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

Emanuela Caci,1 Chiara Folli,1 Olga Zegarra-Moran,1 Tonghui Ma,2 Mark F. Springsteel,3 Robert E. Sammelson,3 Michael H. Nantz,3 Mark J. Kurth,3 A. S. Verkman,2 and Luis J. V. Galietta1

1Laboratorio di Genetica Molecolare, Istituto Giannina Gaslini, 16148 Genova, Italy; 2Departments of Medicine and Physiology, University of California, San Francisco 94143; and 3Department of Chemistry, University of California, Davis, California 95616

Submitted 21 October 2002 ; accepted in final form 12 March 2003


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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 µM) being much better than other flavones, such as apigenin. The benzimidazolone UCCF-853 gave similar Isc but with lower potency (5–20 µM). 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 µM with increasing basal Isc from ~4 µA/cm2 to ~12 µA/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.

airway epithelium; chloride secretion; drug discovery


CYSTIC FIBROSIS (CF), one of the most common and severe inherited diseases, is associated with defective fluid transport in various epithelia. The defective protein in CF is the Cl--permeable ion channel CFTR, the cystic fibrosis transmembrane conductance regulator, which is activated by cAMP-dependent phosphorylation (20). CFTR channel opening and closing also require the binding and hydrolysis of ATP at its two nucleotide binding domains (5, 23). CFTR mutations in CF patients may impair channel gating, channel conductance, and/or CFTR protein trafficking.

Current therapeutic approaches for CF involve the control of chronic bacterial colonization in the airways and the improvement of mucus clearance. There is no therapy yet to treat the primary CF functional defect in vivo. However, several small organic compounds are able to increase CFTR Cl- conductance in vitro, such as the flavonoids apigenin and genistein (1, 13, 14, 29), the xanthines 8-cyclopentyl-1,3-dipropyl xanthine and IBMX (10, 11, 25), the substituted benzo[c]quinolizinium MPB-07 (2), and the benzimidazolone NS004 (1, 9; see also for review Refs. 12, 16, and 22). It is believed that selective and potent CFTR activators may be beneficial in CF to correct electrolyte/fluid transport abnormalities.

We recently used a lead-based combinatorial synthesis approach and high-throughput screening to identify a novel class of potent CFTR activators, the 7,8-benzoflavones (8). In a follow-up study, several isoxazoles and benzimidazolones were identified by a similar approach (17). To assess the potency of these new compounds in native CFTR-expressing epithelia, we report short-circuit current (Isc) measurements in primary human bronchial cell cultures. The best 7,8-benzoflavone (UCCF-029) and the best benzimidazolone (UCCF-853) were characterized in their ability to activate Cl- conductance in combination with cAMP agonists and each other. An interesting observation was that the activating potency of these compounds depended on basal CFTR function/phosphorylation state. Further studies of CFTR specificity were done in CFTR-expressing Fischer rat thyroid (FRT) cells in which Isc was measured after basolateral membrane permeabilization and a transepithelial Cl- gradient.


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Cell culture. Human bronchi were obtained from lung resections or lung transplants. Bronchial epithelial cells were detached by overnight digestion with protease XIV and cultured on petri dishes or flasks in a serum-free medium as previously described (6, 7). The collection and processing of human cells was approved by the local ethics committee. When needed, cells were plated on Snapwell inserts (12-mm diameter, 0.45-µm pore size, Corning Costar) in serum- and hormone-supplemented medium to generate polarized monolayers suitable for the measurement of transepithelial Na+ and Cl- transport (6, 7). The development of transepithelial electrical resistance and electric potential difference was monitored daily with an epithelial voltohmeter (World Precision Instruments). Experiments were performed 10–12 days after plating the cells on Snapwell inserts, when the electrical resistance was 800–1,000 {Omega} · cm2.

FRT cells stably transfected with wild-type CFTR were cultured as previously described (29). For Isc experiments, FRT cells were plated on Snapwell inserts and used after 7–9 days. The FRT monolayers had a resistance >2,000 {Omega} · cm2.

Isc experiments. Snapwell inserts were detached from their supports and mounted in a Vertical Diffusion Chamber (Corning Costar). For bronchial epithelial cells, the two hemichambers were filled with 5 ml each of a solution containing (in mM): 126 NaCl, 0.38 KH2PO4, 2.13 K2HPO4, 1 MgSO4, 1 CaCl2, 24 NaHCO3, and 10 glucose. During experiments, each hemichamber was bubbled with 5% CO2-95% air to give pH 7.3.

For FRT cells, the basolateral solution contained (in mM): 130 NaCl, 2.7 KCl, 1.5 KH2PO4, 1 CaCl2, 0.5 MgCl2, 10 sodium-HEPES (pH 7.3), and 10 glucose. The apical solution was modified by replacing half of NaCl with sodium gluconate and by increasing CaCl2 to 2 mM to compensate for Ca2+ buffering caused by gluconate (15). The basolateral membrane of FRT cells was permeabilized with 250 µg/ml of amphotericin B for 30 min before starting the recording. The ionophore was kept in the basolateral chamber for the duration of the experiment. Both hemichambers were bubbled with air. All experiments were performed at 37°C.

The transepithelial potential difference was short circuited with a voltage clamp (DVC-1000, World Precision Instruments) connected to apical and basolateral chambers with Ag-AgCl electrodes and agar bridges. The apical side was taken for reference. Positive currents mean Cl- moving from the basolateral to the apical chamber. The Isc was recorded with a MacLab/200 data recording system using a 0.2-Hz sampling frequency. During experiments, forskolin and 8-(4-chlorophenylthio)-cAMP (CPT-cAMP) were added at the indicated concentrations on both sides of bronchial and FRT epithelia. CFTR activators were added to the apical side only. In most experiments, glibenclamide (400–500 µM) was added to both sides of the epithelium to block CFTR currents.

Dose responses from each experiment were fitted with the Hill equation using Igor software (Wavemetrics). Data are reported as representative traces/dose responses or as means ± SE. Where indicated, the difference between groups of data was evaluated using the Student's t-test.

Synthesis of UCCF-853. The synthesis of 1-(3-chlorophenyl)-5-trifluoromethyl-3-hydrobenzimidazol-2-one (UCCF-853, Fig. 1) was accomplished in three steps from 3-chloroaniline and 4-fluoro-3-nitrobenzotrifluoride via nucleophilic aromatic substitution, nitro-to-amino reduction, and subsequent benzimidazolone formation. The aniline and benzotrifluoride were coupled with potassium carbonate in dimethylformamide at 80°C. Standard reduction conditions with stannous chloride (SnCl2 · 2H2O) gave the corresponding diamine. Reaction with N,N'-carbonyldiimidazole in tetrahydrofuran yielded target benzimidazolone. After purification using silica gel flash chromatography, the identity and purity of UCCF-853 (>95%) were confirmed by nuclear magnetic resonance and thin-layer chromatography.



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Fig. 1. A: chemical structures of 4',5,7-trihydroxy-flavone (apigenin), 2-(4-pyridyl)benzo[h]4H-chromen-4-one (UCCF-029), and 1-(3-chlorophenyl)-5-trifluoromethyl-3-hydrobenzimidazol-2-one (UCCF-853). B: representative trace showing activity of UCCF-029 on human bronchial epithelia after blocking the Na+ channel with 10 µM amiloride. C: dose-response relationship for the 3 compounds. Each point is the mean ± SE of 8–14 experiments. Isc, short-circuit current.

 


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Figure 1A shows the structures of the CFTR activators used in our study. Apigenin is a flavone-type CFTR activator (13, 14). The 7,8-benzoflavone UCCF-029 was the most potent CFTR activator, identified recently in a screening of flavone and benzo[c]quinolizinium analogs (8). Subsequent screening of other lead-based libraries have led to the identification of the benzimidazolone UCCF-853 (17).

We tested these three compounds on human bronchial epithelia after blocking the Na+ channel with 10 µM amiloride. Under these conditions, UCCF-029 induced a dose-dependent increase in Isc with concentrations as low as 200 nM being able to induce a sustained effect (Fig. 1B). Apigenin and UCCF-853 were also effective, but micromolar concentrations were required to evoke a significant current activation. The half-maximal activation obtained after averaging all data was 0.22, 7.1, and 5.3 µM for UCCF-029, apigenin, and UCCF-853, respectively (Fig. 1C). At very high concentrations (20–100 µM), the CFTR activators, particularly apigenin, induced a partial current block (see also Fig. 2A).



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Fig. 2. Activation of Isc in human bronchial epithelial cells. A: representative experiments in which UCCF-029 (left) and apigenin (right) were given at indicated concentrations to epithelia with high (top) and low (bottom) amiloride-insensitive current. Amiloride (10 µM) and glibenclamide (400 µM) were added to block epithelial Na+ channel and CFTR, respectively. Inset: a representative experiment in which glibenclamide was added before UCCF-029. B: dose-response relationships for UCCF-029 (left) and apigenin (right) from representative experiments with high baseline and low baseline Isc. C: apparent activating potencies (Kd) vs. the baseline (amiloride-insensitive) current for all experiments with UCCF-029 and apigenin (left). Maximal current (Imax, drug-activated plus baseline current) vs. baseline (Ibase, right).

 

We noted significant variability in the apparent affinities of activators in different cell preparations. Figure 2A shows representative experiments with dose responses for UCCF-029 and apigenin. The top traces in Fig. 2A show examples of experiments from a cell preparation with "high-affinity" behavior. The bottom traces are from epithelia with "low-affinity" behavior. We found that within each set of experiments, UCCF-029 was consistently more potent than apigenin, with an apparent activating potency (Kd) down to the nanomolar range. In contrast, apigenin did not elicit a significant response at this concentration. Maximal effect (6–14 µA/cm2) was obtained with 1–5 µM UCCF-029 and 20–50 µM apigenin. The maximal current induced by both flavonoids was 60–80% of that obtained with 500 µM CPT-cAMP, which caused full activation of CFTR in our cell preparation (7). From measurements on different cultures, we noted a strict trend in that the activating potency strongly correlated with basal (amiloride-insensitive) Isc. For example, Fig. 2A (left) shows two experiments in which the apparent affinity for UCCF-029 was in the nanomolar and micromolar range and baseline was ~10 and 4 µA/cm2, respectively. Similarly, apigenin also showed this inverse relationship (Fig. 2A, right). Dose-response curves for these two pairs of experiments are summarized in Fig. 2B. The fit of experimental data gave a Hill coefficient close to 1 for UCCF-029 (1.14 ± 0.05; n = 14). On the contrary, apigenin showed a higher coefficient (1.65 ± 0.11; n = 8). In particular, we noted a trend in which experiments with higher Kd for apigenin were also characterized by a Hill coefficient closer to 2 (not shown).

Figure 2C (left) shows the apparent Kd of UCCF-029 and apigenin vs. the size of the baseline current for many cultures. Apparent Kd decreased substantially, with increasing baseline Isc for both compounds. At corresponding values of baseline current, UCCF-029 was much more potent than apigenin. Another interesting observation was that high baseline Isc correlated with a larger total current, i.e., the baseline current plus the current activated by openers (Fig. 2C, right).

Addition of the CFTR blocker glibenclamide after stimulation by UCCF-029, apigenin, or other flavonoids reduced Isc to near zero, regardless of the magnitude of the baseline or the amiloride-inhibited Isc (Fig. 2A). To test whether the amiloride-insensitive Isc was CFTR dependent, glibenclamide was added before UCCF-029 (Fig. 2A, inset). Glibenclamide decreased Isc to near zero and abolished the activation by UCCF-029.

The results in nonpermeabilized bronchial epithelial cells suggested that the baseline current was due to CFTR activity. To test this hypothesis, we performed Isc measurements on FRT cells stably expressing CFTR in which the basolateral membrane was permeabilized with a Cl- gradient (65 mM apical/130 mM basolateral). Under these conditions, Isc reflects CFTR activity and is not influenced by other channels and transporters. Dose-response relationships for UCCF-029 and apigenin were generated under basal conditions and after stimulation with submaximal concentrations of CPT-cAMP (3–30 µM) to give different levels of CFTR phosphorylation, equivalent to different basal activities. As shown in Fig. 3A (top traces), in the absence of prestimulation, relatively high concentrations of UCCF-029 and apigenin were needed to induce significant CFTR Cl- currents. The apparent affinity of these flavonoids was increased by CFTR submaximal stimulation (bottom). Figure 3B shows dose-response curves for these experiments, and Fig. 3C summarizes the apparent Kd vs. CFTR Cl- current measured before flavone addition. In agreement with data obtained in airway epithelial cells, there was an inverse relationship with an increase in activating potency with increasing basal CFTR activity.



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Fig. 3. Activation of Isc in Fischer rat thyroid (FRT) cells expressing CFTR with basolateral permeabilization and asymmetric Cl-. A: representative experiments in which UCCF-029 and apigenin were added in the absence (top) and in the presence (bottom) of 8(4-chlorophenylthio) (CPT)-cAMP (10 µM). B: dose-response relationships for the experiments shown in A. C: apparent Kd vs. baseline current for all experiments with UCCF-029 and apigenin.

 

We also tested the benzimidazolone UCCF-853 that is structurally similar to NS004. UCCF-853 activated CFTR in human bronchial epithelia (Fig. 4A). The half-maximal concentration was variable and generally >5 µM, whereas the maximal current was similar to that induced by UCCF-029 and apigenin. The Hill coefficient arising from dose-response fits was close to 1 (1.11 ± 0.04; n = 9). Similar to the flavonoids, the potency of UCCF-853 was higher in bronchial cell cultures having larger baseline Isc (Fig. 4A, middle). Total current (baseline plus drug-activated current) was also related to baseline current (Fig. 4A, right). We confirmed these findings in FRT cells expressing wild-type CFTR. Isc was increased by UCCF-853 in FRT cells expressing wild-type CFTR (Fig. 4B, left), and the effect was blocked by glibenclamide. The activating potency of the benzimidazolone increased with increasing CFTR prestimulation (Fig. 4B, right).



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Fig. 4. Activity of the benzimidazolone UCCF-853. A, left: representative experiment showing activity of UCCF-853 on amiloride-treated bronchial epithelial cells. Inset: dose-response fit. A, middle: apparent Kd vs. baseline (amiloride-insensitive) current for all experiments on bronchial epithelial cells. A, right: maximal current vs. baseline. B, left: representative experiment showing activity of UCCF-853 on FRT cells expressing CFTR. Inset: dose-response fit. Cells were prestimulated with 3 µM CPT-cAMP. B, right: apparent Kd vs. baseline current for all experiments.

 

Additional experiments were done to investigate possible additivity/synergy by combined stimulation with compounds and CPT-cAMP. After maximal stimulation of CFTR by 500 µM CPT-cAMP, there was no further effect of high concentrations of UCCF-029 or apigenin (Fig. 5A, left). In contrast, after full stimulation with CPT-cAMP, there was a further increase of Isc induced by UCCF-853 (Fig. 5A, middle). The net Isc increase was ~50% of that elicited by the cAMP analog alone (Fig. 5A, right). Costimulation of cells with the flavones apigenin and UCCF-029 did not produce additive effects (Fig. 5B, left). However, addition of UCCF-029 to cells that were maximally stimulated by UCCF-853 further increased Isc by >80% compared with either compound alone (Fig. 5B, middle and right). The addition of compounds in reverse order, i.e., first UCCF-029 followed by UCCF-853, was also effective (not shown).



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Fig. 5. Additivity of benzoflavone and benzimidazolone effects on CFTR-dependent Cl- secretion in airway epithelial cells. A, left and middle: representative experiments showing costimulation with CPT-cAMP and UCCF-029 or UCCF-853. A, right: summary of data expressed in percent of the current activated by CPT-cAMP. Each bar is the mean ± SE of 4–6 experiments. *Significant difference with respect to CPT-cAMP alone (P < 0.01). B: response to UCCF-029 after maximal stimulation with apigenin (left) or UCCF-853 (right). B, right: summary of data. The values are expressed as percent of the current activated by UCCF-853 alone (for UCCF-853 plus UCCF-029) or by apigenin alone (for apigenin plus UCCF-029). Each bar is the mean ± SE of 4–6 experiments. The current activated by UCCF-853 plus UCCF-029 is significantly larger than with UCCF-853 alone (P < 0.01).

 

To further investigate the basis of the additive effects seen in airway epithelial cells, we performed similar experiments on FRT cells after permeabilization of basolateral membrane. Cells were maximally stimulated with 500 µM CPT-cAMP and then treated with high concentrations of UCCF-029 or UCCF-853. Both compounds were ineffective in causing potentiation of the Isc under these conditions (Fig. 6A). UCCF-853 (50 µM) produced a small inhibition. We also tested the effect of combined stimulation by UCCF-029 and UCCF-853. Interestingly, the order of addition was important in determining the type of response. After a maximal concentration of UCCF-029, UCCF-853 was not effective and gave a small inhibition (Fig. 6B, left). In the reverse experiment, cells stimulated with maximal UCCF-853 responded to UCCF-029 (Fig. 6B, right).



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Fig. 6. Effect of combined stimulation in permeabilized FRT cells. A: representative traces showing lack of response to UCCF-029 and UCCF-853 after maximal stimulation with 500 µM CPT-cAMP. B, top: representative experiments showing the effect of addition of UCCF-029 and UCCF-853 in different orders. Forskolin (fsk) concentration was 0.5 µM. Bottom: summary of experiments of the type shown above. Each bar is the mean ISC ± SE of 4 experiments. The stimulation with UCCF-029 (5 µM) after UCCF-853 (50 µM) evoked a current that was significantly larger than with the benzimidazolone alone (P < 0.01).

 


    DISCUSSION
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Recent screening performed using an assay based on the halide-sensitive yellow fluorescent protein and CFTR-transfected FRT cells identified novel classes of CFTR activators, including 7,8-benzoflavones with improved potency compared with known flavonoids such as genistein and apigenin (8). The initial purpose of the present study was to test the activity of these compounds in a native expression system, airway epithelial cells, studying their ability to produce sustained transepithelial Cl- transport.

Isc measurements indicated that amiloride-treated bronchial epithelia responded to 7,8-benzoflavones with the activation of a glibenclamide-sensitive CFTR current. UCCF-029 was very potent with a half-effective concentration down to the nanomolar range, below that of known flavonoids, such as apigenin. We also tested a novel benzimidazolone, UCCF-853, which was recently identified in a screening of a combinatorial library (17). UCCF-853 also activated CFTR currents, although with a potency lower than UCCF-029.

Interestingly, we found that the apparent affinities of UCCF-029, apigenin, and UCCF-853 were dependent on the size of the baseline current that remained after blocking Na+ absorption with amiloride. This basal transport is probably due to resting CFTR activity since it is strongly inhibited by glibenclamide and is absent in cells from {Delta}F508 CF patients (not shown). Therefore, our data suggest that the level of CFTR basal phosphorylation affects the response to the three CFTR activators we tested. This was confirmed in FRT cells in which CFTR activity was set using different submaximal concentrations of the cAMP analog CPT-cAMP. Our observations are in agreement with results published previously by other investigators that indicated that CFTR activators require a basal level of CFTR phosphorylation to be active. Indeed, it was found that channel activation by NS004 in excised membrane patches requires previous cell stimulation with forskolin (9). Also, genistein does not activate CFTR in excised membrane patches without previous in vitro phosphorylation (28). Furthermore, the activity of flavonoids in airway epithelia was enhanced by treating the cells with forskolin (13). For example, the half-maximal concentration for apigenin decreased from 11.2 to 3.4 µM after stimulation with forskolin. Our data confirm these observations, suggesting that the dependence on basal phosphorylation is a general characteristic of CFTR openers. It is possible that phosphorylation of R domain affects CFTR conformation, allowing better access of activating compounds to their binding site(s). We also found, as previously reported by Illek and Fischer (13), that apigenin activation kinetics show cooperativity with two possible binding sites. Actually, these authors observed that the degree of cooperativity and the number of binding sites decrease with increasing CFTR phosphorylation. Our results show that, in contrast to apigenin, UCCF-029 and UCCF-853 have activation kinetics consistent with a single binding site.

The reason for variable CFTR activity under resting conditions in bronchial epithelial cells could be due to autocrine/paracrine factors that might affect the basal cAMP levels or phosphatase activity, altering the extent of phosphorylation. Indeed, CFTR has multiple phosphorylation sites. Increasing phosphorylation at the R domain of CFTR gradually increases its open channel probability (5).

There was a strong correlation between the baseline current and total current (baseline plus current stimulated by CFTR agonists). In other words, the fraction of CFTR that is active under basal conditions increases along with the total amount of functional CFTR protein in the membrane. We have no clear explanation for this phenomenon. It is possible that unknown factors affect basal CFTR phosphorylation as well as the number of CFTR channels in the plasma membrane. For example, higher basal levels of intracellular cAMP in some cell cultures could lead to increased CFTR phosphorylation as well as increased CFTR gene transcription (18).

Combined stimulation by UCCF-853 and UCCF-029 gave an additive effect, suggesting different mechanisms of activation. This was confirmed by the finding that UCCF-853, but not UCCF-029, activated CFTR after maximal stimulation by CPT-cAMP. To investigate this mechanism, experiments were done on FRT cells by using the two agonists together or in combination with CPT-cAMP. In contrast to the results obtained in bronchial cells, UCCF-853 was ineffective after maximal cell stimulation with CPT-cAMP or UCCF-029. These data are probably explained by the effects that UCCF-853 might have on other channels and transporters. It was reported that benzimidazolones are able to activate both CFTR and K+ channels (3, 4, 9, 19, 26, 27). In particular, Devor and colleagues (3, 4) found that the benzimidazolone 1-ethyl-2-benzimidazolinone induces Cl- secretion in intestinal and airway epithelial cells by activating CFTR and a basolateral K+ channel. The combined activity on these two types of channels maximizes the effect on transepithelial Cl- transport. In fact, the hyperpolarization of the basolateral membrane caused by K+ channel activation increases the driving force for Cl- efflux through the apical membrane. Therefore, it is possible that the activity of UCCF-853 in airway epithelial cells is due, in part, to stimulation of the basolateral K+ conductance. We postulate that flavonoids such as UCCF-029 and apigenin activate only CFTR, whereas the benzimidazolone UCCF-853 also activates a basolateral K+ channel. This would explain why the benzimidazolone is effective in the intact airway epithelium when CFTR is maximally stimulated with forskolin or UCCF-029. In agreement with this hypothesis, UCCF-853 is ineffective in permeabilized FRT cells when CFTR is fully activated with the cAMP analog or with UCCF-029. Under these conditions, the possible electrical contribution of basolateral K+ channels is neutralized by the presence of amphotericin B. However, we also noted in FRT cells that a maximal concentration of UCCF-853 does not completely prevent the response to UCCF-029. Therefore, it appears that the order of addition of activators in FRT cells is important in determining the type of response. In other words, UCCF-853 behaves as a partial agonist with respect to UCCF-029. This characteristic was not observed in airway epithelial cells probably because of the ability of the benzimidazolone to work as a K+ channel activator. This property would increase the Cl- secretion to levels comparable with those induced by the benzoflavone.

At the moment, we have no evidence of how the new compounds identified by high-throughput screening activate CFTR. Because these compounds do not increase cAMP and do not inhibit endogenous phosphatases, we hypothesize that they could bind and directly activate CFTR. However, a confirmation of this hypothesis will require measurements of channel activity in membrane patches. It is possible that different CFTR activators share activation mechanisms. For example, it has been reported that genistein, NS004, and NS1619 increase the channel open time and decrease the channel closed time in a similar way (1). It has been proposed that these compounds act by stabilizing the open state through an inhibition of ATP hydrolysis at the second nucleotide binding fold (1).

In conclusion, our experiments indicate that the new benzoflavone UCCF-029 and the new benzimidazolone UCCF-853 are strong activators of CFTR-dependent Cl- secretion in the intact human airway epithelium. Further studies are needed to assess the efficacy of CFTR openers on bronchial epithelia from CF patients. In addition to CF, potent stimulators of fluid secretion in the airways could be beneficial in pathological conditions, such as chronic obstructive pulmonary disease, in which suboptimal CFTR activity may be a risk factor (21, 24).


    ACKNOWLEDGMENTS
 
This work was supported by a drug discovery grant from the Cystic Fibrosis Foundation and by Telethon-Italy Grant GP0296Y01.


    FOOTNOTES
 

Address for reprint requests and other correspondence: L. J. V. Galietta, Laboratorio di Genetica Molecolare, Istituto Giannina Gaslini, L.go Gerolamo Gaslini, 5, 16148 Genova, Italy (E-mail: galietta{at}unige.it).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


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