Identification of natural coumarin compounds that rescue defective DeltaF508-CFTR chloride channel gating.
ABSTRACT 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.
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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 (E(Cl)) 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 (EC(50) 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(-) (EC(50) 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(-) homeostasis and functions of Cl(-)-selective channels under different physiological and pathological conditions.Frontiers in Molecular Neuroscience 12/2009; 2:15. DOI:10.3389/neuro.02.015.2009
- Clinical and Experimental Pharmacology and Physiology 09/2008; 35(8):861-2. DOI:10.1111/j.1440-1681.2008.04953.x · 2.41 Impact Factor
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ABSTRACT: Frederick Kaplan – The University of Pennsylvania School of Medicine, Department of Orthopaedic Surgery, Philadelphia, PA 19104, USA Amber Salzman – Cardiokine, Inc., Philadelphia, PA 19102, USADrug Discovery Today Therapeutic Strategies 12/2008; 5(4):243-248. DOI:10.1016/j.ddstr.2008.10.004