Rescuing the CFTR protein function: Introducing 1,3,4-oxadiazoles as translational readthrough inducing drugs

Article abstract of DOI:10.1016/j.ejmech.2018.09.057

Nonsense mutations in the CFTR gene prematurely terminate translation of the CFTR mRNA leading to the production of a truncated protein that lacks normal function causing a more severe form of the cystic fibrosis (CF) disease. About 10% of patients affected by CF show a nonsense mutation. A potential treatment of this alteration is to promote translational readthrough of premature termination codons (PTCs) by Translational Readthrough Inducing Drugs (TRIDs) such as PTC124. In this context we aimed to compare the activity of PTC124 with analogues differing in the heteroatoms position in the central heterocyclic core. By a validated protocol consisting of computational screening, synthesis and biological tests we identified a new small molecule (NV2445) with 1,3,4-oxadiazole core showing a high readthrough activity. Moreover, we evaluated the CFTR functionality after NV2445 treatment in CF model systems and in cells expressing a nonsense-CFTR-mRNA. Finally, we studied the supramolecular interactions between TRIDs and CFTR-mRNA to assess the biological target/mechanism and compared the predicted ADME properties of NV2445 and PTC124.

Full text of DOI:10.1016/j.ejmech.2018.09.057

Accepted Manuscript
Rescuing the CFTR protein function: Introducing 1,3,4-oxadiazoles as translational
readthrough inducing drugs
Ivana Pibiri, Laura Lentini, Raffaella Melfi, Marco Tutone, Sara Baldassano, Paola
Ricco Galluzzo, Aldo Di Leonardo, Andrea Pace
PII:
S0223-5234(18)30835-3
10.1016/j.ejmech.2018.09.057
EJMECH 10768
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 10 August 2018
Revised Date: 21 September 2018
Accepted Date: 24 September 2018
Please cite this article as: I. Pibiri, L. Lentini, R. Melfi, M. Tutone, S. Baldassano, P.R. Galluzzo,
A. Di Leonardo, A. Pace, Rescuing the CFTR protein function: Introducing 1,3,4-oxadiazoles as
translational readthrough inducing drugs, European Journal of Medicinal Chemistry (2018), doi: https://
doi.org/10.1016/j.ejmech.2018.09.057.
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ACCEPTED MANUSCRIPT

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Rescuing the CFTR protein function: introducing
1
,3,4-oxadiazoles as translational readthrough
inducing drugs
‡a
‡a
a
a
a
a
Ivana Pibiri* , Laura Lentini* , Raffaella Melfi , Marco Tutone , Sara Baldassano , Paola Ricco Galluzzo , Aldo
a,b
a
Di Leonardo and Andrea Pace
a
Dipartimento di Scienze e Tecnologie Biologiche, Chimiche e Farmaceutiche (STEBICEF), Università degli Studi
b
di Palermo, Viale delle Scienze Ed. 16-17, 90128, Palermo, Italy. Centro di OncoBiologia Sperimentale (COBS)
via San Lorenzo Colli 90145 Palermo, Italy.
KEYWORDS. Cystic Fibrosis, Premature Termination Codon, Nonsense Mutation, Genetic
Disorder, Oxadiazole.
1
ABSTRACT. Nonsense mutations in the CFTR gene prematurely terminate translation of the
CFTR mRNA leading to the production of a truncated protein that lacks normal function causing
a more severe form of the cystic fibrosis (CF) disease. About 10% of patients affected by CF
ABBREVIATIONS: CCR2, CC chemokine receptor 2; CCL2, CC chemokine ligand 2; CCR5, CC chemokine
receptor 5; TLC, thin layer chromatography.
1

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show a nonsense mutation. A potential treatment of this alteration is to promote translational
readthrough of premature termination codons (PTCs) by Translational Readthrough Inducing
Drugs (TRIDs) such as PTC124. In this context we aimed to compare the activity of PTC124
with analogues differing in the heteroatoms position in the central heterocyclic core. By a
validated protocol consisting of computational screening, synthesis and biological tests we
identified a new small molecule (NV2445) with 1,3,4-oxadiazole core showing a high
readthrough activity. Moreover, we evaluated the CFTR functionality after NV2445 treatment in
CF model systems and in cells expressing a nonsense-CFTR-mRNA. Finally, we studied the
supramolecular interactions between TRIDs and CFTR-mRNA to assess the biological
target/mechanism and compared the predicted ADME properties of NV2445 and PTC124.
INTRODUCTION
Cystic fibrosis (CF) is caused by mutations in the gene encoding the cystic fibrosis trans-
membrane conductance regulator (CFTR). About 2000 different mutations are involved in the
CF pathology [1], the most common being a three base pair deletion causing the loss of
phenylalanine at position 508 (∆F508) and a missense mutation at position 551 (G551D).
Nonsense-mutations in the CFTR gene represent approximately 10% of the CF cases, their
presence produce a truncated protein that is rapidly degraded, so the patients lack the functional
protein and suffer a more severe form of the disease.
Pharmaceutical approaches targeting the specific genetic defect have been faced and
heterocyclic scaffolds rule the personalized medicinal approach, as proved by many studies in
the CF field [2-6].
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Concerning nonsense mutations, in the last ten years the only treatment proposed for these
patients was the translational readthrough of the premature termination codon (PTC) to
selectively promote the bypass of the PTC so that the expression of a functional protein is
restored to some extent. To this aim aminoglycosides (e.g. gentamicin, tobramycin,
paromomycin, geneticin reported in Figure 1) were previously used to suppress the normal
proof-reading function of the ribosome [7,8] leading to the insertion of a near-cognate amino
acid at the PTC site thus allowing the translation of the remainder of the open reading frame.
However, severe side-effects caused by prolonged treatments with aminoglycosides including
renal, auditory, and vestibular toxicities have been reported [9], limiting their widespread clinical
use for this purpose. Despite these issues, very recently a new aminoglycoside showed potential
activity as TRID and was launched in phase II clinical trials [10].
PTC124 (Figure 1), also known as Ataluren, was launched by PTC Therapeutics to promote the
readthrough of premature but not normal termination codons in HEK293 cells [11]. PTC124
does not possess the toxicity of an aminoglycoside and has been suggested as a potential
treatment of genetic disorders caused by nonsense mutations, particularly those involving the
UGA premature codon [12]. Although results of the phase II studies showed improvement in
markers of CFTR function, no improvements in sweat chloride levels or nasal potential
difference were reported in a new phase III trial [13]. In May 2014, PTC Therapeutics concluded
phase III Clinical trials for FC and DMD patients, in order to evaluate long-term safety of
PTC124 and supported the registration of the drug to the regulatory authorities. At conclusion of
this study it was evidenced that, although Ataluren (PTC124) did not improve lung function in
the overall population of nonsense-mutation cystic fibrosis patients who received this treatment,
it can be considered beneficial for patients not taking chronic inhaled tobramycin. The
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researchers' hypothesis was that PTC124 and tobramycin, both by acting at the level of the
ribosome are in competition with each other. To confirm these data an additional confirmation
Phase III clinical study was started but it was suspended in April 2017 due to conflicting results
[14, 15]. Therefore, today, Ataluren is among the prospective lead-like compounds despite its
real efficacy in CF patients and its effective biological target are still not clear [16].
Figure 1. Structures of reported Translational Readthrough Inducing Drugs
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Our previous results obtained by virtual mutagenesis of the CFTR mRNA and molecular
dynamic simulation in presence of PTC124, indicated that the bases surrounding the premature
stop codon in the mRNA could affect the readthrough mechanism [17, 18]. Indeed, we
hypothesized that PTC124 could interact differently with different type of nonsense mutations of
the CFTR gene (G542X, R1162X, W1282X etc.) depending on the genetic context. These
observations could explain why the efficacy of PTC124 in CF is still debated and highlights the
importance of the identification of new readthrough agents.
Oxadiazoles are one of the main characters in the drug discovery scene; being often used as
bioisosteric replacements for ester and amide functionalities. In a work by Boström et al. [19]
comparing 1,2,4- and 1,3,4-oxadiazoles, it has been shown a systematic trend, regardless of
substitution patterns, in the pharmaceutical properties of these two closely related molecules. In
virtually all cases, the 1,3,4-oxadiazole isomer shows an order of magnitude lower lipophilicity,
increased aqueous solubility, decreased hERG inhibition, and improved metabolic stability as
compared to the 1,2,4 isomer. In this context, considering that PTC124 has a 1,2,4-oxadiazole as
a core, we performed a virtual screening on public and in-house structure libraries and focused
further experimental studies on a selected set of small molecules containing a 1,3,4-oxadiazole
heterocyclic core.
Further selection was based on biological screening using the firefly luciferase reporter, while
the activity of the most promising compound as readthrough promoter was confirmed by
orthogonal assays. Further computational studies simulated the interactions of the most active
molecule with the specific PTC on the mRNA and predicted ADME properties.
Finally, further in vitro assays were performed to evaluate the efficacy of the most active
among the synthesized compounds in re-establishing the production of a functional CFTR
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protein in cells engineered to express a nonsense CFTR mRNA relative to the G542X mutation
as well as in immortalized and primary cells isolated from cystic fibrosis patients.
RESULTS AND DISCUSSION
Virtual Screening. The virtual screening campaign started with the development of
pharmacophore models on the basis of previous results [20] and with the refinement of obtained
models by means of 3D-QSAR (quantitative structure-activity relationships) analysis. The
endpoint to build QSAR models were the firefly luciferase activity (FLuc) data converted to
logFLuc values (Table 1). In order to find the common pharmacophore hypothesis, we divided a
set of twenty-four compounds in three sub-sets of different ranges of logFLuc activity values
(Pharm set). We considered as active compounds those with logFLuc values higher than the
value found for PTC124 (logFLuc > 4.528); as inactive compounds those with logFLuc ≤ 4.181,
and as compounds of moderate activity, those with 4.182 ≤ logFLuc < 4.527 (Table 1). The
entire Pharm set was randomly split into a training set (19 compounds) for the generation of
model hypotheses (Hypo), and a test set (5 compounds) for the validation of the developed
model, as reported in Table 1.
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Table 1. Pharm set for pharmacophore model development
logFLuc
RLU
Compound
PTC124
QSAR set
RLU values values
Pharm Set
Fitness
Exp.
Pred.
4.58
4.41
4.27
4.64
4.40
4.80
4.21
4.32
4.42
4.23
4.39
4.31
4.18
4.09
4.25
4.25
4.24
4.40
4.37
4.37
4.19
4.54
4.27
3.99
Test
4.528
4.532
4.477
4.628
4.397
4.788
4.125
4.103
4.311
4.269
4.439
4.414
4.178
4.008
4.171
4.363
4.250
4.411
4.428
4.250
4.181
4.551
4.253
4.000
Active
3.00
2.94
2.79
2.82
2.87
2.90
2.78
2.91
2.91
2.76
2.90
2.80
1.65
2.71
2.74
2.77
2.76
2.78
2.76
2.78
2.74
2.76
2.80
1.66
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
-NV1103
-NV1127
-NV1133
-NV1128
-NV1173
-NV1153
-NV1149
-NV1170
-NV1175
0-NV1171
1-NV1174
2-NV1798
3-NV1751
4-NV1954
5-NV1859
6-NV1861
7-NV1879
8-NV1883
9-NV1894
0-NV1905
1-NV1898
2-NV1919
3-NV1940
Training
Training
Training
Test
Active
Moderate
Active
Moderate
Active
Training
Training
Training
Training
Training
Training
Training
Training
Training
Test
Inactive
Inactive
Moderate
Moderate
Moderate
Moderate
Inactive
Inactive
Inactive
Moderate
Moderate
Moderate
Moderate
Moderate
Inactive
Active
Training
Training
Training
Training
Test
Training
Training
Test
Moderate
Inactive
Training
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Table 2. Statistical parameters for Hypo models comparison.
2
2
2
Model
ID
2
Pearson-
R
2
Q
2
’2
2
(R -
(R -
‘
R
SD
F
RMSE
Q
R
R
R
m
2
2
’2
2
k
k
0
0
ext
R )/R R )/R
0
0
Hypo.7 0.805 0.102 20.7 0.0672 0.9482
Hypo.8 0.798 0.104 19.8 0.0941 0.9077
0.72
0.714 0.851 0.821 0.617 -0.064 -0.026
0.987 1.012
0.982 1.017
0.451 0.611 1.263 0.708 0.253 -0.583 0.112
SD: standard deviation; F: Fisher test; RMSE: root mean square error.
The models have been robustly validated (Table 2), and Hypo.7 showed the best predictive
capability (Figure 2 and 3).
Figure 2. Test-set (left) and training-set (right) plots for Hypo.7
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Figure 3. Validated Hypo.7 aligned with PTC124
Hypo.7 has been used as starting point to perform a high-throughput virtual screening on public
databases together with an “in-house” library of compounds available in our laboratories. From
the top 5% of Hypo.7 retrieved hits, we selected seven synthetically accessible 1,3,4-oxadiazoles
to which we added 2-amino-5-methyl-1,3,4-oxadiazole (NV924) as a representative non-arylated
1
,3,4-oxadiazole with apolar (CH ) and H-bond acceptor/donor (NH ) substituents (Figure 4).
3 2
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F
N N
O
N N
COOCH3
O
COOCH3
NV2347
NV2653
N N
N N
O
H C
3
O
NH2
F
NV924
NV1191
F
N N
O
N
N
COOCH3
O
F
COOH
F
NV2445
NV2712
F
F
N N
O
N N
O
COOH
COOH
F
F
NV2702
NV2729
Figure 4. Selected set of synthetically accessible 1,3,4-oxadiazoles
Synthesis. The synthetic protocol used to obtain compounds having as common feature the
,3,4-oxadiazole core is the following (see Scheme 1): The first step is a solvent free reaction
performed in a microwave oven at 900W, by reacting substituted-benzoic acid with hydrazine. In
1
1
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a following step the so obtained products were further reacted with the appropriate aldehyde in
refluxing ethanol, finally cyclization at 100°C was realized by stirring the open-chain
compounds in the presence of molecular iodine and potassium carbonate in DMSO. In the case
of ester compounds, further hydrolysis with lithium hydroxide in THF produced the
corresponding acids.
Scheme 1. Synthetic path to 1,3,4-oxadiazoles
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Experimental screening of the readthrough activity by the FLuc assay. To ascertain the
effectiveness of the newly synthesized PTC124 analogues in promoting the readthrough of
premature termination codons we used the FLuc cell-based assay [17, 18]. To this aim HeLa
cells were transfected transiently with the plasmids pFLuc-WT (control) and pFLuc-opal (UGA
stop mutation) [20]. FLuc gene expression was then measured by luminescence. HeLa cells
transfected with the pFluc-WT plasmid showed high levels of luciferase activity confirming the
functioning of the assay (Figure 5). On the contrary, pFluc-opal transfected HeLa cells did not
show any activity. However, when these pFluc-opal cells were treated with PTC124 and 8
different analogues we observed a significant increase of luciferase activity in comparison to
untreated cells specifically with the analogues: NV2445>NV2712>2347 (Figure 5).
Figure 5. The new synthetized molecules show readthrough activity in FLuc cell model
system. Histogram showing luciferase (FLuc) activity after 24 hours of exposition to PTC124
and its analogues (all at the concentration of 12 µM) in HeLa FLuc-opal transfected cells.
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Next, we focused on the compound NV2445 that showed enhanced readthrough activity. To
confirm the results for NV2445 we used HeLa cells stably transfected with a vector harboring
the UGA mutation in the coding sequence of the H2BGFP cDNA [21]. Detection of green
fluorescent cells after 24 hours of treatment indicates that NV2445, allows PTCs readthrough
and the translation of the full length H2B-GFP fusion protein. PTC124 and G418 were used as
positive control (Figure 6).
Figure 6. NV2445 induces H2BGFP expression. Immunofluorescence analysis of the H2BGFP
fusion protein in H2BGFP-Opal transfected HeLa cells after 24 hours of exposition to PTC124,
G418 (positive controls) and the newly synthesized molecule NV2445.
Induced Fit Docking and Molecular Dynamics on NV2445-mRNA. Similarly, to our previous
study [21], and based on the experimental data obtained in this work, we decided to perform
computational simulations modeling the interaction of a 33-nucleotides CFTR mRNA fragment,
harboring a UGA premature stop codon, with NV2445. Induced Fit docking run followed by a
1
00 ns MD simulation were performed. In the Induced Fit docking (IFD) analysis, the docking
box was centered on the equilibrium pose of PTC124 extracted from our previous MD study
21]. Obtained results from IFD showed as NV2445 pose is similar to PTC124 with some
[
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difference in terms of interactions. In fact, despite PTC124-mRNA interaction is stabilized by a
H-bond with A18, NV2445 establishes H-bond with U12. π-π stacking between G10 and the
three aromatic rings of NV2445, and G17-1,3,4-oxadiazole ring, are the essential interactions.
Despite these different binding modes, docking scores of the two molecules are almost equal
(∆docking score = 0.6 kcal/mol) (Figure 7).
Figure 7. IFD poses of NV2445 (light blue) compared with PTC124 equilibrium pose (grey).
Dashed light blue lines represent the π-π stacking interaction.
With the aim to explore how the NV2445-mRNA interactions vary during the time, and to
compare with our previous study, we performed 100ns MD run starting from the NV2445 pose
obtained by means of IFD. NV2445 remains in the RNA loop for the whole simulation time.
Nevertheless, we observed a particular behavior of NV2445, with substantial difference from
PTC124 with the UGA stop codon. After about 1 ns, NV2445 moves inside the nucleotide
sequence, assuming a parallel pose among A18 and G19. This orientation of NV2445 is
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maintained along the rest of the simulation, stabilized by π-π stacking occurring between the aryl
moiety bearing the carboxyl group and A18, and between the tolyl moiety with G19. This
particular pose puts the carboxyl group directed towards the phosphate chain, establishing H-
bonding with the water molecules. While the oxadiazole ring remains in close contact with A18.
In a such orientation the nitrogens of the 1,3,4-oxadiazole moiety are in close contact with the
N1 and the amino group of the adenine 18, which are responsible of the H-bonds that guarantee
the anticodon recognition (Figure 8).
This insertion could primarily lead to mRNA:tRNA mispairing at codon position 3 and reflects
the proposed mechanism of action for PTC124 by Jacobson and coworkers [22]. In fact, they
experimentally proved mispairing and insertion of the near-cognate tRNA, in particular, they
demonstrated that, at position 3, multiple nonstandard mispairings are tolerated including A-C,
G-G, A-G, and possibly others.
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Figure 8. MD snapshots of NV2445 inserted into A18 and G19 of the mRNA. Dashed light blue
lines represent the π-π stacking interactions.
We extended our investigation with the MM-GBSA calculations to get quantitative estimates
for the binding free energies of the NV2445-mRNA complex compared with PTC124-mRNA
complex. By means of such analysis, we are able to evaluate the free energy components such as
van der Waals (vdW), electrostatic, and solvation contributions to provide detailed molecular
information regarding the complexes under investigation (Table 3). The analysis of the MM-
GBSA terms indicates that, even though, the two compounds have high similarity (shape
similarity = 0.83) the physico-chemical properties that have a preponderant role in the potency
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are quite different. For PTC124, vdW and electrostatic contributes are prominent, while for
NV2445, vdW is prominent but the major contribute is due to solvation term as observed by the
H-bonding of carboxyl group with water molecules. However, the binding affinity can be the
result of the combination of the particular effect of these features.
Table 3. Predicted free-energies (kcal/mol) and individual energy terms of mRNA-
NV2445/PTC124 complexes
Molecule
NV2445
PTC124
∆Gcalc
∆GvdW
-24.554
-13.994
∆GCoul
98.285
-16.292
∆Gsolv
∆∆Gcalc
-1.213
ref
-14.638
-13.425
-89.253
15.821
ADME-Tox predictions for NV2445. In the attempt to predict absorption, distribution,
metabolism, and excretion (ADME) for NV2445, this one was submitted to Qikprop calculations
and toxicity prediction such as physics-based membrane permeability and structure based P450
sites of metabolism prediction. Regarding ADME predictions the final result is expressed in
terms of #stars, Number of property or descriptor values that fall outside the 95% range of
similar values for known drugs large number of stars suggests that a molecule is less drug-like
than molecules with few stars. The following properties and descriptors are included in the
determination of #stars: MW, dipole, IP, EA, SASA, FOSA, FISA, PISA, WPSA, PSA, volume,
#
rotor, donorHB, accptHB, glob, QPpolrz, QPlogPC16, QPlogPoct, QPlogPw, QPlogPo/w, logS,
QPLogKhsa, QPlogBB, #metabol. (Table 4). All the calculated values for NV2445 were
compared with the calculated values for PTC124.
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Table 4. Predictive ADME-Tox comparison between PTC124 and NV2445
FISA
(7.0-
#
stars
MW
(130-725)
Dipole
(1.0-12.5)
IP(eV)
(7.9-10.5) (-0.9-1.7)
EA(eV)
SASA
(300-1000) (0-750)
FOSA
molecule
(
0-5)
3
30)
NV2445
PTC124
0
1
280.3
284.2
6.2
1.9
9.5
9.7
1.2
1.3
536.0
520.1
73.4
0
144.2
154.6
Volume
(500-
Glob
(0.75-
0.95)
0.84
PISA
WPSA
(0-175)
PSA
(7-200)
#rotor
(0-15)
donorHB
(0-6)
accptHB
(2-20)
molecule
(
0-450)
2
000)
NV2445 318.4
0
82.5
85.1
903.0
1
1
1
1
4.5
5
PTC124
333.4
32.1
866.8
0.84
QPpolrz QPlogPC16 QPlogPoct QPlogPw QPlogPo/w QPlogKhsa QPlogBB #metab
molecule
(
13-70)
(4-18)
(8-35)
(4-45)
(-2.0-6.5)
(-1.5-1.5)
(-3.0-1.2) (1-8)
NV2445 32.38
10.01
15.28
9.83
2.898
0.01
-0.85
2
PTC124 31.08
9.63
14.80
10.47
2.6
-0.17
-0.86
0
MW, molecular weight; IP, PM3 calculated ionization potential (negative of HOMO energy);
EA, PM3 calculated electron affinity (negative of LUMO energy); SASA, Total solvent
accessible surface area (SASA) in square angstroms using a probe with a 1.4 Å radius; FOSA,
Hydrophobic component of the SASA (saturated carbon and attached hydrogen); FISA,
Hydrophilic component of the SASA (SASA on N, O, H on heteroatoms, carbonyl C); PISA,
(carbon and attached hydrogen) component of the SASA; WPSA, Weakly polar component of
the SASA (halogens, P, and S); Volume, Total solvent-accessible volume in cubic angstroms
using a probe with a 1.4 Å radius; #rotor, number of rotatable bonds; donorHB, number of h-
2
bond donor, accptHB, number of H-bond acceptor; glob, Globularity descriptor, (4π
r
) ⁄ (SASA
)
,
where
r
is the radius of a sphere with a volume equal to the molecular volume; QPpolrz,
Predicted polarizability in cubic angstroms, QPlogPC16, Predicted hexadecane/gas partition
coefficient; QPlogPoct, Predicted octanol/gas partition coefficient; QPlogPw, Predicted
water/gas partition coefficient; QPlogPo/w, Predicted octanol/water partition coefficient.;
QPlogKhsa, Prediction of binding to human serum albumin; QPlogBB, Predicted brain/blood
partition coefficient; #metab, Number of likely metabolic reactions.
Furthermore, NV2445 respects the Lipinski’s rule of five, and it has a predicted human oral
absorption >80%. CYPP450 sites of metabolism for isoform 2C9 were also calculated for
NV2445, and reported in Figure 9. Combined results show as the tolyl ring could represents a
good site of metabolism for NV2445.
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Figure 9. P450 site of metabolism (SOM). Intrinsic reactivity: positive values show high
reactive site, negative values show weak reactive site; Fe-accessibility: positive values show
high accessibility, negative values show poor accessibility, Combined reactivity: the number of
rays indicates the values of the Fe-accessibility from IFD calculation, The circle encode 3
separate pieces of data.1) the size of the circles indicates the overall SOM score – the larger the
circle the better the score, 2) the color of the circle indicates the atomic intrinsic reactivity – the
redder the circle, the higher the intrinsic reactivity, 3) a blu ring around the circle indicates that
the atom passed through the CYP filtering stage.
Detection of CFTR re-expression and localization on the cellular membrane of IB3.1
(CFTR ꢀF508/W1282X) cells after treatment with PTC124 analogues. To evaluate if
NV2445, besides allowing the readthrough of PTCs, was able to restore CFTR proper
localization to the plasma membranes, a biological test involving a human IB3.1 cell line has
been performed.
The IB3.1 is an immortalized human cell line from FC patient harboring two mutant alleles of
CFTR (ꢀF508 and W1282X) causing impaired plasma membrane normal localization. The
CFTR protein was detected in cells not permeabilized by a specific antibody targeting its first
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external portion. The IB3.1cells were treated with G418 (430 µM), PTC124 and NV2445 (both
at 12 µM). Immunofluorescence microscopy images show that after a 24 hours treatment CFTR
actually localize at the cell membrane suggesting the translation readthrough occurrence,
CFBE41o-cells transfected with a CFTR WT construct was used as positive control of the CFTR
expression (Figure 10A-B).
Figure 10. Immunofluorescence shows expression of the CFTR protein following the
readthrough of the UGA stop codon in CFTR of IB3.1 human cells. A) Expression of CFTR
in CFBE41o- (CFTRwt; positive control) cells and in B) IB3.1 cells untreated (untr; negative
control), treated with G418 (positive control), with PTC124 and NV2445 for 24 hours. CFTR
protein was revealed by a specific antibody targeting its first external loop (green, Alexa-488).
Nuclei (blue) were DAPI stained. The cell membrane and Golgi apparatus were stained in red
(
WGA-Alexa 594).
To quantify the CFTR protein level after exposure to NV2445 we treated IB3.1 cells with
PTC124 and NV2445 for 24 hours and extracted and purified the membrane protein fractions
that were analyzed by western blot (Figure 11A-B). For the Western blot, we used a CFTR C-
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terminal specific antibody. The treatment with the newly synthesized molecule NV2445
dramatically increased the presence of the CFTR protein in IB3.1 cell membrane fraction when
compared to untreated (figure 11A-B).
A
B
Figure 11. A) Western blot showing CFTR protein levels in membrane extracts of IB3.1 cells
untreated (lane: 1) or treated with the indicated compounds (lane 2: PTC124; lane 3: NV2445).
Ponceau red was used as a loading control. B) Histogram of the densitometry of the Western
blotting bands. A primary antibody raised against the C-terminus of CFTR was used.
A successive step was to measure the efficacy of the readthrough activity of NV2445 in a
primary human bronchial epithelial cell line (genotype: L1077P/W1282X) from CF patients, the
results (SM section 1 and Figure S1) confirmed the results we observed in IB3.1 cells.
Treatment of the IB3.1 cells with NV2445 induces a functional CFTR protein. To ascertain
the functionality of the re-expressed CFTR protein, produced after exposure to NV2445, a
quench-EYFP assay was performed. This assay is based on iodide-mediated quenching rates of
an ectopically expressed mutant form of the Yellow fluorescent protein (YFP). To this aim,
IB3.1 and CFBE41o- cells were transfected with the plasmid pCDNA3.1 EYFP (M148Q; I152L)
and selected for the resistance to the G418 antibiotic. Figure 12 shows the results of the EYFP
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assay done in IB3.1 cells after 24 hours of exposition to PTC124 and NV2445. Cells were plated
in a 6 well plate one day before the experiment, they were stimulated with forskolin (20 µM) for
2
0 min and placed in a buffer containing iodide (PBS1x similar buffer, NaCl is replaced by
equimolars of NaI ). Changes in fluorescence intensity were monitored by a Zeiss fluorescence
microscope and the images were recorded every 1.5 s with a CCD digital camera (AxioCam,
Zeiss).
Upon replacement of chloride for iodide we observed a rapid decline in EYFP fluorescence
(
Figure 12) in the IB3.1 cells treated with NV2445 and in positive control cells (PTC124; G418
treated cells).
Figure 12. Exposure of IB3.1 cells to NV2445 induces functional expression of the CFTR
protein channel. EYFP assay in CFBE41o- and IB3.1 cells transfected with the pcDNA3 YFP-
H148Q-I152L vector, untreated or treated with the indicated compounds for 24 hour (G418: 430
µM; PTC124 and NV2445: 12 µM).
Despite the good results obtained on IB3 cells, during the YFP assay the long exposition to
PBS/forskolin induced the detachment of many of them from the plate, reducing the number of
analyzable cells. Moreover, the genotype of IB3 cells was not a homozygous for the stop
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mutation, precisely the IB3.1 cells used in our experiment showed a CFTR allele with the ∆F508
mutation. Therefore, to confirm results observed with IB3.1 cells, we shifted to another cell line.
To this aim we generated Fisher rat thyroid cell harboring the mutated (G542X) CFTR obtained
by Site directed mutagenesis (see SM, Section 2, Figures S2, S3 and S4 for detailed
information).
Production of FRT cells expressing nonsense-CFTR (nsCFTR) and evaluation of the CFTR
expression after treatment with NV2445. To further evaluate the efficacy of NV2445 FRT
WT
cells were transfected with the pTracer vectors containing either the WT-CFTR (CFTR ) or the
(
G542X-opal)
G542X-CFTR (nsCFTR
) human cDNA and selected by Zeocin resistance. The
advantage of the use of these cells is that they do not express cAMP dependent channels (as
WT
(G542X-opal)
CFTR) or CFTR protein. The expression of the CFTR and nsCFTR
in transfected
FRT cells was evaluated by qRT-PCR and Western blot (Figure 13).
Figure 13. A) qRT-PCR to detect CFTR transcript expression after Zeocin selection in FRT
WT
cells. B) Western blot to detect CFTR protein in FRT transfected cells (CFTR
and
(
G542X-opal)
nsCFTR
). β-tubulin was used as control for protein loading.
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The FRT cells were then treated with NV2445 (12 µM), PTC124 (12 µM) and G418 (430
WT
µM) (as positive controls) for 24 hours and the expression of the human CFTR
and
(
G542X-opal)
nsCFTR
was evaluated by immunofluorescence and Western blot analyses (Figure 14
A-C). Immunofluorescence microscopy images allow to visualize CFTR localization at the cell
membrane suggesting the occurrence of translation readthrough and proper localization also in
this cell type after NV2445 exposure (Figure 14 A). Similar results were obtained by the Western
blot analysis. After 24 hours of treatment with NV2445 CFTR protein levels appreciably
increase when compared with controls (Untreated and PTC124) (Figure 14 B-C).
Figure 14. A) Immunofluorescence analysis to detect the CFTR protein following the
(
opal)
readthrough of the UGA stop codon in nsCFTR
FRT cells untreated (untr), treated with
G418 (positive control) and with PTC124 and NV2445 for 24 h. CFTR protein was revealed by a
specific antibody targeting its first external portion and a secondary antibody (SC2092-red).
Nuclei (blue) were DAPI stained. B) Western blot analysis to detect CFTR protein in transfected
(
gG542X-opal)
FRT cells (FRT-CFTR
) left untreated and treated with PTC124 and NV2445 for 24 h
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β-tubulin was used as control for the protein loading. C) Quantization of the gel bands by ImageJ
software.
Molecular and functional analyses by record in vitro of the transepithelial CFTR-mediated Cl-
current and EYFP assay after NV2445 exposure indicated functional CFTR channel production
(
opal)
in FRT cells. Our results showed that the exposition of nsCFTR
FRT cells to the NV2445
induced the CFTR full-lenght protein expression and its membrane localization. Therefore, the
next step was to analyse the functionality of the CFTR channel in our cell model system by two
different approaches.
The first approach was to detect CFTR activity by the EYFP quenching assay. FRT cells
ectopically expressing the EYFP protein, were used to perform the “halide sensor fluorescent
WT
(opal)
assay”. To evaluate CFTR activity/functionality, FRT EYFP cells (CFTR and nsCFTR
were treated with NV2445 and then analysed by fluorescent microscopy and fluorimetric assays
Figure 15 A-B).
)
(
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WT
opal
Figure 15. A) EYFP assay in FRT EYFP-CFTR and CFTR cells, untreated or treated with
G418 (430 µM), PTC124 (12 µM) and NV2445 (12 µM) for 24 hours, pre-stimulated with
forskolin (20 µM) for 20 min, before and post addition of NaI 137 mM to quench the EYFP
fluorescence in presence of a functional CFTR channel. B) FRT EYFP-CFTR opal cells,
untreated (only forskolin 20 µM for 20 min) or treated with G418 (430 µM), PTC124 (12 µM)
and NV2445 (12 µM) for 24 hours, stimulated by forskolin 20 min before of the assay (with or
without NaI addition).
By the second approach we measured the activity of the CFTR channel by using FRT cells that
are able to differentiate in a tight epithelium thus allowing evaluation of CFTR channel
-
functionality by trans-epithelial CFTR-mediated Cl current.
Cells were plated on Snapwell permeable supports for one week (sufficient time to induce
epithelium formation) and then exposed to NV2445 and PTC124 for 24 hours (apical and basal).
WT
(G542X-opal)
CFTR FRT cells were used as positive controls while untreated nsCFTR
FRT cells
as negative controls.
-
In order to determine restoration of CFTR channel function, transepithelial CFTR-mediated Cl
currents across cell monolayer was measured by Ussing Chamber. Under basal condition, there
WT
was no significantly difference in basal Isc among the samples (Figure 16). In CFTR FRT
epithelium, stimulation of monolayers with the adenylate cyclase activator FSK (20 µM)
2
(opal)
increased basal Isc by 27.5 µA/cm . Application of FSK in nsCFTR
FRT treated with
NV2445 and PTC124 produced a substantial increase in basal CFTR-dependent Isc by 17
2
2
(G542X- opal)
µA/cm and 11 µA/cm respectively, whereas untreated nsCFTR
FRT cells did not
produce any increase in forskolin-stimulated chloride currents (Figure 16).
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(
G542Xopal)
Figure 16. Characterization of CFTR-dependent Isc in nsCFTR
FRT epithelium.
-
Average of the basal current and of transepithelial CFTR-mediated Cl currents after application
of FSK (20 µM) in WT-CFTR-FRT (positive control) and in nsCFTR
untreated or treated for 24 hours with PTC124 (12 µM) and NV2445 (12 µM).
(
opal)
FRT epithelium
In vitro analysis of the safety profile of NV2445. Primary human fibroblasts IMR90 were used
to determine the possible cytotoxic effects of NV2445 and the impact on cell proliferation. In
figure 17A-B are reported the percentages of dead cells and proliferating cells after treatment
with 12 µM NV2445, 12 µM PTC124 and 430 µM G418. Our results showed that NV2445 and
PTC124 induce an increase in dead cells at 24h (PTC124:30%; NV2445: 18%) and 72h
(PTC124: 30%; NV2445: 21%) that was constant in the time. In contrast the G418 induced the
3
5% of dead cells at 24 hours and this percentage increased at 72 hours (51%). We observed that
IMR90 cells proliferate as untreated cells when treated daily (every 24h) for 10 days with
NV2445 (figure 17 C).
(
G542X- opal)
Similar results were observed by MTT assay performed nsCFTR
FRT cells to
evaluate the effects on cell viability at three different concentrations (12, 24, 48 µM) of the
NV2445 after 24 and 72 hours of treatment (See SM Section 3 Figure S5).
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The possible genotoxic effect of NV2445 was evaluated by comparing the expression of a
DNA damage marker (γH2AX) after treatment with NV2445, PTC124 and Doxorubicin (DNA
damage agent used by us as positive control). By immunofluorescence analysis using a primary
antibody against γH2AX phosphorylated in Ser139 we detected γH2AX positive cells only in
doxorubicin treated IMR90 (Figure 17 D).
Figure 17. A) Percentages of live and dead IMR90 cells untreated or treated with G418 (430
µM), PTC124 (12 µM) and NV2445 (12 µM) for 24-72hours; and in B) the relative count of cell
proliferation at the indicated time. C) Representative images of the primary human fibroblasts
IMR90 after ten days of daily treatment (every 24h) with the indicated drugs. D) Evaluation of
DNA damage (genotoxic effect) in IMR90 cells untreated or treated with Doxorubicin (430
µM), PTC124 (12 µM) and NV2445 (12 µM) for 72hours (Doxorubicin 0,2 µg/ml was used as
positive control).
This result was confirmed by another analysis performed to identify a form of p53
phosphorylated in Ser15, specific for DNA damage response.
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