1 Laboratoire de Physiologie des Régulations Cellulaires, Unité Mixte de Recherche 6558, 86022 Poitiers, and 2 Laboratoire de Chimie Organique, Faculté de Médecine et de Pharmacie, 86005 Poitiers, France; 3 Laboratorio di Genetica Molecolare, Istituto G. Gaslini, Genoa 16148, Italy; 4 Durham Veterans Affairs and Duke University Medical Center, Durham, North Carolina; and 5 Kinderklinik im Dr. von Haunerschen Kinderspital, Ludwig-Maximilians-Universität, Munich, Germany
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ABSTRACT |
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We have designed and synthesized benzo[c]quinolizinium derivatives and evaluated their effects on the activity of G551D cystic fibrosis transmembrane conductance regulator (CFTR) expressed in Chinese hamster ovary and Fisher rat thyroid cells. We demonstrated, using iodide efflux, whole cell patch clamp, and short-circuit recordings, that 5-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 G551D NBD1/R/GST fusion proteins or on the ATPase, GTPase, and adenylate kinase activities of purified NBD2. The activation of CFTR by MPB-91 is independent of phosphorylation because 1) kinase inhibitors have no effect and 2) the compound still activated CFTR having 10 mutated protein kinase A sites (10SA-CFTR). The new pharmacological agent MPB-91 may be an important candidate drug to ameliorate the ion transport defect associated with CF and to point out a new pathway to modulate CFTR activity.
pharmacology; disease-causing mutation; cystic fibrosis; nucleotide binding domains; cystic fibrosis transmembrane conductance regulator
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INTRODUCTION |
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CYSTIC FIBROSIS (CF), the most common lethal autosomal recessive genetic disease, is caused by mutations of the CF gene (25, 33), which normally encodes a multifunctional and integral apical membrane chloride channel: the CF transmembrane conductance regulator (CFTR) (25). CFTR mediates chloride transepithelial transport (7, 13, 29), and is activated by phosphorylation (4, 7, 31) and gated by ATP hydrolysis (for review, see Refs. 10 and 15). More than 900 CF mutations are known (www.genet.sickkids.on.ca), and a lot of them generate defective chloride transport and perturb the quantity and composition of epithelial fluids. This results in the manifestations of the disease, which include airway obstruction and infection, pancreatic failure, male infertility, and elevated levels of salt in sweat (reviewed in Ref. 12).
The main mutations are a deletion of the phenylalanine at
position 508 (F508, class II mutation), which represents
~66% of the mutated chromosomes, and a glycine-to-aspartic acid
missense mutation at codon 551 (G551D, class III mutation) with a
frequency of 2-5% (33). Both mutations are located
in the first nucleotide binding domain (NBD1) (21), but,
whereas
F508-CFTR is not correctly addressed toward the
membrane (8, 13), the processing, maturation, and assigned
apical location of G551D CFTR are not affected (30, 33).
However, in cells expressing the G551D mutant, chloride transport
cannot be activated by cAMP-elevating agents (1, 11, 33).
Although the regulatory domain of CFTR could be normally phosphorylated
(6), a decreased nucleotide binding (21) and ATPase activity at NBD1 (14, 19) have been demonstrated.
We have recently shown that benzo[c]quinolizinium compounds (MPB-07 and MPB-27) are activators of wild-type CFTR (2). We have further performed a structure-activity study and identified 5-butyl-6- hydroxy-10-chlorobenzo[c]quinolizinium chloride (MPB-91), a compound able to restore chloride secretion in G551D and wild-type CFTR-expressing cells. The role of ATPase activity and phosphorylation in the MPB-dependent activation of CFTR was investigated.
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METHODS |
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MPB-91.
The methods for chemical synthesis of MPB-91 were similar to those
previously described (2). The starting material,
2-pentylpyridine, was obtained by condensation of 2-picolyllithium with
1-bromobutane. Then 4.48 g of 2-pentylpyridine (0.03 mol) in
tetrahydrofuran (30 ml) were metalated at 0°C with lithium
diisopropylamide (0.033 mol) and cooled to 40°C, and 3.08 g of
methyl-2,3-dichlorobenzoate (0.015 mol) in tetrahydrofuran (15 ml) were
added. The mixture was stirred for 1 h at
40°C and hydrolyzed
at 20°C with 10 ml of water. The product was warmed to 200°C for
1 h. The residue was chromatographed to obtain a cream powder
[melting point = 160°C, yield = 15%; analysis of
C17H17N1O1Cl2:
C, 63.37; H, 5.32; N, 4.35; found: C, 63.36; H, 5.27; N, 4.32; infrared
(KBr): 3439, 3121, 2951, 2923, 2895, 2863, 2496, 2344, 1629, 1589, 1502, 1457, 1488, 1400; 1H-NMR
(Me2SO-d6):
9.25 (d, J = 7 Hz, 1H, H1), 8.5 (d, J = 7 Hz, 1 H), 8.1-6.9 (m, 5H + OH), 2.9 (t, J = 4 Hz, 2H, CH2), 1.5-1.3 (m, 4H,
2 CH2), 0.9 (t, 3H, CH3); mass spectrum (ion
chromatography mass spectrometry): 286 (M-HCl)].
Cell culture.
Chinese hamster ovary (CHO) cells stably transfected with pNUT vector
alone (CHO pNUT) or containing wild-type CFTR [CFTR(+) CHO], G551D
CFTR (G551D CHO), or 10SA CFTR (10SA CHO) were provided by J. R. Riordan and X.-B. Chang (Scottsdale, AZ) (6, 31). Cells
cultured at 37°C in 5% CO2 were maintained in MEM
containing 7% fetal bovine serum, 0.5% L-glutamine, 0.5%
antibiotics (50 IU/ml penicillin and 50 µg/ml streptomycin), and
20-100 µM methotrexate. For detailed procedures see elsewhere
(2, 32). Calu-3 (American Type Culture Collection), a cell
line of human pulmonary origin (9), was cultured at 37°C
in 5% CO2 and maintained in DMEM Ham's F-12 Nutritif Mix
(1:1) supplemented by 10% FCS and 1% antibiotics (50 IU/ml penicillin
and 50 µg/ml streptomycin).
Iodide efflux experiments and patch-clamp experiments.
CFTR chloride channel activity was assayed at 37°C as previously
described (2, 5) by measuring the rate of iodide
(125I) efflux (5, 32) from CHO and Calu-3
cells cultured in multiwell plates to perform parallel experiments and
comparison analysis. Briefly, the fraction of initial intracellular
125I lost during each time point was determined, and
time-dependent rates of 125I efflux were calculated from
ln(125It1/125It2)/(t1 t2), where
125It is the intracellular
125I at time t, and t1
and t2 are successive time points
(32). Curves were constructed by plotting rate of
125I vs. time. All comparisons were based on maximal values
for the time-dependent rates (peak rates), excluding the points used to establish the baseline (5). In Fig. 6 the relative rates
(r) correspond to r peak/r basal. In
experiments in which the chloride transport inhibitors DIDS,
glibenclamide, DPC, and Calixarene or the protein kinase
inhibitors
N-(2-[p-bromocinnamylamino]ethyl)- 5-isoquinolinesulfonamide
(H-89) and
1,2-dimethoxy-N-methyl-[1,3]-benzodioxolo[5,6-c]phenantridinium chloride (chelerythrine chloride) were used, these agents were present
in the loading solution and in the efflux buffer as indicated in
RESULTS.
Ussing chamber experiments in G551D Fisher rat thyroid cells. The plasmid vector for expression of human CFTR gene in Fisher rat thyroid (FRT) cells was constructed by cloning the CFTR cDNA encoding sequence in the expression vector pTracer-cytomegalovirus (CMV; Invitrogen). Expression of the CFTR gene is controlled by the CMV promoter, whereas synthesis of the green fluorescent protein-Zeocin fusion protein is controlled by the SV40 promoter. Site-directed mutagenesis was done using the GeneEditor kit (Promega). FRT cells were cultured on 60-mm petri dishes with Coon's modified F-12 medium containing 5% serum, 2 mM L-glutamine, 50 U/ml penicillin, and 50 µg/ml streptomycin. Cells were stably transfected using 10 µl Effectene (Qiagen) with 1 µg of the construct according to the manufacturer's instructions. Clones were selected and maintained in 800 or 600 µg/ml Zeocin, respectively.
For Ussing chamber experiments, cells were seeded at high density (5 × 105 cells/cm2) on Snapwell inserts (Costar, Corning), maintained at 37°C, and gassed with 5% CO2-95% air. The medium was replaced every 48 h. Transepithelial resistance was measured daily with an epithelial voltohmeter (Millipore-ERS, Millipore) using chopstick-like electrodes. After 4-7 days, FRT monolayers developed a transepithelial resistance in the range of 2-4 kATPase assays of fusion protein. Production of the fusion protein NBD1/R/GST and corresponding enzyme assays were performed as previously described (14, 22, 27). A second fusion protein (MBP/NBD2) of the maltose-binding protein (MBP) and the human CFTR protein sequence from Gly-1208 to Leu-1399 (numbering according to Ref. 25), enclosing the whole predicted second nucleotide binding domain of CFTR (NBD2), was expressed, purified, and characterized in Escherichia coli (23, 24); all enzymatic assays were performed as described in Refs. 23 and 24.
Chemicals.
Forskolin, 1,9-dideoxyforskolin,
8-(4-chlorophenylthio)adenosine-3',5'-cyclic monophosphate (CPT-cAMP),
glibenclamide, DPC, DIDS, H-89, and chelerythrine chloride were from
Sigma Chemical (St. Louis, MO).
5,11,17,23-Tetrasulfonato-25,26,27,28-tetramethoxy-calix[4]arene (TS-TM calix[4]arene) was a generous gift of
Drs. Singh and Bridges (University of Pittsburgh, Pittsburgh). All
other products were from Sigma, except MEM and DMEM/Ham Nutritif Mix
F-12, which were from Fisher PAA and GIBCO BRL.
Statistics. Results are expressed as means ± SE of n observations. To compare sets of data, we used either an ANOVA or Student's t-test. Differences were considered statistically significant when P < 0.05 [nonsignificant (NS) difference was P > 0.05]. All tests were performed using GraphPad Prism version 3.0 for Windows (GraphPad Software, San Diego, CA).
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RESULTS |
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Activation of wild-type CFTR channels by MPB-91 in Calu-3 and
CFTR(+) CHO cells.
We previously found that several substitutions within the
benzo[c]quinolizinium (MPB) skeleton are crucial for CFTR activation (2). These are the hydroxyl at position 6 and the chlorine atom at positions 7 (MPB-27) or 10 (MPB-07). Here we performed chemical
modifications of the MPB-07 structure by keeping the 10-chlorine and
6-hydroxyl substituents and changing the substituent at position 5. Through a screening process using a cell-based iodide efflux assay, we
identified the 5-butyl-6-hydroxy-10-chlorobenzo[c]quinolizinium derivative named MPB-91 (Fig.
1A) and found that it
activates wild-type CFTR in CFTR(+) CHO cells and in the human
airway epithelial Calu-3 cell lines (Fig. 1, B, C, and
F). Relative to control rates in the absence of agonists
(i.e., in the presence of DMSO alone), rates of MPB-91-dependent iodide
efflux were stimulated in CFTR(+) CHO cells 1.93 ± 0.15-fold
(n = 8, P < 0.01) and in Calu-3 cells 1.91 ± 0.12-fold (n = 20, P < 0.001). An EC50 of 46.7 ± 1.3 µM (n = 3) was determined in iodide efflux experiments on Calu-3 cells. We
found it necessary to study the additive stimulatory effect of
forskolin and MPB-91 in CFTR(+) CHO cells, since the effect on G551D
CFTR had been tested in the same cell line. In CFTR(+) CHO cells
stimulated by low concentrations of forskolin (500 nM, peak rate
0.20 ± 0.02 min1, n = 4, vs.
control without forskolin: 0.01 ± 0.01 min
1,
n = 4, P < 0.01), 250 µM MPB-91
further stimulated CFTR (peak rates: MPB-91 + forskolin, 0.33 ± 0.01 min
1, n = 4, P < 0.01; Fig. 1E). Similar results were observed in Calu-3
cells (not shown). Additional control experiments have been performed
on cells that do not express CFTR (CHO pNUT; Fig. 1D).
MPB-91 at 250 µM failed to stimulate any significant iodide efflux
(relative rate: 1.11 ± 0.14 min
1, n = 8), even in the presence of forskolin (not shown). Figure 1F presents peak rates of MPB-91-induced iodide efflux in
each cell line vs. control condition.
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MPB-91 but not MPB-07 is an activator of G551D CFTR.
Because little is known about the pharmacology of G551D CFTR, we
determined the effects of MPB-07 and MPB-91 on the activity of G551D
expressed in CHO cells using iodide efflux and whole cell patch-clamp
methods. Control experiments confirmed that forskolin indeed failed to
stimulate iodide efflux from G551D-CFTR-expressing cells (peak
rate-to-basal: 0.10 ± 0.01 min1,
n = 14; 10 µM forskolin: 0.10 ± 0.01 min
1, n = 14). We found that MPB-07 (250 µM) either alone (peak rate: 0.09 ± 0.01 min
1,
n = 4) or in combination with 10 µM forskolin (peak
rate: 0.09 ± 0.01 min
1, n = 4)
failed to stimulate the iodide efflux in G551D cells. In contrast, the
substituted compound MPB-91 (250 µM) dramatically stimulated iodide
efflux in the presence of forskolin (peak rate with 250 µM MPB-91:
0.21 ± 0.01 min
1, n = 52; peak rate
without MPB-91: 0.09 ± 0.01 min
1, n = 14, P < 0.001). This efflux was inhibited by 61%
and 68% with, respectively, 100 µM glibenclamide (P < 0.001, n = 8) or 250 µM DPC (P < 0.001, n = 8), but was not affected by 200 µM DIDS
(NS, n = 8). When added simultaneously with the
inactive form of forskolin, 1,9-dideoxyforskolin (10 µM,
n = 4), MPB-91 did not stimulate iodide efflux, whereas
in the presence of CPT-cAMP (200 µM, n = 4), iodide
efflux was recovered (not shown).
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Role of ATPase activities at both NBD1 and NBD2 in the activation
of CFTR by MPB-91.
Because NBD1 and NBD2 can bind and hydrolyze ATP to gate the CFTR
channel (10), we investigated the effect of MPB-91 on both
NBD1 and NBD2 ATPase activities. First, wild-type and G551D NBD1/R/GST
ATPase activities were measured at their respective Michaelis-Menten
constant (Km) values (70 and 250 µM; Ref.
14) and at 1 mM ATP. G551D NBD1 has a significantly
reduced ATPase activity compared with wild-type NBD1 (Fig.
5A) as expected from previous
studies (14, 19). MPB-91 does not affect the ATPase activity of wild-type or G551D NBD1 at any ATP and MPB-91
concentrations tested. Figure 5A shows results obtained with
MPB-91 at 1 mM. We also determined the enzymatic activities of a
recombinant NBD2 polypeptide fused to maltose-binding protein
(23, 24). MPB-07, MPB-27, and MPB-91 (at 200 µM) did not
influence ATPase activity, as shown Fig. 5B. A similar lack
of effect was determined for GTPase and adenylate kinase activities of
MBP/NBD2 (Table 1). Concerning MBP/NBD2
ATPase activity, they neither affected the Km
for ATP (Km values: control, 783 ± 28 µM; MPB-07, 756 ± 16 µM; MPB-27, 738 ± 86; MPB-91,
740 ± 40 µM; n 2 for each condition), which,
under the assumption of an equilibrium-binding model, mainly reflects
the affinity of MBP/NBD2 for ATP, nor the catalytic rates (Vmax: control, 0.95 ± 0.01; MPB-07,
0.94 ± 0.01; MPB-27, 0.93 ± 0.03; MPB-91, 0.92 ± 0.01 µmol ATP · min
1 · µmol
MPB
1; n
2 for each condition).
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Effect of protein kinase A and protein kinase C inhibitors.
To determine whether wild-type and G551D CFTR activation by MPB-91
involved phosphorylation of the channel, the effects of two kinase
inhibitors were investigated. H-89 (10 µM) and chelerythrine chloride
(1 µM) were chosen as potent inhibitors of protein kinase A (PKA) and
protein kinase C (PKC), respectively. Because our multiwell assay for
CFTR activity allowed us to perform parallel experiments, the effect of
kinase inhibitors was conducted in paired experiments including both
forskolin and MPB-91 as agonists. We first investigated the effect of
kinase inhibitors on CFTR activity in Calu-3 and CFTR(+) CHO cells. As
expected, inhibition of forskolin-induced CFTR-mediated efflux was
observed in the presence of H-89 in both CFTR(+) CHO and Calu-3 cells
(85% and 90%, respectively, P < 0.001, n = 8 for each experiment; Fig. 6, A and B). We
also observed inhibition with chelerythrine chloride, which was found
more efficiently on CFTR(+) CHO cells than on Calu-3 cells [54%
inhibition (P < 0.01) vs. 20% inhibition
(P < 0.01), n = 8 for each condition,
Fig. 6, A and B]. In contrast, results presented
in Fig. 6C showed the lack of significant effect of both
kinase inhibitors on MPB-91-stimulated iodide efflux in Calu-3 cells
(P > 0.05, for both inhibitors). Similar results were
observed in CFTR(+) CHO cells (not shown). Finally, experiments were
performed on G551D CFTR cells exposed to forskolin (10 µM) and MPB-91
(250 µM). We observed <1% inhibition using H-89, which was not
different from control without H-89 (P > 0.05, n = 8). Chelerythrine chloride affected 22% of efflux
stimulated by forskolin + MPB-91 (P < 0.01, n = 8, Fig. 6D). MPB-91 was also tested on CHO cells having 10 PKA consensus sites converted into alanine (10SA
CHO cells). Chang et al. (6) reported that, with
the 10SA-CFTR mutant, no [32P]orthophosphate could be
incorporated in the presence of PKA, although a moderate CFTR channel
activity could still be recorded. Accordingly, we observed an
attenuated iodide efflux in the presence of a high dose (10 µM) of
forskolin (peak rate: 0.21 ± 0.02 min1,
n = 8, vs. with 5 µM forskolin on wild-type CFTR:
0.46 ± 0.03 min
1, n = 8, P < 0.01). However, when 250 µM MPB-91 was added to
the 10SA-CFTR cell preparation, a significant stimulation of the iodide efflux was still observed in eight of eight experiments (Fig. 7) with a peak rate value (0.195 ± 0.01 min
1, n = 8) similar to that of
MPB-91 on wild-type CFTR (compare with Fig. 1F).
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DISCUSSION |
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The development of specific CFTR modulators (2, 3, 5, 17, 26) is of key importance for our understanding of the role of chloride channels in the physiology of epithelia and in other tissues including heart and nephron. It is also crucial for the physiopathology of CF, chronic obstructive pulmonary disease, and other diseases of electrolyte transport.
Activation of CFTR chloride channel by MPB-91. Our study first illustrated that investigation of the structure-activity relationship (SAR) of CFTR modulators may lead to the discovery of more potent compounds. Indeed, we found that MPB-07, which is able to activate wild-type CFTR (2), is not effective as a G551D CFTR activator. Modification of the chemical structure using SAR identified an important position within the MPB skeleton, leading to MPB-91, a compound that kept its ability to activate wild-type CFTR but more importantly acquired the property to activate G551D CFTR. We report here that this compound modulates chloride secretion in airway epithelial cells and, in a recombinant system, activates the CFTR chloride channel. The chloride transport observed in Calu-3 cells by iodide efflux and by patch-clamp experiments presents all the characteristics of CFTR (i.e., kinetics properties and pharmacological profile). We demonstrated that MPB-91 restores CFTR-mediated chloride secretion in cells expressing G551D CFTR. The inhibitory profile of G551D CFTR channel activity is similar to that described for wild-type CFTR (13, 26).
Role of NBD1 domains in MPB-91-mediated CFTR activation. It is generally accepted that ATP binding and hydrolysis are crucial steps leading to CFTR chloride channel activation (10). Electrophysiological data indicates that hydrolysis at NBD1 is a prerequisite to channel opening, whereas ATP hydrolysis at NBD2 is implicated in channel closure (10). However, it has been recently reported that ATP binding alone could be sufficient to promote CFTR opening (15). Because ATP hydrolysis at NBD1 has been linked to CFTR activation, we initially hypothesized that MPB-91 could modulate wild-type ATPase and/or could restore normal G551D ATPase function (e.g., by reducing the Km value for ATP). To study this, we first measured the ATPase activity of wild-type NBD1 in the presence of MPB-91. Our results clearly demonstrated that MPB-91 did not compete with ATP binding, did not modulate ATPase activity, and had no significant effect on reduced ATPase activity of G551D NBD1. We then concluded that the mutation G551D interferes with the mechanism of channel opening that is dependent of ATP hydrolysis at NBD1. Because the compound MPB-91 was found to be able to activate wild-type CFTR and G551D CFTR without directly affecting NBD1 ATPase activity, these observations strongly suggest that an alternative pathway of activation exists that bypasses the defective ATPase activity of G551D CFTR proteins.
Role of NBD2 domains in MPB-91-mediated CFTR activation. Recent studies have proposed that genistein could activate CFTR (16) by a direct binding to NBD2 associated to inhibition of ATPase activity, which in turn prolongs the open time of the channel (23). We performed an analysis of ATPase activity at NBD2 and showed that MPB-91 (and other MPB derivatives) affected neither Vmax nor Km of the reaction. This clearly indicated that our compounds did not activate CFTR by interfering with NBD2 (and NBD1) ATPase activity, although we cannot exclude that these compounds act on NBD by interactions that could change the conformation of the channel.
Our results also demonstrated an important difference between MPB-91 and genistein. For example, these two drugs are both able to activate wild-type and G551D CFTR, but probably through two distinct mechanisms. At least two different classes of activators of CFTR began to emerge: those (like genistein) opening CFTR (16) and altering its ATPase activity at NBD2 (23) but not at NBD1 (14) and another class of compounds (like MPB) opening CFTR without affecting its enzymatic activity (this report). It is interesting to note that none of the modulators of CFTR studied so far interacted with ATPase activity at NBD1 (14). It may be speculated that compounds interfering with the ATPase activity of NBD1 would be inhibitors of CFTR channel activity.Role of phosphorylation in the mechanism of activation of CFTR by MPB-91. One major difference currently observed between wild-type and G551D CFTR is the role of phosphorylation. In wild-type CFTR cells, stimulation of the cAMP pathway is sufficient to open CFTR chloride channels (10, 29). Although G551D CFTR could be normally phosphorylated (11) and despite the fact that phosphorylation by itself is not sufficient to activate G551D CFTR (11, 33), it is required for CFTR to be opened by MPB-91 and other compounds like genistein (17). Thus one hypothesis is that MPB-91 directly interacts with the phosphorylated protein itself rather than with associated kinases or phosphatases. Indeed, we have shown that MPB compounds have no effect on various phosphatases or kinases implicated in CFTR activation (2). In addition, MPB-91 does not alter the cAMP levels in both Calu-3 and G551D CHO cells (T. Métayé, R. Dérand, L. Bulteau, and F. Becq, unpublished data). Although activity of CFTR is critically dependent of its phosphorylation status (18, 20, 31), MPB-91 activates CFTR in low phosphorylation conditions (low concentrations of forskolin), in variants having 10 mutated PKA sites, and even in the presence of kinase inhibitors. In the presence of H-89 or chelerythrine chloride (to prevent CFTR phosphorylation), MPB-91 still activates CFTR channels, probably through a phosphorylation-independent pathway.
In G551D CHO cells we found, however, that MPB-91 alone is not sufficient to stimulate the chloride current. A similar effect was observed using genistein (17). However, once phosphorylated, G551D CFTR can be activated by MPB-91. Surprisingly, when H-89 or chelerythrine chloride were added, we never observed an inhibition of the CFTR activity. These results suggest that phosphorylation of G551D CFTR may act only as a switch, making the mutated protein responsive or not to MPB-91. On the basis of all our results, we believe that within the CFTR architecture, a binding site for MPB-91 is unmasked following phosphorylation. When MPB-91 occupies the putative site, inhibiting the kinases (PKA or PKC) has no effect. Further experiments are needed to clear this point. In conclusion, in this report we have presented the potential interest of MPB-91 as a putative drug in CF, since it not only modulates wild-type CFTR but also activates CFTR having the disease-causing mutation G551D. We previously predicted that the benzo[c] quinolizinium compounds were good candidates for pharmacological CFTR activation in airways (2). By designing new derivatives based on the MPB-07 skeleton, we have identified an important site on the MPB skeleton (position 5) and have obtained a new compound that is more potent on CFTR channel activity (normal and mutated). The pathway to activate CFTR with MPB drugs appears to be different (no direct effect on NBD domains) from that involved with genistein. In addition, studying ATPase dependence of CFTR activation with a given modulator may thus reveal novel mechanisms to control the channel opening, which may help to better understand the underlying molecular mechanism leading to activation of normal and mutated CFTR. ![]() |
ACKNOWLEDGEMENTS |
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We thank Dr. P. Fanen from Institut National de la Santé et de la Recherche Médicale U468, Créteil, France, for plasmid construction. CHO cells were a generous gift of J. R. Riordan and X.-B. Chang, Scottsdale, AZ.
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FOOTNOTES |
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This work was supported by an Association Française de Lutte contre la Mucoviscidose Grant to R. Dérand and L. Bulteau-Pignoux, a National Institutes of Health Grant to J. A. Cohn, a Cystic Fibrosis Foundation Grant to L. D. Howell, and a Deutsche Forschungsgemeinschaft Grant to C. Randak.
Address for reprint requests and other correspondence: F. Becq, Laboratoire de Physiologie des Régulations Cellulaires, UMR 6558, 40 Ave. du Recteur Pineau, 86022 Poitiers, France (E-mail: frederic.becq{at}univ-poitiers.fr).
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.
Received 19 April 2001; accepted in final form 16 July 2001.
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