Development of substituted Benzo[c]quinolizinium compounds as novel activators of the cystic fibrosis chloride channel.

Chloride channels play an important role in the physiology and pathophysiology of epithelia, but their pharmacology is still poorly developed. We have chemically synthesized a series of substituted benzo[c]quinolizinium (MPB) compounds. Among them, 6-hydroxy-7-chlorobenzo[c]quinolizinium (MPB-27) and 6-hydroxy-10-chlorobenzo[c]quinolizinium (MPB-07), which we show to be potent and selective activators of the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel. We examined the effect of MPB compounds on the activity of CFTR channels in a variety of established epithelial and nonepithelial cell systems. Using the iodide efflux technique, we show that MPB compounds activate CFTR chloride channels in Chinese hamster ovary (CHO) cells stably expressing CFTR but not in CHO cells lacking CFTR. Single and whole cell patch clamp recordings from CHO cells confirm that CFTR is the only channel activated by the drugs. Ussing chamber experiments reveal that the apical addition of MPB to human nasal epithelial cells produces a large increase of the short circuit current. This current can be totally inhibited by glibenclamide. Whole cell experiments performed on native respiratory cells isolated from wild type and CF null mice also show that MPB compounds specifically activate CFTR channels. The activation of CFTR by MPB compounds was glibenclamide-sensitive and 4, 4'-diisothiocyanostilbene-2,2'-disulfonic acid-insensitive. In the human tracheal gland cell line MM39, MPB drugs activate CFTR channels and stimulate the secretion of the antibacterial secretory leukoproteinase inhibitor. In submandibular acinar cells, MPB compounds slightly stimulate CFTR-mediated submandibular mucin secretion without changing intracellular cAMP and ATP levels. Similarly, in CHO cells MPB compounds have no effect on the intracellular levels of cAMP and ATP or on the activity of various protein phosphatases (PP1, PP2A, PP2C, or alkaline phosphatase). Our results provide evidence that substituted benzo[c]quinolizinium compounds are a novel family of activators of CFTR and of CFTR-mediated protein secretion and therefore represent a new tool to study CFTR-mediated chloride and secretory functions in epithelial tissues.     

Benzoflavone activators of the cystic fibrosis transmembrane conductance regulator: towards a pharmacophore model for the nucleotide-binding domain

Our previous screen of flavones and related heterocycles for the ability to activate the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel indicated that UCCF-029, a 7,8-benzoflavone, was a potent activator. In the present study, we describe the synthesis and evaluation, using cell-based assays, of a series of benzoflavone analogues to examine structure-activity relationships and to identify compounds having greater potency for activation of both wild type CFTR and a mutant CFTR (G551D-CFTR) that causes cystic fibrosis in some human subjects. Using UCCF-029 as a structural guide, a panel of 77 flavonoid analogues was prepared. Analysis of the panel in FRT cells indicated that benzannulation of the flavone A-ring at the 7,8-position greatly improved compound activity and potency for several flavonoids. Incorporation of a B-ring pyridyl nitrogen either at the 3- or 4-position also elevated CFTR activity, but the influence of this structural modification was not as uniform as the influence of benzannulation. The most potent new analogue, UCCF-339, activated wild-type CFTR with a K(d) of 1.7 microM, which is more active than the previous most potent flavonoid activator of CFTR, apigenin. Several compounds in the benzoflavone panel also activated G551D-CFTR, but none were as active as apigenin. Pharmacophore modeling suggests a common binding mode for the flavones and other known CFTR activators at one of the nucleotide-binding sites, allowing for the rational development of more potent flavone analogues.     

CFTR activation in human bronchial epithelial cells by novel benzoflavone and benzimidazolone compounds

Activators of the CFTR Cl- channel may be useful for therapy of cystic fibrosis. Short-circuit current (Isc) measurements were done on human bronchial epithelial cells to characterize the best flavone and benzimidazolone CFTR activators identified by lead-based combinatorial synthesis and high-throughput screening. The 7,8-benzoflavone UCcf-029 was a potent activator of Cl- transport, with activating potency (<1 microM) being much better than other flavones, such as apigenin. The benzimidazolone UCcf-853 gave similar Isc but with lower potency (5-20 microM). In combination, the effect induced by maximal UCcf-029 and UCcf-029, UCcf-853, and apigenin increased strongly with increasing basal CFTR activity: for example, Kd for activation by UCcf-029 decreased from >5 to <0.4 microM with increasing basal Isc from approximately 4 microA/cm2 to approximately 12 microA/cm2. This dependence was confirmed in permeabilized Fischer rat thyroid cells stably expressing CFTR. Our results demonstrate efficacy of novel CFTR activators in bronchial epithelia and provide evidence that activating potency depends on basal CFTR activity.     

Activation of G551D CFTR channel with MPB-91: regulation by ATPase activity and phosphorylation

We have designed and synthesizedbenzo[c]quinolizinium derivatives and evaluated their effects on theactivity of G551D cystic fibrosis transmembrane conductance regulator(CFTR) expressed in Chinese hamster ovary and Fisher ratthyroid cells. We demonstrated, using iodide efflux, whole cell patchclamp, and short-circuit recordings, that5-butyl-6-hydroxy-10-chlorobenzo[c]quinolizinium chloride (MPB-91)restored the activity of G551D CFTR (EC₅₀ = 85 µM)and activated CFTR in Calu-3 cells (EC₅₀ = 47 µM).MPB-91 has no effect on the ATPase activity of wild-type and G551DNBD1/R/GST fusion proteins or on the ATPase, GTPase, and adenylatekinase activities of purified NBD2. The activation of CFTR by MPB-91 isindependent of phosphorylation because 1) kinase inhibitors have no effect and 2) the compound still activated CFTRhaving 10 mutated protein kinase A sites (10SA-CFTR). The newpharmacological agent MPB-91 may be an important candidate drug toameliorate the ion transport defect associated with CF and to point outa new pathway to modulate CFTR activity.

     

Regulatory domain phosphorylation to distinguish the mechanistic basis underlying acute CFTR modulators

Modulator compounds intended to overcome disease-causing mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) show significant promise in clinical testing for cystic fibrosis. However, the mechanism(s) of action underlying these compounds are not fully understood. Activation of CFTR ion transport requires PKA-regulated phosphorylation of the regulatory domain (R-D) and dimerization of the nucleotide binding domains. Using a newly developed assay, we evaluated nine compounds including both CFTR potentatiators and activators discovered via various high-throughput screening strategies to acutely augment CFTR activity. We found considerable differences in the effects on R-D phosphorylation. Some (including UC(CF)-152) stimulated robust phosphorylation, and others had little effect (e.g., VRT-532 and VX-770). We then compared CFTR activation by UC(CF)-152 and VRT-532 in Ussing chamber studies using two epithelial models, CFBE41o(-) and Fischer rat thyroid cells, expressing various CFTR forms. UC(CF)-152 activated wild-type-, G551D-, and rescued F508del-CFTR currents but did not potentiate cAMP-mediated CFTR activation. In contrast, VRT-532 moderately activated CFTR short-circuit current and strongly potentiated forskolin-mediated current. Combined with the result that UC(CF)-152, but not VRT-532 or VX-770, acts by increasing CFTR R-D phosphorylation, these findings indicate that potentiation of endogenous cAMP-mediated activation of mutant CFTR is not due to a pathway involving augmented R-D phosphorylation. This study presents an assay useful to distinguish preclinical compounds by a crucial mechanism underlying CFTR activation, delineates two types of compound able to acutely augment CFTR activity (e.g., activators and potentiators), and demonstrates that a number of different mechanisms can be successfully employed to activate mutant CFTR.     

Genistein modifies the activation kinetics and magnitude of phosphorylated wild-type and G551D-CFTR chloride currents

We have studied the mechanism by which genistein activates cystic fibrosis transmembrane conductance regulator (CFTR) in CHO cells expressing wild type or G551D-CFTR. In wild-type CHO cells, after exposure to 2.5 microM forskolin, 25 microM genistein induced a further 2-fold and rapid increase of the forskolin-activated CFTR current. In both types of cells, when forskolin was added after genistein preincubation, whole-cell current density was greatly reduced compared to that measured when genistein was added after phosphorylation of CFTR, and all activation kinetic parameters were significantly altered. Genistein had no effect on the adenylate cyclase activity. Our results suggest that the occupancy of a putative genistein binding site is critical for the gating mechanism of CFTR chloride channels, which, depending on the phosphorylation status of the R-domain, drives CFTR either into a refractory state or alternatively to a highly activated state.     

The glycine residues G551 and G1349 within the ATP-binding cassette signature motifs play critical roles in the activation and inhibition of cystic fibrosis transmembrane conductance regulator channels by phloxine B

The cystic fibrosis transmembrane conductance regulator (CFTR) protein contains a canonical ATP-binding cassette (ABC) signature motif, LSGGQ, in nucleotide binding domain 1 (NBD1) and a degenerate LSHGH in NBD2. Here, we studied the contribution of the conserved residues G551 and G1349 to the pharmacological modulation of CFTR chloride channels by phloxine B using iodide efflux and whole-cell patch clamp experiments performed on the following green fluorescent protein (GFP)-tagged CFTR: wild-type, delF508, G551D, G1349D, and G551D/G1349D double mutant. We found that phloxine B stimulates and inhibits channel activity of wild-type CFTR (Ks = 3.2 +/- 1.6 microM: , Ki = 38 +/- 1.4 microM: ) and delF508 CFTR (Ks = 3 +/- 1.8 microM: , Ki = 33 +/- 1 microM: ). However, CFTR channels with the LSGDQ mutated motif (mutation G551D) are activated (Ks = 2 +/- 1.13 microM: ) but not inhibited by phloxine B. Conversely, CFTR channels with the LSHDH mutated motif (mutation G1349D) are inhibited (Ki = 40 +/- 1.01 microM: ) but not activated by phloxine B. Finally, the double mutant G551D/G1349D CFTR failed to respond not only to phloxine B stimulation but also to phloxine B inhibition, confirming the importance of both amino acid locations. Similar results were obtained with genistein, and kinetic parameters were determined to compare the pharmacological effects of both agents. These data show that G551 and G1349 control the inhibition and activation of CFTR by these agents, suggesting functional nonequivalence of the signature motifs of NBD in the ABC transporter CFTR.     

Genistein improves regulatory interactions between G551D-cystic fibrosis transmembrane conductance regulator and the epithelial sodium channel in Xenopus oocytes

The cystic fibrosis transmembrane conductance regulator (CFTR) in addition to its well defined Cl(-) channel properties regulates other ion channels. CFTR inhibits epithelial Na(+) channel (ENaC) currents in many epithelial and non-epithelial cells, whereas the presence of ENaC increases CFTR functional expression. This interregulation is reproduced in Xenopus oocytes where both the open probability and surface expression of wild type CFTR Cl(-) channels are increased when CFTR is co-expressed with alphabetagamma-mouse ENaC (mENaC) and conversely when the activity of mENaC is inhibited after wild type CFTR activation. Using the Xenopus oocyte expression system, different functional regulatory interactions were observed between G551D-CFTR and alphabetagamma-mENaC. The co-expression of G551D-CFTR and alphabetagamma-mENaC resulted in a 5-fold increase in G551D-CFTR Cl(-) current compared with oocytes expressing G551D-CFTR alone. Oocytes co-injected with both G551D-CFTR and ENaC expressed an amiloride-sensitive whole cell current that was similar to that observed before and after G551D-CFTR activation with forskolin/isobutylmethylxanthine. Treatment with genistein both enhanced the functional expression of G551D-CFTR and improved regulatory interactions between G551D-CFTR and ENaC. These data suggest that genistein may be useful in patients with cystic fibrosis and the G551D-CFTR mutation.     

Dual activity of aminoarylthiazoles on the trafficking and gating defects of the cystic fibrosis transmembrane conductance regulator chloride channel caused by cystic fibrosis mutations

A large fraction of mutations causing cystic fibrosis impair the function of the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel by causing reduced channel activity (gating defect) and/or impaired exit from the endoplasmic reticulum (trafficking defect). Such defects need to be treated with separate pharmacological compounds termed potentiators and correctors, respectively. Here, we report the characterization of aminoarylthiazoles (AATs) as compounds having dual activity. Cells expressing mutant CFTR were studied with functional assays (fluorescence-based halide transport and short circuit current measurements) to assess the effect of acute and chronic treatment with compounds. We found that AATs are effective on F508del, the most frequent cystic fibrosis mutation, which is associated with both a gating and a trafficking defect. AATs are also effective on mutations like G1349D and G551D, which cause only a gating defect. Evaluation of a panel of AAT analogs identified EN277I as the most effective compound. Incubation of cells expressing mutant CFTR with EN277I caused a strong stimulation of channel activity as demonstrated by single channel recordings. Compounds with dual activity such as AATs may be useful for the development of effective drugs for the treatment of cystic fibrosis.     

Regulation of the cystic fibrosis transmembrane conductance regulator channel by beta-adrenergic agonists and vasoactive intestinal peptide in rat smooth muscle cells and its role in vasorelaxation

The signaling events that regulate vascular tone include voltage-dependent Ca(2+) influx and the activities of various ionic channels; which molecular entities are involved and their role are still a matter of debate. Here we show expression of the cystic fibrosis transmembrane conductance regulator (CFTR) Cl(-) channel in rat aortic smooth muscle cells. Immunoprecipitation and in vitro protein kinase A phosphorylation show the appearance of mature band C of CFTR. An immunohistochemistry study shows CFTR proteins in smooth muscles of aortic rings but not in skeletal muscles. Using the iodide efflux method, a combination of agonists and pharmacological agents was used to dissect the function of CFTR. Agonists of the cAMP pathway, the beta-adrenergic agonist isoproterenol, and the neuropeptide vasoactive intestinal peptide activate CFTR-dependent transport from cells maintained in a high but not low extracellular potassium-rich saline, suggesting that depolarization of smooth muscle is critical to CFTR activation. Smooth muscle CFTR possesses all of the pharmacological attributes of its epithelial homologues: stimulation by the CFTR pharmacological activators MPB-07 (EC(50) = 158 microm) and MPB-91 (EC(50) = 20 microm) and inhibition by glibenclamide and diphenylamine-2-carboxylic acid but not by 5,11,17,23-tetrasulfonato-25,26,27,28-tetramethoxy-calix[4]arene. Contraction measurements on isolated aortic rings were performed to study the contribution of CFTR to vascular tone. With aortic rings (without endothelium) preconstricted by high K(+) saline or by the alpha-adrenergic agonist norepinephrine, CFTR activators produced a concentration-dependent relaxation. These results identify for the first time the expression and function of CFTR in smooth muscle where it plays an unexpected but fundamental role in the autonomic and hormonal regulation of the vascular tone.     

The cystic fibrosis mutation G551D alters the non-Michaelis-Menten behavior of the cystic fibrosis transmembrane conductance regulator (CFTR) channel and abolishes the inhibitory Genistein binding site

Loss of cystic fibrosis transmembrane conductance regulator (CFTR) channel activity explains most of the manifestations of the cystic fibrosis (CF) disease. To understand the consequences of CF mutations on CFTR channel activity, we compared the pharmacological properties of wild-type (wt) and G551D-CFTR. Dose-dependent relationships of wt-CFTR activated by genistein follows a non-Michaelis-Menten behavior consistent with the presence of two binding sites. With phosphorylated CFTR, a high affinity site for genistein is the activator (K(s) approximately 3 microm), whereas a second site of low affinity (K(i) approximately 75 microm) is the inhibitor. With non-phosphorylated CFTR, K(s) was increased (K(s) approximately 12 microm), but K(i) was not affected (K(i) approximately 70 microm). In G551D-CFTR cells, channel activity was recovered by co-application of forskolin and genistein in a dose-dependent manner. A further stimulation of G551D-CFTR channel activity was measured at concentrations from 30 microm to 1 mm. The dose response is described by a classical Michaelis-Menten kinetics with only a single apparent site (K(m) approximately 11 microm). Our results suggest glycine 551 in NBD1 as an important location within the low affinity inhibitory site for genistein and offers new evidence for pharmacological alteration caused by an NBD1 mutation of CFTR. This study also reveals how a mutation of an ion channel converts a non-Michaelis-Menten behavior (two binding sites) into a classical Michaelis-Menten model (one binding site).     

Cystic fibrosis mice carrying the missense mutation G551D replicate human genotype-phenotype correlations.

We have generated a mouse carrying the human G551D mutation in the cystic fibrosis transmembrane conductance regulator gene (CFTR) by a one-step gene targeting procedure. These mutant mice show cystic fibrosis pathology but have a reduced risk of fatal intestinal blockage compared with 'null' mutants, in keeping with the reduced incidence of meconium ileus in G551D patients. The G551D mutant mice show greatly reduced CFTR-related chloride transport, displaying activity intermediate between that of cftr(mlUNC) replacement ('null') and cftr(mlHGU) insertional (residual activity) mutants and equivalent to approximately 4% of wild-type CFTR activity. The long-term survival of these animals should provide an excellent model with which to study cystic fibrosis, and they illustrate the value of mouse models carrying relevant mutations for examining genotype-phenotype correlations.     

Properties of CFTR activated by the xanthine derivative X-33 in human airway Calu-3 cells

The pharmacological activation of thecystic fibrosis gene protein cystic fibrosis transmembrane conductanceregulator (CFTR) was studied in human airway epithelial Calu-3 cells,which express a high level of CFTR protein as assessed by Western blotand in vitro phosphorylation. Immunolocalization shows that CFTR islocated in the apical membrane. We performed iodide efflux, whole cell patch-clamp, and short-circuit recordings to demonstrate that the novelsynthesized xanthine derivative 3,7-dimethyl-1-isobutylxanthine (X-33)is an activator of the CFTR channel in Calu-3 cells. Whole cell currentactivated by X-33 or IBMX is linear, inhibited by glibenclamide anddiphenylamine-2-carboxylate but not by DIDS or TS-TM calix[4]arene.Intracellular cAMP was not affected by X-33. An outwardly rectifyingCl current was recorded in the absence of cAMP and X-33stimulation, inhibited by DIDS and TS-TM calix[4]arene. With the useof short-circuit recordings, X-33 and IBMX were able to stimulate alarge concentration-dependent CFTR transport that was blocked byglibenclamide but not by DIDS. Our results show that manipulating thechemical structure of xanthine derivatives offers an opportunity toidentify further specific activators of CFTR in airway cells.

     

Potentiation of ΔF508- and G551D-CFTR-Mediated Cl- Current by Novel Hydroxypyrazolines

The most common mutation of CFTR, affecting approximately 90% of CF patients, is a deletion of phenylalanine at position 508 (F508del, ΔF508). Misfolding of ΔF508-CFTR impairs both its trafficking to the plasma membrane and its chloride channel activity. To identify small molecules that can restore channel activity of ΔF508-CFTR, we synthesized and evaluated eighteen novel hydroxypyrazoline analogues as CFTR potentiators. To elucidate potentiation activities of hydroxypyrazolines for ΔF508-CFTR, CFTR activity was measured using a halide-sensitive YFP assay, Ussing chamber assay and patch-clamp technique. Compounds 7p, 7q and 7r exhibited excellent potentiation with EC₅₀ value <10 μM. Among the compounds, 7q (a novel CFTR potentiator, CP7q) showed the highest potentiation activity with EC₅₀ values of 0.88 ± 0.11 and 4.45 ± 0.31 μM for wild-type and ΔF508-CFTR, respectively. In addition, CP7q significantly potentiated chloride conductance of G551D-CFTR, a CFTR gating mutant; its maximal potentiation activity was 1.9 fold higher than the well-known CFTR potentiator genistein. Combination treatment with CP7q and VX-809, a corrector of ΔF508-CFTR, significantly enhanced functional rescue of ΔF508-CFTR compared with VX-809 alone. CP7q did not alter the cytosolic cAMP level and showed no cytotoxicity at the concentration showing maximum efficacy. The hydroxypyrazolines may be potential development candidates for drug therapy of cystic fibrosis.     

ATP hydrolysis by a CFTR domain: pharmacology and effects of G551D mutation

Residues 417-830 of the cystic fibrosis transmembrane conductance regulator (CFTR) were expressed as a glutathione-S-transferase fusion protein. This fusion protein, NBD1/R/GST, contains the regulatory and first nucleotide binding domains of CFTR. NBD1/R/GST hydrolyzed ATP with a K(M) (60 microM) and V(max) (330 nmol/min/mg) that differed from those reported for CFTR and for a peptide containing CFTR residues 433-589. The ATPase inhibitor profile of NBD1/R/GST indicates that CFTR resembles P-glycoprotein with respect to the NBD1 ATPase catalytic mechanism. ATP hydrolysis by NBD1/R/GST was unaffected by genistein, glybenclamide, and other agents known to affect CFTR's chloride channel function, suggesting that these agents do not act by directly influencing the ATPase function of NBD1. The disease-causing mutation, G551D, reduced ATP hydrolysis by NBD1/R/GST by increasing the K(M) for ATP fourfold. This suggests that when G551D occurs in patients with cystic fibrosis, it affects CFTR function by reducing the affinity of NBD1 for ATP.     

Role of protein phosphatases in the activation of CFTR (ABCC7) by genistein and bromotetramisole

Genistein and bromotetramisole(Br-t) strongly activate cystic fibrosis transmembrane conductanceregulator (CFTR; ABCC7) chloride channels on Chinese hamster ovarycells and human airway epithelial cells. We have examined the possiblerole of phosphatases in stimulation by these drugs using patch-clampand biochemical methods. Genistein inhibited the spontaneous rundown ofchannel activity that occurs after membrane patches are excised fromcAMP-stimulated cells but had no effect on purified protein phosphatasetype 1 (PP1), PP2A, PP2B, PP2C, or endogenous phosphatases when assayed as [³²P]PO₄ release from prelabeled casein,recombinant GST-R domain fusion protein, or immunoprecipitatedfull-length CFTR. Br-t also slowed rundown of CFTR channels, but, inmarked contrast to genistein, it did inhibit all four proteinphosphatases tested. Half-maximal inhibition of PP2A and PP2C wasobserved with 0.5 and 1.5 mM Br-t, respectively. Protein phosphataseswere also sensitive to (+)-p-Br-t, a stereoisomer of Br-tthat does not inhibit alkaline phosphatases. Br-t appeared to actexclusively through phosphatases since it did not affect CFTR channelsin patches that had low apparent endogenous phosphatase activity (i.e.,those lacking spontaneous rundown). We conclude that genistein and Br-tact through different mechanisms. Genistein stimulates CFTR withoutinhibiting phosphatases, whereas Br-t acts by inhibiting amembrane-associated protein phosphatase (probably PP2C) that presumablyallows basal phosphorylation to accumulate.

     

Defective function of the cystic fibrosis-causing missense mutation G551D is recovered by genistein

The patch-clamp technique was used to investigate the effects ofthe isoflavone genistein on disease-causing mutations (G551D andF508) of the cystic fibrosis transmembrane conductance regulator (CFTR). In HeLa cells recombinantly expressing thetrafficking-competent G551D-CFTR, the forskolin-stimulated Cl currentswere small, and average open probability of G551D-CFTR wasP_o = 0.047 ± 0.019. Addition of genistein activated Cl currents~10-fold, and the P_o of G551D-CFTRincreased to 0.49 ± 0.12, which is aP_o similar towild-type CFTR. In cystic fibrosis (CF) epithelial cells homozygous forthe trafficking-impaired F508 mutation, forskolin and genistein activated Cl currents only after 4-phenylbutyrate treatment. These datasuggested that genistein activated CFTR mutants that were present inthe cell membrane. Therefore, we tested the effects of genistein in CFpatients with the G551D mutation in nasal potential difference (PD)measurements in vivo. The perfusion of the nasal mucosa of G551D CFpatients with isoproterenol had no effect; however, genisteinstimulated Cl-dependent nasal PD by, on average, 2.4 ± 0.6 mV, which corresponds to 16.9% of the responses (to -adrenergicstimulation) found in healthy subjects.

     

A quantitative description of the activation and inhibition of CFTR by potentiators: Genistein

The CFTR, encoded by the gene mutated in cystic fibrosis (CF) patients, is responsible for cAMP dependent chloride transport in epithelia. Substances that activate CFTR have been suggested as possible CF therapy. Most substances investigated so far exert a dual effect on the CFTR: low concentrations stimulate CFTR, whereas higher concentrations inhibit CFTR. Besides, the CFTR phosphorylation level determines the apparent affinity of the drug. We have studied the properties of genistein, the well known CFTR potentiator, by measuring apical membrane current on epithelia formed by cells stably transfected with CFTR and stimulated with different concentrations of CPTcAMP. We propose a quantitative model to describe the activatory and inhibitory effect of genistein, accounting also for the cAMP dependent activation.     

Cell-based assay for high-throughput quantitative screening of CFTR chloride transport agonists

Drug discovery by high-throughput screening is a promising approach to develop new therapies for the most common lethal genetic disease, cystic fibrosis. Because disease-causing mutations of the cystic fibrosis transmembrane conductance regulator (CFTR) protein produce epithelial cells with reduced or absent Cl(-) permeability, the goal of screening is to identify compounds that restore cell Cl(-) transport. We have developed a rapid, quantitative screening procedure for analysis of CFTR-mediated halide transport in cells with the use of a conventional fluorescence plate reader. Doubly transfected cell lines were generated that express wild-type or mutant CFTR together with a yellow fluorescent protein (YFP)-based halide sensor. CFTR function was assayed from the time course of cell fluorescence in response to extracellular addition of 100 mM I(-) followed by forskolin, resulting in decreased YFP fluorescence due to CFTR-mediated I(-) entry. Cell lines were chosen, and conditions were optimized to minimize basal halide transport to maximize assay sensitivity. In cells cultured on 96-well plastic dishes, the assay gave reproducible halide permeabilities from well to well and could reliably detect a 2% activation of CFTR-dependent halide transport produced by low concentrations of forskolin. Applications of the assay are shown, including comparative dose-dependent CFTR activation by genistein, apigenin, 8-cyclopentyl-1,3-dipropylxanthine, IBMX, 8-methoxypsoralen, and milrinone as well as activation of alternative Cl(-) channels. The fluorescence assay and cell lines should facilitate the screening of novel CFTR activators and the characterization of alternative Cl(-) channels and transporters.