1. Deletion of phenylalanine at position 508 (DeltaF508) of the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel is the most common mutation causing cystic fibrosis (CF). Effective pharmacological therapy of CF caused by the DeltaF508-CFTR mutation requires the rescue of both intracellular processing and channel gating defects. 2. We identified a class of natural coumarin compounds that can correct the defective DeltaF508-CFTR chloride channel gating by screening a collection of 386 single natural compounds from Chinese medicinal herbs. Screening was performed with an iodide influx assay in Fischer rat thyroid epithelial cells coexpressing DeltaF508-CFTR and an iodide-sensitive fluorescent indicator (YFP-H148Q/I152L). 3. Dose-dependent potentiation of defective DeltaF508-CFTR chloride channel gating by five coumarin compounds was demonstrated by the fluorescent iodide influx assay and confirmed by an Ussing chamber short-circuit current assay. Activation was fully abolished by the specific CFTR inhibitor CFTR(inh)-172. Two potent compounds, namely imperatorin and osthole, have activation K(d) values of approximately 10 micromol/L, as determined by the short-circuit current assay. The active coumarin compounds do not elevate intracellular cAMP levels. Activation of DeltaF508-CFTR by the coumarin compounds requires cAMP agonist, suggesting direct interaction with the mutant CFTR molecule. Kinetics analysis indicated rapid activation of DeltaF508-CFTR by the coumarin compounds, with half-maximal activation of < 5 min. The activating effect was fully reversed for all five active compounds 45 min after washout. 4. In conclusion, the natural coumarin DeltaF508-CFTR activators may represent a new class of natural lead compounds for the development of pharmacological therapies for CF caused by the DeltaF508 mutation.
"In a previous study, we identified a family of natural coumarin compounds that can potentiate Cl– transport through ΔF508-CFTR by screening a collection of 386 natural single compounds from Chinese medicinal herbs (Xu et al., 2008). However, the in vivo effects of these coumarin compounds have not been determined. "
[Show abstract][Hide abstract] ABSTRACT: Mutations of cystic fibrosis (CF) transmembrane conductance regulator (CFTR) cause lethal hereditary disease CF that involves extensive destruction and dysfunction of serous epithelium. Possible pharmacological therapy includes correction of defective intracellular processing and abnormal channel gating. In a previous study, we identified five natural coumarin potentiators of ΔF508-CFTR including osthole, imperatorin, isopsoralen, praeruptorin A, and scoparone. The present study was designed to determine the activity of these coumarine compounds on CFTR activity in animal tissues as a primary evaluation of their therapeutic potential. In the present study, we analyzed the affinity of these coumarin potentiators in activating wild-type CFTR and found that they are all potent activators. Osthole showed the highest affinity with K(d) values <50 nmol/L as determined by Ussing chamber short-circuit current assay. Stimulation of rat colonic mucosal secretion by osthole was tested by the Ussing chamber short-circuit current assay. Osthole reached maximal activation of colonic Cl(-) secretion at 5 μmol/L. Stimulation of mouse tracheal mucosal secretion was analyzed by optical measurement of single gland secretion. Fluid secretion rate of tracheal single submucosal gland stimulated by osthole at 10 μmol/L was three-fold more rapid than that in negative control. In both cases the stimulated secretions were fully abolished by CFTR(inh)-172. In conclusion, the effective stimulation of Cl(-) and fluid secretion in colonic and tracheal mucosa by osthole suggested the therapeutic potential of natural coumarin compounds for the treatment of CF and other CFTR-related diseases.
Frontiers in Pharmacology 09/2011; 2:52. DOI:10.3389/fphar.2011.00052 · 3.80 Impact Factor
"Because there is insufficient information at the present time to design rationally CFTR correctors and potentiators, the strategy of choice to identify drug-like small molecule CFTR modulators is high-throughput screening (HTS)45. HTS exploits a reliable, sensitive, cost-effective assay to screen libraries of chemically diverse small molecules (eg approved drugs51, drug-like chemicals52 and natural products53) to identify lead compounds for medicinal chemistry optimization. For example, Alan Verkman (UCSF, San Francisco, USA) used Fischer rat thyroid cells, epithelial cells devoid of CFTR expression and cAMP-stimulated Cl− currents54 engineered to co-express recombinant human CFTR and a green fluorescent protein (GFP) with ultra high halide sensitivity in a halide flux assay (eg Yang et al52). "
[Show abstract][Hide abstract] ABSTRACT: The mutation F508del is the commonest cause of the genetic disease cystic fibrosis (CF). CF disrupts the function of many organs in the body, most notably the lungs, by perturbing salt and water transport across epithelial surfaces. F508del causes harm in two principal ways. First, the mutation prevents delivery of the cystic fibrosis transmembrane conductance regulator (CFTR) to its correct cellular location, the apical (lumen-facing) membrane of epithelial cells. Second, F508del perturbs the Cl(-) channel function of CFTR by disrupting channel gating. Here, we discuss the development of rational new therapies for CF that target F508del-CFTR. We highlight how structural studies provide new insight into the role of F508 in the regulation of channel gating by cycles of ATP binding and hydrolysis. We emphasize the use of high-throughput screening to identify lead compounds for therapy development. These compounds include CFTR correctors that restore the expression of F508del-CFTR at the apical membrane of epithelial cells and CFTR potentiators that rescue the F508del-CFTR gating defect. Initial results from clinical trials of CFTR correctors and potentiators augur well for the development of small molecule therapies that target the root cause of CF: mutations in CFTR.
"To screen potential activators of CFTR-mediated Cl − fl ux, a YFP mutant with higher halide sensitivity (YFP-H148Q/I152L) has been co-expressed with defective ΔF508-CFTR in FRT epithelial cells (Yang et al., 2003; Xu et al., 2008). It has been shown that natural cumarine compounds rescue defective ΔF508-CFTR chloride channel gating (Xu et al., 2008). A mutant with enhanced sensitivity to halides (YFP-V163S) has been used in M1 cortical collecting-duct cells to monitor changes in Cl − mediated by CFTR or by stimulation with cAMP-and Ca 2+ increasing agonists (Adam et al., 2005). "
[Show abstract][Hide abstract] ABSTRACT: This review briefly discusses the main approaches for monitoring chloride (Cl−), the most abundant physiological anion. Noninvasive monitoring of intracellular Cl− ([Cl−]i) is a challenging task owing to two main difficulties: (i) the low transmembrane ratio for Cl−, approximately 10:1; and (ii) the small driving force for Cl−, as the Cl− reversal potential (ECl) is usually close to the resting potential of the cells. Thus, for reliable monitoring of intracellular Cl−, one has to use highly sensitive probes. From several methods for intracellular Cl− analysis, genetically encoded chloride indicators represent the most promising tools. Recent achievements in the development of genetically encoded chloride probes are based on the fact that yellow fluorescent protein (YFP) exhibits Cl−-sensitivity. YFP-based probes have been successfully used for quantitative analysis of Cl− transport in different cells and for high-throughput screening of modulators of Cl−-selective channels. Development of a ratiometric genetically encoded probe, Clomeleon, has provided a tool for noninvasive estimation of intracellular Cl− concentrations. While the sensitivity of this protein to Cl− is low (EC50 about 160 mM), it has been successfully used for monitoring intracellular Cl− in different cell types. Recently a CFP–YFP-based probe with a relatively high sensitivity to Cl− (EC50 about 30 mM) has been developed. This construct, termed Cl-Sensor, allows ratiometric monitoring using the fluorescence excitation ratio. Of particular interest are genetically encoded probes for monitoring of ion channel distribution and activity. A new molecular probe has been constructed by introducing into the cytoplasmic domain of the Cl−-selective glycine receptor (GlyR) channel the CFP–YFP-based Cl-Sensor. This construct, termed BioSensor-GlyR, has been successfully expressed in cell lines. The new genetically encoded chloride probes offer means of screening pharmacological agents, analysis of Cl− homeo
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