QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Abstract Full Text (PDF) All Versions of this Article: jpet.106.104521v1 319/1/349    most recent Submit a response Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Noel, S. Articles by Becq, F. Search for Related Content PubMed PubMed Citation Articles by Noel, S. Articles by Becq, F.

CELLULAR AND MOLECULAR

Discovery of Pyrrolo[2,3-b]pyrazines Derivatives as Submicromolar Affinity Activators of Wild Type, G551D, and F508del Cystic Fibrosis Transmembrane Conductance Regulator Chloride Channels

Institut de Physiologie et Biologie Cellulaires Centre National de la Recherche Scientifique (CNRS) Unité Mixte de Recherche 6187, Université de Poitiers, Poitiers, France (S.N., C.F., C.N., C.R., Y.M., F.B.); and Faculté de Médecine et Pharmacie, Poitiers cedex, France (C.F., Y.M.)

Received March 15, 2006; accepted July 6, 2006.

	Abstract

Top Abstract Materials and Methods Results Discussion References

 
The cystic fibrosis transmembrane conductance regulator (CFTR) represents the main Cl^- channel in the apical membrane of epithelial cells for cAMP-dependent Cl^- secretion. Here we report on the synthesis and screening of a small library of 6-phenylpyrrolo[2,3-b]pyrazines (named RP derivatives) evaluated as activators of wild-type CFTR, G551D-CFTR, and F508del-CFTR Cl^- channels. Iodide efflux and whole-cell patch-clamp recordings analysis identified RP107 [7-n-butyl-6-(4-hydroxyphenyl)[5H]-pyrrolo[2,3-b]pyrazine] as a submicromolar activator of wild-type (WT)-CFTR [human airway epithelial Calu-3 and WT-CFTR-Chinese hamster ovary (CHO) cells], G551D-CFTR (G551D-CFTR-CHO cells), and F508del-CFTR (in temperature-corrected human airway epithelial F508del/F508del CF15 cells). The structural analog RP108 [7-n-butyl-6-(4-chlorophenyl)[5H]pyrrolo[2,3-b]pyrazine], contrary to RP107, was a less potent activator only at micromolar concentrations. RP107 and RP108 did not have any effect on the cellular cAMP level. Activation was potentiated by low concentration of forskolin and inhibited by glibenclamide and CFTR_inh-172 [3-[(3-trifluoromethyl)phenyl]-5-[(4-carboxyphenyl-)methylene]-2-thioxo-4-thiazolidinone]but not by calixarene or DIDS (4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid). Finally, we found significant stimulation of short circuit current (I_sc) by RP107 (EC₅₀ = 89 nM) and RP108 (EC₅₀ = 103 µM) on colon of Cftr^+/+ but not of Cftr^-/- mice mounted in Ussing chamber. Stimulation of I_sc was inhibited by glibenclamide but not affected by DIDS. These results show that RP107 stimulates wild-type CFTR and mutated CFTR, with submicromolar affinity by a cAMP-independent mechanism. Our preliminary structure-activity relationship study identified 4-hydroxyphenyl and 7-n-butyl as determinants required for activation of CFTR. The potency of these agents indicates that compounds in this class may be of therapeutic benefit in CFTR-related diseases, including cystic fibrosis.

Identification of a selective agonist for a particular ion channel without secondary effect on other channels or unrelated proteins is critical for use as a biological tool or pharmaceutical drug but is a high-cost process that usually takes several years. However, a number of compounds have been identified during the last 15 years, activating wild-type and mutated CFTR by mechanisms that do not involve an increase of intracellular cAMP (reviewed in Becq, 2006). Our laboratory develops and synthesizes original small molecules and evaluates them for their ability to activate CFTR using a simple and robust robotic cell-based assay (Marivingt-Mounir et al., 2004). In this report, we present the 6-phenylpyrrolo[2,3-b]pyrazines, a family of derivatives (also named Aloisines) initially described as CDK/GSK-3 inhibitors (Mettey et al., 2003). One of these compounds, RP107 [7-n-butyl-6-(4-hydroxyphenyl)[5H]pyrrolo[2,3-b]pyrazine], activates CFTR-dependent Cl^- secretion with submicromolar affinity in various epithelial cell types. Moreover, RP107 activates two CF-associated CFTR mutants (G551D-CFTR and F508del-CFTR mutants). The potency of these agents indicates the possibility that compounds in this class may be of therapeutic benefit in CF.

	Materials and Methods

Top Abstract Materials and Methods Results Discussion References

 
Chemistry. The 6-phenylpyrrolo[2,3-b]pyrazines derivatives shown in Table 1 were synthesized as presented in Fig. 1 from alkylpyrazines and aromatic nitriles, as described previously (Vierfond et al., 1981). Demethylation of methoxycompounds (1, 3, 5, 7, and 9) was obtained by heating in refluxing HBr. Synthesis and spectral data of compounds 1 through 8 and 11 through 12 (Table 1) are described elsewhere (Mettey et al., 2003).

View this table: [in this window] [in a new window]   TABLE 1 Structure, yields, and physical data of compounds 1 through 12

View larger version (7K): [in this window] [in a new window]   Fig. 1. Scheme for the synthesis of 6-phenylpyrrolo[2,3-b]pyrazines.

Compounds used are as follows: 6-(4-methoxyphenyl)[5H]pyrrolo-[2,3-b]pyrazine (1, RP11); 6-(4-hydroxyphenyl)[5H]pyrrolo[2,3-b]-pyrazine (2, RP26); 6-(4-methoxyphenyl)-7-methyl[5H]pyrrolo[2,3-b]pyrazine (3, RP95); 6-(4-hydroxyphenyl)-7-methyl[5H]pyrrolo[2,3-b]pyrazine (4, RP96); 6-(4-methoxyphenyl)-7-propyl[5H]pyrrolo[2,3-b]pyrazine (5, RP127); 6-(4-hydroxyphenyl)-7-propyl[5H]pyrrolo[2,3-b]pyrazine (6, RP132); 7-n-butyl-6-(4-methoxyphenyl)[5H]pyrrolo[2,3-b]pyrazine (7, RP106); 8, RP107; 6-(4-chlorophenyl)[5H]-pyrrolo[2,3-b]pyrazine (11, RP14); and 7-n-butyl-6-(4-chlorophenyl)[5H]pyrrolo[2,3-b]pyrazine (12, RP108). Similar chemical synthesis method has been used for compounds 9 and 10. The corresponding elemental analyses are provided below.

Compound 9, RP149. m.p. 144.2°C; IR 3143, 3059, 2959, 2927, 2858 cm^-1; ¹H NMR (60 MHz, DMSO-d₆), 11.75 (bs, 1H), 8.40 and 8.20 (2d, 1H each, J = 2.6 Hz), 7.70 and 7.10 (2bd, 2H each, J = 8 Hz), 3.90 (s, 3H), 3.20-2.80 (m, 2H), 2.00-1.1 (m, 6H), 0.9 (t, 3H, J = 7.2 Hz). Analog calculated for C₁₈H₂₁N₃O (295.39), calculated for C₁₈H₂₁N₃O: C, 73.19; H, 7.17; N, 14.22. Found: C, 73.06; H, 7.26; N, 14.09.

Compound 10, RP150. m.p. 270.7°C. IR 3224, 3150, 3060, 2949, 2924, 2861 cm^-1. ¹H NMR (60 MHz, DMSO-d₆) 11.85 (bs, 1H), 9.90 (bs, 1H), 8.35 and 8.15 (2d, 1H each, J = 2.6 Hz), 7.50 and 6.95 (2d, 2H each, J = 7 Hz), 3.10-1.70 (m, 2H), 2.00-1.05 (m, 6H), 0.80 (t, 3H, J = 7 Hz). Analog calculated for C₁₇H₁₉N₃O (281.36): C, 72.57; H, 6.81; N, 14.93. Found: C, 72.35; H, 6.78; N, 14.75.

Other Chemicals. TS-TM calix[4]arene (5,11,17,23-tetrasulfonato-25,26,27,28-tetramethoxy-calix[4]arene), an inhibitor of outwardly rectifying Cl^- channels (Singh et al., 1995), was generously provided by A. Singh and R. J. Bridges (University of Pittsburgh, Pittsburgh, PA). The specific CFTR inhibitor 3-[(3-trifluoromethyl)-phenyl]-5-[(4-carboxyphenyl)methylene]-2-thioxo-4-thiazolidinone (CFTR_inh-172) (Ma et al., 2002) and forskolin were purchased from VWR International (Fontenay/bois, France) and PKC Pharmaceuticals, Inc. (Woburn, MA), respectively. All other chemicals were from Sigma (St Louis, MO). All chemical agents, including pyrrolo[2,3-b]pyrazines compounds, were dissolved in DMSO (final concentration 0.1%), with the exception of TS-TM calix[4]arene, which was dissolved in H₂O. The currents were not altered by DMSO alone.

Cell Culture. All cell lines were grown at 37°C in 5% CO₂ under standard culture conditions as follows. CHO cells stably transfected with pNUT vector alone (mock-CHO) or containing wild-type CFTR (WT-CFTR-CHO) and the mutant G551D-CFTR were provided by J. R. Riordan and X. B. Chang (Mayo Clinic of Scottsdale, Scottsdale, AZ) (Tabcharani et al., 1991; Becq et al., 1994, 1999). They were maintained in -minimal essential medium-GlutaMAX containing 7% FBS, 50 IU/ml penicillin and 50 µg/ml streptomycin, and methotrexate for cell selection (WT-CFTR-CHO, 100 µM; G551D-CHO, 20 µM; pNUT-CHO, 20 µM) (Tabcharani et al., 1991; Becq et al., 1994). The human pulmonary epithelial cell line Calu-3 cell (American Type Culture Collection, Manassas, VA) (Shen et al., 1994) was maintained in Dulbecco's modified Eagle's medium-Ham's F-12 (1:1) nutritive mix supplemented by 10% FBS and 100 IU/ml penicillin and 100 µg/ml streptomycin (Dérand et al., 2004). The human nasal epithelial JME/CF15 cell line, derived from a F508del homozygous patient (Jefferson et al., 1990), was maintained in Dulbecco's modified Eagle's medium-Ham's F-12 (3:1) nutritive mix supplemented by 10% FBS, 100 IU/ml penicillin and 100 µg/ml streptomycin, 5 µg/ml insulin, 5 µg/ml transferrin, 5.5 µM epinephrine, 180 µM adenine, 1.64 nM epidermal growth factor, 2 nM T3 (3,3',5-triiodo-L-thyronine sodium salt), and 1.1 µM hydrocortisone (Norez et al., 2006). All culture media and antibiotics were from Invitrogen (Cergy-Pontoise, France), and FBS was from PerbioScience (Brebières, France). Hormones and growth factors were from Sigma. Cells were seeded in 24-well plates for iodide efflux and [cAMP] measurements and in 35-mm plastic dishes for whole-cell patch-clamp recordings. Medium was renewed at 2-day intervals.

[cAMP] Measurements. Calu-3 cells were incubated for 5-10 min at 37°C in culture medium with drugs at indicated concentration. Supernatants were collected, and cells were lysed with 12% trichloroacetic acid. The different collected fractions were homogenized and centrifuged. The cAMP was evaluated with radioimmunoassay ¹²⁵I-cAMP kit (PerkinElmer Life Sciences, Courtaboeuf, France). The radioactivity was counted by gamma Cobra II counter (PerkinElmer Life Sciences). The cAMP values were determined following the manufacturer's instructions.

Iodide Efflux. Screening of small molecules and concentration-response curves were determined by measuring the rate of ¹²⁵I efflux with a high-capacity robotic system (MultiProbe II EXT; PerkinElmer Life Sciences) adapted to the determination of iodide efflux as described previously (Marivingt-Mounir et al., 2004). Our protocol of screening is as follows. Four different cell types, WT-CFTR-CHO, G551D-CFTR CHO, Calu-3, and CF-15, were incubated in Multiwell plates at 37°C in Krebs' solution containing 1 µM KI and 1 µCi/ml Na¹²⁵I (NEN, Boston, MA) for 30 min (CHO cells) or 1 h (Calu-3 and CF15 cells) to permit the ¹²⁵I to reach equilibrium. The first three aliquots were used to establish a stable baseline in ice-cold Krebs' buffer (from t₀ to t₂). A medium containing the appropriate drug was then used for the remaining aliquots from t₃ to t₈. Residual radioactivity was extracted at the end of the experiment, with a mixture of 0.1 N NaOH and 0.1% SDS and determined using a gamma counter (Cobra II; PerkinElmer Life Sciences). The fraction of initial intracellular ¹²⁵I lost during each time point was determined, and time-dependent rates (k = peak rate, min^-1)of ¹²⁵I efflux were calculated from the following equation: k = ln (¹²⁵I_t1/¹²⁵I_t2)/(t₁ - t₂), where ¹²⁵I_t is the intracellular ¹²⁵I at time t and t₁ and t₂ are successive time points (Marivingt-Mounir et al., 2004). Relative rates were calculated: k_peak (peak rate of efflux) - k_basal (basal rate of efflux) (min^-1), i.e., the maximal value for the time-dependent rate (k_peak, min^-1) excluding the third point used to establish the baseline (k_basal, min^-1). Concentration-dependent activation curves were constructed as percentage maximal activation (set at 100%) transformed from the calculated relative rates. In some experiments, chloride transport inhibitors were present in the loading solution and in the efflux buffer. The activity of CFTR-dependent iodide efflux was stimulated either by 1 µM forskolin or by pyrrolo[2,3-b]pyrazines derivatives or by a cocktail containing 1 µM forskolin + pyrrolo[2,3-b]pyrazines. Other details are elsewhere (Marivingt-Mounir et al., 2004).

Whole-Cell Patch-Clamp Recordings. Whole-cell patch-clamp experiments were performed on Calu-3 and WT-CFTR-CHO cells at room temperature. Currents were recorded with a RK-400 patch-clamp amplifier (Biologic, Grenoble, France). I-V relationships were built by clamping the membrane potential to -40 mV and by pulses from -80 mV to +80 mV (20-mV increments). Pipettes with a resistance of 3 to 4 M were pulled from borosilicate glass capillary tubing (GC150-TF10; Clark Electromedical Inc., Reading, UK) using a two-step vertical puller from Narishige (Tokyo, Japan). They were filled with the following solution: 1 mM NaCl, 113 mM L-aspartic acid, 113 mM CsOH, 1 mM MgCl₂, 27 mM CsCl, 1 mM EGTA, 10 mM TES, and 3 mM MgATP (pH 7.2; 285 mOsm). They were connected to the head of the patch-clamp amplifier through an Ag-AgCl pellet. Seal resistances ranging from 6 to 20 G were obtained. Results were analyzed with the pClamp 6.0.2 package software (Molecular Devices, Sunnyvale, CA). The external bath solution contained 145 mM NaCl, 4 mM CsCl, 1 mM CaCl₂, 1 mM MgCl₂, 10 mM glucose, and 10 mM TES (pH 7.4; 340 mOsm with mannitol). The liquid potential was corrected before seal establishment. Pipette capacitances were electronically compensated in cell-attached mode. To standardize experiments, recordings were performed only when the input resistance had a value 10 M. The mean value of access resistance was 7.3 ± 1.1 M (n = 5) for Calu-3 cells and 7.9 ± 0.8 M (n = 5) for WT-CFTR-CHO cells. Membrane capacitances were measured in the whole-cell mode by fitting capacitance currents obtained in response to a hyperpolarization of 6 mV, with a first-order exponential, and by integrating the surface of the capacitance current. Mean values of membrane capacitance were 52.2 ± 12.2 pF (n = 5) for Calu-3 cells and 63.7 ± 11.6 pF (n = 5) for WT-CFTR-CHO cells. For graphic representations, I-V relationship was normalized to 1 pF to remove variability due to differences in cell sizes. For time-course experiments, current amplitude measured at +40 mV was plotted each 15 s.

Short-Circuit Current Measurement. Experiments were carried out on the colon epithelium of wild-type (Cftr^+/+) or CFTR knock-out (Cftr^-/-) mice B6 129-CFTR^tm1Unc (Snouwaert et al., 1992) obtained from CNRS-CDTA (Center de Distribution, Typage et Archivage Animal, Orléans, France). Animals were killed by cervical dislocation, a procedure approved by the local animal ethic committee of the University of Poitiers. We used one animal per experiment (n is the number of animals). The ascending colon was removed, opened longitudinally, and washed in ice-cold phosphate-buffered saline. The muscle layers and connective tissue were dissected away from the colon epithelium. The epithelium was mounted in a vertical Ussing chamber (0.26 cm² surface area). Luminal and serosal sides were bathed at 37°C, with a nutrient buffer containing 120 mM NaCl, 1.2 mM CaCl₂, 1.2 mM MgCl₂, 0.8 mM K₂HPO₄, 3.3 mM KH₂PO₄, 25 mM NaHCO₃, and 10 mM D-glucose. The pH value of this solution was 7.4 when gassed with 95% O₂-5% CO₂ at 37°C. The upper level of fluids in both luminal and serosal reservoirs was identical. After mounting tissues in Ussing chamber, an equilibration period 40 min, was allowed for stabilization of basal electrical properties. Transepithelial potential difference (V_TE) was measured by the Ag/AgCl electrodes placed as close as possible to (and on either side of) the epithelium to reduce the magnitude of the solution series resistance. They were connected to the preamplifier headstage of an amplifier (EC-825 Epithelial Voltage-Clamp; Warner Instruments Corporation, Hamden CT). Potential difference values were corrected for the junction potential between the luminal and serosal solutions when no tissue was mounted in the chamber. The polarity of I_sc and V_TE was referred to serosal side of the epithelium. An apical anion secretion was indicated by a decrease in I_sc. Transepithelial resistances (R_TE) were determined according to Ohm's law, from the voltage deflection (V_TE) due to pulsed current injection (1 s) of 0.5 µA and subtraction of the empty chamber resistance. In our experiments, the range of colonic epithelia R_TE was 580 to 2000 · cm^-2, and the means were 1113 ± 157 · cm^-2 (N = 20) for Cftr^-/- mice colon and 1388 ± 251 · cm^-2 (N = 26) for wild-type mice colon (no significant difference). Data were collected with the Chart version 4.2.2 package software (ADInstruments Phy Ltd, Castle Hill, NSW, Australia). All experiments were carried out in the presence of amiloride (500 µM) in the apical solution to prevent sodium transport.

View larger version (27K): [in this window] [in a new window]   Fig. 2. Screening of pyrrolo[2,3-b]pyrazines derivatives. A, structure of the 12 pyrrolo[2,3-b]-pyrazines compounds tested (numbered 1 to 12). B, effects of these agents on iodide efflux in WT-CFTR-CHO cells. All compounds were tested (n = 4) in presence of 1 µM fsk at 100 µM, with the exception of RP149 and RP150 (300 µM). The corresponding effect was normalized to that of 1 µM fsk (100%, indicated by the dashed line). C, effect of the two active derivatives RP107 and RP108 (at 100 µM) on [cAMP]i of Calu-3 cells in the presence or absence of forskolin at 1 µM and compared with the effect at 10 µM fsk (n = 6 each). **, P < 0.01; ***, P < 0.001. ns, nonsignificant.

	Results

Top Abstract Materials and Methods Results Discussion References

 
Our study was carried out with the 12 pyrrolo[2,3-b]pyrazines derivatives shown in Fig. 2A. These agents are named RP derivatives (see Table 1) to facilitate the reading. These compounds were selected from a library of more than 100 derivatives synthesized in our laboratory, because they share structural determinants with other CFTR activators (reviewed in Becq, 2006).

Identification of cAMP-Independent CFTR Activators. We first examined the effects of 100 µM of each compound on CHO cells stably expressing WT-CFTR with our robotic cell-based primary screening assay using iodide efflux measurement. This assay allowed the rapid detection of CFTR channel activity from cells cultured in 24-well plates. Several control experiments have been first performed on resting cells (noted ctrl for control, corresponding to 0%) or on cells stimulated with a submaximal concentration (1 µM) of the adenylate cyclase activator forskolin (hereafter noted fsk, corresponding to 100%, gray bar in Fig. 2B). This minimal concentration of fsk was chosen, because it allows low stimulation of CFTR-dependent iodide efflux as reported previously (Marivingt-Mounir et al., 2004). Among these compounds only RP107 and RP108 activated an iodide efflux above the control (Fig. 2B). The remaining 10 RP compounds did not stimulate iodide efflux (dashed line in Fig. 2B was set at the level of fsk response). To begin to characterize the mechanism of activation, we determined the cellular cAMP level in WT-CFTR-CHO cells in the following experimental conditions, with cells stimulated by 1 µM fsk alone or stimulated by 1 µM fsk plus 100 µM RP107 or plus 100 µM RP108 and with cells stimulated by 10 µM fsk. Figure 2C shows that neither RP107 nor RP108 potentiated the cAMP level measured with 1 µM fsk alone. In addition, the compounds alone have no effect on the basal cellular cAMP level.

We then determined the concentration-response relationships in presence of 1 µM fsk plus increasing concentrations of each RP in WT-CFTR-CHO cells (Fig. 3). The iodide secretion peaked and then fell rapidly. For RP107, the peak rate (k_peak) of iodide efflux occurred within the first 2 min after adding the compound. In control condition (DMSO), k_peak was 0.065 ± 0.003 min^-1 in the presence of 1 µM fsk, and k_peak increased to 0.108 ± 0.005 min^-1 and was significantly potentiated to 0.22 ± 0.008 min^-1 after the addition of 100 µM RP107 (n = 4 for each condition). The decline of the efflux rate could be attributed either to tracer depletion (because of a finite source of ¹²⁵I at the beginning of each experiment), local ATP depletion, or desensitization. With 1 µM fsk present, the maximal stimulation (plateau) was achieved at 3 µM (Fig. 3, A and B), and the half-maximal effective concentration was EC₅₀ = 152 ± 1.2 nM for RP107 (Fig. 3B). For RP108, the corresponding EC₅₀ value was much higher (24 ± 1.2 µM; Fig. 3C). No stimulation in the presence of either RP107 or RP108 was noted with mock-CHO cells, i.e., nonexpressing CFTR cells (not shown). Thus, RP107 selectively stimulates CFTR-dependent iodide efflux with submicromolar concentrations, whereas its analog RP108 seems approximately 158-fold less potent.

View larger version (25K): [in this window] [in a new window]   Fig. 3. Concentration-response relationships for RP107 and RP108 on iodide efflux in WT-CFTR-CHO cells. A, time-dependent iodide efflux experiments performed in presence of increasing concentrations of RP107 as indicated. The control is 1 µM fsk. The horizontal bar above traces in A indicates the presence of agonists. Each data point is mean ± S.E.M. of n = 4. In B and C, concentration-response curves for RP107 and RP108, respectively. n = 4 for each concentration.

In the following experiments, we determined the pharmacological profile for inhibition of the RP-stimulated iodide efflux in WT-CFTR-CHO cells. The peak of iodide efflux stimulated by 1 µM fsk + 1 µM RP107 (k_peak = 0.207 ± 0.003 min^-1, n = 8) was fully abolished by 100 µM glibenclamide (k_peak = 0.063 ± 0.003 min^-1, n = 8) and 10 µM CFTR_inh-172 (k_peak = 0.055 ± 0.003 min^-1, n = 8), two well known CFTR inhibitors (Fig. 4A, right traces). In contrast, fsk/RP107 response was not affected by 100 nM TS-TM calix[4]arene (k_peak = 0.2 ± 0.004 min^-1, n = 8) or 500 µM DIDS (k_peak = 0.213 ± 0.011 min^-1, n = 8), two non-CFTR channel inhibitors (Fig. 4A, left traces). A summary of the data are presented in Fig. 4B with statistical analysis. We then determined the effect of RP107 in Calu-3 cells, a model for human airway epithelial cells endogenously expressing WT-CFTR (Shen et al., 1994; Dérand et al., 2004). Figure 5A shows stimulation by RP107 of iodide efflux in Calu-3 cells in the absence of fsk. As can be seen in Fig. 5A, the latency to reach the maximal efflux response (k_peak) was reduced at higher RP107 concentrations (t₅ at 100-300 nM but t₃ at 1-10 µM). The corresponding half-maximal effective concentration was EC₅₀ = 303 ± 1.5 nM (n = 4; Fig. 5B, right). In similar experiment but in the presence of submaximal concentration of fsk (1 µM), RP107 activated the efflux with a greater affinity (EC₅₀ = 140 ± 2.6 nM, n = 4; Fig. 5B, left). These results suggest that the stimulation of CFTR by RP107 depends on the phosphorylation state of the channels.

View larger version (28K): [in this window] [in a new window]   Fig. 4. Pharmacological profile of RP107 induced iodide efflux on WT-CFTR-CHO cells. Inhibitors were preincubated 30 min before experiment and iodide efflux was stimulated by 1 µM fsk + 1 µM RP107. A, effects of 100 nM calixarene, 500 µM DIDS (left traces), 100 µM glibenclamide, and 10 µM CFTRinh-172 (right traces). Control (ctrl), vehicle only. Each data point is mean ± S.E.M. of n = 8. The horizontal bar above traces in A indicates the presence of agonists, inhibitors, or vehicle (noted control). B, bar graphs of the normalized results obtained in A (n = 8 for each condition). ***, P < 0.001; ns, nonsignificant.

View larger version (26K): [in this window] [in a new window]   Fig. 5. Stimulation of iodide efflux by RP107 in Calu-3 cells. A, effect of increasing concentrations of RP107 on the iodide efflux. The horizontal bar above traces indicates the presence of RP107 at the indicated concentration. No forskolin is present. Each data point is mean ± S.E.M. of n = 4. B, plots of the concentration-response effects of RP107 alone (right) or RP107 in presence of 1 µM fsk (left). Each data point is mean ± S.E.M. of n = 4.

View larger version (26K): [in this window] [in a new window]   Fig. 6. Effect of RP107 on class II and class III CFTR mutants. A, effect of RP107 in G551D-CFTR-CHO cells. Iodide efflux stimulated in presence of increasing concentrations of RP107 and in presence of 10 µM fsk (left). Plots of the concentration-response effect are presented on the right. B, effect of RP107 in human airway epithelial F508del-CFTR CF15 cells. Iodide efflux stimulated in presence of increasing concentrations of RP107 and in presence of 1 µM fsk (left). Plots of the concentration-response effects are presented on the right (each data point is mean ± S.E.M. of n = 4). For the experiments on CF15 cells (F508del-CFTR), cells were incubated for 24 h at low temperature (n = 4 for each condition). The horizontal bar above traces indicates the presence of RP107 or fsk at the indicated concentration.

View larger version (28K): [in this window] [in a new window]   Fig. 7. Whole-cell patch-clamp experiments were performed on Calu-3 cells. All experiments were performed in the presence of 100 nM calixarene and 200 µM DIDS in the bath to inhibit non-CFTR chloride channels. A, currents recorded in control condition. B, currents recorded after the addition of 1 µM RP107 to the bath. C, currents recorded after the addition of 100 µM glibenclamide to the bath already containing 1 µM RP107. D, plots of the mean ± S.E.M. current density/voltage in the three experimental conditions (n = 5 for each). ***, P < 0.001; ns, nonsignificant compared with control.

To further demonstrate that the chloride current activated by RP107 was due to CFTR, we added 100 µM glibenclamide to the bath after stable and maximal activation of the current (Fig. 7C). The current density significantly decreased in the presence of glibenclamide to 3.1 ± 1.5 pA/pF (n = 5, P < 0.001 compared with RP107 alone and not significant compared with control; Fig. 7D). Whole-cell patch-clamp experiments were also conducted in WT-CFTR-CHO cells. As expected, RP107 also stimulated CFTR currents. An example of the concentration- and time-dependent activation of CFTR Cl^- currents (representative of five separate cells) is presented in Fig. 8. As for Calu-3 cells, we incubated the cells in a mixture of calixarene/DIDS (control in Fig. 8A). In this basal condition, the current amplitude was 103 pA at +40mV. Increasing the concentrations of RP107 in the bath from 10 nM to 10 µM activated a linear Cl^- current (Fig. 8B), with an amplitude (at +40 mV) of 309 pA at 10 nM, 633 pA at 100 nM, and 1077 pA at 1 µM (Fig. 8A). At 1 µM RP107, the CFTR Cl^- current was maximal; with 10 µM of the current amplitude remained unchanged (1064 pA at +40 mV). The current was fully and rapidly (140 pA at +40 mV after 2 min) inhibited after the addition to the bath of 10 µM CFTR_inh-172 (in the presence of 10 µM RP107). Figure 8B presents the corresponding time course of activation for the current traces shown in Fig. 8A. These results show that, in WT-CFTR-CHO and Calu-3 cells, RP107 activates CFTR Cl^- currents inhibited by CFTR_inh-172 and glibenclamide.

View larger version (36K): [in this window] [in a new window]   Fig. 8. Whole-cell patch-clamp experiments performed on WT-CFTR-CHO cells. All experiments (n = 5) were in the presence of 100 nM calixarene and 200 µM DIDS in the bath to inhibit non-CFTR chloride channels. A, whole-cell chloride currents recorded in control condition after adding increasing concentrations of RP107 to the bath from 10 nM to 10 µM as indicated above current traces or after adding 10 µM CFTRinh-172 to the bath already containing 10 µM RP107. B, corresponding time course of whole-cell current. Note the presence of calixarene/DIDS during all of the experiment and of CFTRinh-172 only at the end.

View larger version (19K): [in this window] [in a new window]   Fig. 9. Ussing chamber recordings of Isc in mice ascending colon. A and B, effect of 500 µM amiloride, 10 µM fsk, and 500 µM glibenclamide on WT-CFTR (A) and WT-CFTR null (B) mice colon. C and D, effect of cumulative concentrations of RP107 and glibenclamide on WT-CFTR (C) and WT-CFTR null (D) mice colon. In C and D, we first added 500 µM amiloride before increasing concentrations of RP107 were applied to serosal side of the two types of tissue. Note that glibenclamide was only added on wild-type mice colon. E, effect of RP107 on the change in Isc (Isc) on wild-type (open bars) or CFTR null (filled bars) mice colon. The change of Isc was defined as a difference of current between the sustained phase of the response and the respective baseline (microamperes/centimeter2) for each experiment. Each bar is mean ± S.E.M. (N = 8 for CFTR null mice colon and N = 8 for wild-type mice colon, unpaired experiments). F, corresponding plots of the concentration-response (Isc) for wild-type mice colon. ***, P < 0.001; ns, non significant.

The experiments described below were conducted after inhibition of the ENaC current by 500 µM amiloride. The addition of RP107 to either apical and basolateral or basolateral only produced the same response in CFTR wild-type mice colon (Fig. 9C). Increasing concentrations of RP107 induced a change of I_sc, as for the fsk-activated I_sc, corresponding to an apical Cl^- secretion. The maximal RP107-dependent response I_sc = 7.27 ± 2.4 µA · cm^-2 (N = 8; Fig. 9C) was fully reversed after bilateral application of 1 mM glibenclamide (I_sc = 1.16 ± 1.45 µA · cm^-2, N = 8, P < 0.001; Fig. 9C) but was not affected by 500 µM DIDS (I_sc = 7.47 ± 0.44 µA · cm^-2, N = 3; data not shown). In contrast, in CFTR null mice colon, no change of I_sc was obtained with RP107 used at the same concentrations (N = 8; Fig. 9D). Figure 9E indicates I_sc for each concentration of RP107 tested on the colon of Cftr^+/+ and Cftr^-/- mice (N = 8 animals for each). We determined an EC₅₀ of 89 ± 6 nM for Cftr^+/+ mice (N = 8; Fig. 9F). Similar experiments have been performed in the presence of 1 µM fsk to mimic the protocol of iodide efflux experiments. After the addition of amiloride, the tissue was first stimulated by 1 µM fsk and then by RP107. After stable stimulation by fsk of the apical Cl^- secretion, the response was further potentiated by RP107, with an EC₅₀ of 173 ± 36 nM (N = 6). Finally, with RP108, we found stimulation of a glibenclamide-sensitive apical Cl^- secretion in Cftr^+/+ but not Cftr^-/- mice, with an EC₅₀ of 103 ± 9 µM (N = 6).

	Discussion

Top Abstract Materials and Methods Results Discussion References

 
In the present study, we report on the discovery of 6-phenylpyrrolo[2,3-b]pyrazines, a novel family of wild-type CFTR, G551D, and F508del-CFTR activators. These agents have been identified with our CF drug discovery program using a simple and robust robotic cell-based assay, allowing the discovery of small correctors and potentiators of CFTR chloride channels. Two agents that we described here are RP107 and RP108. RP107 is the most potent with an affinity of 140 to 152 nM on wild-type CFTR (Calu-3 and WT-CFTR-CHO cells), 1.5 nM on G551D-CFTR, and 111 nM on temperature-corrected F508del-CFTR. The Cl^- secretion was stimulated in proximal colon of mice, with an affinity of approximately 90 nM for RP107 and 103 µM for RP108. The mechanism of action, although still unknown, is independent of cAMP but depends on the phosphorylation state of CFTR, because these agents have a greater affinity for the phosphorylated channels. RP107 and RP108 are selective for CFTR because, in nonexpressing CFTR cells (mock-CHO cells), in F508del cells at 37°C (CF15 cells), and in the colon of Cftr^-/- mice, these agents have no effect on iodide efflux, whole-cell Cl^- currents, or I_sc. Although we have no evidence for a direct activation of CFTR, it is reasonable to conclude that 6-phenylpyrrolo[2,3-b]pyrazines selectively activate CFTR but not other Cl^- channels.

6-Phenylpyrrolo[2,3-b]pyrazines (Aloisines). This family of compounds has been first described as CDK and GSK-3 inhibitors (Mettey et al., 2003). Aloisines act by competitive inhibition of ATP binding to the catalytic subunit of the kinases and may be of therapeutic value in Alzheimer's disease because implication of CDK5 and GSK-3 has been suggested in the disease (Knockaert et al., 2002). All of the compounds listed in Fig. 2 and tested on CFTR function are active on CDK1, 2, and 5 at submicromolar concentrations (Mettey et al., 2003), but not all of them are CFTR activators. This indicates that CFTR activation and CDK inhibition are probably not related, which also suggests that the design of a CFTR-specific activator may be achieved after careful determination of the structural determinants of Aloisines.

Structural Determinants for CFTR Activation. To begin to define a structure-activity relationship with 6-phenylpyrrolo[2,3-b]pyrazines tailored to CFTR activation, we compared the chemical structure of RP107 and RP108 with the 10 inactive related derivatives (Fig. 2A) and with several known CFTR activators. First, within the 6-phenylpyrrolo[2,3-b]pyrazines family, we observed that RP108 bearing a 4-chlorophenyl substituent is less potent than RP107 having 4-hydroxyphenyl substituent. In addition, 4-methoxyphenyl compounds (RP11, RP95, RP127, RP106, and RP149) are inactive. Therefore, one favorable determinant for CFTR activation is the phenyl ring substituted at the 4-position, with the following potency: OH>Cl>>OCH₃. Interestingly, a hydroxyphenyl substituent appears also for NS004, NS1619, genistein, apigenin, and benzo[c]quinolizinium compounds (reviewed in Becq, 2006). A chlorine atom is also commonly found for NS004, benzo[c]quinolizinium, and 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid diamides (Gribkoff et al., 1994; Marivingt-Mounir et al., 2004; Hirth et al., 2005). Some agents possess both OH and Cl on the same ring (NS004) or on different rings (MPB-91 and MPB-104). In a previous structure-activity relationship of the CFTR activators, benzo[c]quinolizinium salts (Marivingt-Mounir et al., 2004), we observed that alkyl substituent and especially butyl chain improved the efficacy of the drugs. Interestingly, the substitution of the pyrrolopyrazines core with butyl in R₁ (Fig. 1) is also present in RP107 and RP108 (Fig. 2A). Therefore, a second favorable determinant for CFTR activation is the length of the alkyl chain with the following potency: (CH₂)₃-CH₃>> H CH₃ (CH₂)₂-CH₃ (CH₂)₄-CH₃ as found in compounds RP107, RP108 >> RP26 RP96 RP132 RP150. Finally, compounds RP26 having the OH-phenyl but not the butyl chain, RP106 having the butyl chain but not the OH-phenyl, or RP14 having the Cl-phenyl but not the butyl chain are not activators. These observations suggest that both determinants (OH-phenyl and butyl chain) are required for CFTR activation. These very preliminary data provide some interesting clues to improve the potency of future 6-phenylpyrrolo[2,3-b]pyrazines.

Toward a New Scaffold Structure for CFTR Activation. The CFTR activators of the first generation were originally identified after investigations of the intracellular signaling pathways (phosphodiesterases and phosphoprotein phosphatases) (reviewed in Becq 2006). This field has profoundly changed the last decade with the achievement of specific and new screening assays developed by universities (Chappe et al., 1998; Galietta et al., 2001; Ma et al., 2002; Sammelson et al., 2003; Yang et al., 2003; Marivingt-Mounir et al., 2004; Szkotak et al., 2004) and biopharmaceutical companies (Hirth et al., 2005; Van Goor et al., 2006).

Several commercial agents, such as phenanthrolines and benzoquinolines, have been reported to activate epithelial Cl^- secretion with EC₅₀ from 612 to 34 µM (Duszyk et al., 2001; Cuthbert, 2003; Cuthbert and MacVinish, 2003). The 4-chloro-benzo[f]isoquinoline has an EC₅₀ of 4 µM in Calu-3 cells (Murthy et al., 2005). Introducing a halogen atom (Cl) in position 4 increased the efficacy of the agent compared with other benzoisoquinolines (Szkotak et al., 2004; Murthy et al., 2005). A similar effect after the addition of a halogen atom has been noted earlier for benzoquinoliziniums (Marivingt-Mounir et al., 2004). Other agents were discovered by high-throughput screening (Galietta et al., 2001; Ma et al., 2002; Sammelson et al., 2003; Yang et al., 2003), like for example 3-(2-benzyloxyphenyl)isoxazoles and 3-(2-benzyloxyphenyl)-isoxazolines (Sammelson et al., 2003). Agents with tetrahydrocarbazole, hydroxycoumarin, and thiazolidine core structures reversibly activated CFTR with K_d as low as 200 nM (Ma et al., 2002). It is noteworthy that trifluoromethylphenylbenzamine activated G551D-CFTR channels with a K_d > 10 µM (Ma et al., 2002). Stimulation of F508del-CFTR activity was also reported with tetrahydrocarbazole and N-phenyltriazine with K_d below 1 µM (Ma et al., 2002). Further high throughput screening efforts also identified 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid diamides as CFTR modulators with nanomolar potency (Hirth et al., 2005) and quinazolinone from a library of 164,000 synthetic compounds (Van Goor et al., 2006).

In conclusion, we have identified 6-phenylpyrrolo[2,3-b]pyrazines as a new scaffold structure for the selective activation of CFTR, with submicromolar affinity on wild-type CFTR, G551D-, and F508del-CFTR, two of the most common CF mutations. Although the mechanism of activation is still unknown, the challenge will be to design more potent CFTR-specific activators and to determine their potential and optimal use for therapeutic application in cystic fibrosis and CFTR-related diseases.

	Acknowledgements

 
We thank Nathalie Bizard, Patricia Léon and James Habrioux for excellent technical assistance, Drs. Singh and Bridges for generous gift of TS-TM calix[4]arene and Laurent Meijer for discussions.

	Footnotes

 
This work was supported by the French Association Vaincre la Mucoviscidose (VLM), ABCF Proteins, and CNRS. S.N. was supported by a thesis studentship from MucoVie 66. C.N. was supported by a thesis studentship from VLM. C.F. was supported by a specific grant from CNRS. Part of this work has already been published in an abstract form, in Noel S, Mettey Y, Norez C, Rogier C, and Becq F (2005) Activation of CFTR-dependent chloride secretion by a novel activator with nanomolar affinity. Pediatr Pulmonol 39 (Suppl. 28):205.

Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.

doi:10.1124/jpet.106.104521.

ABBREVIATIONS: CF, cystic fibrosis; CDK, cyclin-dependent kinase; CFTR, cystic fibrosis transmembrane conductance regulator; CFTR_inh-172, 3-[(3-trifluoromethyl)phenyl]-5-[(4-carboxyphenyl)methylene]-2-thioxo-4-thiazolidinone; CHO, Chinese hamster ovary; DIDS, 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid; DMSO, dimethyl sulfoxide; ENaC, epithelial sodium channel; fsk, forskolin; GSK-3, glycogen synthase kinase-3; RP107, 7-n-butyl-6-(4-hydroxyphenyl)[5H]pyrrolo[2,3-b]pyrazine; RP108, 7-n-butyl-6-(4-chlorophenyl)[5H]pyrrolo[2,3-b]pyrazine; R_TE, transepithelial resistance; RP11, 6-(4-methoxyphenyl)[5H]pyrrolo[2,3-b]pyrazine; RP26, 6-(4-hydroxyphenyl)[5H]pyrrolo[2,3-b]pyrazine; RP95, 6-(4-methoxyphenyl)-7-methyl[5H]pyrrolo[2,3-b]pyrazine; RP96, 6-(4-hydroxyphenyl)-7-methyl[5H]pyrrolo[2,3-b]pyrazine; RP127, 6-(4-methoxyphenyl)-7-propyl[5H]pyrrolo[2,3-b]pyrazine; RP132, 6-(4-hydroxyphenyl)-7-propyl[5H]pyrrolo[2,3-b]pyrazine; RP106, 7-n-butyl-6-(4-methoxyphenyl)[5H]-pyrrolo[2,3-b]pyrazine; RP149, 7-n-pentyl-6-(4-methoxyphenyl)[5H]pyrrolo[2,3-b]pyrazine; RP150, 7-n-pentyl-6-(4-hydroxyphenyl)[5H]pyrrolo[2,3-b]pyrazine; RP14, 6-(4-chlorophenyl)[5H]pyrrolo[2,3-b]pyrazine; TES, N-tris[hydroxymethyl]methyl-2-aminoethane-sulfonic acid; TS-TM calix[4]arene, 5,11,17,23-tetrasulfonato-25,26,27,28-tetramethoxy-calix[4]arene; V_TE, transepithelial potential difference; WT, wild type; FBS, fetal bovine serum; T3, 3,3',5-triiodo-L-thyronine sodium salt; NS004, (5-trifluoromethyl-(5-chloro-2-hydroxyphenyl)-1,3-dihydro-2H-benzimidazol-2-one; NS1619, (1-(2'-hydroxy-5-trifluoromethylphenyl)-5-trifluoromethyl-1,3-dihydro-2H-benzimidazole-2-one; MPB-91, 5-butyl-10-chloro-6-hydroxybenzo[c]quinolizinium chloride; MPB-104, 5-butyl-7-chloro-6-hydroxybenzo[c]quinolizinium chloride.

Address correspondence to: Pr. Frédéric Becq, IPBC CNRS UMR 6187, Université de Poitiers, 40 Avenue du Recteur Pineau, 86022 Poitiers, France. E-mail: frederic.becq{at}univ-poitiers.fr

	References

Top Abstract Materials and Methods Results Discussion References

 

Anderson MP, Berger HA, Rich DP, Gregory RJ, Smith AE, and Welsh MJ (1991) Nucleotide triphosphate are required to open the CFTR chloride channel. Cell 67: 775-784.[CrossRef][Medline]

Becq F, Jensen TJ, Chang XB, Savoia A, Rommens JM, Tsui LC, Buchwald M, Riordan JR, and Hanrahan JW (1994) Phosphatase inhibitors activate normal and defective CFTR chloride channels. Proc Natl Acad Sci USA 91: 9160-9164.[Abstract/Free Full Text]

Becq F, Mettey Y, Gray MA, Galietta LJ, Dormer RL, Merten M, Metaye T, Chappe V, Marvingt-Mounir C, Zegarra-Moran O, et al. (1999). Development of substituted benzo[c]quinolizinium compounds as novel activators of the cystic fibrosis chloride channel. J Biol Chem 274: 27415-27425.[Abstract/Free Full Text]

Becq F (2006) On the discovery and development of CFTR chloride channel activators. Curr Pharm Des 12: 471-484.[CrossRef][Medline]

Chappe V, Mettey Y, Vierfond JM, Hanrahan JW, Gola M, Verrier B, and Becq F (1998) Structural basis for specificity and potency of xanthine derivatives as activators of the CFTR chloride channel. Br J Pharmacol 123: 683-693.[CrossRef]

Cuthbert AW (2003) Benzoquinolines and chloride secretion in murine colonic epithelium. Br J Pharmacol 138: 1528-1534.[CrossRef]

Cuthbert AW and MacVinish LJ (2003) Mechanisms of anion secretion in Calu-3 human airway epithelial cells by 7,8-benzoquinoline. Br J Pharmacol 140: 81-90.[CrossRef][Medline]

Davis ML, Wakefield BJ, and Wardell JA (1992) Reactions of -(lithiomethyl)azines with nitriles as rate to pyrrolo-pyridines, -quinolines, -pyrazines, quinoxalines and pyrimidines. Tetrahedron Lett 48: 939-952.

De Jonge HR, Ballmann M, Veeze H, Bronsveld I, Stanke F, Tummler B, and Sinaasappel M (2004) Ex vivo CF diagnosis by intestinal current measurements (ICM) in small aperture, circulating Ussing chambers. J Cyst Fibrosis 3: 159-163.

Denning GM, Anderson MP, Amara JF, Marshall J, Smith AE, and Welsh MJ (1992) Processing of mutant cystic fibrosis transmembrane conductance regulator is temperature-sensitive. Nature (Lond) 358: 761-764.[CrossRef][Medline]

Dérand R, Montoni A, Bulteau-Pignoux L, Janet T, Moreau B, Muller JM, and Becq F (2004) Activation of VPAC1 receptors by VIP and PACAP-27 in human bronchial epithelial cells induces CFTR-dependent chloride secretion. Br J Pharmacol 141: 698-708.[CrossRef][Medline]

Duszyk M, MacVinish L, and Cuthbert AW (2001) Phenanthrolines, a new class of CFTR chloride channel openers. Br J Pharmacol 134: 853-864.[CrossRef]

Galietta LV, Jayaraman S, and Verkman AS (2001) Cell-based assay for high-throughput quantitative screening of CFTR chloride transport agonists. Am J Physiol 281: C1734-C1742.

Gray MA, Greenwell JR, and Argent BE (1988) Secretin-regulated chloride channel on the apical plasma membrane of pancreatic duct cells. J Membr Biol 105: 131-142.[CrossRef][Medline]

Gribkoff VK, Champigny G, Barbry P, Dworetzky SI, Meanwell NA, and Lazdunski M (1994) The substituted benzimidazolone NS004 is an opener of the cystic fibrosis chloride channel. J Biol Chem 269: 10983-81096.[Abstract/Free Full Text]

Hirth BH, Qiao S, Cuff LM, Cochran BM, Pregel MJ, Gregory JS, Sneddon SF, and Kane JL Jr (2005) Discovery of 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid diamides that increase CFTR mediated chloride transport. Bioorg Med Chem Lett 15: 2087-2091.[CrossRef][Medline]

Illek B, Zhang L, Lewis NC, Moss RB, Dong JY, and Fischer H (1999) Defective function of the cystic fibrosis-causing missense mutation G551D is recovered by genistein. Am J Physiol 277: C833-C839.[Medline]

Jefferson DM, Valentich JD, Marini FC, Grubman SA, Iannuzzi MC, Dorkin HL, Li M, Klinger KW, and Welsh MJ (1990) Expression of normal and cystic fibrosis phenotypes by continuous airway epithelial cell lines. Am J Physiol 259: L496-L505.[Medline]

Knockaert M, Greengard P, and Meijer L (2002) Pharmacological inhibitors of cyclin-dependent kinases. Trends Pharmacol Sci 23: 417-425.[CrossRef][Medline]

Ma T, Thiagarajah JR, Yang H, Sonawane ND, Folli C, Galietta LJ, and Verkman AS (2002) Thiazolidinone CFTR inhibitor identified by high-throughput screening blocks cholera toxin-induced intestinal fluid secretion. J Clin Investig 110: 1651-1658.[CrossRef][Medline]

Marivingt-Mounir C, Norez C, Derand R, Bulteau-Pignoux L, Nguyen-Huy D, Viossat B, Morgant G, Becq F, Vierfond JM, and Mettey Y (2004) Synthesis, SAR, crystal structure, and biological evaluation of benzoquinoliziniums as activators of wild-type and mutant cystic fibrosis transmembrane conductance regulator channels. J Med Chem 47: 962-972.[CrossRef][Medline]

Mettey Y, Gompel M, Thomas V, Garnier M, Leost M, Ceballos-Picot I, Noble M, Endicott J, Vierfond JM, and Meijer L (2003) Aloisines, a new family of CDK/GSK-3 inhibitors: SAR study, crystal structure in complex with CDK2, enzyme selectivity, and cellular effects. J Med Chem 46: 222-236.[CrossRef][Medline]

Murthy M, Pedemonte N, MacVinish L, Galietta L, and Cuthbert A (2005) 4-Chlorobenzo[f]isoquinoline (CBIQ), a novel activator of CFTR and DeltaF508 CFTR. Eur J Pharmacol 516: 118-124.[CrossRef][Medline]

Norez C, Antigny F, Becq F, and Vandebrouck C (2006) Maintaining low calcium level in the endoplasmic reticulum restores abnormal endogenous F508del-CFTR trafficking in airway epithelial cells. Traffic 7: 562-573.[CrossRef][Medline]

Quinton P (1983) Chloride impermeability in cystic fibrosis. Nature (Lond) 301: 421-422.[CrossRef][Medline]

Ratjen F and Doring G (2003) Cystic fibrosis. Lancet 361: 681-689.[CrossRef][Medline]

Riordan JR, Rommens JM, Kerem B, Alon N, Rozmahel, Grzelczak Z, Zielenski J, Lok S, Playsic N, Chou JL, et al. (1989) Identification of the cystic fibrosis gene: cloning and characterization of complementary DNA. Science (Wash DC) 245: 1066-1073.[Abstract/Free Full Text]

Sammelson RE, Ma T, Galietta LJ, Verkman AS, and Kurth MJ (2003) 3-(2-Benzyloxyphenyl)isoxazoles and isoxazolines: synthesis and evaluation as CFTR activators. Bioorg Med Chem Lett 13: 2509-2512.[CrossRef][Medline]

Shen BQ, Finkbeiner WE, Wine JJ, Mrsny RJ, and Widdicombe JH (1994) Calu-3: a human airway epithelial cell line that shows cAMP-dependent Cl^- secretion. Am J Physiol 266: L493-L501.[Medline]

Sheppard DN and Welsh MJ (1999) Structure and function of the CFTR chloride channel. Physiol Rev 79: S23-S45.[Medline]

Singh AK, Venglarik CJ, and Bridges RJ (1995) Development of chloride channel modulators. Kidney Int 48: 985-993.[Medline]

Snouwaert JN, Brigman KK, Latour AM, Malouf NN, Boucher RC, Smithies O, and Koller BH (1992) An animal model for cystic fibrosis made by gene targeting. Science (Wash DC) 257: 1083-1088.[Abstract/Free Full Text]

Szkotak AJ, Murthy M, MacVinish LJ, Duszyk M, and Cuthbert AW (2004) 4-Chloro benzo[f]isoquinoline (CBIQ) activates CFTR chloride channels and KCNN4 potassium channels in Calu-3 human airway epithelial cells. Br J Pharmacol 142: 531-542.[CrossRef]

Tabcharani JA, Chang XB, Riordan JR, and Hanrahan JW (1991) Phosphorylation-regulated Cl^- channel in CHO cells stably expressing the cystic fibrosis gene. Nature (Lond) 352: 628-631.[CrossRef][Medline]

Van Goor F, Straley KS, Cao D, Gonzalez J, Hadida S, Hazlewood A, Joubran J, Knapp T, Makings LR, Miller M, et al. (2006) Rescue of delF508 CFTR trafficking and gating in human cystic fibrosis airway primary culture by small molecule. Am J Physiol 290: 1117-1130.

Vierfond JM, Mettey Y, Mascrier-Demagny L, and Miocque M (1981) Cyclisation par amination intramoléculaire dans la série de la pyrazine. Tetrahedron Lett 22: 1219-1222.

Welsh MJ and Smith AE (1993) Molecular mechanisms of CFTR chloride channel dysfunction in cystic fibrosis. Cell 73: 1251-1254.[CrossRef][Medline]

Yang H, Shelat AA, Guy RK, Gopinath VS, Ma T, Du K, Lukacs GL, Taddei A, Folli C, Pedemonte N, et al. (2003) Nanomolar affinity small molecule correctors of defective Delta F508-CFTR chloride channel gating. J Biol Chem 278: 35079-35085.[Abstract/Free Full Text]

This article has been cited by other articles:

				S. Noel, M. Wilke, A. G. M. Bot, H. R. De Jonge, and F. Becq Parallel Improvement of Sodium and Chloride Transport Defects by Miglustat (n-Butyldeoxynojyrimicin) in Cystic Fibrosis Epithelial Cells J. Pharmacol. Exp. Ther., June 1, 2008; 325(3): 1016 - 1023. [Abstract] [Full Text] [PDF]

				R. Robert, G. W. Carlile, C. Pavel, N. Liu, S. M. Anjos, J. Liao, Y. Luo, D. Zhang, D. Y. Thomas, and J. W. Hanrahan Structural Analog of Sildenafil Identified as a Novel Corrector of the F508del-CFTR Trafficking Defect Mol. Pharmacol., February 1, 2008; 73(2): 478 - 489. [Abstract] [Full Text] [PDF]

				C. Routaboul, C. Norez, P. Melin, M.-C. Molina, B. Boucherle, F. Bossard, S. Noel, R. Robert, C. Gauthier, F. Becq, et al. Discovery of {alpha}-Aminoazaheterocycle-Methylglyoxal Adducts as a New Class of High-Affinity Inhibitors of Cystic Fibrosis Transmembrane Conductance Regulator Chloride Channels J. Pharmacol. Exp. Ther., September 1, 2007; 322(3): 1023 - 1035. [Abstract] [Full Text] [PDF]

This Article Abstract Full Text (PDF)