cytochalasin-b and Cystic-Fibrosis

Small molecules that correct the folding defects and enhance surface localization of the F508del mutation in the Cystic Fibrosis Transmembrane conductance Regulator (CFTR) comprise an important therapeutic strategy for cystic fibrosis lung disease. However, compounds that rescue the F508del mutant protein to wild type (WT) levels have not been identified. In this report, we consider obstacles to obtaining robust and therapeutically relevant levels of F508del CFTR. For example, markedly diminished steady state amounts of F508del CFTR compared to WT CFTR are present in recombinant bronchial epithelial cell lines, even when much higher levels of mutant transcript are present. In human primary airway cells, the paucity of Band B F508del is even more pronounced, although F508del and WT mRNA concentrations are comparable. Therefore, to augment levels of "repairable" F508del CFTR and identify small molecules that then correct this pool, we developed compound library screening protocols based on automated protein detection. First, cell-based imaging measurements were used to semi-quantitatively estimate distribution of F508del CFTR by high content analysis of two-dimensional images. We evaluated ~2,000 known bioactive compounds from the NIH Roadmap Molecular Libraries Small Molecule Repository in a pilot screen and identified agents that increase the F508del protein pool. Second, we analyzed ~10,000 compounds representing diverse chemical scaffolds for effects on total CFTR expression using a multi-plate fluorescence protocol and describe compounds that promote F508del maturation. Together, our findings demonstrate proof of principle that agents identified in this fashion can augment the level of endoplasmic reticulum (ER) resident "Band B" F508del CFTR suitable for pharmacologic correction. As further evidence in support of this strategy, PYR-41-a compound that inhibits the E1 ubiquitin activating enzyme-was shown to synergistically enhance F508del rescue by C18, a small molecule corrector. Our combined results indicate that increasing the levels of ER-localized CFTR available for repair provides a novel route to correct F508del CFTR.

Topics: Alleles; Benzoates; Cells, Cultured; Cystic Fibrosis; Cystic Fibrosis Transmembrane Conductance Regulator; Endoplasmic Reticulum; Furans; Gene Deletion; HEK293 Cells; HeLa Cells; High-Throughput Screening Assays; Humans; Hydroxamic Acids; Microscopy, Fluorescence; Protein Folding; Protein Structure, Tertiary; Pyrazoles; RNA, Messenger; Small Molecule Libraries; Ubiquitination; Vorinostat

A disorder of calcium homeostasis has been related to the pathogenesis of Cystic Fibrosis (CF). The Authors have studied the relationship between the cytosolic free calcium concentration ([Ca2+]i), the amount of Ca2+ released from endogenous stores and the secretory response in CF neutrophils. Significantly elevated resting [Ca2+]i and depressed Ca2+ release induced by the chemotactic peptide N-formyl-L-methionyl-L-leucyl-L-phenylalanine (FMLP) is present in CF neutrophils. In the absence of exogenous Ca2+ the secretory response of CF neutrophils after a weak stimulus such as Cytochalasin B (CB) is greater than in normal neutrophils, while a depressed secretion of azurophilic granules is evident in CF neutrophils stimulated by CB + FMLP. The data confirm the hypothesis of an altered Ca2+ homeostasis in CF cells. Cystic Fibrosis (CF), an autosomal recessive exocrinopathy, is characterized by secretory abnormalities and ion transport dysfunctions (for review see 1,2). Since intracellular Ca2+ seems to play a role in stimulus-secretion coupling and ion movements, several aspects of Ca2+ homeostasis have been investigated in CF. The total Ca2+ content has been reported to be increased in fibroblast cultures and in lymphocytes (3,4,5) and mitochondrial Ca2+ uptake was found elevated in fibroblast cultures (6). An elevated free cytosolic calcium concentration ([Ca2+]i) has been recently reported in buccal epithelial cells (7), while normal concentration has been found in lymphocytes and Epstein Barr virus transformed lymphoblasts (5,8). The present paper shows the results of a study in human neutrophils, a cell whose several functions such as secretion, movement and respiratory burst are in some way regulated by Ca2+. The data report that in neutrophils of CF patients the resting [Ca2+]i is higher and the secretory response is partly modified.

Topics: Aminoquinolines; Calcium; Cystic Fibrosis; Cytochalasin B; Cytosol; Humans; N-Formylmethionine Leucyl-Phenylalanine; Neutrophils

Lysosomal enzyme secretion by skin fibroblasts derived from cystic fibrosis homozygotes and normal donors of the same range of age, sex and culture passage number were compared. No differences were detected between these groups in their rates of spontaneous lysosomal enzyme release. Incubation of either normal or cystic fibrosis fibroblasts with cystic fibrosis serum did not alter these secretion rates. The lysosomal enzyme secretion of both normal and cystic fibrosis fibroblasts responded similarly to incubation with NH4Cl, monensin, A23187, trifluoperazine or Cytochalasin B. It was concluded that lysosomal enzyme hypersecretion, which has previously been suggested to occur in cystic fibrosis, is unlikely to provide a reliable diagnostic test for the disease.

Topics: Acetylglucosaminidase; Ammonium Chloride; Cell Line; Cystic Fibrosis; Cytochalasin B; Fibroblasts; Hexosaminidases; Humans; Kinetics; Lysosomes; Monensin

The ability of epsilon-amino caproic acid (EACA)-treated normal serum and of cystic fibrosis (CF)-affected and carrier sera to promote the release of lysosomal enzymes from sensitized human polymorphonuclear leukocytes (PMN) was assessed through the measurement of beta-glucuronidase and myeloperoxidase activity after exposure of these cells to the various test sera. This study was initiated to extend the analogies between preciliary dyskinesia factor (pre-CDF), separated from the cell-free media of cultures derived from CF homozygous and heterozygous individuals, and C3a anaphylatoxin. The extent of lysosomal degranulation of human PMN exposed to fresh untreated sera of each of five controls, seven CF homozygotes, and eight heterozygotes, as expressed by the amount of beta-glucuronidase releases, was 7.84% (+/- 0.934) for countrol sera, 14.01% (+/- 1.79) for CF-affected sera, and 10.61% (+/- 1.43) for heterozygous sera. The difference between CF homozygotes and control subjects is significatn (P less than 0.0001), as is the difference between CF-affected and carrier individuals (0.001 less than P less than 0.005) and between control subjects and carriers (0.001 less than P less than 0.005), when beta-glucuronidase. However, the differences between control subjects and CF heterozygous individuals are not significant. Treatment of these sera with 1 M EACA gave values for beta-glucuronidase and myeloperoxidase release which are slightly reduced when compared with those obtained with fresh, untreated samples. EACA apparently reduces the activity of beta-glucuronidase released from PMN. Amicon filtration studies of these serum samples demonstrated that degranulating ability and the presence of cilicary dyskinesia, as assessed by rabbit tracheal bioassay, are not always associated. Therefore, the relationship between pre-CDF and the degranulator activity in native CF-affected and carrier sera is unclear, in part because of the limitations inherent in the test systems employed.

Topics: Aminocaproates; Animals; Carrier State; Cilia; Cystic Fibrosis; Cytochalasin B; Cytoplasmic Granules; Glucuronidase; Heterozygote; Homozygote; Humans; Immunoglobulin G; Neutrophils; Peroxidase; Peroxidases; Trachea