Enhancement of premature stop codon readthrough in the CFTR gene by Ataluren (PTC124) derivatives

Article abstract of DOI:10.1016/j.ejmech.2015.06.038

Abstract Premature stop codons are the result of nonsense mutations occurring within the coding sequence of a gene. These mutations lead to the synthesis of a truncated protein and are responsible for several genetic diseases. A potential pharmacological

Full text of DOI:10.1016/j.ejmech.2015.06.038

European Journal of Medicinal Chemistry 101 (2015) 236e244
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Research paper
Enhancement of premature stop codon readthrough in the CFTR gene
by Ataluren (PTC124) derivatives
,
b
,
,
, b, *
Ivana Pibiri a 1, Laura Lentini a 1, Raffaella Melfi a, Giulia Gallucci a, Andrea Pace a
,
Angelo Spinello a, Giampaolo Barone a b, Aldo Di Leonardo a
,
, c, **
a
ꢀ
Dipartimento di Scienze e Tecnologie Biologiche, Chimiche e Farmaceutiche (STEBICEF), Universita degli Studi di Palermo, Viale delle Scienze Ed. 17, 90128
Palermo, Italy
b Istituto EuroMediterraneo di Scienza e Tecnologia (IEMEST), Via Emerico Amari 123, 90139 Palermo, Italy
c Centro di OncoBiologia Sperimentale (COBS), Via San Lorenzo Colli, 90145 Palermo, Italy
a r t i c l e i n f o
a b s t r a c t
Article history:
Premature stop codons are the result of nonsense mutations occurring within the coding sequence of a
gene. These mutations lead to the synthesis of a truncated protein and are responsible for several genetic
diseases. A potential pharmacological approach to treat these diseases is to promote the translational
readthrough of premature stop codons by small molecules aiming to restore the full-length protein. The
compound PTC124 (Ataluren) was reported to promote the readthrough of the premature UGA stop
codon, although its activity was questioned. The potential interaction of PTC124 with mutated mRNA was
recently suggested by molecular dynamics (MD) studies highlighting the importance of H-bonding and
Received 16 March 2015
Received in revised form
14 June 2015
Accepted 19 June 2015
Available online 21 June 2015
Keywords:
Cystic fibrosis
stacking
pep interactions. To improve the readthrough activity we changed the fluorine number and
position in the PTC124 fluoroaryl moiety. The readthrough ability of these PTC124 derivatives was tested
in human cells harboring reporter plasmids with premature stop codons in H2BGFP and FLuc genes as
well as in cystic fibrosis (CF) IB3.1 cells with a nonsense mutation. Maintaining low toxicity, three of
these molecules showed higher efficacy than PTC124 in the readthrough of the UGA premature stop
codon and in recovering the expression of the CFTR protein in IB3.1 cells from cystic fibrosis patient.
Molecular dynamics simulations performed with mutated CFTR mRNA fragments and active or inactive
derivatives are in agreement with the suggested interaction of PTC124 with mRNA.
PTCs readthrough
CFTR gene
Green fluorescent protein
Nonsense mutation
Fluorinated oxadiazole
© 2015 Elsevier Masson SAS. All rights reserved.
1. Introduction
dystrophy and cystic fibrosis are genetic diseases for which these
approaches have been tested. Cystic fibrosis (CF) is caused by mu-
Nonsense mutations are single nucleotide changes in the gene
that result in in-frame UGA (opal), UAG (amber), and UAA (ochre)
premature termination codons (PTCs) in the coding region of the
mRNA. These PTCs cause inappropriate termination of translation
leading to a truncated protein unable to fulfill its functions, and
promote mRNA destabilization by the nonsense-mediated mRNA
decay (NMD) pathway [1]. Over the years, several strategies have
been suggested to facilitate the readthrough of PTCs [2e4], thus
restoring the synthesis of a full-length protein. Duchenne muscular
tations in the gene encoding the cystic fibrosis transmembrane
conductance regulator (CFTR). More than 2000 disease-causing
mutations in CFTR have been identified and can be divided into
six major classes [5]. The most commonly observed mutations
include three base pair deletion causing the loss of phenylalanine at
position 508 (delta-F508), a missense mutation at position 551 and
a nonsense mutation at position 542 (G542X) known as a class I
mutation type. Generally, CF patients with nonsense-mutation in
the CFTR gene produce no CFTR protein thus suffering from a more
severe form of the disease. A potential treatment for these patients
is to selectively promote translational readthrough of the PTC so
that the expression of a functional protein will be restored to some
extent. To this aim aminoglycosides antibiotics (gentamicin,
tobramycin, paromomycin, geneticin etc.) were previously
employed to suppress the normal proof-reading function of the
ribosome [6,7] leading to the insertion of a near-cognate amino acid
* Corresponding author. Dipartimento di Scienze e Tecnologie Biologiche, Chi-
ꢀ
miche e Farmaceutiche (STEBICEF), Universita degli Studi di Palermo, Viale delle
Scienze Ed. 16, 90128 Palermo, Italy.
** Corresponding author. Centro di OncoBiologia Sperimentale (COBS) via San
Lorenzo Colli 90145 Palermo, Italy.
E-mail addresses: andrea.pace@unipa.it (A. Pace), aldo.dileonardo@unipa.it
(A. Di Leonardo).
1
I.P. and L.L.: these authors contributed equally to this study.
http://dx.doi.org/10.1016/j.ejmech.2015.06.038
0223-5234/© 2015 Elsevier Masson SAS. All rights reserved.

I. Pibiri et al. / European Journal of Medicinal Chemistry 101 (2015) 236e244
237
at the PTC site, thus allowing the translation of the remainder of the
open reading frame. This “translational readthrough” of premature
stop codons, but not of normal termination codons, has been
shown to partially restore protein function in a number of pre-
clinical settings [8e10]. However, severe side-effects caused by
prolonged treatments with aminoglycosides including renal,
auditory, and vestibular toxicities have been reported [11], along
with the reduced read-through ability at subtoxic doses in a mouse
model [12] and in clinical trials [13]. These results have limited the
widespread clinical use of aminoglycosides for this purpose. The
compound PTC124, also known as Ataluren, was originally reported
to promote the readthrough of premature but not normal termi-
nation co-dons in HEK293 cells transfected with a luciferase re-
porter gene (LUC190) harboring a PTC at Thr190, where the normal
ACA codon was replaced with either UAA, UAG, or UGA codon [14].
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 pre-
mature codon [12,14,15]. Although earlier studies [16e19] ques-
tioned whether Ataluren promoted readthrough, this issue has now
been addressed by other independent publications demonstrating
this compound's readthrough activity in diverse cellular and animal
models [20e26]. In this context of contrasting results, obtained
from both in vitro and in vivo experiments, PTC124's mechanism of
action still remains not well established [4]. Therefore, it is crucial
that claims regarding the readthrough activity of PTC124 or of its
derivatives are confirmed by experiments on at least two reporters
(orthogonal assay) [27,28]. In our previous study, we attempted to
rationalize PTC124's high selectivity toward the readthrough of opal
with respect to amber and ochre nonsense mutations. Computa-
tional results on the hypothesized supramolecular interaction be-
tween PTC124 and mRNA highlighted that a selective interaction
with the premature UGA codon occurs through hydrogen bonding
of PTC124 carboxylic moiety and stacking interactions involving its
aromatic rings. On this basis, we were prompted to investigate
variations of the PTC124 scaffold, particularly considering the
esterification of the carboxylic moiety as well as the number and
position of fluorine atoms as factors able to strongly affect the
PTC124's ability to interact with its biological target. These modi-
fications, some of which were previously pointed out by PTC
Therapeutics [29], would allow us to focus our biological assays to
rationalize structure-activity relationships (SAR). Synthesized de-
rivatives were first tested for their readthrough activity with a
luciferase-based reporter. Most active compounds were further
tested with a green fluorescent protein (GFP) based reporter and
evaluated for the suppression of nonsense mutations in the CFTR
gene in the human bronchial epithelial cell line IB3.1 (CFTR geno-
type W1282X/F508del).
tests by HPLC and NMR. In all the cases purity was higher than 95%.
HRMS spectra were recorded by analyzing a 50 ppm solution of the
compound in a 6540 UHD Accurate-Mass Q-TOF LC/MS (Agilent
Technologies) equipped with a Dual AJS ESI source.
2.1.1. General procedure for the synthesis of compounds 3 and 4a-l
2 mmol of either amidoxime 1 [30] or 2 [26] were dissolved in
acetone (200 mL) in a round-bottomed flask; then, K2CO3 (0.35 g;
2.5 mmol) and the corresponding aroyl chloride (2.5 mmol), were
added to the reaction mixture and stirred for 24 h at room tem-
perature. The solvent was removed under vacuum and the residue
treated with water and refluxed for 30 min 1,2,4-Oxadiazoles
products were obtained by filtration and further purified by
chromatography.
2.1.1.1. 3-Toluyl, 5-(2-fluorophenyl)-1,2,4-oxadiazole (3a). Yield 70%;
m.p. 93e95 ꢀC (lit. 93 ꢀC) [31]. HRMS for C15H11FN2O found
255.0924 [MþH]þ (Calcd 255.0928).
2.1.1.2. Methyl 3-(5-(2-fluorophenyl)-1,2,4-oxadiazol-3-ꢁyl1)benzoate)
(4a). Yield 80%; m.p. 134e136 ꢀC; IR (nujol): 1723 cm
;
1H NMR
(300 MHz, CDCl3):
(m, 2H), 7.59 e 7.52 (m, 2H), 7.32 e 7.20 (m, 2H), 3.91 (s, 3H). 13
NMR (75 MHz, CDCl3): (ppm) 173.8, 168.8, 167.0, 163.6, 159.4,
d (ppm) 8.78 (m, 1H), 8.31 (m, 1H), 8.18 e 8.12
C
d
149.6, 135.4, 132.9, 132.4, 131.7, 129.6, 127.9, 125.4, 117.9, 115.0, 53.0.
GCeMS: m/z (%) 298 (Mþ, 60), 267(70),177 (35),146 (60),130 (100),
102 (60), 75 (45), 44 (75). HRMS for C16H11FN2O3 found 299.0817
[MþH]þ (Calcd 299.0826).
2.1.1.3. Methyl 3-(5-(3-fluorophenyl)-1,2,4-oxadiazol-3-ꢁyl1)benzoate)
(4b). Yield 78%; m.p. 124e126 ꢀC; IR (nujol): 1733 cm
;
1H NMR
(300 MHz, CDCl3):
d
(ppm) 8.84 (s, 1H), 8.37 (d, 1H, J ¼ 7.2 Hz), 8.21
(d, 1H, J ¼ 8.4 Hz), 8.04 (d, 1H, J ¼ 6.9 Hz), 7.94 (d, 1H, J ¼ 9 Hz), 7.64
e 7.53 (m, 2H), 7.37 e 7.26 (m, 1H), 3.99 (s, 3H). GCeMS: m/z (%)
298 (Mþ, 60), 267(90), 177 (25), 146 (65), 130 (100), 102 (60), 75
(45), 44 (60). HRMS for C16H11FN2O3 found 299.0823 [MþH]þ
(Calcd 299.0826).
2.1.1.4. Methyl 3-(5-(4-fluorophenyl)-1,2,4-oxadiazol-3-ꢁyl1)benzoate)
(4c). Yield 86%; m.p. 164e166 ꢀC; IR (nujol): 1733 cm
;
1H NMR
(300 MHz, CDCl3):
d (ppm) 8.84 (m, 1H), 8.36 (m, 1H), 8.28 e 8.19
(m, 3H), 7.64 e 7.58 (m, 1H), 7.29 e 7.23 (m, 2H), 3.98 (s, 3H).
GCeMS: m/z (%) 298 (Mþ, 80), 267(100), 177 (35), 146 (80), 130
(85), 102 (55), 88 (40), 75 (50). HRMS for C16H11FN2O3 found
299.0812 [MþH]þ (Calcd 299.0826).
2.1.1.5. Methyl 3-(5-(2,3-difluorophenyl)-1,2,4-oxadiazol-3-yl)ben-
zoate) (4d). Yield 82%; m.p. 146e148 ꢀC; IR (nujol): 1722 cmꢁ1; 1H
NMR (300 MHz, CDCl3): d (ppm) 8.85 (m, 1H), 8.38 (m, 1H), 8.22 (m,
2. Materials and methods
1H), 8.04 e 7.99 (m, 1H), 7.63 (m, 1H), 7.47 e 7.44 (m, 1H), 7.32 e
7.26 (m, 1H), 3.98 (s, 3H). GCeMS: m/z (%) 316 (Mþ, 70), 285(100),
146 (60), 130 (45), 113 (30), 102 (40), 88 (30). HRMS for
2.1. Chemistry
C
16H10F2N2O3 found 317.0723 [MþH]þ (Calcd 317.0732).
IR spectra were registered with a Shimadzu FTIR-8300 instru-
ment. 1H NMR of all compounds and 13C NMR spectra of repre-
sentative 4a and 5b,i were recorded on a Bruker 300 Avance
spectrometer, operating at the indicated frequency, with TMS as an
internal standard. Flash chromatography was performed by using
silica gel (Merck, 0.040e0.063 mm) and mixtures of ethyl acetate
and petroleum ether (fraction boiling in the range of 40e60 ꢀC) in
various ratios. GCeMS determinations were carried out on a Shi-
madzu GCMS-QP2010 system. All solvents and reagents were ob-
tained from commercial sources. All synthesized compounds were
purified by chromatography and analyzed by IR, GCeMS and NMR.
Purity of synthesized compounds was verified prior to biological
2.1.1.6. Methyl 3-(5-(2,4-difluorophenyl)-1,2,4-oxadiazol-3-yl)ben-
zoate) (4e). Yield 92%; m.p. 126e128 ꢀC; IR (nujol): 1730 cmꢁ1; 1H
NMR (300 MHz, CDCl3):
d (ppm) 8.84 (s, 1H), 8.26 e 8.20 (m, 3H),
7.61 (m, 1H), 7.14 e 7.02 (m, 2H), 3.98 (s, 3H). GCeMS: m/z (%) 316
(Mþ, 55), 285(65), 177 (30), 139 (60), 130 (100), 113 (30), 102 (65),
88 (40), 75 (40). HRMS for C16H10F2N2O3 found 317.0719 [MþH]þ
(Calcd 317.0732).
2.1.1.7. Methyl 3-(5-(2,5-difluorophenyl)-1,2,4-oxadiazol-3-yl)ben-
zoate) (4f). Yield 78%; m.p. 161e163 ꢀC; IR (nujol): 1714 cmꢁ1 1H
;
NMR (300 MHz, CDCl3):
d (ppm) 8.84 (s, 1H), 8.38 (m, 1H), 8.22 (m,

238
I. Pibiri et al. / European Journal of Medicinal Chemistry 101 (2015) 236e244
1H), 7.98 e 7.93 (m, 1H), 7.62 (m, 1H), 7.34 e 7.27 (m, 2H), 3.99 (s,
3H). GCeMS: m/z (%) 316 (Mþ, 65), 285(100), 146 (60), 130 (70), 113
(30), 102 (40), 88 (30), 75 (25). HRMS for C16H10F2N2O3 found
317.0724 [MþH]þ (Calcd 317.0732).
2.1.2.3. 3-(5-(Perfluorophenyl)-1,2,4-oxadiazol-3-yl)benzoic
(5l). Yield 78%, m.p. 202e203 ꢀC, IR (nujol): 3250, 1715 cmꢁ1
acid
1H
;
NMR (300 MHz, DMSO-d6):
d (ppm) 8.67 (s, 1H), 8.39 (d, 1H,
J ¼ 7.8 Hz), 8.25 (d, 1H, J ¼ 7.8 Hz), 7.83 (m, 1H), 6.58 (bs, OH). HRMS
for C15H5F5N2O3 found 357.0285 [MþH]þ (Calcd 357.0293).
2.1.1.8. Methyl 3-(5-(2,6-difluorophenyl)-1,2,4-oxadiazol-3-yl)ben-
zoate) (4g). Yield 88%; m.p. 136e138 ꢀC; IR (nujol): 1734 cmꢁ1; 1H
2.2. Biology
NMR (300 MHz, CDCl3):
d (ppm) 8.84 (m, 1H), 8.40 e 8.37 (m, 1H),
2.2.1. Cell culture conditions and transfection of reporter plasmids
HeLa and IB3.1 cells were cultured in DMEM supplemented with
FBS 10% (GIBCO) in a humidified atmosphere of 4% CO2 in air at
37 ꢀC. HeLa cells plated in a 6 well plate at a density of 2 ꢂ 105/mL
were transfected with the reporter plasmids by using lipofectamine
2000 (Invitrogen). HeLa stably transfected cells were selected by
8.23 e 8.20 (m, 1H), 7.64 e 7.59 (m, 2H), 7.14 (m, 2H), 3.97 (s, 3H).
GCeMS: m/z (%): 316 (Mþ, 40), 285 (60), 177 (30), 139 (65), 130
(100), 102 (60), 88 (40), 75 (40). HRMS for C16H10F2N2O3 found
317.0721 [MþH]þ (Calcd 317.0732).
2.1.1.9. Methyl 3-(5-(3,4-difluorophenyl)-1,2,4-oxadiazol-3-yl)ben-
zoate) (4h). Yield 79%; m.p. 145e147 ꢀC; IR (nujol): 1728 cmꢁ1; 1H
NMR (300 MHz, CDCl3):
8.20 (m, 1H), 8.12 e 8.00 (m, 2H), 7.61 (m, 1H), 7.42 e 7.34 (m, 1H),
3.98 (s, 3H). GCeMS: m/z (%) 316 (Mþ, 70), 285 (90), 161 (45), 139
(65), 130 (100), 113 (50), 102 (60), 88 (45), 75 (50). HRMS for HRMS
for C16H10F2N2O3 found 317.0723 [MþH]þ (Calcd 317.0732).
blasticidin (5 mg/mL) and 15 clones of H2BGFP-opal, 5 clones of
H2BGFP-amber and 2 clones of H2BGFP-ochre were isolated. mRNA
levels of the H2BGFP mutated genes were verified by Real-Time
qRT-PCR (details in Supplementary Data).
d (ppm) 8.82 (m, 1H), 8.35 (m, 1H), 8.23-
2.2.2. Construction of reporter plasmids by site directed
mutagenesis and clone screening
Reporter plasmids harboring PTCs were constructed by using
the pBOS-H2BGFP plasmid [32,33]. The tryptophan codon (TGG) at
position 1197-9 of the GFP coding sequence was mutagenized to
introduce a stop codon (TGA, TAG or TAA). Four clones from each
reaction of mutagenesis were screened by “selective PCR”
(Supporting Information). Plasmid DNA from positive clones was
purified with NucleoSpin Plasmid miniprep kit (Macherey-Nagel)
and the mutations were verified by sequencing (Eurofins MWG
Operon).
2.1.1.10. Methyl
benzoate) (4i). Yield 81%; m.p. 132e134 ꢀC; IR (nujol): 1716 cmꢁ1
1H NMR (300 MHz, CDCl3):
(ppm) 8.83 (m, 1H), 8.36 (m, 1H), 8.22
3-(5-(2,4,5-trifluorophenyl)-1,2,4-oxadiazol-3-yl)
;
d
(m, 1H), 8.16 e 8.08 (m, 1H), 7.65 e 7.59 (m, 1H), 7.23 e 7.14 (m, 1H),
3.99 (s, 3H). GCeMS: m/z (%) 334 (Mþ, 55), 303 (60), 159 (50), 146
(60), 130 (100), 102 (45), 88 (45), 75 (40). HRMS for HRMS for
C
16H9F3N2O3 found 335.0638 [MþH]þ (Calcd 335.0633).
2.1.1.11. Methyl 3-(5-(perfluorophenyl)-1,2,4-oxadiazol-3-yl)benzo-
ate) (4l). Yield 83%; m.p. 109e110 ꢀC; IR (nujol): 1725 cmꢁ1 1H
NMR (300 MHz, CDCl3):
(ppm) 8.83 (s, 1H), 8,38 (d, 1H, J ¼ 7.8 Hz),
;
2.2.3. Genomic DNA purification and reporter gene control
d
To isolate genomic DNA from the selected clones we used the
PureLink Genomic DNA Kit (Invitrogen). 3 ꢂ 106 cells were lysed
and the DNA was rapidly purified using a spin column based
centrifugation procedure. To verify the integrity of the H2BGFP
reporter gene 130 ng of genomic DNA were used as template for a
8.24 (d, 1H, J ¼ 7.8 Hz), 7.64 (m, 1H); GCeMS: m/z (%) 370 (Mþ, 25),
339 (50), 193 (40), 161 (20), 146 (30), 130 (100), 102 (65), 88 (25), 75
(30), 51 (15), 44 (65). HRMS for HRMS for C16H7F5N2O3 found
371.0439 [MþH]þ (Calcd 371.0449).
PCR reaction. We used the EF1a forward primer annealing to the
2.1.2. General procedure for the synthesis of 5b,i,l
gene promoter region and the GFP30reverse primer annealing to the
Similarly to what reported for the synthesis of PTC124 5a [26],
either methylester 4b, 4i, or 4l (0.34 mmol) were dissolved in
benzene (40 mL) in a round-bottomed flask; then, BBr3 (4 mL;
13.7 mmol) was added to the reaction mixture and stirred at 80 ꢀC
for 4 h. The solvent was removed under vacuum and the residue
treated with water, extracted with ethyl acetate and dried over
anhydrous sodium sulphate. After removal of the solvent under
vacuum acids 5b, 5i, and 5l were purified by chromatography.
30end of the gene. After 35 cycles of amplification 5
ml of PCR
product were run on 1% agarose gel.
2.2.4. Measurement of luciferase activity by luminescence
HeLa cells were plated in a 12 well plate at a density of 1 ꢂ 105/
mL and transfected with WT (Fluc) and mutant (Fluc-opal) plas-
mids, by using lipofectamine 2000 (Invitrogen). Cells were incu-
bated for 24 h and PTC124 (5a) and its derivatives (12 mM) were
added for 24 h. Next, cells were washed with PBS, incubated with
the detection mix Steady-Glo luciferase reagent (Promega) and
2.1.2.1. 3-(5-(3-Fluorophenyl)-1,2,4-oxadiazol-3-yl)benzoic acid (5b).
Yield 70%; m.p. 252e254 ꢀC; IR (nujol): 1698 cmꢁ1 1H NMR
;
200
ml of cell suspension were plated in triplicate in a 96 well.
(300 MHz, DMSO-d6):
8.17 (d, 1H, J ¼ 7.8 Hz), 8.07- 7.97 (m, 2H), 7.76 e 7.69 (m, 2H), 7.64 e
7.57 (m, 1H). 13C NMR (75 MHz, CDCl3):
(ppm) 174.7, 167.9, 166.6,
d
(ppm) 8.61 (s, 1H), 8.31 (d, 1H, J ¼ 7.8 Hz),
Luciferase activity was measured on a luminometer (Promega). This
experiment was performed three times and quantitative data
illustrated in Fig. 1 are the average of the repeated experiments.
d
164.3, 160.4, 132.4, 132.0, 131.1, 129.8, 127.9, 126.5, 125.3, 124.4,
120.5, 114.8. HRMS for C15H9FN2O3 found 285.0662 [MþH]þ (Calcd
285.0670).
2.2.5. Immunofluorescence microscopy
To visualize GFP and CFTR proteins cells were grown on rounded
glass coverslips and fixed with methanol for 2 min. The cell
membrane and Golgi apparatus were stained by the Wheat Germ
Agglutinin (WGA) Alexa 594 (Life Technologies). For GFP-detection
cells were permeabilized with 0.01% TritonX (Sigma-Aldrich) in PBS
(15 min) and blocked with 0.1% BSA (30 min) both at RT. Cover slips
were incubated with a mouse monoclonal antibody against GFP
2.1.2.2. 3-(5-(2,4,5-Trifluorophenyl)-1,2,4-oxadiazol-3-yl)benzoic
acid (5i). Yield 70%; m.p. 248e250 ꢀC; IR (nujol): 1685 cmꢁ1 1H
;
NMR (300 MHz, DMSO-d6):
2H), 8.24 (d, 1H, J ¼ 7.8 Hz), 8.08 e 8.03 (m, 1H), 7.84 e 7.79 (m, 1H).
13C NMR (75 MHz, DMSO-d6):
(ppm) 171.3, 167.6, 166.7, 158.3,
d (ppm) 8.69 (s, 1H), 8.47 e 8.37 (m,
d
154.5, 150.7, 144.7, 132.4, 131.0, 129.8, 127.9, 126.2, 118.8, 108.6,
108.2. HRMS for C15H7F3N2O3 found 320.0399 [MþH]þ (Calcd
320.0409).
(Sigma-Aldrich, 5 mg/mL) and a mouse monoclonal antibody (CF3)
that recognizes the first extracellular loop of human CFTR (Abcam,
1:500) overnight at 4 ꢀC, followed by a goat anti-mouse IgG-FITC

I. Pibiri et al. / European Journal of Medicinal Chemistry 101 (2015) 236e244
239
PTC124 derivatives above. The starting position of both ligands was
retained from the equilibrium geometry reported for the analogous
PTC124-mRNA complex [26]. A cubic box filled with TIP3P water
molecules was added around the RNA and the ligand to a depth
0.8 nm on each side of the solute. Naþ counterions were added to
neutralize the negative charges of the RNA backbone, other Naþ
and Clꢁ ions were added to achieve a solution ionic strength of
about 0.15 M. Simulations were performed in the NPT ensemble, at
the temperature of 300 K. All covalent bonds were constrained with
the LINCS algorithm. Energy minimization was run for 5000 steps
using the steepest descend algorithm. In a 500 ps equilibration the
oligonucleotides were harmonically restrained with a force con-
stant of 1000 kJ molꢁ1 nmꢁ2 at 300 K, which was gradually lowered
until no restrains were applied. The binding free energy was
calculated using the g_mmpbsa [42], with snapshots extracted
every 0.5 ns from the production trajectory. Simulations were also
conducted mimicking a 9 mM magnesium ion concentration
without observing any difference in the interaction between UGA
and 5i.
3. Results
Generally, 1,2,4-oxadiazoles biological activity is related to
their bioisosterism with amides and esters [43], and to the H-
bond accepting nature of the ring heteroatoms [44]. Recently, the
carboxylic moiety of PTC124 has been reported to be involved as
H-bond acceptor in relevant snapshots of MD simulation of the
selective interaction with the premature UGA codon in a 33-nt
long mRNA fragment coding for CFTR [26]. Therefore, by
following the amidoxime route for the synthesis of fluoroarylated
1,2,4-oxadiazoles [45e49], we synthesized the corresponding
methyl 3a and methylester 4a derivatives of PTC124 (Scheme 1)
that were tested for readthrough activity with a Fluc-based re-
porter (Fig. 1).
Fig. 1. Luciferase activity after treating HeLa Fluc190UGA transfected cells with PTC124
and its derivatives 3a, 4 and 5 in comparison with wt Fluc and untransfected or un-
treated cells (HeLa).
(Sigma-Aldrich, 1:200) and goat polyclonal to mouse Alexa-Fluor-
488 (Abcam, 1:1000) secondary antibodies for 1 h at 37 ꢀC. Nuclei
were visualized with DAPI. Cells were examined under a Zeiss
Axioskop microscope equipped for fluorescence.
For the preliminary evaluation of the readthrough activity of
compounds 3e5, we used the FLuc cell-based assay [14,26]. HeLa
cells were transiently transfected with the plasmids pFLuc-WT and
pFLuc-opal, and FLuc gene expression was measured by lumines-
2.2.6. Western blotting
cence. The dose used (12 mM) was chosen on the basis of our pre-
Proteins (50 mg) were separated by 10% SDS-PAGE and 4e12%
vious results on PTC124 [26]. The three most active compounds 4a,
SDS-PAGE (Bolt, Life Technologies) containing 0.1% SDS and trans-
ferred to Hybond-C nitrocellulose membranes (Amersham Life
Science) by electroblotting [34] The membrane was incubated with
anti-GFP mouse (Sigma-Aldrich, 2 mg/mL), as primary antibody and
HRP-conjugated anti-mouse IgG (Abcam, 1:5000), as secondary
antibody. For CFTR detection the membrane was incubated with a
goat polyclonal antibody antieCFTR (C-19, Santa Cruz 1:500) raised
against a peptide mapping near the C-terminus of CFTR of human
origin, and HRP-conjugated anti-goat (Abcam, 1:5000). The target
protein was detected by ECL reagents (Pierce). We used b-tubulin
antibody (mouse; Sigma-Aldrich 1:10.000) to confirm equal pro-
teins loading. Gel bands were quantified by Image Lab software
(BioRad). WB assays were repeated at least three times and were
reproducible within 5% variations. Representative WB images and
data have been selected for Figs. 3 and 5A.
2.3. Computational details
The topology of 5i and 4d ligands was generated using ACPYPE
[35e37] and Molecular Dynamics (MD) simulations were per-
formed with the GROMACS 4.6.6 suite [38,39] using the AMBER
ff99SB force field [40] with parmBsc0 nucleic acid torsions [41]. MD
simulations of 50 ns were conducted to assess the supramolecular
interactions between the mRNA fragment described above and two
Scheme 1. Synthesis of PTC124's derivatives.

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I. Pibiri et al. / European Journal of Medicinal Chemistry 101 (2015) 236e244
Fig. 2. Immunofluorescence of HeLa cells stably transfected with the H2BGFP-opal (A), H2BGFP-amber (B) and H2BGFP-ochre (C) plasmid untreated (Untr) and treated with the
indicated compounds for 24 h. H2BGFP fusion protein (green) was detected with a specific anti-GFP antibody revealed by a FITC-conjugated antibody. Nuclei (blue) were DAPI
stained. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
5b and 5i showed readthrough activity also at lower concentrations
(3 M and 6 M) with 5i showing the higher activity (see Fig. 5 in
monofluorinated derivatives 4a-c, the position of fluorine in the
ring can cause a dramatic change in activity.
m
m
Supplementary Data). Detection of high levels of luciferase showed
by HeLa cells transfected with the pFLuc-WT plasmid indicated the
correct functioning of this assay.
The three most active compounds, ester 4a and acids 5b and 5i,
together with the least active ester 4d, and PTC124 5a, were
subjected to further evaluation by using a GFP-based assay as
previously reported [26]. For this purpose, live cell fluorescence
microscopy is not always able to detect low level of H2BGFP re-
expression in live cells because of the intrinsic low amount of
recoded mRNA by readthrough of premature stop codons [26].
Therefore, to enhance H2BGFP detection, we investigated the
readthrough ability of PTC124's derivatives by indirect immuno-
fluorescence analysis in HeLa cells stably transfected with the
pBOS-H2BGFP wild-type (wt) plasmid (control) and the opal-,
amber-, and ochre-mutated pBOS-H2BGFP plasmids. Immunoflu-
orescence microscopy done in H2BGFPeopal, -amber, -ochre
transfected HeLa cells revealed the presence of positive cells for
the H2BGFP protein induced by PTC124 and by derivatives 5b, 4a,
5i (Fig. 2) when compared to untreated cells. These immunoflu-
orescence experiments confirmed that the readthrough of the
UGA premature stop codon (opal) resulted, at least partially, in a
Despite the absence of the carboxyl moiety, compound 3a
retained some activity, although it was significantly lower
compared to PTC124. This suggests that H-bonding from the
carboxyl moiety is important but not crucial for activity. Interest-
ingly, the activity of the methylester 4a was very similar to that of
PTC124 thus confirming the role of the carboxyl moiety as H-bond
acceptor. Therefore, we focused on the fluoroaryl ring by synthe-
sizing a series of methylester derivatives of PTC124 where the
number and the position of fluorine atoms have been varied
(Scheme 1). PTC124 5a and carboxylic acids 5b,i,l were obtained by
demethylation of the corresponding esters 4a,b,i,l with boron
tribromide.
Luciferase activity data illustrated in Fig. 1 clearly pointed out
that in the methylester series the activity is not enhanced by
increasing the number of fluorine atoms and that, for

I. Pibiri et al. / European Journal of Medicinal Chemistry 101 (2015) 236e244
241
distant goal.
These results were confirmed by western blotting analysis that
detected a similar amount of full-length H2BGFP protein in HeLa
cells transfected with the reporter harboring the premature stop
codon and treated with compounds 4a, and 5b,i, PTC124, and the
known readthrough promoter G418 (Fig. 3).
Since compounds 4a and 5b,i performed well in both Fluc and
GFP reporters, we tested them for nonsense suppression in CF
bronchial epithelial cell line IB3.1 derived from a CF patient (CFTR
genotype W1282X/F508del). HT29 cells were used as a positive
control for the CFTR protein expression (see Fig.
6 in
Supplementary Data). Fluorescence microscopy confirmed that
compounds 4a and 5b,i were able to readthrough the UGA pre-
mature stop codon in IB3.1 cells as showed by the detection of
increased level of the CFTR protein (Fig. 4A). Tests performed on
compound 4d, which was the least active with the Fluc and GFP
reporters, confirmed its inactivity (data not reported). Comparison
of 5i activity with G418, that is known to do the readthrough of
premature stop codons, confirmed this result (Fig. 4B).
To confirm immunofluorescence results we evaluated the CFTR
levels in IB3.1 cells by Western blotting (Fig. 5A). The IB3.1 cells
were treated with G418, PTC124 (5a) and the derivatives 4a, and
5b,i for 24 h. Western blot results indicated that the treatment with
G418 and PTC124's derivatives induced the recovery of the CFTR
protein expression. With respect to the CFTR protein expressed in
the untreated cells, data show a five-fold increase for compounds 5i
and 4a and a sixfold increase for compound 5b, while PTC124
induced a less pronounced protein recovery (Fig. 5A). Interestingly,
ester 4a, which was chemically stable in the presence of water
under neutral conditions, produced a higher level of CFTR protein
than its corresponding acid 5a. However, at this stage we cannot
exclude that such activity derives from a higher cell penetration of
4a followed by its enzymatic hydrolysis into the active acid 5a.Also,
we evaluated the CFTR mRNA levels after the treatment with G418,
PTC124 (5a), 5i, 5b, and 4a. As shown in Fig. 5B, the mRNA levels in
cells treated with G418 were higher than those detected in cells
treated with PTC124 and its derivatives, and did not correlate with
Fig. 3. Western blot showing H2BGFP protein levels in H2BGFPeopal transfected HeLa
cells left untreated (untr.) or treated with PTC124 (5a), G418 and the indicated PTC124
derivatives. Protein extracts of HeLa H2BGFP-wt cells were used as a control for the
H2BGFP-wt protein, b-tubulin was used as a loading control. The histogram below the
WB quantitates the Western bands by densitometry by using the Image Lab software
(BioRad). A primary antibody targeting the C-terminus of GFP protein was used.
functional protein since the green fluorescence was mainly
observed in the nucleus, with compounds 4a and 5b showing a
brighter green fluorescence (Fig. 2A). Interestingly, cells trans-
fected with the plasmids bearing the UAA (amber) stop codon
showed some fluorescence when treated with the compound 5b
(Fig. 2B), while treatment with 4a resulted in weak fluorescence in
cells transfected with the plasmid bearing the UAG (ochre) stop
codon (Fig. 2C). This may suggest that selectivity towards a given
PTC could be driven by very small changes in the scaffold struc-
ture, although specific readthrough optimization still remains a
Fig. 4. A) Immunofluorescence assay to detect readthrough of the UGA stop codon in CFTR of IB3.1 human cells untreated (untr), treated with G418 (positive control) and with
PTC124 (5a) and its derivatives: 5b, 5i and 4a for 24 h. CFTR protein was revealed by a specific antibody targeting its first external portion (green, Alexa-488). Nuclei (blue) were
DAPI stained. B) Cells untreated and treated with G418 and 5i for 24 h. CFTR protein was revealed by a specific antibody targeting its external portion when located on the
membrane (green, Alexa 488). Nuclei (blue) were DAPI stained the cell membrane and Golgi apparatus was stained in red (WGA-Alexa 594). (For interpretation of the references to
color in this figure legend, the reader is referred to the web version of this article.)

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I. Pibiri et al. / European Journal of Medicinal Chemistry 101 (2015) 236e244
Fig. 5. A) Western blot (bottom panel) showing CFTR protein levels in IB3.1 cells untreated (untr.) or treated with the indicated compounds. HeLa cells were used as a control for the
CFTR protein. The upper panel shows the quantitation of the bands by densitometry by using the Image Lab software (BioRad). A primary antibody targeting the C-terminus of CFTR
was used, b-tubulin was used as a loading control. B) Real Time RT PCR showing CFTR mRNA levels in IB3.1 cells untreated (untr.) or treated with the indicated compounds.
the protein levels detected by Western Blot.
remains in the RNA loop for the whole simulation time.
Additionally, a cell viability assay was performed to determine if
the new molecules affected cell survival. The assay showed that, at
the used doses, only G418 resulted toxic for the cells (Fig. 6) while
no cytotoxic effects were observed in IB3.1 cells treated for 10 days
with both PTC124 and its derivatives (data not shown).
Finally, similarly to our previous study [26] and based on the
experimental data obtained in this work with various PTC124 de-
rivatives, we decided to perform a molecular dynamics simulation
modeling the interaction of a 33-nucleotides CFTR mRNA fragment,
harboring a UGA premature stop codon, with the most active 5i and
the least active, 4d, derivatives. The trifluorinated derivative, 5i,
was posed in the same orientation of the equilibrium structure of
PTC124 [26] and a 50 ns MD simulation was performed. The ligand
As shown in Fig. 7 (left), there are at least two stabilizing in-
teractions in the 5i/mRNA complex. Indeed, besides stacking in-
teractions, the occurrence of hydrogen bond between the
heterocyclic fluorine atoms and the oxadiazole heterocyclic nitro-
gen with the amino group on the nucleobases contributes signifi-
cantly to the complex stability. The binding free energy of the
complex between 5i and the mRNA fragment is ꢁ128.1 kJ/mol,
similar to that observed for the PTC124/mRNA complex [26].
Interestingly, under the same simulation conditions, inactive
compound 4d readily moves far away from the UGA codon.
4. Discussion
Nonsense mutations are detected in ~10% of Cystic Fibrosis pa-
tients representing one of the most severe forms of the disease. A
potential therapeutic strategy for these patients is to promote the
readthrough of premature stop codons to restore a full length CFTR
protein [50]. We used Ataluren as a starting point for selected
modifications of moieties possibly involved in its readthrough ac-
tivity, particularly focusing on the carboxylic moiety, and on the
number and relative position of fluorine substituents. In fact, the
carboxymethylic moiety could positively influence the absorption
process by decreasing the hydrophilicity induced by the carboxylic
group. In monofluorinated derivatives, we varied the position of
fluorine considering that the electronic demand of the fluorine
atom is balanced by the electron-withdrawing feature of the 1,2,4-
oxadiazole ring exerted through its C(5) position. These effects can
be either counterposed to each other or coupled depending on their
relative position. This phenomenon affects the overall polarization
of the aromatic rings involved in stacking interactions with the
biological target. Finally, due to their strong electronegativity, the
introduction of additional fluorine atoms affects the electronic
distribution affecting the molecule's lipophilicity that is strictly
connected to its pharmacokinetics.
Fig. 6. Cell viability assay shows that only G418 resulted toxic after 72 h of treatment.

I. Pibiri et al. / European Journal of Medicinal Chemistry 101 (2015) 236e244
243
derivatives. It is worth noting that some monofluorinated de-
rivatives of PTC124, differing from our compounds only for the
position of the carboxylic moiety on the phenyl ring linked to the
C(3) of the 1,2,4-oxadiazole, showed no readthrough activity in a
previous SAR study using a luciferase reporter from Renilla reni-
formis (Rluc) [16]. This apparent contrast with our results with Fluc,
GFP and CFTR remarks a few important principles that should be
considered in the roadmap to nonsense mutation suppressors,
especially in a context where the mechanism of action is not
established: i) negative results can stem from a fault in any of the
three steps of target-reaching, effective target-ligand interaction,
measurable effects; ii) both positive and negative results may suffer
from specificity towards the experimental setup; iii) indirectly
detected readthrough activity (e.g. by fluorimetric methods) should
be accompanied by supporting Western Blotting experiments for
the specific quantitation of recovered protein; iv) positive results
should further be tested for functionality of the recovered protein,
e.g. by in vivo-in vitro studies; v) ideally, recovered functional
proteins should be fully sequenced to identify the type of occurred
repair among amino acid skip, substitution, or correct introduction.
Importantly, cell viability studies confirmed the lack of toxicity of
PTC124's derivatives compared with G418. In our opinion, toxicity
data together with the differences in size and shape with amino-
glycosides, and the opposite activity of G418 and PTC124 de-
rivatives reported in some cases for the same cellular setup, suggest
that PTC124 and its derivatives may not share the same biological
target. Unfortunately, while the interaction of G418 with its target is
known [8,53], other challenges are still open to understand the
mechanism of action of small molecules promoting premature stop
codons' readthrough [50]. From a mechanistic point of view, our
simulations suggest that mRNA could be one of the possible targets
for PTC124-like readthrough promoters. However, neither PTC124
positioning in the ribosomal machinery nor its supposed interac-
tion with mRNA have been experimentally proven and studies in
these directions should be encouraged.
Fig. 7. Representative snapshot showing the interaction of 5i with the UGA codon. The
hydrogen bond between a fluorine atom and the amino group of G17 is evidenced with
a blue line. (For interpretation of the references to color in this figure legend, the
reader is referred to the web version of this article.)
To validate the efficacy of the synthesized compounds we used
orthogonal cell-based assays employing two different reporter
systems: the plasmid pBOS-H2BGFP and the luciferase reporter
gene (FLuc190) [26]. Indeed, the use of orthogonal assays is
considered a powerful approach, which uses different reporters to
verify that positive results of a compound's biological activity do
not depend on the detection method used [27,28,51,52]. Our
immunofluorescence experiments showed that three of the PTC124
derivatives promoted the readthrough of the nonsense mutations
present in the H2BGFP reporter plasmid in HeLa transfected cells.
The readthrough ability of PTC124's derivatives was confirmed by
using an additional assay based on IB3.1 cells derived from a CF
patient and harboring a nonsense mutation in the CFTR gene.
Treatment of IB3.1 cells with compounds 4a, 5b, and 5i induced the
readthrough of the premature translation termination codon
encoded by the hCFTR-W1282X nonsense mutation present in
these cells, resulting in the partial restoration of the CFTR protein.
Real time RT-PCR experiments indicated that there was a lower
increase of the CFTR mRNA after the treatment with the PTC124's
derivatives in comparison to G418, while Western Blotting experi-
ments showed that the amount of CFTR protein after the treatment
with the PTC124's derivatives was greater than that showed by
G418 and PTC124 treatment in IB3.1 cells. This last finding re-
inforces the hypothesis that readthrough, and not stabilization of
the protein encoded by the reporter (H2B-GFP-opal), is the mech-
anism responsible for the recoding of the internal TGA premature
stop codon. The slight increase (though statistically not significant)
in CFTR mRNA observed after treatments also suggests that
combining PTC124 derivatives with NMD inhibitors could have a
synergistic effect in recovering CFTR.
Conflict of interest
The authors declare that they have no conflict of interest.
Acknowledgments
We thank Prof. J. Inglese, NIH Chemical Genomics Center, Na-
tional Institutes of Health, Bethesda, for kindly providing us with
the FLuc190UGA plasmid. We are very grateful to Allan Jacobson,
University of Massachusetts Medical School, Worcester, for his
valuable comments on the manuscript. This work was funded by
the Italian Cystic Fibrosis Research Foundation, grant FFC#2/2011,
with the contribution of Delegazione di Lecce, Delegazione di Vit-
toria (Ragusa) to Prof. Aldo Di Leonardo and grant FFC#1/2014 with
the contribution of Delegazioni FFC di Palermo, Vittoria (Ragusa)
and Catania 2 to Dr. Laura Lentini.
Finally, we decided to verify if our previous computational
approach [26] was able to discriminate between active and inactive
compounds. MD simulations were performed with representative
active (5i) and inactive (4d) derivatives in the presence of a 33-
bases fragment of the CFTR mRNA sequence containing a prema-
ture UGA codon. Interestingly, during the simulation, compound 4d
moves away from its potential UGA target. On the other hand, the
simulation reproduced a UGA binding interaction only in the case of
active compound 5i as observed previously for PTC124 [26].
Appendix A. Supplementary data
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.ejmech.2015.06.038.
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