High-Throughput Screening for Modulators of CFTR Activity Based on Genetically Engineered Cystic Fibrosis Disease-Specific iPSCs.

Organotypic culture systems from disease-specific induced pluripotent stem cells (iPSCs) exhibit obvious advantages compared with immortalized cell lines and primary cell cultures, but implementation of iPSC-based high-throughput (HT) assays is still technically challenging. Here, we demonstrate the development and conduction of an organotypic HT Cl-/I- exchange assay using cystic fibrosis (CF) disease-specific iPSCs. The introduction of a halide-sensitive YFP variant enabled automated quantitative measurement of Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) function in iPSC-derived intestinal epithelia. CFTR function was partially rescued by treatment with VX-770 and VX-809, and seamless gene correction of the p.Phe508del mutation resulted in full restoration of CFTR function. The identification of a series of validated primary hits that improve the function of p.Phe508del CFTR from a library of ∼42,500 chemical compounds demonstrates that the advantages of complex iPSC-derived culture systems for disease modeling can also be utilized for drug screening in a true HT format.

Speeding Up the Identification of Cystic Fibrosis Transmembrane Conductance Regulator-Targeted Drugs: An Approach Based on Bioinformatics Strategies and Surface Plasmon Resonance.

Cystic fibrosis (CF) is mainly caused by the deletion of Phe 508 (ΔF508) in the cystic fibrosis transmembrane conductance regulator (CFTR) protein that is thus withheld in the endoplasmic reticulum and rapidly degraded by the ubiquitin/proteasome system. New drugs able to rescue ΔF508-CFTR trafficking are eagerly awaited. An integrated bioinformatics and surface plasmon resonance (SPR) approach was here applied to investigate the rescue mechanism(s) of a series of CFTR-ligands including VX809, VX770 and some aminoarylthiazole derivatives (AAT). Computational studies tentatively identified a large binding pocket in the ΔF508-CFTR nucleotide binding domain-1 (NBD1) and predicted all the tested compounds to bind to three sub-regions of this main pocket. Noticeably, the known CFTR chaperone keratin-8 (K8) seems to interact with some residues located in one of these sub-pockets, potentially interfering with the binding of some ligands. SPR results corroborated all these computational findings. Moreover, for all the considered ligands, a statistically significant correlation was determined between their binding capability to ΔF508-NBD1 measured by SPR and the pockets availability measured by computational studies. Taken together, these results demonstrate a strong agreement between the in silico prediction and the SPR-generated binding data, suggesting a path to speed up the identification of new drugs for the treatment of cystic fibrosis.

The TMEM16A chloride channel as an alternative therapeutic target in cystic fibrosis.

Cystic fibrosis (CF), a multiorgan genetic disease, is caused by loss of function of CFTR, a cAMP-regulated anion channel. In CF airway epithelia, defective Cl(-) and bicarbonate secretion impairs mucociliary clearance and other innate defense mechanisms, favoring the colonization of the lungs by highly virulent bacteria. The airway epithelium expresses TMEM16A, a second type of Cl(-) channel that is activated by cytosolic Ca(2+). TMEM16A is particularly expressed in goblet cells. This specific localization could be important in the release and hydration of mucins. Activation of TMEM16A with pharmacological agents could circumvent the primary defect in CF. This strategy needs to be carefully designed and tested to avoid possible undesired effects due to the expression of TMEM16A in other cell types such as bronchial smooth muscle cells. This article is part of a Directed Issue entitled: Cystic Fibrosis: From o-mics to cell biology, physiology, and therapeutic advances.

High-throughput screening of libraries of compounds to identify CFTR modulators.

Small molecules acting as selective activators (potentiators), inhibitors, or "correctors" of the CFTR chloride channel represent candidate drugs for various pathological conditions including cystic fibrosis and secretory diarrhea. The identification of CFTR pharmacological modulators may be achieved by screening highly diverse synthetic or natural compound libraries using high-throughput methods. A convenient assay for CFTR function is based on the halide sensitivity of the yellow fluorescent protein (YFP). CFTR activity can be simply assessed by measuring the rate of YFP signal decrease caused by iodide influx. This assay can be automated to test thousands of compounds per day.

Cell-based imaging of sodium iodide symporter activity with the yellow fluorescent protein variant YFP-H148Q/I152L.

The sodium iodide symporter (NIS) mediates iodide (I(-)) transport in the thyroid gland and other tissues and is of increasing importance as a therapeutic target and nuclear imaging reporter. NIS activity in vitro is currently measured with radiotracers and electrophysiological techniques. We report on the development of a novel live cell imaging assay of NIS activity using the I(-)-sensitive and genetically encodable yellow fluorescent protein (YFP) variant YFP-H148Q/I152L. In FRTL-5 thyrocytes stably expressing YFP-H148Q/I152L, I(-) induced a rapid and reversible decrease in cellular fluorescence characterized by 1) high affinity for extracellular I(-) (35 muM), 2) inhibition by the NIS inhibitor perchlorate, 3) extracellular Na(+) dependence, and 4) TSH dependence, suggesting that fluorescence changes are due to I(-) influx via NIS. Individual cells within a population of FRTL-5 cells exhibited a 3.5-fold variation in the rate of NIS-mediated I(-) influx, illustrating the utility of YFP-H148Q/I152L to detect cell-to-cell difference in NIS activity. I(-) also caused a perchlorate-sensitive decrease in YFP-H148Q/I152L fluorescence in COS-7 cells expressing NIS but not in cells lacking NIS. These results demonstrate that YFP-H148Q/I152L is a sensitive biosensor of NIS-mediated I(-) uptake in thyroid cells and in nonthyroidal cells following gene transfer and suggest that fluorescence detection of cellular I(-) may be a useful tool by which to study the pathophysiology and pharmacology of NIS.

Discovery of glycine hydrazide pore-occluding CFTR inhibitors: mechanism, structure-activity analysis, and in vivo efficacy.

The cystic fibrosis transmembrane conductance regulator (CFTR) protein is a cAMP-regulated epithelial Cl- channel that, when defective, causes cystic fibrosis. Screening of a collection of 100,000 diverse small molecules revealed four novel chemical classes of CFTR inhibitors with Ki < 10 microM, one of which (glycine hydrazides) had many active structural analogues. Analysis of a series of synthesized glycine hydrazide analogues revealed maximal inhibitory potency for N-(2-naphthalenyl) and 3,5-dibromo-2,4-dihydroxyphenyl substituents. The compound N-(2-naphthalenyl)-[(3,5-dibromo-2,4-dihydroxyphenyl)methylene]glycine hydrazide (GlyH-101) reversibly inhibited CFTR Cl- conductance in <1 min. Whole-cell current measurements revealed voltage-dependent CFTR block by GlyH-101 with strong inward rectification, producing an increase in apparent inhibitory constant Ki from 1.4 microM at +60 mV to 5.6 microM at -60 mV. Apparent potency was reduced by lowering extracellular Cl- concentration. Patch-clamp experiments indicated fast channel closures within bursts of channel openings, reducing mean channel open time from 264 to 13 ms (-60 mV holding potential, 5 microM GlyH-101). GlyH-101 inhibitory potency was independent of pH from 6.5-8.0, where it exists predominantly as a monovalent anion with solubility approximately 1 mM in water. Topical GlyH-101 (10 microM) in mice rapidly and reversibly inhibited forskolin-induced hyperpolarization in nasal potential differences. In a closed-loop model of cholera, intraluminal GlyH-101 (2.5 microg) reduced by approximately 80% cholera toxin-induced intestinal fluid secretion. Compared with the thiazolidinone CFTR inhibitor CFTR(inh)-172, GlyH-101 has substantially greater water solubility and rapidity of action, and a novel inhibition mechanism involving occlusion near the external pore entrance. Glycine hydrazides may be useful as probes of CFTR pore structure, in creating animal models of CF, and as antidiarrheals in enterotoxic-mediated secretory diarrheas.

High-affinity activators of cystic fibrosis transmembrane conductance regulator (CFTR) chloride conductance identified by high-throughput screening.

Cystic fibrosis (CF) is caused by mutations in the CF transmembrane conductance regulator (CFTR) protein that reduce cAMP-stimulated Cl(-) conductance in airway and other epithelia. The purpose of this investigation was to identify new classes of potent CFTR activators. A collection of 60,000 diverse drug-like compounds was screened at 10 microm together with a low concentration of forskolin (0.5 microm) in Fisher rat thyroid epithelial cells co-expressing human CFTR and a green fluorescent protein-based Cl(-) sensor. Primary screening yielded 57 strong activators (greater activity than reference compound apigenin), most of which were unrelated in chemical structure to known CFTR activators, and 284 weaker activators. Secondary analysis of the strong activators included analysis of CFTR specificity, forskolin requirement, transepithelial short-circuit current, activation kinetics, dose response, toxicity, and activation mechanism. Three compounds, the most potent being a dihydroisoquinoline, activated CFTR by elevating cellular cAMP, probably by phosphodiesterase inhibition. Fourteen compounds activated CFTR without cAMP elevation or phosphatase inhibition, suggesting direct CFTR interaction. The most potent compounds had tetrahydrocarbazol, hydroxycoumarin, and thiazolidine core structures. These compounds induced CFTR Cl(-) currents rapidly (<5 min) with K(d) down to 200 nm and were CFTR-selective, reversible, and nontoxic. Several compounds, the most potent being a trifluoromethylphenylbenzamine, activated the CF-causing mutant G551D, but with much weaker affinity (K(d) > 10 microm). When added for 10 min, none of the compounds activated DeltaPhe(508)-CFTR in transfected cells grown at 37 degrees C (with DeltaPhe(508)-CFTR trapped in the endoplasmic reticulum). However, after correction of trafficking by 48 h of growth at 27 degrees C, tetrahydrocarbazol and N-phenyltriazine derivatives strongly stimulated Cl(-) conductance with K(d) < 1 microm. The new activators identified here may be useful in defining molecular mechanisms of CFTR activation and as lead compounds in CF drug development.