Stimulation of Wild-Type, F508del- and G551D-CFTR Chloride Channels by Non-Toxic Modified pyrrolo[2,3-b]pyrazine Derivatives

Article (PDF Available)inFrontiers in Pharmacology 2:48 · August 2011with25 Reads
DOI: 10.3389/fphar.2011.00048 · Source: PubMed
Abstract
Cystic fibrosis (CF) is a major inherited disorder involving abnormalities of fluid and electrolyte transport in a number of different organs due to abnormal function of cystic fibrosis transmembrane conductance regulator (CFTR) protein. We recently identified a family of CFTR activators, which contains the hit: RP107 [7-n-butyl-6-(4-hydroxyphenyl)[5H]-pyrrolo[2,3-b]pyrazine]. Here, we further evaluated the effect of the chemical modifications of the RP107-OH radical on CFTR activation. The replacement of the OH radical by a fluorine atom at position 2 (RP193) or 4 (RP185) significantly decreased the toxicity of the compounds without altering the ability to activate CFTR, especially for RP193. The non-toxic compound RP193 has no effect on cAMP production but stimulates the channel activity of wild-type CFTR in stably transfected CHO cells, in human bronchial epithelial NuLi-1 cells, and in primary culture of human bronchial epithelial cells (HBEC). Whole-cell and single patch-clamp recordings showed that RP193 induced a linear, time- and voltage-independent current, which was fully inhibited by two different and selective CFTR inhibitors (CFTRinh-172 and GP(inh)5a). Moreover, RP193 stimulates CFTR in temperature-rescued CuFi-1 (F508del/F508del) HBEC and in CHO cells stably expressing G551D-CFTR. This study shows that it is feasible to reduce cytotoxicity of chemical compounds without affecting their potency to activate CFTR and to rescue the class 2 F508del-CFTR and class 3 G551D-CFTR CF mutant activities.
ORIGINAL RESEARCH ARTICLE
published: 23 August 2011
doi: 10.3389/fphar.2011.00048
Stimulation of wild-type, F508del- and G551D-CFTR
chloride channels by non-toxic modified
pyrrolo[2,3-b]pyrazine derivatives
Luc Dannhoffer *, Arnaud Billet, Mathilde Jollivet, Patricia Melin-Heschel, Christelle Faveau and
Frédéric Becq
Institut de Physiologie et Biologie Cellulaires, UMR 6187, Université de Poitiers, CNRS, Poitiers, France
Edited by:
Jean-François Desaphy, University of
Bari Aldo Moro, Italy
Reviewed by:
Giovanni Zifaelli, Institute of
Biophysics, National Research
Council, Italy
Marc Chanson, University of Geneva,
Switzerland
*Correspondence:
Luc Dannhoffer, Institut de
Physiologie et Biologie Cellulaires,
UMR 6187, Université de Poitiers,
CNRS, Pôle Biologie Santé Bât B36,
BP 633, 1 rue Georges Bonnet, 86022
Poitiers, France.
e-mail: luc.dannhoffer@ext.
univ-poitiers.fr
Cystic fibrosis (CF) is a major inherited disorder involving abnormalities of fluid and elec-
trolyte transport in a number of different organs due to abnormal function of cystic
fibrosis transmembrane conductance regulator (CFTR) protein. We recently identified a
family of CFTR activators, which contains the hit: RP107 [7-n-butyl-6-(4-hydroxyphenyl)[5H]-
pyrrolo[2,3-b]pyrazine]. Here, we further evaluated the effect of the chemical modifications
of the RP107-OH radical on CFTR activation. The replacement of the OH radical by a flu-
orine atom at position 2 (RP193) or 4 (RP185) significantly decreased the toxicity of the
compounds without altering the ability to activate CFTR, especially for RP193. The non-
toxic compound RP193 has no effect on cAMP production but stimulates the channel
activity of wild-type CFTR in stably transfected CHO cells, in human bronchial epithelial
NuLi-1 cells, and in primary culture of human bronchial epithelial cells (HBEC). Whole-
cell and single patch-clamp recordings showed that RP193 induced a linear, time- and
voltage-independent current, which was fully inhibited by two different and selective CFTR
inhibitors (CFTRinh-172 and GP
inh
5a). Moreover, RP193 stimulates CFTR in temperature-
rescued CuFi-1 (F508del/F508del) HBEC and in CHO cells stably expressing G551D-CFTR.
This study shows that it is feasible to reduce cytotoxicity of chemical compounds without
affecting their potency to activate CFTR and to rescue the class 2 F508del-CFTR and class
3 G551D-CFTR CF mutant activities.
Keywords: cystic fibrosis, CFTR, activator and inhibitor, ion transport, epithelial cells
INTRODUCTION
Cystic fibrosis (CF; MIM#219700) disease results from mutations
in the gene encoding the cystic fibrosis transmembrane con-
ductance regulator (CFTR) protein. CFTR, which is especially
expressed at the apical membrane of the airway epithelial cells
(Kreda et al., 2005; Regnier et al., 2008), acts as cAMP-activated
chloride channel as well as a regulator of other ion channels like
the canonical Transient Receptor Potential 6, TRPC6 (Antigny
et al., 2011). In the lung, CFTR dysfunctions carry abnormal ion
transports leading to airway surface liquid volume depletion in
proximal and distal airways allowing chronic bacterial infections
and finally severe lung damages (Boucher, 1999; Tarran, 2004;
Blouquit et al., 2006).
Over1600 mutations of the CFTR gene havebeen identified and
several classes of mutations according to the fate of the final prod-
ucthavebeenproposed(Welsh and Smith, 1993). Based on this
classification, it is in theory possible to predict a strategy for devel-
oping a personalized CF therapy corresponding to the class of the
CFTR mutation (Becq, 2010). The majority of severe CFTR muta-
tions belongs to class 1–3 leading to reduced chloride permeability
in affected epithelia in response to cAMP agonists. Class 1 includes
non-sense, frame shift, and splice site mutations (e.g., stop muta-
tions such as G542X) with unstable transcripts and failure of CFTR
translation. Class 2 includes the most common CFTR mutation
F508del. The biogenesis of F508del mutants is extremely ineffi-
cient with its retention in the endoplasmic reticulum (ER) but it
is feasible to rescue the abnormal trafficking by in vitro cooling
CF cells or pharmacological correctors (reviewed in Becq, 2010).
Importantly, sufficient amount of rescued F508del-CFTR at the
plasma membrane conduct chloride ions despite a gating defect
(Dalemans et al., 1991; Li et al., 1993; Dormer et al., 2005). Patients
with a class 3 mutation of CFTR (e.g., G551D, G1349D) have also a
severe form of CF, because they do not have or reduced activation
of the CFTR chloride function at the plasma membrane. Class 4
mutants present defective permeation, class 5 mutants a reduced
synthesis, and class 6 is characterized by altered apical membrane
residence time of CFTR channels at the membrane (Kreindler,
2010).
Such classification could be very helpful for mutation-oriented
search of personalized CFTR modulators. For example, the identi-
fication of pharmacological correctors of the processing defect of
CFTR could be developed only for patients diagnosed with class 2
CFTR mutations such as carrying at least one F508del mutation.
Agents directly stimulating the ion channel activity of CFTR might
be adapted to class 3 or 4 mutations and to class 2 in combination
with a trafficking corrector.
www.frontiersin.org August 2011 | Volume 2 | Article 48 | 1
Dannhoffer et al. RP193 stimulates CFTR in human epithelia
Our groupis interestedin developingnovelmodulators for class
2 and 3 CFTR defect activity. One family of CFTR modulators,
the pyrrolo[2,3-b]pyrazines, which belong to a type of derivatives
initially described as cyclin-dependent kinase (CDK)/glycogen
synthase kinase-3 (GSK-3) inhibitors, which act by competing
with ATP for binding to the kinase active site, has been identi-
fied (Mettey et al., 2003; Noel et al., 2006). One compound of this
family, RP107 [7-n-butyl-6-(4-hydroxyphenyl)[5H]-pyrrolo[2,3-
b]pyrazine], has been more particularly investigated (Noel et al.,
2006) because we observed that the length of the alkyl chain is
essential for CFTR activation with the following potency: (CH
2
)
3
-
CH
3
H
=
CH
3
=
(CH
2
)
2
-CH
3
=
(CH
2
)
4
-CH
3
. Based on these
observations, we decided to conserve the butyl chain in R1 posi-
tion and to modify only determinants on the phenyl ring. Four
different compounds (RP146, 173, RP185, and RP193) have been
synthesized and their effect on CFTR activity measured.
MATERIALS AND METHODS
CHEMISTRY
1
H NMR spectra were recorded on a Bruker Advance DPX 300
spectrometer at 300 mHz. Chemical shifts (ppm) were reported
relative to tetramethylsilane (TMS). Coupling constants (J )were
reported in Hertz (Hz), and s, d, t, q, m, and bs referred to singlet,
doublet, triplet, quartet, multiplet, and broad singlet, respectively.
Infrared spectra (IR) were recorded on an ATI Mattson genesis
series FTIR. Elemental analyses were indicated by the symbol of
the elements, and the results were within ±0.4% (for C, H, N)
of the theoretical values unless otherwise noted; they were per-
formed on a Perkin Elmer Elemental Analyzer (2400). Melting
points were measured in open capillary tubes on an Electrothermal
9200 apparatus and are uncorrected.
All experiments involving butyllithium (BuLi) were carried out
in dried apparatus under an atmosphere of dry oxygen-free nitro-
gen. Tetrahydrofuran (THF) was distilled from benzophenone–
sodium. Diisopropylamine and heterocycles were distilled and
stored over barium oxide. Butyllithium (2.5 M solution in hexane)
was supplied by Acros. Alkylpyrazines and alkylpyridines were
prepared according to usual procedures. Sodium hydride was an
95% dispersion in mineral oil. Matrex silica gel 60 Å, 20–45 μm,
Merck alumina gel 90 Å, 63–200 μm were employed for column
chromatographies.
GENERAL PROCEDURE FOR THE PREPARATION OF 4,7-DIAZAINDOLES
These compounds were prepared as previously described (Mettey
et al., 2003). Physical and spectral data for compound RP108 has
been reported (Mettey et al., 2003; Noel et al., 2006).
7-n-butyl-6-(4-fluorophenyl)-5H-pyrrolo[2,3-b]pyrazine (RP185)
Yield 18%; white cotton; mp 161.9˚C; IR (KBr, cm-1) 3164, 3053,
2958, 2851; 1H NMR (CDCl3) δ (ppm) 11.63 (bs, 1H), 8.46 (s,
1H), 8.05 (s, 1H), 7.70–7.74 (m, 2H), 7.29 (t, J = 8.1 Hz, 2H), 2.99
(t, J = 7.7 Hz, 2H), 1.71–1.79 (m, 2H), 1.39–1.46 (m, 2H), 0.92 (t,
J = 7.2 Hz, 3H). Anal. (C16H16N3F) C, H, N.
7-n-butyl-6-(2-fluorophenyl)-5H-pyrrolo[2,3-b]pyrazine (RP193)
Yield 15%; white powder; mp 136.3˚C; IR (KBr, cm-1) 3131, 3058,
2959, 2853, 1469, 1224, 755; 1H NMR (CDCl3) δ (ppm) 10.81
(bs, 1H), 8.45 (s, 1H), 8.11 (s, 1H), 7.63 (t, J = 7.4 Hz, 1H),
7.46–7.53 (m, 1H), 7.26–7.37 (m, 2H), 2.93 (t, J = 7.8 Hz, 2H),
1.7–1.8 (m, 2H), 1.32–1.44 (m, 2H), 0.88 (t, J = 7.3 Hz, 3H). Anal.
(C16H16N3F) C, H, N.
GENERAL PROCEDURE FOR THE PREPARATION OF COMPOUNDS, FROM
THEIR METHOXY PRECURSORS
Demethylation was performed with HBr, according to our previ-
ously described method (Mettey et al., 2003). Physical and spectral
data for compound RP107 has been reported (Mettey et al., 2003;
Noel et al., 2006).
7-n-Butyl-6-(3-hydroxyphenyl)-5H-pyrrolo[2,3-b]pyrazine
hydrobromide (RP146)
Yield 71%; mp 196.0˚C (dec); IR (KBr, cm-1) 3249, 3092, 2954,
2924, 2859, 2729; 1H NMR (DMSO-d6) δ (ppm) 12.97 (s, 1H),
8.95 (bs, 1H), 8.63 (d, J = 3 Hz, 1H), 8.60 (d, J = 3 Hz, 1H),
7.50 (t, J = 7.9 Hz, 1H), 7.27 (d, J = 7.6 Hz, 1H), 7.26 (s, 1H),
7.05 (dd, J = 7.6 and 1.6 Hz, 1H), 3.01 (t, J = 7.7 Hz, 2H), 1.66–
1.95 (m, 2H), 1.38–1.51 (m, 2H), 0.97 (t, J = 7.4 Hz, 3H). Anal.
(C16H17N3O, HBr) C, H, N.
7-n-Butyl-6-(2-hydroxyphenyl)-5H-pyrrolo[2,3-b]pyrazine
hydrobromide (RP173)
Yield 80%; yellow powder; mp 243.0˚C (dec); IR (KBr, cm-1) 3072,
2951, 2639; 1H NMR (DMSO-d6) δ (ppm) 12.58 (bs, 1H), 9.95
(bs, 1H), 8.49 (d, J = 3 Hz, 1H), 8.45 (d, J = 3 Hz, 1H), 7.34–7.38
(m, 2H), 7.06 (d, J = 8.5 Hz, 1H), 6.98 (t, J = 7.5 Hz, 1H), 5.15 (bs,
1H), 2.76 (t, J = 7.6 Hz, 2H), 1.51–1.61 (m, 2H), 1.17–1.29 (m,
2H), 0.78 (t, J = 7.3 Hz, 3H). Anal. (C16H17N3O, HBr) C, H, N.
Elemental analyses
(RP173) Calcd for C16H17N3O, HBr: C, 55.18; H, 5.21; N, 12.07
Found: C, 55.02; H, 5.21; N, 12.02
(RP185) Calcd for C16H16N3F: C, 71.35; H, 5.99; N, 15.60
Found: C, 71.29; H, 5.99; N, 15.50
(RP193) Calcd for C16H16N3F: C, 71.35; H, 5.99; N, 15.60
Found: C, 71.13; H, 5.94; N, 15.58
CELL LINE CULTURE
All cell cultures were grown at 37˚C in 5% CO
2
in standard
culture conditions as follows. CHO cells stably transfected with
pNUT vector alone or containing wild-type CFTR (wt-CFTR-
CHO) and the mutant G551D-CFTR (Tabcharani et al., 1991;
Becq et al., 1994, 1999) were cultured in α-minimal essen-
tial medium-GlutaMAX containing 7% of fetal bovine serum,
50 IU/ml penicillin and 50 μg/ml streptomycin, and methotrex-
ate for cell selection (wt-CFTR-CHO: 100 μM; G551D-CHO:
20 μM; pNUT-CHO: 20 μM). CuFi-1 and NuLi-1 cells derived
from human bronchial epithelium from a CF patient (CuFi-1,
F508del/F508del-CFTR mutant genotype) and a non-CF subject
(NuLi-1, wild-type CFTR). These cell lines were grown on human
placental collagen type VI (Sigma, St. Louis, MO, USA) coated
flasks in BEGM medium (Cambrex Bio Science Walkersville, MD,
USA), as previously described (Zabner et al., 2003). Cells were
seeded in 24-well plates for iodide efflux and in 35-mm plastic
dishes for whole-cell and cell-attached patch-clamp recordings.
Culture media were renewed every 2 days.
Frontiers in Pharmacology | Pharmacology of Ion Channels and Channelopathies August 2011 | Volume 2 | Article 48 | 2
Dannhoffer et al. RP193 stimulates CFTR in human epithelia
PRIMARY CULTURES OF HUMAN BRONCHIAL EPITHELIAL CELLS
Fragments of non-CF lungs were obtained from three patients
who underwent lung resection for cancer (three males, mean
age: 61.3 ± 5.5 years). Tissues were received and processed within
2 h after surgery. All procedures were performed in compliance
with the current French legislation. Human bronchi were dis-
sected free of lung parenchyma. Bronchial epithelial cells were
harvested using enzymatic isolation procedures to establish pri-
mary cultures, as previously described (Blouquit et al., 2002;
Dannhoffer et al., 2009). Briefly, fragment of bronchi were incu-
bated 24 h at 4˚C with 0.1% protease and 0.01% deoxyribonuclease
in DMEM/Hams F-12 medium. Enzymatic digestion was neu-
tralized by 10% fetal bovine serum; cells were then centrifuged
at 600 rpm for 6 min at room temperature (RT). Epithelial iso-
lated cells were seeded at high density on home-made permeable
collagen supports affixed to an orifice drilled in polycarbon-
ate cups (Blouquit et al., 2002; Dannhoffer et al., 2009). They
reached confluence between 8 and 12 days after seeding. The
culture medium consisted of a 1:1 mix of Dulbeccos modi-
fied Eagle’s medium-Hams F-12 medium supplemented with:
5 μg/ml insulin, 7.5 μg/ml transferrin, 10
6
M hydrocortisone,
2 μg/ml endothelial cell growth supplement, 25 ng/ml epithelial
growth factor, 3.10
8
M triiodothyronine, 2.5 mM l-glutamine,
and 100 IU/ml penicillin and 100 μg/ml streptomycin.
IODIDE EFFLUX
Screening of molecules and concentration response curves were
determined by measuring the rate of
125
I efflux with a high-
capacity robotic system (MultiProbe II EXT; PerkinElmer Life Sci-
ences) adapted to the determination of iodide efflux as described
previously (Marivingt-Mounir et al., 2004). Time-dependent rates
(k = peak rate, min
1
)of
125
I efflux were calculated from the fol-
lowing equation: k = ln (
125
I
t1
/
125
I
t2
)/(t
1
t
2
), where
125
I
t
is the
intracellular
125
I at time t and t
1
and t
2
are successive time points.
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 activa-
tion (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 by 1 μM forskolin
or by a cocktail containing 1 μM forskolin +100 μMRPderiva-
tives (146, 173, 185, or 193). The effect of RP193 100 μM without
forskolin on CFTR activity was also investigated.
CYTOTOXICITY ASSAY
To determine effects of compounds on wt-CFTR-CHO cells viabil-
ity, colorimetric 3-(4,5-dimethylthiazol-2-yl)2,5-diphenyltetra-
zolium bromide (MTT, Sigma-Aldrich) assay was performed as
previously described (Routaboul et al., 2007). Cell survival was
evaluated by measuring the mitochondrial-dependent reduction
of MTT. Cells seeded in 96-well plates were incubated during 24 h
in the presence of 100 μM of the test compound (RP107, RP108,
RP146, RP173, RP185, or RP193) at 37˚C, and the MTT test was
performed the day after. At each point of treatment, cells were
washed with PBS (in mM: 130 NaCl, 2 KCl, 1.5 Na
2
HPO
4
, and 8
KH
2
PO
4
, pH 7.4). The medium was removed and 100 μlofMTT
solution (0.5 mg/ml in PBS) were added to each well. After 4 h of
incubation at 37˚C, the supernatant was removed and the purple
formazan crystals were dissolved by addition of 100 μl dimethyl-
sulfoxide (DMSO, Sigma-Aldrich). The plates were agitated, and
the optical density was read at the wavelength of 570 nm and a
reference wavelength at 630 nm in a microplate reader (Spectra-
Count microplate photometer, Packard). Report values were the
mean of three replicates and are expressed as percentage of the
control values.
DETERMINATION OF cAMP
cAMP levels were measured using the cAMP-Screen ELISA System
(Applied Biosystems, Bedford, MA, USA). Wt-CFTR-CHO cells
were incubated with drugs for 10 min at 37˚C. Reactions were ter-
minated, processed, and quantitated according to manufacturer’s
instructions.
WHOLE-CELL PATCH-CLAMP RECORDINGS
Whole-cell patch-clamp experiments were performed on wt-
CFTR-CHO and K1-CHO, which did not expressed CFTR, cells at
RT. Currents were recorded with a RK-400 patch-clamp amplifier
(Biologic, Claix, France). Current–voltage (I/V) relationships were
constructed by clamping the membrane potential to 40 mV and
by pulses from 100 to +100 mV with 20 mV increments. Pipettes
were pulled from borosilicate glass capillary tubing (GC150-TF10;
Clark Electromedical Inc., Reading, UK) using a two-step ver-
tical puller from Narishige (Tokyo, Japan) and had a resistance
from3to5MΩ. They were filled with the following solution
(in mM: 1 NaCl, 113 l-aspartic acid, 113 CsOH, 1 MgCl
2
,27
CsCl, 1 EGTA, 10 TES, and 3 MgATP, ex temporane, pH 7.2; 285
mOsm). They were connected to the head of the patch-clamp
amplifier through an Ag–AgCl pellet. The bath solution con-
tained in mM: 145 NaCl, 4 CsCl, 1 CaCl
2
, 1 MgCl
2
, 10 glucose,
and 10 TES (pH 7.4; 340 mOsm). The liquid potential was cor-
rected before seal establishment. Seal resistances ranging from
5to10GΩ were obtained. Pipette capacitances were electroni-
cally compensated in cell-attached mode. Membrane capacitances
were measured in the whole-cell mode by fitting capacitance
currents obtained in response to a hyperpolarization of 10 mV,
with a first-order exponential, and by integrating the surface of
the capacitance current. Sample frequency was 3 kHz. Results
were analyzed with the pClamp9 package software (Molecular
Devices, Sunnyvale, CA, USA). Mean values of membrane capac-
itance were 39.1 ± 6.0 pF (n = 14) for wt-CFTR-CHO cells. For
graphic representations, I/V relationship was normalized to 1 pF
to removevariability due to differences in cell sizes. For time course
experiments, current amplitude measured at +40 mV was plotted
each 10 s.
SINGLE-CHANNEL PATCH-CLAMP RECORDINGS
Single-channel currents were recorded from cell-attached patches.
Pipettes were pulled from borosilicate glass capillary tubing
(GC150-F10; Clark Electromedical Inc., Reading, UK) using a
two-step vertical puller from Narishige (Tokyo, Japan) had a resis-
tance of 15–30 MΩ. Seal resistances ranging from 10 to 20 GΩ
were obtained. Experiments were performed at RT. Cells were
stimulated with forskolin (1 μM) and RP193 (10 μM). Results
www.frontiersin.org August 2011 | Volume 2 | Article 48 | 3
Dannhoffer et al. RP193 stimulates CFTR in human epithelia
were displayed conventionally with inward currents (outward flow
of anions) indicated by downward deflections. In all the figures,
dashed lines give the zero current baseline when the channels are
in the closed state. Potentials were expressed as the bath potential
minus the patch electrode potential. Single-channel recordings
were sampled at 3 kHz and filtered at 100 Hz with an eight-pole
Bessel filter. The pipette solution contained in mM: 150 NaCl, 2
MgCl
2
, and 10 TES (pH = 7.4); the bath contained: 145 NaCl, 4
KCl, 2 MgCl
2
, and 10 TES (pH = 7.4).
Transepithelial ion transports
Confluent human bronchial epithelial cell cultures were mounted
in Ussing chambers, bathed on both sides with Krebs bicarbonate
Ringer (KBR) solution warmed at 37˚C, and gassed with 95% O
2
-
5% CO
2
. KBR composition was in mM: 119.7 NaCl, 15 NaHCO
3
,
10 glucose, 4.5 KCl, 2 CaCl
2
, 1.5 NaH
2
PO
4
, 0.7 Na2HPO4, 0.23
MgCl
2
. The short-circuit current (I
sc
) was monitored continu-
ously using a VCC MC2 voltage clamp (Physiologic Instruments
Inc., San Diego, USA). One voltage-sensing electrode and one
current-passing electrode were placed on each side of the culture.
The transepithelial resistance (R
t
) was determined by clamping
the potential difference (PD) to +2 mV at 90 s intervals, recording
the deflection in short-circuit current (I
sc
), and applying Ohms
law. Cell cultures were allowed to equilibrate until stabilization
of bioelectric variables, which required approximately 20–30 min.
Basal bioelectric activity was monitored for an additional 10 min
period prior to the addition of drugs. The pharmacological agent
amiloride (an inhibitor of the epithelial sodium channel, ENaC)
and forskolin (an activator of adenylate cyclase) were then added
sequentially, and the bioelectric activity was allowed to stabilize
10–15 min before addition of the subsequent agent. Amiloride
(10 μM) was added to the apical bath, forskolin (1 μM) was
added to the apical and the basolateral baths and RP193 (100 and
200 μM) was added to the basolateral bath. Changes in I
sc
were
calculated as the difference between the values measured immedi-
ately before the addition of reagents, and the values corresponding
to the plateau phase after the addition of amiloride, forskolin, and
RP193.
CHEMICALS
All culture media, hormones, growth factors, or antibiotics
were from Invitrogen (Cergy-Pontoise, France) or Sigma (Saint
Quentin Fallavier, France), and FBS was from PerbioScience (Bre-
bières, France). Unless otherwise stated, chemicals were purchased
from Sigma.
RESULTS
EFFECT OF THE NEWLY SYNTHESIZED COMPOUNDS ON CFTR
We first examined the effects of the modified pyrrolo[2,3-
b]pyrazine derivatives (Figure 1A) on CHO cells stably express-
ing wt-CFTR with a cell-based primary screening assay using
iodide efflux measurement (Marivingt-Mounir et al., 2004). All
compounds stimulated CFTR with various half-maximal effec-
tive concentrations. For the most potent compound, RP193 we
determined an EC
50
of 270 nM. Then, for RP173 we obtained an
EC
50
of 0.2 μM. Finally for the two remaining compounds we
found much higher values with or EC
50
= 78 μM for RP185 and
EC
50
= 97 μM for RP146 (Figure 1B). Before extensive analysis
of the activation of CFTR by these compounds, their cytotoxicity
has been evaluated by measuring cellular dehydrogenase activ-
ity using the water-soluble tetrazolium salt MTT. Importantly,
we observed that the two compounds with a fluorine atome on
the phenyl ring, at the position 2 (RP193) or 4 (RP185) are not
cytotoxic (Figure 1C). These experiments also revealed that the
others compounds (RP107, RP108, RP146, and RP173) have a
significantly higher cytotoxicity compared to the control condi-
tions. Thus, among the pyrrolo[2,3-b]pyrazine derivatives tested
here and previously (Noel et al., 2006) the compound 7-n-butyl-
6-(2-fluorophenyl)-5H-pyrrolo[2,3-b]pyrazine named RP193 is
the less toxic derivative and the most potent activator of wt-
CFTR. Iodide efflux experiments were then performed with RP193
without forskolin 1 μM on wt-CFTR-CHO cells and revealed no
stimulation of CFTR activity (Figure 1D). Further experiments
revealed that potentiation of CFTR activity by RP193 (Figure 1D)
was independent of intracellular cAMP levels (Figure 1E).
ACTIVATION OF A LINEAR CFTR Cl
CURRENT BY RP193
We performed whole-cell patch-clamp experiments to record
CFTR chloride currents after stimulation of wt-CFTR-CHO
cells by RP193. Characteristic whole-cell currents and the cor-
responding current/voltage plots in the absence and presence
of RP193 are presented Figure 2. In the absence of any acti-
vator, no or very small current was recorded (Figures 2A,B,D,
noted basal). Addition of low concentration of forskolin (1 μM)
into the bath stimulated a time- voltage-independent and lin-
ear conductance with small amplitude, suggesting the presence
of a functional CFTR (Figures 2A,B,D, noted fsk). Addition
of 10 μM RP193 (Figures 2A,B,D, RP193) in the presence of
1 μM fsk induced a large and significant increase of the CFTR
current amplitude compared to basal current and to forskolin-
induced current. When measured at +40 mV the amplitude val-
ues are: basal, 1.8 ± 0.4 pA/pF; fsk, 18.4 ± 6.2 pA/pF; fsk + RP193,
113.8 ± 15.1 pA/pF. This current was fully and rapidly inhibited
by two selective CFTR inhibitors; CFTR-
inh
172 (10 μM, current
amplitude at +40 mV: 8.2 ± 5.2 pA/pF) and GP
inh
5a (100 pM,cur-
rent amplitude at +40 mV: 11.6 ± 7.7 pA/pF; Figures 2A,B,D).
As expected, we observed no effect of either forskolin (1 μM)
or RP193 (10 μM) in CHO-K1 (Figure 2C). The time course
of the activation and inhibition of CFTR currents from two dif-
ferent whole-cell recordings is shown in Figure 3. Addition of
RP193, in presence of forskolin, induced a significant increase of
the current within 5–7 min, which was fully and rapidly inhibited
by the addition of either CFTR-
inh
172 (Figure 3A)orGP
inh
5a
(Figure 3B).
ACTIVATION OF CFTR CHLORIDE CHANNELS BY RP193
We performed single-channel patch-clamp experiments to record
CFTR activity in the presence of RP193. In basal condition no
spontaneous channel activity was detected in cell-attached con-
figuration (Figure 4A, left part of the exemplar tracing). In the
presence in the bath of a low concentration of forskolin (1 μM)
we recorded little CFTR activity (Figure 4A, middle part of
the exemplar tracing). Following addition to the bath of RP193
(10 μM) progressive and sustained CFTR activity was recorded
Frontiers in Pharmacology | Pharmacology of Ion Channels and Channelopathies August 2011 | Volume 2 | Article 48 | 4
Dannhoffer et al. RP193 stimulates CFTR in human epithelia
FIGURE1|(A)Chemical structure of the 2 pyrrolo[2,3-b]pyrazines previously
tested compounds (RP107 and RP108) and newly synthesized compounds
(RP146, 173, 185, and 193). (B) EC
50
of different compounds tested
determinate by iodide efflux in wt-CFTR-CHO. (C) Evaluation of the
cytotoxicity of the six different derivatives. The toxicity of the compounds was
evaluated as described in the experimental section. Results are presented as
percentage of cell viability for wt-CFTR-CHO cells treated 24 h with 100 μMof
each compound; n = 20 for each compound, except for RP146, n = 3 and for
DMSO 100% and DMSO 0.1%, n = 10. (D) Bar chart showing RP193
(100 μM), forskolin (1 μM), and forskolin (1 μM) + RP193 (100 μM) dependent
iodide efflux in wt-CFTR-CHO cells. n = 4 for each. (E) Bar chart showing
cAMP levels in wt-CFTR-CHO cells stimulated by RP193 (100 μM), forskolin
(1 μM) + RP193 (100 μM), or forskolin (1 μM). Data are expressed as the
mean ± SEM of 8–16 measurements normalized to cAMP levels in the
presence of 10 μM forskolin (100%) and are representative of two
independent experiments. Ns: no significant differences with respect to
experimental condition in which DMSO 0.1% was added, ***p < 0.001 using
the t-test.
(Figures 4A,B). After excision of the membrane in the presence of
1 μM forskolinand 10 μM RP193 in the bath,we observed the typ-
ical run down (Becq, 1996) of CFTR channels (Figure 4B) leading
to complete closure of CFTR channels. The average unitary con-
ductance of 8.1 ± 0.0 pS calculated in these experiments (n = 4) is
consistent with previous data obtained using cAMP agonists (Becq
et al., 1999; Kammouni et al., 1999; Figures 4C,D).
RP 193 ACTIVATES TWO CFTR MUTANTS G551D- AND F508del-CFTR
CAUSING SEVERE CF DISEASE
To determine whether RP193 could activate CFTR mutants, we
performed iodide efflux experiments with cells expressing two of
the most frequent CF mutations, the class 3 CF mutation G551D-
CFTR, studied in CHO cells stably expressing G551D-CFTR, and
the class 2 CF mutation F508del-CFTR, studied in CuFi-1 cells
derived from a human CF bronchial epithelium (F508del/F508del
genotype). As CuFi-1 cells are CF bronchial epithelial cells, we
also compared the effect of RP193 on NuLi-1 cells derived from
a non-CF human bronchial epithelium. RP193 (100 μM) stim-
ulated CFTR in NuLi-1 cells in presence of forskolin (10 μM;
Figures 5A,B). This effect was abolished by the selective CFTR
inhibitor CFTR-
inh
172 (10 μM, Figure 5B). RP193 did not stimu-
late iodide efflux in CuFi-1 cells with the F508del-CFTR mutation
cultivated at 37˚C (Figure 5C). Then, CuFi-1 cells were incubated
www.frontiersin.org August 2011 | Volume 2 | Article 48 | 5
Dannhoffer et al. RP193 stimulates CFTR in human epithelia
FIGURE 2 | Activation of CFTR current in CHO cells. Representative
current traces for CFTR are shown in the basal state and after successive
addition of forskolin (1 μM), RP193 (10 μM), and specific CFTR inhibitor
CFTR-
inh
172 (10 μM) (A) or GP
inh
5a (100 pM) (B). (C) Representative current
traces recorded after the successive addition of forskolin (1 μM) and RP193
(10 μM) in CHO-K1. Step protocol consisted of 300 ms voltage from 100
to +100 mV from a holding potential of 40 mV. (D) I/V curves (mean ± SE)
for CFTR current in the four experimental conditions for the three different
protocols used.
FIGURE 3 | Representative time course of whole-cell activation and
inhibition of CFTR current at +40 mV. (A) Sequential addition of Fsk
(1 μM), RP193 (10 μM), and CFTR-
inh
172 (10 μM) and (B) sequential addition
of Fsk (1 μM), RP193 (10 μM), and GP
inh
5a (100 pM). Horizontal bars
represent time of perfusion of different drugs.
for 24 h at low temperature (27˚C) to rescue F508del-CFTR (Den-
ning et al., 1992). In these conditions of culture and in pres-
ence of forskolin 10 μM, RP193 induced an iodide efflux signifi-
cantly higher than with forskolin alone (Figures 5D,E). In further
experiments, CuFi-1 cells were incubated 24 h at 37˚C with the
pharmacological corrector miglustat in order to rescue F508del-
CFTR (Norez et al., 2006). Following this treatment of CuFi-1
cells, RP193 significantly induced a higher iodide efflux than in
basal conditions or with forskolin alone (Figure 5F), confirming
the functional rescue of F508del-CFTR proteins. This efflux was
fully inhibited in presence of the CFTR
inh
-172 (10 μM, data not
FIGURE 4 | Single CFTR chloride channel activation by RP193 in
wt-CFTR-CHO cells. (A) Continuous cell-attached recording obtained on a
wt-CFTR-CHO showing the activation of CFTR chloride channel by 10 μMof
RP193 in the bath in presence of forskolin (1 μM) at the potential of
+60 mV. (B) Representative recording of rundown of CFTR after channel
excision in presence of RP193 (10 μM). Insert depicts 1 min of channel
opening recording in cell-attached configuration. (C) Representative
recordings at various patch potentials as indicated in the presence of RP193
at (10 μM) in the bath and the associated I/V curve (D).
shown), and no effect of RP193 was observed in CuFi-1 cells non-
treated with miglustat (Figure 5F). Thus RP193 is able to stimulate
F508del-CFTR activity after maneuvers leading to the correction
of its abnormal trafficking.
We also performed iodide efflux experiments on G551D-CFTR-
CHO cells. No significant stimulation was observed compared
to basal state with 10 μM forskolin alone (Figure 5G). However,
RP193 10 μM stimulated an efflux in G551D-CFTR-CHO cells in
presence of forskolin 10 μM(n = 4, Figure 5G) while RP193 alone
did not have any effect (not shown). Taken together these exper-
iments revealed that RP193 is able not only to activate wt-CFTR
but also rescued F508del-CFTR and G551D-CFTR.
ACTIVATION OF CFTR CHLORIDE CHANNELS BY RP193 IN PRIMARY
CULTURES OF HUMAN BRONCHIAL EPITHELIAL CELLS
Ten primary cultures of human bronchial epithelial cells (HBEC)
werestudied in Ussing chambers. The mean PD, I
sc
,and R
t
of these
cultures were respectively: 4.7 ± 0.6 mV, 53.4 ± 7.2 μA/cm
2
,
and 98.8 ± 14.8 Ω cm
2
. Addition of amiloride (10 μM) to the
luminal bath significantly reduced I
sc
in the preparations
(29.5 ± 4.5 μA/cm
2
). In the presence of amiloride, the addition
Frontiers in Pharmacology | Pharmacology of Ion Channels and Channelopathies August 2011 | Volume 2 | Article 48 | 6
Dannhoffer et al. RP193 stimulates CFTR in human epithelia
FIGURE 5 | Examples of iodide efflux curves as function of time on
NuLi-1 cells (A), CuFi-1 (C) cultured at 37˚C and CuFi-1 treated by low
temperature (24 h, 27˚C) (D) in basal conditions, stimulated by forskolin
(10 μM), or by forskolin (10 μM) and RP193 (100 μM). Bar chart showing
iodide efflux experiments on NuLi-1 (B), CuFi-1 treated by low temperature
(24 h, 27˚C) (E), CuFi-1 treated with miglustat (24 h, 100 μM) (F) and
G551D-CHO cells (G). Cells were successively treated with forskolin
(10 μM) and RP193 (100 μM) and NuLi-1 cells were equally treated by
CFTR-
inh
172 (10 μM), n = 4 for each. *p < 0.05 and **p < 0.01 using the
t-test.
www.frontiersin.org August 2011 | Volume 2 | Article 48 | 7
Dannhoffer et al. RP193 stimulates CFTR in human epithelia
FIGURE 6 | Bar chart representing I
sc
variations in primary culture of
human bronchial epithelial cells in response to RP193 (100 and
200 μM) in presence of amiloride 10 μM and forskolin 1 μM, n = 10 for
each. ***p < 0.001 using the t -test.
of forskolin (1 μM) induced a significant increase in I
sc
in HBEC
cultures (0.4 ± 0.1 μA/cm
2
). Successive addition of RP193 (100
and 200 μM) increased significantly I
sc
from respectively 0.5 ± 0.2
and 1.1 ± 0.3 μA/cm
2
(Figure 6) indicating that RP193 is able to
increased I
sc
in primary cultures of HBEC.
DISCUSSION
Based on previously published data from our laboratory, one of
the main objective of this ligand-based study was to evaluate the
effects of the modification of determinants on the phenyl ring
of the pyrrolo[2,3-b]pyrazines family on their potency to acti-
vate CFTR. Firstly, we translocated the 4-OH substituent of the
phenyl ring of RP107, in position 2 (compound RP173) and in
the position 3 (compound RP146). Secondly, based on results
obtained with a 4-Cl substituent (RP108; Noel et al., 2006), we
tested another halogen substituent at the same position on the
phenyl ring: 4-F (RP185) and in position 2-F (RP193). Ability
of these newly synthesized compounds to activate wt-CFTR, was
assessed by iodide efflux, which allowed us to identify RP193 as a
new cAMP-independent hit of the pyrrolo[2,3-b]pyrazines family
(EC
50
= 270 nM). Most importantly,RP193 appeared to be signifi-
cantly less toxic than compounds developed previously as revealed
by cytotoxicity assays. To complete our study of RP193 com-
pound, we performed patch-clamp experiment in the whole-cell
and the cell-attached configurations, which confirmed its ability to
activate wt-CFTR. As lung pathology represents the most drastic
syndrome in CF (Boucher, 1999), another important objective of
this work was to evaluate the effect of RP193 on CFTR activity in
cultures of human bronchial non-CF and CF epithelial cells. We
then performed iodide efflux experiment on cells, which expressed
two of the most common mutation encountered in CF patients.
Our data demonstrated that RP193 was able to stimulate rescued
F508del-CFTR and G551D-CFTR.
A recent study carried out in our laboratory demonstrated the
ability of RP compounds previously described as CDK/GSK-3
inhibitors, to activate CFTR with a strong affinity (Noel et al.,
2006). It was shown that RP107 was able to stimulate wt-CFTR
with a submicromolar affinity by a cAMP-independent mecha-
nism. The importance of the length of the alkyl chain was observed
with the best activation of CFTRobtained with compounds having
a butyl chain. This property was conserved in the newly synthe-
sized compounds, and only the nature and/or the position of the
substituent on the phenyl ring were changed. Firstly, transloca-
tion of the OH substituent at position 2 (RP173) or at position
3 (RP146) reduced the potency of the compound suggesting that
the position of this substituent on the phenyl ring is an important
determinant for the ability to activate CFTR.
Experiments performed with RP108 demonstrated efficiency
to activate wt-CFTR of compound with a halogen substituent
(Noel et al., 2006). Other studies demonstrated efficiency
of compounds, like the benzimidazolone NS004 or 1,2,3,4-
tetrahydroisoquinoline-3-carboxylic acid diamides, to activate
CFTR with different halogen substituent in their structure
(Gribkoff et al., 1994; Devor et al., 2000; Hirth et al., 2005; Murthy
et al., 2005) suggesting that these derivatives could play an impor-
tant role in the activation of CFTR. Substitution of the chlorine
atom by another halogen, a fluorine atom, at position 4 (RP185)
or position 2 (RP193) of the phenyl ring was then performed.
It was shown that fluoride, the reduced form of fluorine, stimu-
lated phosphorylated CFTR most probably by direct interaction
with NBD2 domain and by slowing CFTR channel closure (Berger
et al., 1998). Caci et al. (2003) also demonstrated activation of
CFTR by a compound containing F residue. In their study, these
authors demonstrated that UC
CF
-853 is an activator of CFTR-
dependent Cl
secretion in intact human airway epithelium.
Iodide efflux experiments revealed that our compounds poten-
tiated CFTR activity with a higher potency for RP193. Marivingt-
Mounir et al. (2004) already tried to introduce a fluorine atom
in another class of CFTR activators, the benzo[c]quinoliziniums.
However, no activation of CFTR was found with these compounds.
Nevertheless, contrary to MPB104, the most active compound in
their study, MPB with a fluorine substituent did not have a butyl
chain,which appeared to be a key determinant for CFTR activation
by RP agents.
Interestingly, RP107 and RP193 presented similar EC
50
values
for CFTR potentiation in iodide efflux (EC
50
are respectively 150
and 270 nM). Nevertheless, cytotoxicity assays revealed that com-
pounds with chlorine atom or hydroxyl radical are more toxic than
compounds with fluorine atom whatever the position of the sub-
stituent is (2 or 4 on the phenyl ring). To our knowledge, there
are no comparative studies of cell toxicity of compounds with
or without a fluorine atom. The addition of the fluorine atom
could then reduce cell toxicity of compounds that are still able to
activate phosphorylated CFTR. These observations could provide
some clue to generate new potent CFTR activators (importance
of the OH radical) with less cytotoxicity (addition of a fluorine
atom).
We demonstrated that RP193 is able to potentiate wt-CFTR
in CHO cell line. It appeared then important to test this com-
pound on CFTR mutant. First, we performed experiments on
human bronchial epithelial cell expressing endogenous WT-CFTR
or F508del-CFTR, the most common mutation in CF patients.
RP193 potentiated WT-CFTR activity, as observed in other cell
type, but it was also able to potentiate F508del-CFTR corrected
Frontiers in Pharmacology | Pharmacology of Ion Channels and Channelopathies August 2011 | Volume 2 | Article 48 | 8
Dannhoffer et al. RP193 stimulates CFTR in human epithelia
either by low temperature incubation or by a 24-h-miglustat-
treatment. Secondly,we performed experiments on G551D-CFTR,
a class 3 mutation retrieved in patients with severe disease and
which present no or reduced activation of the CFTR chloride
function. Our studies revealed that in presence of a low forskolin
concentration, RP193 potentiates the activity of G551D-CFTR. It
should be noted that forskolin alone did not have any effect as pre-
viously demonstrated (Becq et al., 1994; Illek et al., 1999). Thus, the
ability of RP193 compound to activate two of the most common
CFTR mutants is very interesting in the perspective of develop-
ing CF pharmacotherapy. A recent study demonstrated that the
use of pharmacological activator of CFTR could, in particular,
improve lung function in patients with G551D-CFTR showing that
pharmacological treatment in CF is a realistic objective (Accurso
et al., 2010). Our approach developed here is clearly in this
line.
Inconclusion,our study emphasizes the importance of the pres-
ence of an OH radical in compounds in order to activate CFTR.
We equally revealed that presence of a fluorine atom could reduce
cytotoxicity without altering efficiency of the compound to stim-
ulate phosphorylated CFTR. Finally, we identified RP193 as a new
compound able to stimulate, through a cAMP-independent path-
way, wt-CFTR, rescued F508del-CFTR, and G551D-CFTR activity
in HBEC.
ACKNOWLEDGMENTS
The authors thank S. Mirval for technical assistance. This work
was funded by the association Vaincre la mucoviscidose.
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Conflict of Interest Statement: The
authors declare that the research was
conducted in the absence of any
commercial or financial relationships
that could be construed as a potential
conflict of interest.
Received: 08 June 2011; accepted: 03
August 2011; published online: 23 August
2011.
Citation: Dannhoffer L, Billet A, Jol-
livet M, Melin-Heschel P, Faveau
C and Becq F (2011) Stimulation
of wild-type, F508del- and G551D-
CFTR chloride channels by non-toxic
modified pyrrolo[2,3-b]pyrazine deriv-
atives. Front. Pharmacol. 2:48. doi:
10.3389/fphar.2011.00048
This article was submitted to Frontiers
in Pharmacology of Ion Channels and
Channelopathies, a specialty of Frontiers
in Pharmacology.
Copyright © 2011 Dannhoffer, Billet , Jol-
livet , Melin-Heschel, Faveau and Becq.
This is an open-access article subject
to a non-exclusive license between the
authors and Frontiers Media SA, which
permits use, distribution and reproduc-
tion in other forums, provided the original
authors and source are credited and other
Frontiers conditions are complied with.
Frontiers in Pharmacology | Pharmacology of Ion Channels and Channelopathies August 2011 | Volume 2 | Article 48 | 10
    • "In CHO cells expressing wt CFTR, 15b enhanced iodide flux with an EC 50 value of 270 nM. Iodide flux measurements also showed the effects of RP193 (15b) on the mutants F508del and G551D, but high concentrations (100 µM) of the agent were necessary [65]. Hence, the pyrolopyrazines have emerged as interesting activators of wt and mutant CFTRs and constitute a promising scaffold for further optimization and eventually clinical development . "
    [Show abstract] [Hide abstract] ABSTRACT: Introduction: Cystic fibrosis (CF) is the most prevalent, recessively inherited, disease in the western world. It is characterized by gene mutations in CF transmembrane conductance regulator (CFTR), a transmembrane ion channel that is responsible for chloride secretion in the airway passages. Although much is known about the defects in CFTR and the consequences of these mutations, CF therapy currently focuses on the secondary outcomes and symptoms of the disease. However, developments in CFTR modulators may bring about new therapeutic options. Areas covered: The authors discuss CFTR defects, as a molecular basis, before presenting and discussing CFTR modulators including correctors and potentiators. Specifically, the authors review promising CFTR modulators currently in preclinical and clinical development along with their medicinal chemistry and structure-activity relationships (SARs) and their in vitro and in vivo pharmacology. Expert opinion: Although the development of CFTR-targeting agents has little access to structural information from crystal structures, several promising compounds have been discovered so far. Advanced virtual models of CFTR and high-throughput assays have helped the developmental programs. While Ivacaftor, the first of the CFTR potentiators, has now reached clinical use, CFTR corrector development has not been successful thus far. However, intense research of the mutation F508del, the mutation considered the most frequent in CF, could provide new causal treatment options in the future. Furthermore, the eventual synergy with multiple correctors may bring further success. CFTR modulators provide a new personalized therapeutic option where CF therapy is based on the mutations patients carry rather than by simply their symptoms.
    Article · Apr 2013
  • [Show abstract] [Hide abstract] ABSTRACT: INTRODUCTION ENaC and CFTR are coexpressed in epithelia and have positive or negative functional interactions. In addition, ENaC and CFTR promote migration in placental trophoblastic cells and human airway cells, respectively. Here we tested the idea if CFTR is functionally expressed in BeWo cells, a trophoblastic cell line, and if it is involved in their migratory behavior. METHODS CFTR expression was studied in BeWo cells with RT-PCR, biotinylation and Western blot. Ion currents were analyzed with patch clamp, and cell migration with the wound healing method. RESULTS The mature CFTR 160-kDa band was present, and its localization at the surface membrane was confirmed. Forskolin (20 μM), an adenylate cyclase activator, was used for channel activation, and subsequently CFTRinh-172 (2 μM) for its inhibition. The conductances in the presence of CFTRinh-172 plus forskolin (16.0 ± 0.7 pS/ pF and 32.6 ± 1.5 pS/ pF) were significantly lower than in presence of only forskolin (29.7 ± 0.9 and 47.0 ± 2.0 pS/ pF). The conductance of CFTRinh-172 inhibited currents was 14.9 ± 0.7 pS/ pF with a linear I-V relationship illustrating the nonrectifying properties of the CFTR. Cell migration was measured and covered 11.2 ± 0.4, 24.0 ± 1.7 and 13.9 ± 1.0 % of the wound when cells were cultivated under control, forskolin, and forskolin plus CFTRinh-172, respectively. Proliferation was not changed by any of the treatments. CONCLUSIONS Our results shows that BeWo cells functionally express the CFTR which plays a role in the wound healing increasing the cell migration process.
    Article · Jan 2013
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    Full-text · Article · Apr 2014
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