in the presence of tropomyosin in an in vitro motility assay.
Glucocerebrosidase is a lysosomal enzyme that catalyzes the hydrolysis of glucosylceramide to form ceramide and glucose. A deficiency of lysosomal glucocerebrosidase due to genetic mutations results ...in Gaucher disease, in which glucosylceramide accumulates in the lysosomes of certain cell types. Although enzyme replacement therapy is currently available for the treatment of type 1 Gaucher disease, the neuronopathic forms of Gaucher disease are still not treatable. Small molecule drugs that can penetrate the blood-brain barrier, such as pharmacological chaperones and enzyme activators, are new therapeutic approaches for Gaucher disease. Enzyme assays for glucocerebrosidase are used to screen compound libraries to identify new lead compounds for drug development for the treatment of Gaucher disease. But the current assays use artificial substrates that are not physiologically relevant. We developed a glucocerebrosidase assay using the natural substrate glucosylceramide coupled to an Amplex-red enzyme reporting system. This assay is in a homogenous assay format and has been miniaturized in a 1,536-well plate format for high throughput screening. The assay sensitivity and robustness is similar to those seen with other glucocerebrosidase fluorescence assays. Therefore, this new glucocerebrosidase assay is an alternative approach for high throughput screening.
Fabry disease is inherited as an X-linked disorder secondary to deficiency of α-galactosidase A, resulting in abnormal metabolism of substances containing α-D-galactosyl moieties. As a consequence, a ...multisystem disorder develops, culminating in strokes, progressive renal, and cardiac dysfunction. Signs and symptoms of Fabry disease become manifest in childhood, but diagnosis is often delayed. Thirteen children with Fabry disease (age range, 6.5-17 years) were studied as part of a 6-month open-label study of enzyme replacement therapy (ERT) with agalsidase alfa. Paired serum samples were drawn at the start of the study and after 6 months of ERT. Global protein changes in paired samples were compared by using differential stable isotope labeling of peptide lysine residues with O-methylisourea and subsequent nanoHPLC-tandem MS. Statistically significant decreases were observed for five proteins following ERT: α₂-HS glycoprotein, vitamin D-binding protein, transferrin, Ig-α-2 C chain, and α-2-antiplasmin. The presence of low levels of α-2-antiplasmin and plasminogen was confirmed by alternate means in 34 consecutive patients, including four of five ERT-naïve subjects. Decreased α-2-antiplasmin was associated with a parallel increase in circulating VEGF. Soluble VEGF receptor-2 was significantly elevated in plasma of patients compared with pediatric controls and decreased with ERT. These results suggest previously unknown abnormalities of fibrinolysis and angiogenesis factors in Fabry disease. We demonstrated the feasibility of identifying treatment-specific alterations in a small number of subjects that point to previously unsuspected disease-related biological abnormalities.
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