Recovery of Δf508-CFTR function by analogs of hyaluronan disaccharide

Article abstract of DOI:10.1002/jcb.23339

We recently discovered that hyaluronan was exported from fibroblasts by MRP5 and from epithelial cells by cystic fibrosis (CF) transmembrane conductance regulator (CFTR) that was known as a chloride channel. On this basis we developed membrane permeable a

Full text of DOI:10.1002/jcb.23339

Journal of Cellular
Biochemistry
ARTICLE
Journal of Cellular Biochemistry 113:156–164 (2012)
Recovery of DF508-CFTR Function by Analogs of
Hyaluronan Disaccharide
Ewa Nowakowska,1 Tobias Schulz,1 Natalia Molenda,2 Hermann Schillers,2 and
1
*
Peter Prehm
1Muenster University Hospital, Institute of Physiological Chemistry and Pathobiochemistry, Waldeyerstr. 15,
D-48129 Muenster, Germany
2Muenster University Hospital, Institute of Physiology II, Robert-Koch-Str. 27b, D-48129 Muenster, Germany
ABSTRACT
We recently discovered that hyaluronan was exported from fibroblasts by MRP5 and from epithelial cells by cystic fibrosis (CF)
transmembrane conductance regulator (CFTR) that was known as a chloride channel. On this basis we developed membrane permeable
analogs of hyaluronan disaccharide as new class of compounds to modify their efflux. We found substances that activated hyaluronan export
from human breast cancer cells. The most active compound 2-(2-acetamido-3,5-dihydroxyphenoxy)-5-aminobenzoic acid (Hylout4) was
tested for its influence on the activity of epithelial cells. It activated the ion efflux by normal and defective DF508-CFTR. It also enhanced the
plasma membrane concentration of the DF508-CFTR protein and reduced the transepithelial resistance of epithelial cells. In human trials of
healthy persons, it caused an opening of CFTR in the nasal epithelium. Thus compound Hylout4 is a corrector that recovered DF508-CFTR
from intracellular degradation and activated its export function. J. Cell. Biochem. 113: 156–164, 2012. ß 2011 Wiley Periodicals, Inc.
KEY WORDS: CYSTIC FIBROSIS; CFTR; ION TRANSPORT; HYALURONAN
ystic fibrosis (CF) is one of the most common inherited
diseases, afflicting 1 in approximately 2,500 white individu-
membrane-rescued DF508-CFTR protein remains defective such
that its open probability after cAMP stimulation is reduced by more
than threefold compared with that of wild-type CFTR [Dalemans
et al., 1991; Haws et al., 1996]. Small-molecule correctors of
defective DF508-CFTR folding/cellular processing (‘‘correctors’’)
and channel gating (‘‘potentiators’’) may provide a strategy for
therapy of CF that corrects the underlying defect. A potential
advantage of pharmacotherapy for defective DF508-CFTR proces-
sing and gating is that it minimizes concerns about treating the
wrong cells or losing physiological CFTR regulation, as might occur
with gene therapy or activation of alternative chloride channels. A
number of small-molecule DF508-CFTR potentiators [Drumm et al.,
1991; Hwang et al., 1997; Al-Nakkash and Hwang, 1999] and
correctors [Yang et al., 2003; Pedemonte et al., 2005; Carlile et al.,
2007; Wang et al., 2007; Loo et al., 2008; Yoo et al., 2008] have been
identified. These potentiators and correctors were mostly discovered
by high-throughput screening for activation of the chloride channel.
Recently, we discovered that hyaluronan is exported from
fibroblasts by MRP5 [Schulz et al., 2007] and from epithelial cells by
CFTR [Schulz et al., 2010]. These studies added hyaluronan as
another important natural substrate to CFTR in addition to chloride.
C
als [Bobadilla et al., 2002]. The primary cause of morbidity and
mortality in CF is chronic lung infection and deterioration of
lung function. CF is caused by mutations in the CF transmembrane
conductance regulator (CFTR) gene, which encodes a cAMP-
regulated chloride channel expressed at the apical membrane of
epithelial cells in the airways, pancreas, testis, and other tissues
[Pilewski and Frizzell, 1999; Sheppard and Welsh, 1999]. The most
common CFTR mutation producing CF is deletion of phenylalanine
at residue 508 (DF508) in its amino acid sequence, which is present
in at least 1 allele in approximately 90% of CF subjects [Bobadilla
et al., 2002]. The DF508-CFTR protein is misfolded and retained at
the endoplasmic reticulum (ER), where it is degraded rapidly
[Denning et al., 1992; Lukacs et al., 1994; Du et al., 2005]. The
misfolding of DF508-CFTR is thought to be mild, because it can be
‘‘rescued’’ in cell culture models by incubation for 18 h or more at
reduced (<308C) temperature or with chemical chaperones such as
glycerol [Sato et al., 1996] or phenylbutyrate [Rubenstein et al.,
1997], which results in partial restoration of DF508-CFTR plasma
membrane expression. However, channel gating of the plasma
Grant sponsor: Deutsche Forschungsgemeinschaft; Grant number: SFB 492.
*Correspondence to: Peter Prehm, Muenster University Hospital, Institute of Physiological Chemistry and
Pathobiochemistry, Waldeyerstrasse 15, D-48149 Muenster, Germany. E-mail: prehm@uni-muenster.de
Received 3 November 2010; Accepted 22 August 2011 ꢀ DOI 10.1002/jcb.23339 ꢀ ß 2011 Wiley Periodicals, Inc.
Published online 31 August 2011 in Wiley Online Library (wileyonlinelibrary.com).
156

They also suggested a compromise in the controversy of the CF
pathology between the hydration and the salt hypothesis [Guggino,
1999], because hyaluronan fulfills both properties being an
extremely hydrated salt. Hyaluronan is not only exported from
bronchial epithelia by CFTR upon inflammation, but also from
human breast carcinoma cells, because they have an epithelial
origin. Hyaluronan export by CFTR can best be analyzed on breast
carcinoma cell lines, because bronchial epithelial cell lines lose the
hyaluronan synthesizing capacity.
homogenized by ultrasonication three times for 10 s and undis-
solved materials were removed by centrifugation for 50 min at
48,000g. The membranes containing pellets were solubilized in
200 ml of 0.5% Triton X-100, 50 mM imidazol, 150 mM NaCl, 1 mM
EDTA, 1 mM phenylmethylsulfonylfluoride, 1 mg/ml leupeptin, and
1 mg/ml pepstatin pH 7.0. Undissolved proteins were removed by
centrifugation for 50 min at 48,000g. Equal aliquots were loaded
onto a 10% SDS polyacrylamide gel and analyzed by Western
blotting with mouse-anti-CFTR-IgM. The blots were probed using an
ECL light-based immunodetection system (Amersham Biosciences,
Inc.).
Therefore, we intended to chemically synthesized compounds
that modulate hyaluronan production. These compounds should be
useful as drugs, be membrane permeable and mimic important
structural features of the terminal ends of the hyaluronan chain.
These prerequisites guided us to the synthesis of diaryls substituted
with hydroxyl-, acetamido-, and carboxyl groups in positions as in
hyaluronan disaccharides. Here we describe a new class of
compounds that activate chloride export from bronchial epithelial
cells and hyaluronan export from breast carcinomal cells.
CHEMICAL SYNTHESIS OF Hylout4
Nitrophloroglucinol (1 g, 6.5 mM) was dissolved in 10 ml of
methanol and hydrogenated in a hydrogen atmosphere in the
presence of 0.1 g of 10% Pd/C overnight at room temperature. The
catalyst was removed by filtration and the solvent was evaporated to
obtain aminophloroglucinol. It was dissolved in a 1:1 mixture
of water and ethanol and an equimolar amount of acetic anhydride
was added at 608C. After 1 h at 608C the solution was concentrated
and N-acetylaminophloroglucinol was obtained. N-Acetylamino-
phloro-glucinol (6 mM) was dissolved in 12 ml of dimethylforma-
mide, mixed with 2-chloro-5-nitrobenzoic acid (1.2 g; 6 mM), 1.7 g
of K2CO3, 0.18 g of copper powder, and 0.18 g of CuCl and refluxed
for 3 h. After cooling to room temperature, the solution was filtered
through celite. Water (120 ml) was added and the solution was
acidified with concentrated HCl to pH 2, and the product was
extracted with 120 ml of ethylacetate. The organic phase was dried
over Na2SO4 and evaporated. The product was dissolved in 12 ml
of methanol, 0.1 g of palladium (10% on charcoal) was added
and hydrogenated in a hydrogen atmosphere overnight at room
temperature. The catalyst was removed by centrifugation, and the
solvent was evaporated to obtain Hylout4. It was further purified by
chromatography on silica gel with ethylacetate as solvent.
MATERIALS AND METHODS
MATERIALS
Mouse-anti-CFTR-IgM was from Acris Antibodies, Hiddenhausen,
Germany. Other chemical were from Sigma–Aldrich Chemical
Corporation.
CELL LINES
The epithelial breast cancer cells HMT3552 [Jojovic et al., 2002]
have been described. Human epithelial cells containing wild-type
CFTR (16HBE14oꢁ) and the mutant cell line containing DF508-CFTR
(CFBE41oꢁ) were kindly provided by Dr. D.C. Gruenert [Kunzelmann
et al., 1993]. The cell line 16HBE14oꢁ is an SV40 large T-antigen
transformed epithelial cell line derived from human bronchial
epithelium which retains differentiated epithelial morphology and
functions. The cell line CFBE41oꢁ are transformed human airway
epithelial cells carrying a homozygous mutation for DeltaF508. The
cells were grown in Dulbecco’s medium supplemented with
streptomycin/penicillin (100 units of each/ml) and 10% foetal calf
serum.
TRANSEPITHELIAL RESISTANCE (TER)
Human bronchial epithelium cells (16HBE14oꢁ and CFBE41oꢁ)
were seeded on a 12-well plate ThinCertTM cell culture inserts (area
1.13 cm2, pore size 0.4 mm, pore density 108/cm2, Greiner Bio-One,
Germany) and cultured for 7–10 days at 378C, 5% CO2. We
developed a system which allows measuring the TER of 8 culture
inserts simultaneously at time intervals of 20 s or 5 min. Each well
was equipped with six titanium electrodes, three to inject current
and three to measure voltage. Electrodes were arranged in a way that
resulted in a fairly homogenous electrical field. Current was injected
at the given time interval for 1 s and voltage was measured. Data
acquisition and processing was done by 2-channel-PowerLab
system (26 Series, ADInstruments GmbH, Germany). 8cpt-cAMP
(8-(4-chlorophenylthio)-adenosine-30,50-cyclic monophosphate) or
Hylout4 were added simultaneously to both compartments (final
concentration 100 mM). TER was measured inside of the incubator at
378C and 5% CO2. TER data are expressed as relative values to allow
appropriate comparisons between the different series of experi-
ments. The value measured directly before adding any substances
was set as 100% (reference value).
CYTOTOXICITY ASSAYS
The cytotoxicity was measured by the Alamar blue1 assay which
determined the cell viability by reduction of resazurin with NADH
[Nakayama et al., 1997].
WESTERN BLOTTING OF CFTR
The amounts of CFTR in membranes of CFBE41oꢁ cells were
analyzed by Western blotting. Briefly, equal amounts of cells were
incubated with increasing concentrations of 2-(2-acetamido-3,5-
dihydroxyphenoxy)-5-aminobenzoic acid (Hylout4) for 24 h,
washed washed twice with ice-cold phosphate-buffered saline,
scrapped off with 1 ml of ice-cold phosphate-buffered saline and
sedimented for 5 min at 1,000g. The cell pellets were suspended in
200 ml of homogenizing buffer containing 0.1 M sodium acetate,
0.2 M NaCl, 1 mM EDTA, 1 mM phenylmethylsulfonylfluoride, 1 mg/
ml leupeptin, and 1 mg/ml pepstatin pH 6.0. The suspensions were
JOURNAL OF CELLULAR BIOCHEMISTRY
HYALURONAN DISACCHARIDE ANALOGS AS CFTR CORRECTORS 157

DETERMINATION OF HYALURONAN EXPORT
DETERMINATION OF THE NASAL TRANSEPITHELIAL
POTENTIAL DIFFERENCE
The breast cancer cells HMT3552 were incubated for 24 h at 378C,
the media were replaced with fresh media and after additional
24 h aliquots (5 and 20 ml) of the culture medium were used for
measurement of the hyaluronan concentration in the cell culture
medium by an ELISA. The wells of a 96 well Covalink-NH-microtiter
plate (NUNC) were coated with 100 ml of a mixture of 100 mg/ml of
hyaluronan (Healon1), 9.2 mg/ml of N-hydroxysuccin-imide-3-
sulfonic acid and 615 ml/ml of 1-ethyl-3-(3-dimethylaminopropyl)-
carbodiimide for 2 h at room temperature and overnight at 48C.
The wells were washed three times with 2 M NaCl, 41 mM MgSO4,
0.05% Tween-20 in 50 mM phosphate buffered saline pH 7.2
(buffer A) and once with 2 M NaCl, 41 mM MgSO4, in phosphate
buffered saline pH 7.2. Additional binding sites were blocked
by incubation with 300 ml of 0.5% bovine serum albumin in
phosphate buffered saline for 30 min at 378C. Calibration of the
assay was performed with standard concentrations of hyaluronan
ranging from 15 ng/ml to 6,000 ng/ml in equal volumes of culture
medium as used for measurement of the cellular supernatants. A
solution (50 ml) of the biotinylated hyaluronan binding fragment
of aggrecan (Applied Bioligands Corporation, Winnipeg, Canada)
in 1.5 M NaCl, 0.3 M guanidinium hydrochloride, 0.08% bovine
serum albumin 0.02% NaN3, 25 mM phosphate buffer pH 7.0 was
preincubated with 50 ml of the standard hyaluronan solutions or
cellular supernatants for 1 h at 378C. The mixtures were transferred
to the hyaluronan-coated test plate and incubated for 1 h at
378C. The microtiter plate was washed three times with buffer A
and incubated with 100 ml/well of a solution of streptavidin-
horseradish-peroxidase (Amersham) at a dilution of 1:100 in
phosphate buffered saline, 0.1% Tween-20 for 30 min at room
temperature. The plate was washed five times with buffer A and the
color was developed by incubation with a 100 ml/well of a solution
of 5 mg o-phenylenediamine and 5 ml 30% H2O2 in 10 ml of 0.1 M
citrate-phosphate buffer pH 5.3 for 25 min at room temperature.
The adsorption was read at 490 nm. The concentrations in the
samples were calculated from a logarithmic regression curve of
the hyaluronan standard solutions.
The effect of compound Hylout4 on the transepithelial nasal
potential difference (NPD) was measured with healthy human
¨
volunteers by a modified method of Schuler et al. [Schuler et al.,
2004]. The nasal mucosa was perfused at a rate of 5 ml/min. Baseline
PD was measured after perfusion of the nasal epithelium with
isotonic NaCl solution (147 mM NaCl, 2 mM CaCl2). PD changes
were recorded during these perfusion protocols:
(1) Isotonic NaCl (147 mM NaCl, 2 mM CaCl2).
(2) No chloride solution (147 mM Na-gluconate, 2 mM CaCl2).
(3) No chloride solution þ 100 mM Hyalout4.
(4) No chloride solution þ 10 mM isoprenaline.
(5) Isotonic NaCl.
In order to test, if the different effects of Hyalout4 and
isoprenaline on nasal transepithelial PD, step 3 and 4 of this
perfusion protocol were interchanged for the second experiment.
Solutions were changed as soon as a steady voltage tracing was
achieved for at least 30 s, and the differences in PD values were
measured between the plateaus of the corresponding solutions.
RESULTS
HYALURONAN DISACCHARIDE ANALOGS
In attempts to develop specific membrane permeable inhibitors of
hyaluronan export, we synthesized the diaryl analogs of the two
possible hyaluronan dissacharides 1 and 2 with the non-reducing
GlcNac terminus and a non-reducing GlcA terminus, respectively
(Fig. 1) that were designed as membrane permeable analogs:
2-(2-acetamido-3-hydroxyphenoxy)benzoic acid (Hylout2) and
2-(2-acetamidophenoxy)-6-hydroxybenzoic acid (Hylout3). The
structures differ only in the position of one hydroxyl group being
in o-position of the acetylamido group in Hylout2 or in o-position
of the carboxyl group in Hylout3. Thus these compounds
resemble the non-reducing end of a hyaluronan chain with a
terminal N-acetylamino group for Hylout2 and with a terminal
glucuronic acid for Hylout3. These compounds were tested for their
effect on hyaluronan export. Much to our surprise and contrary
to our expectation, compound Hylout2 was activating at low
micromolar concentrations, whereas Hylout3 was inactive. Then we
modified the lead compound Hylout2 by introducing additional
hydroxyl, amino, or hydrophobic groups. All of these compounds
also activated and the most active one was Hylout4 (Fig. 2). The
toxicity of compound Hylout4, in concentrations up to of 400 mM,
was measured by the Alamar blue assay and it was found to be
not toxic (data not shown).
IODIDE EFFLUX
Iodide efflux experiments were performed as described [Lansdell
et al., 1998]. Briefly, 16HBE14oꢁ or CFBEoꢁ cells (80–90%
confluent) were incubated for 1 h in a loading buffer containing
136 mM NaI, 3 mM KNO3, 2 mM Ca(NO3)2, 11 mM glucose,
and 20 mM Hepes, adjusted to pH 7.4 with NaOH. To remove
extracellular iodide, cells were thoroughly washed with efflux buffer
(136 mM NaNO3 replacing 136 mM NaI in the loading buffer) and
then equilibrated in 2.5 ml efflux buffer for 1 min. The efflux buffer
was changed at 1 min intervals over the duration of the experiment.
Four minutes after anion substitution, cells were exposed to
Hylout4. The amount of iodide in each 2.5 ml sample of efflux buffer
was determined using an iodide-selective electrode (HNU Systems
Ltd, Warrington, UK). In a voltage clamp experiment, the cells
were incubated in the presence of 100 mM KCl and 10 mM
valinomycin. Cells were loaded and experiments performed at
room temperature.
IODIDE EFFLUX
CFTR is known as a channel that does not only export chloride
and hyaluronan, but also other ions such as iodide. Iodide is a
convenient ion to determine the functional activity of CFTR in living
cells, because its concentration can be determined by an iodide
selective electrode. We used the iodide efflux technique to assess
the effect of Hylout4 on epithelial cell lines 16HBE14oꢁ and
158 HYALURONAN DISACCHARIDE ANALOGS AS CFTR CORRECTORS
JOURNAL OF CELLULAR BIOCHEMISTRY

Fig. 1. Structures of hyaluronan disaccharide and analogs.
CFBE41oꢁ cells which express the intact and DF508-defective CFTR
channel, respectively. Iodide efflux was measured at a concentration
of 100 mM of Hylout4, because this concentration was also
effectively activating hyaluronan export from breast carcinoma
cells in the previous experiment. Figure 3 shows that Hylout4
stimulated a sudden burst of iodide efflux from 16HBE14oꢁ as well
as from CFBE41oꢁ cells. The immediate opening of the channels
indicates that compound Hylout4 functions as an potentiator. The
Fig. 2. Activation of hyaluronan export by Hylout4. Human breast carcinoma
cells HMT3552 were grown to 50% confluence and incubated for 2 days with
increasing concentrations of Hylout4, and the hyaluronan concentrations were
determined in the culture supernatant. The error bars indicate the sd of nine
determinations.
Fig. 2.
JOURNAL OF CELLULAR BIOCHEMISTRY
HYALURONAN DISACCHARIDE ANALOGS AS CFTR CORRECTORS 159

Fig. 3. Compound Hylout4 stimulates iodide efflux. CFTR can also export iodide instead of chloride. We made use of this property to measure the kinetics of export with
an iodide sensitive electrode. 16HBE14oꢁ (A) or CFBE41oꢁ (B) were loaded with iodide and the iodide concentration was measured by an iodide sensitive electrode
in 1 min intervals (&). Parallel cultures were exposed to 100 mM of Hylout4 (&) at time point 0 for 4 min. After 2 min of Hylout4 addition, the CFTR-specific inhibitor
CFTRinh-172 was added to CFBE14oꢁ cells (~). Preincubation for 24 h with Hylout4 and additional exposure at time point 0 for 4 min further activated the iodide efflux of
CFBE41oꢁ cells (~). In a control experiment, the cells were voltage clamped by 100 M KCl and 10 mM valinocin (ꢂ). The error bar indicates the sd of three determinations.
activation of iodide export by Hylout4 was blocked by the specific
CFTRinh-172 inhibitor. When 16HBE14oꢁ cells were preincubated
for 24 h with Hylout4, the iodide efflux from CFBE41oꢁ cells was
even more activated. To eliminated the possibility of an influence of
Hylout4 on the membrane potential rather than opening of CFTR, a
control experiment was performed with voltage clamped cells in the
presence of 100 mM KCl and 10 mM valinomycin. This treatment led
to a similar iodide efflux in CFBE41oꢁ cells.
expressing 16HBE14oꢁ and DF508-CFTR expressing CFBE41oꢁ cell
monolayers in the presence of 100 mM Hylout4 or the membrane
permeable cAMP analog 8cpt-cAMP that is known to activate CFTR
[Moran, 2010]. Figure 4 shows that Hyalout4 exerts the same short
term effect on 16HBE14oꢁ cells as 8cpt-cAMP. In both cases TER
dropped immediately after application. While TER remains low with
8cpt-cAMP even 20 h after application, TER of Hyalout4 treated
16HBE14oꢁ cells recovered to values of untreated cells within 20 h.
In CFBE41oꢁ cells 8cpt-cAMP and compound Hylout4 caused a
transient increase of TER followed by a slow decrease of the
resistance over a period of 20 h. In a separate experiment, the
decrease of resistance in 16HBE14oꢁ cells was determined for
TRANSEPITHELIAL RESISTANCE (TER)
The transport activity of epithelial cells can conveniently be
measured by the TER. We measured the TER of wild-type CFTR
160 HYALURONAN DISACCHARIDE ANALOGS AS CFTR CORRECTORS
JOURNAL OF CELLULAR BIOCHEMISTRY

Fig. 4. Transepithelial resistance. Long time observation of relative TER in 16HBE14oꢁ (A) and CFBE41oꢁ cells (B) in the absence (black) or presence of 100 mM of the
membrane permeable CFTR activator 8cpt-cAMP (red) or 100 mM of Hylout4 (blue). Substances were added after 9 h, the value measured directly before adding any substances
was set as 100% (reference value). The traces were calculated from the mean of three wells and the standard errors were below 5%.
Hylout4 at concentrations of 10, 50, 100, and 200 mM after 20 h and
found to be 94%, 89%, 67%, and 65%, respectively. The initial
absolute resistances were 407 ꢃ 41 V ꢂ cm2 and 440 ꢃ 48 V ꢂ cm2
for 16HBE14oꢁ and CFBE41oꢁ cells, respectively. These results
suggested a long term recovery of functionally active CFTR.
as the appearance of complex glycosylation (i.e., an increase in the
ratio of the C-band to the B-band form of the protein). Western
blotting with anti-CFTR antibodies showed that the amounts of
intact DF508-CFTR increased with Hylout4 (Fig. 5). In a control
ACTION OF Hylout4 ON DF508-CFTR EXPRESSION
The DF508-CFTR mutation impairs maturation and transport
competence at the ER and destabilizes DF508-CFTR in post-Golgi
compartments. Correctors may facilitate post-translational folding
of newly synthesized DF508-CFTR and/or enhance the stability of
mature DF508-CFTR. Therefore, we analyzed the amount of DF508-
CFTR in the cells in the presence of increasing Hylout4 concentra-
tions in CFBE41oꢁ cells. Correction of this primary defect is evident
Fig. 5. Expression of DF508-CFTR. CFBE41oꢁ cells were incubated with
increasing concentrations of Hylout4 for 24 h and the amounts of DF508-
CFTR was analyzed by Western blotting as described in the methods section.
JOURNAL OF CELLULAR BIOCHEMISTRY
HYALURONAN DISACCHARIDE ANALOGS AS CFTR CORRECTORS 161

experiment, we evaluated the level of DF508-CFTR mRNA and
found that it was not altered by Hyalout4. This result indicated that
compound Hylout4 enhanced cellular processing of DF508-CFTR.
ꢁ5 mV. Changing the perfusion to no-chloride caused a decrease of
PD by ꢁ15 mV. This reflects a depolarization of the apical
membrane of epithelial cells caused by chloride efflux which
was induced by an extracellular directed gradient for chloride.
Application of no-chloride solution together with 100 mM Hyalout
caused a further decrease of PD by ꢁ14 mV with a slope smaller than
for no-chloride alone (Fig. 6A). It is likely that Hyalout4 induced a
recruitment of CFTR molecules from submembraneous stores into
the plasma membrane, allowing a further chloride efflux and
subsequently a further depolarization of the apical membrane. The
following perfusion with no-chloride and 10 mM isoprenaline
caused a further PD decrease by ꢁ5 mV by a chloride efflux through
the activation of freshly recruited CFTR channels. In order to test the
reliability of the potential measurement, isotonic NaCl was perfused.
This resulted in a sharp increase of the PD reaching the basal NPD
withing a few minutes (Fig. 6). The change of PD upon maximal
activation of chloride conductance was about ꢁ34 mV, more than
40% caused by Hyalout4. In a second approach, isoprenaline was
perfused before Hyalout4 (Fig. 6B). The decrease of PD was about
ꢁ9 mV for isoprenaline in no-chloride solution. After the maximal
response to isoprenaline (plateau) Hyalout4 in no-chloride solution
caused a decrease of PD by maximum ꢁ10 mV. The pharmacological
induced decrease of PD in both experimental setting (Hyalout4 þ
isoprenaline) was about ꢁ20 mV. The effect of Hyalout4 was more
pronounced when perfused before isoprenaline. This indicates that
Hyalout4 (1) activated CFTR chloride conductance and (2) induced
the insertion of submembraneous stored CFTR molecules into the
plasma membrane. In the case of ‘‘isoprenaline before Hyalout4,’’
the activation of CFTR molecules already present in the plasma
membrane, was done by isoprenaline. Therefore, the portion
of Hyalout4 at the pharmacological induced decrease of PD is
limited to the recruitment and activation of submembraneous
stored CFTR.
NASAL TRANSEPITHELIAL POTENTIAL DIFFERENCE
There is no proper animal model for testing potential drugs that
could correct the defective DF508-CFTR function [Kukavica-Ibrulj
and Levesque, 2008]. Therefore we preliminarily tested Hylout4 on
healthy human volunteers with the approval of the local ethical
committee (Fig. 6). The NPD is used to assess ion conductance in the
upper respiratory epithelium. It is used for diagnosis of CF and to
monitoring the effects of pharmacological agents to correct the
abnormalities of ion transport in CF [Schuler et al., 2004]. During
perfusion with isotonic NaCl, both individuals showed a PD of
DISCUSSION
Recently, we discovered that CFTR can export hyaluronan in
addition to chloride or other halides such as iodide [Schulz et al.,
2010] and that hyaluronan export is defective in epithelial cells
expressing the mutated DF508-CFTR. This defective export could be
responsible for the highly viscous mucous of aggregated hyaluronan
protein mixtures in patients with CF, because hyaluronan is an
extremely hydrated polyanion that is the major water binding
component. In an attempt to modify hyaluronan export, we
synthesized two disaccharide analogs that differed only in the
position of one hydroxyl group mimicking either the non-reducing
terminus GlcNac or GlcA. These compounds were tested on human
breast carcinoma cells for their influence on hyaluronan exporter
CFTR. Surprisingly, the disaccharide with the non-reducing
terminus GlcNac was activating, whereas the non-reducing
terminus GlcA was inactive. Thus the activating property resided
in the position of the hydroxyl group indicating a stringent
specificity. Further modifying the chemical structure of the
activating disaccharide led to the hitherto most activating
compound Hylout4.
Fig. 6. Effect of compound Hylout4 on the nasal transepithelial potential
difference. The effect of Hylout4 was analyzed by the nasal potential difference
(NPD) of healthy individuals (A and B). Basal PD (isotonic NaCl) was ꢁ5 mV for
both individuals. No-chloride causes a decrease of PD by ꢁ15 mV. Perfusion of
Hyalout4 before isoprenaline causes a further decrease of PD by ꢁ14 mV with a
small slope. Isoprenaline causes a further PD decrease by ꢁ5 mV (A). In the case
of ‘‘isoprenaline before Hyalout4’’ (B), the effect of Hyalout4 was smaller
(ꢁ10 mV) than in the ‘‘Hyalout4 before isoprenaline’’ configuration. Perfusion
with isotonic NaCl at the end of the experiment ensures the reliability of the
potential measurement because of a PD increase back to the basal PD within a
few minutes.
162 HYALURONAN DISACCHARIDE ANALOGS AS CFTR CORRECTORS
JOURNAL OF CELLULAR BIOCHEMISTRY

In order to assess whether Hylout4 is a general activator of CFTR
in epithelial cells, we analyzed the effect of compound Hylout4 on
iodide transport as a convenient surrogate of chloride export. It
activated iodide export immediately in wild-type and DF508-CFTR
mutated epithelial cells, and it was CFTR specific, because activation
was reduced by the inhibitor CFTRinh-172. In addition, Hylout4
corrected DF508-CFTR cellular misprocessing and restored expres-
sion and halide permeability. When DF508-CFTR cells were
preincubated with Hylout4 for 24 h to allow full expression and
cellular processing, the iodide outflow increased about 2.6-fold. This
suggested that more functional DF508-CFTR channels were present
in the plasma membranes.
Hylout4 thus appeared superior by orders of magnitude. Another
challenge for therapy discovery has been an inhibition of halide flux
by small molecule correctors such as VRT-325 [Kim Chiaw et al.,
2010]. Hylout4 met this challenge.
ACKNOWLEDGMENTS
¨
The authors thank A. Blanke, R. Schulz, and Mike Walte for
excellent technical assistance.
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