Targeting nonsense: Optimization of 1,2,4-oxadiazole trids to rescue cftr expression and functionality in cystic fibrosis cell model systems

Article abstract of DOI:10.3390/ijms21176420

Cystic fibrosis (CF) patients develop a severe form of the disease when the cystic fibrosis transmembrane conductance regulator (CFTR) gene is affected by nonsense mutations. Nonsense mutations are responsible for the presence of a premature termination c

Full text of DOI:10.3390/ijms21176420

International Journal of
Molecular Sciences
Article
Targeting Nonsense: Optimization of 1,2,4-Oxadiazole
TRIDs to Rescue CFTR Expression and Functionality
in Cystic Fibrosis Cell Model Systems
1
,
1
, Marco Tutone 1 , Aldo Di Leonardo 1,2 , Andrea Pace 1
Ivana Pibiri * , Raffaella Melfi
1
,
and Laura Lentini *
1
Dipartimento di Scienze e Tecnologie Biologiche, Chimiche e Farmaceutiche (STEBICEF), Università degli
Studi di Palermo, Viale delle Scienze Ed. 16-17, 90128 Palermo, Italy; raffaella.melfi@unipa.it (R.M.);
marco.tutone@unipa.it (M.T.); aldo.dileonardo@unipa.it (A.D.L.); andrea.pace@unipa.it (A.P.)
Centro di OncoBiologia Sperimentale (COBS), via San Lorenzo Colli, 90145 Palermo, Italy
Correspondence: ivana.pibiri@unipa.it (I.P.); laura.lentini@unipa.it (L.L.);
2
*
Tel.: +39-091-238-97545 (I.P.); +39-091-238-97341 (L.L.)
ꢀ
ꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀ
ꢁꢂꢃꢄꢅꢆ
Received: 13 July 2020; Accepted: 1 September 2020; Published: 3 September 2020
Abstract: Cystic fibrosis (CF) patients develop a severe form of the disease when the cystic fibrosis
transmembrane conductance regulator (CFTR) gene is affected by nonsense mutations. Nonsense
mutations are responsible for the presence of a premature termination codon (PTC) in the mRNA,
creating a lack of functional protein. In this context, translational readthrough-inducing drugs
(TRIDs) represent a promising approach to correct the basic defect caused by PTCs. By using
computational optimization and biological screening, we identified three new small molecules
showing high readthrough activity. The activity of these compounds has been verified by evaluating
CFTR expression and functionality after treatment with the selected molecules in cells expressing
nonsense–CFTR–mRNA. Additionally, the channel functionality was measured by the halide sensitive
yellow fluorescent protein (YFP) quenching assay. All three of the new TRIDs displayed high
readthrough activity and low toxicity and can be considered for further evaluation as a therapeutic
approach toward the second major cause of CF.
Keywords: premature termination codon; nonsense mutation; genetic disorder; translational
readthrough inducing drugs; oxadiazoles
1
. Introduction
Protein synthesis is a crucial process for life; therefore, gene mutations can seriously compromise
the quality of life and even life expectancy.
A point mutation in the gene can result in a silent, missense, or nonsense mutation. In the
fortunate case of a silent mutation, there are no effects on the amino acid sequence of the resulting
protein. A missense mutation, which alters the primary structure, can cause protein malfunctioning
due to incorrect folding or different conformations in the active site of a specific functional protein [1].
In the worst case, when a nonsense mutation is present in the gene producing a premature
termination codon in the transcript, the protein production is compromised. A surveillance mechanism
named NMD (nonsense-mediated decay) intervenes to destroy aberrant transcripts, and the small
amount of leftover mRNA bearing the PTC will produce truncated polypeptides that, in turn, will be
degraded as aberrant protein [2].
When a PTC occurs in the transcript, a basal readthrough partially restores protein synthesis to a
very small extent, although this is too low to make an improvement in the disease phenotype [3].
Int. J. Mol. Sci. 2020, 21, 6420; doi:10.3390/ijms21176420
www.mdpi.com/journal/ijms

Int. J. Mol. Sci. 2020, 21, 6420
2 of 18
If compared to basal readthrough, drug-induced readthrough allows for the restoration of protein
synthesis to a wider extent [ ]. Both basal and drug-induced readthrough of PTCs are known to occur
by a misreading of the PTC by a near-cognate tRNA [ ]. In particular, it has been suggested that a
4
5–8
tRNA carrying tryptophan, cysteine, or arginine misreads the third base (A/G for tryptophan; A/U,C
for cysteine) or the first base (U/C for arginine) of the UGA premature termination codon. The same
kind of misreading could justify the insertion of glutamine, tyrosine, or lysine during the readthrough
of the other two PTCs (UAG and UAA), as sketched in a genetic language translation roulette in
Figure 1 [3,8].
Figure 1. Codons language and most frequent misreadings for premature termination codon
(PTC)’s readthrough.
The readthrough process depends on the kind of PTC, its location in the gene, and the surrounding
mRNA sequence. Its efficiency is affected by the nucleotide immediately downstream of the PTC;
moreover, the role of ancillary bases upstream of the PTC has recently been noted in the literature
regarding drug-induced readthrough [7–9].
The amount of protein rescued by readthrough can be raised by NMD inhibition, as reported in
many cases [10–14].
The production of a functional protein is guaranteed when the readthrough causes the insertion
of the wild-type amino acid in the correspondence of the PTC. Alternatively, the readthrough can also
skip the nonsense and produce the effect of a missense mutation, as widely discussed [15,16].
Nonsense mutations are found in about 11% of genetic disorders such as cystic fibrosis
(
CF) [17], Duchenne muscular dystrophy (DMD) [18], spinal muscular atrophy [19
neurofibromatosis [21], retinitis pigmentosa [22 24], lysosomal storage disease [13 25 26], Rett
syndrome [27], Shwachman–Diamond syndrome [28 29], and Usher’s syndrome (USH) [30]. In this
,20],
–
, ,
,
context and despite its limits, the translational readthrough of PTCs can represent a valuable
pharmaceutical approach to target the specific genetic defect caused by nonsense mutations.
Among genetic disorders, cystic fibrosis (CF) is not a rare disease as it involves more than
1
00,000 people worldwide and affects approximately 36,000 individuals in the European Union (EU),
while 1000 new cases of CF are diagnosed each year in the US [31].
The cystic fibrosis transmembrane conductance regulator (CFTR) gene encodes for a channel
protein, which is fundamental for cellular homeostasis. CFTR starts its biogenesis by co-translational

Int. J. Mol. Sci. 2020, 21, 6420
3 of 18
insertion into the membrane of the endoplasmic reticulum and, by following the folding of the
cytosolic domains, takes on its native fully folded compact structure. Then, CFTR is modified
at the Golgi apparatus and delivered to the plasmatic membrane. It works as a cAMP- and
phosphorylation-regulated chloride/bicarbonate channel [32]. Both its biogenesis and functional
chloride secretion and hydration determine its overall activity and, thus, contribute to the fine
modulation of the homeostasis of the epithelial surfaces [32].
About 2000 different mutations have been described as being involved in the onset of the
disease [33], some of which are more common, such as the deletion of the triplet encoding for
phenylalanine 508 (
G551D).
In addition to addressing the disease symptomatically, a personalized medicinal approach
targeting the specific protein defect has also been used recently in many studies in the CF field [34 38].
Recently, a pharmacological treatment that combines three different drugs (elexacaftor/tezacaftor/
ivacaftor), which act synergistically, has been approved to overcome the F508 CFTR defect. On the
∆F508) or the missense mutation, which substitutes glycine 551 with aspartic acid
(
–
∆
other hand, however, nonsense mutations, which recur in 10–15% of the CF cases worldwide, are still
lacking an effective pharmacological approach.
Despite the significant efforts devoted to finding a cure for CF, there is no current therapy for the
nonsense mutations of the CFTR gene that cause the lack of CFTR protein and are responsible for a
more severe form of the disease (Figure 2A,B) [39].
(
A)
(
B)
Figure 2. Illustrations of cystic fibrosis transmembrane conductance regulator (CFTR) nonsense
mutations and their effect. ( ) CFTR mRNA sequence of the stop codon G542X and W1282X (in red
A
the PTC UGA codons). ( ) CFTR localization and effect of the nonsense mutation on the CFTR gene,
B
impairing cell homeostasis and causing the accumulation of mucus and the following infection.
A potential therapeutic approach is represented by the development of drugs inducing the
readthrough of nonsense mutations. By promoting a selective bypass of the PTC, the readthrough

Int. J. Mol. Sci. 2020, 21, 6420
4 of 18
approach would restore the expression of the CFTR protein and, once functionality is rescued to a
sufficient extent, improve cell homeostasis.
The first drugs tested as translational readthrough-inducing drugs (TRIDs) were aminoglycosides
(e.g., gentamicin, tobramycin, paromomycin, geneticin; see Figure 3), which have been shown to
suppress the normal proof-reading function of the ribosome [40 41]. These drugs, which were already
,
known for their antibiotic activity, induce the readthrough of the PTCs continuing the translation
process [42]. Unfortunately, the chronic use of high dosages of aminoglycosides is associated with
severe side-effects such as renal, auditory, and vestibular toxicities [43], which is what inhibited their
pharmaceutical implementation. Nevertheless, a new aminoglycoside, showing potential activity as a
TRID and reduced toxicity, has been tested in healthy subjects and is currently proceeding further in
clinical trials [44].
Figure 3. Different structures of proposed translational readthrough-inducing drugs (TRIDs).

Int. J. Mol. Sci. 2020, 21, 6420
5 of 18
To overcome the aminoglycoside toxicity, different molecules such as (+)-negamicine [45],
amlexanox [25], clitocine [46], and PTC124 (also known as ataluren) [47] (Figure 3) have been
proposed as readthrough promoters. Among these, ataluren was able to promote the readthrough
of PTCs, particularly UGA, without affecting the normal termination codons [47], thus being a good
candidate for developing a therapy for nonsense mutations [48].
Phase I and II clinical trials, including healthy subjects and CF and Duchenne muscular dystrophy
(DMD) patients, have evaluated the long-term safety of ataluren.
Trials showed improvement of some markers of CFTR function in CF patients; however, in a
new phase III trial, sweat chloride levels and the nasal potential difference were not improved [49].
Due to conflicting results, the phase III clinical study of PTC124 was suspended in April 2017 for CF
patients, while the drug has been registered with the regulatory authorities and is currently in use for
DMD patients [49,50]. Specifically, during the trial with CF patients, reports have shown that there is
a competition between ataluren and tobramycin (which interacts at the level of the ribosome) when
administered simultaneously [49].
Our previous studies on ataluren and its analogs aimed to look for alternative molecules and to
understand the effective biological target and mechanism of action of TRIDs [9,10,51–54].
We hypothesized that ataluren could interact differently with the PTCs, depending on their
localization in the CFTR gene (e.g., G542X, R1162X, W1282X) and on the genetic context [9].
Our research has focused on oxadiazoles, as they are widely recognized as lead
compounds in medicinal chemistry [55
amide functionalities.
–59], particularly as bioisosteric replacements for ester and
The flaws in the clinical trials for ataluren, as well as the discrete toxicity evidenced in the MTT
test (see Figure S1) for our recently reported lead compound NV2445 [54], pushed us to find new
alternative leads.
By performing an in-silico optimization of the lead molecule NV2445 [54] using public and
“in-house” compound libraries, we designed and synthesized three small molecules containing a
1
,2,4-oxadiazole as the heterocyclic core and evaluated their efficacy as TRIDs.
The preliminary biological screening was performed by using the firefly luciferase reporter; the
activity was confirmed by further assays evaluating the efficacy in promoting the production of a
G542X
W1282X
functional CFTR protein in cells engineered to express the nonsense CFTR
or CFTR
mRNA.
Additional tests were conducted in vitro to evaluate the toxicity of the synthesized molecules.
2
. Results and Discussion
2
.1. Design and Synthesis
The 3D-QSAR pharmacophore modeling was carried out based on our previous results [54],
which allowed the identification of a validated pharmacophore model. This model was used as a query
to screen public and “in-house” compound libraries. From the top 5% of retrieved hits, we selected
three synthetically accessible 1,2,4-oxadiazoles (Figure 4).
Figure 4. 1,2,4-oxadiazoles selected structures.

Int. J. Mol. Sci. 2020, 21, 6420
6 of 18
Interestingly, despite the common oxadiazole core, the suggested structures presented significant
differences with respect to ataluren in terms of the lack of aryl substituents (NV848), the lack of fluorine
and acidic moieties (NV848 and NV930), and the maximized fluorine content (NV914).
The synthesis and crystal structure of 3-acetylamino-5-methyl-1,2,4-oxadiazole, NV848, has been
previously reported in the literature [60–62].
Ethyl 5-phenyl-1,2,4-oxadiazole-3-carboxylate (NV930) was prepared by the classic amidoxime
route, as illustrated in Scheme 1.
Scheme 1. Synthesis of NV930.
2,3,4,5,6-Pentafluoro-N-(5-(perfluorophenyl)-1,2,4-oxadiazol-3-yl)benzamide (NV914) was prepared
by acylation of the amine 2 [60–62] with perfluorobenzoyl chloride, as reported in Scheme 2.
Scheme 2. Synthesis of NV914.
All the synthesized compounds were purified by preparative flash chromatography and
crystallization, analyzed by spectroscopic techniques, and displayed an appropriate purity grade for
the biological assays.
2
.2. Testing Cell Proliferation and Viability
Before testing the activity of the compounds as readthrough promoters, we tested their toxicity.
Cell viability was assessed by the MTT assay after 24 and 72 h of exposition to NV848
The assay showed that cell viability was already affected after 24 h of treatment with either PTC124
48 M) or NV2445 (48 M), used as control. In contrast, NV848 NV914, and NV930 molecules did
, NV91, and NV930.
(
µ
µ
,
not show altered cell viability or proliferation (Figure S1) at the same time and concentration points.
A partial cytotoxic effect was observed at 72 h, especially after the addition of NV930 at the doses of 24
and 48 µM.
2
.3. Experimental Screening of the Readthrough Activity by Luciferase Assays, Immunofluorescence, and
Western Blot Analyses
To ascertain the efficacy of the newly synthesized small molecules in promoting the readthrough
of premature termination codons, we used the FLuc cell-based assay [52,53]. Although it is not usually
reliable as a quantitative assessment, this screening is easy and useful to find potential readthrough
promoters. HeLa cells were transiently transfected with the plasmids pFLuc-WT (positive control)
and pFLuc-opal (UGA stop mutation) [63]. After 24 h, the new molecules were added to the cells and,
finally, 48 h after the transfection, FLuc gene expression was measured by luminescence.

Int. J. Mol. Sci. 2020, 21, 6420
7 of 18
HeLa cells transfected with the pFluc-WT plasmid were used as a positive control of the experiment.
6
These cells showed high levels of luciferase activity (about 5
(
(
×
10 activity/relative luminescence units
RLU)). On the other hand, untreated pFluc-opal transfected HeLa cells did not show any activity
negative control). However, when pFluc-opal cells were treated with PTC124 and the three different
analogs, we observed a significant increase of luciferase activity in comparison to untreated cells
Figure 5).
(
Figure 5. The newly synthesized molecules show readthrough activity in the FLuc cell model system.
Histogram shows luciferase (FLuc) activity (RLU) after 24 h of exposition to PTC124, NV848 NV930
and NV914 (all at the concentration of 12 M) in HeLa FLuc-opal transfected cells. Data were analyzed
by GraphPad Prism 6 software and expressed as mean values standard error of the mean (SEM; n = 3).
,
,
µ
±
Symbol (*) represents the statistical significance of data regarding PTC124, NV848
, NV914, and NV930
versus Untr: ***, p < 0.001 (ANOVA with Dunnett’s posthoc test).
The readthrough activity on the CFTR mRNA was studied in Fisher rat thyroid (FRT) cells. FRT
−
cells do not express endogenous CFRT and other endogenous cAMP-regulated Cl channels. The cells
were transfected with a vector harboring the full-length CFTR cDNA (pTracer-CFTR) [64]. The vector
harbored either a wild-type (WT) or a mutated CFTR cDNA in which we previously introduced one of
the two stop mutations, G542X or W1282X, by site-directed mutagenesis [54].
To evaluate the readthrough activity of the three NV molecules, FRT cells transfected with the
mutated cDNA were treated for 24 h with NV848
, NV914, NV930, or PTC124 (positive control) at a
1
2 µM concentration, chosen for better comparison to ataluren’s reported activity [51]. The expression
G542X
W1282X
of the human CFTR-WT and the rescued CFTR (from CFTR
immunofluorescence and Western blot analyses.
or CFTR
) were detected by
Dose–response activity was tested only on FRT CFTRW1282X, and the maximum readthrough
activity was recorded at 24
was recorded at 48 M, with a very small increase with respect to activity at 24
The experiments were performed in duplicate and allowed us to obtain a preliminary estimate of the
EC50 in the range 6–16 M (see Table S1), although further tests are needed to define the new TRIDs’
µM for NV848 and NV930 (see Figure S2B,D). For NV914, the highest activity
µ
µM
(see Figure S2C).
µ
efficacy, especially for different mutations.
Immunofluorescence microscopy images allowed us to visualize CFTR localization at the cell
G542X
membrane, in particular when the CFTR
FRT cells were exposed to NV848 and NV930, suggesting
the occurrence of translational readthrough after the treatments (Figure 6). On the other hand,
W1282X
in CFTR
FRT cells, the expression of the CFTR protein was significantly evident after treatment
with NV914 and NV930, as shown by immunofluorescence (Figure 7). Similar results were detected
and quantified by Western blot analyses (Figure 8A,B and Figure S3A,B).

Int. J. Mol. Sci. 2020, 21, 6420
8 of 18
Figure 6. Immunofluorescence analysis of the full-length CFTR protein in wild-type (CFTR-WT) and
G542X
CFTR
Fisher rat thyroid (FRT) cells, untreated (Untr) or treated with 12 µM PTC124, NV848,
NV930, or NV914 for 24 h. CFTR protein was revealed by a specific antibody (ab570-green). Nuclei
0
(
blue) were DAPI (4 ,6-diamidino-2-phenylindole)-stained and the cellular membrane was stained by
wheat germ agglutinin (WGA)-Alexa 594 (red).
Figure 7. Immunofluorescence analysis of the full-length CFTR protein in wild-type (CFTR-WT) and
W1282X
CFTR
FRT cells, untreated (Untr) or treated with 12 µM PTC124, NV848, NV930, or NV914 for
2
4 h. CFTR protein was revealed by a specific antibody (ab570-green). Nuclei (blue) were DAPI-stained
and the cellular membrane was stained by WGA-Alexa 594 (red).

Int. J. Mol. Sci. 2020, 21, 6420
9 of 18
Figure 8. Western blot analysis of the CFTR protein in wild-type (WT) CFTR, and CFTRG542X
(A) and
W1282X
CFTR
(
B
) FRT cells, untreated (Untr) or treated with 12
-tubulin was used as control for the protein loading. The graphs show
the quantification of the gel bands by ImageJ software. The experiment was performed in triplicate
µM PTC124 (positive control), NV848,
NV930, or NV914 for 24 h;
β
and data were normalized by
are expressed as mean values
statistical significance of NV848
β
-tubulin expression, analyzed by GraphPad Prism 6 software. Data
standard error of the mean (SEM). Symbols (* and §) represent the
NV914, and NV930 versus Untr (*) and versus PTC124 (§). two
±
,
symbols, p < 0.01; three symbols, p < 0.001 (Student’s t-test and ANOVA with Dunnett’s posthoc test).
2
.4. Testing Functionality of the Recovered CFTR
While the NMD pathway affects the amount of recovered protein, the variability of tRNA insertion
plays a role in the quality of the protein produced by readthrough. In this context, besides measuring the
effective protein production by quantitative methods, it is fundamental to prove protein functionality
after readthrough.
For this purpose, molecular and functional analyses have been conducted by halide-sensitive YFP
assay to evaluate the functionality of the CFTR channel produced in FRT cells after treatment with
NV848, NV914, and NV930.
-
The assay is based on the sensibility of the YFP protein to the I ion. In fact, iodide anions induce
the quenching of YFP fluorescence (Figure 9A), thus evidencing the channel activity [65].
G542X
W1282X
To evaluate the activity of the CFTR channel, CFTR
4 h with either one of the NV848 NV914, or NV930 compounds. After 24 h, cells were prestimulated
with forskolin to activate the channel and observed by microscopy before and after the addition of NaI.
and CFTR
FRT cells were treated for
2
,
G542X
W1282X
Treated CFTR
and CFTR
, as well as WT-CFTR, FRT cells showed a significant quenching of
the YFP fluorescence after the addition of iodide (Figure 9B,C), thus proving the channel functionality.
Interestingly, the response to the YFP assay was different based on the stop codon undergoing the
G542X
readthrough. In fact, CFTR
with NV848
FRT cells clearly showed a YFP fluorescence quenching when treated
,
NV914, or NV930 (Figure 9C, left) while CFTRW1282X FRT cells showed quenching of the
YFP fluorescence when specially treated with NV930 (Figure 9C, right). The same experiments were
performed to measure the YFP fluorescence quenching after treatment with the three molecules in a

Int. J. Mol. Sci. 2020, 21, 6420
10 of 18
fluorescent microplate reader and data are illustrated in Figure 9D as the percentage of fluorescent
cells before (−) and after (+) the addition of NaI.
Figure 9. CFTR functionality in FRT cells measured by the yellow fluorescent protein (YFP) assay
WT
(
pink: cells, green: YFP protein expression, blue: multiwell), (
A
) in YFP-CFTR
µ
FRT, and (
B)
CFTRG542X and CFTRW1282X FRT cells, untreated or treated with 12
M PTC124, NV848, NV914, or
NV930 for 24 h (
C
). Cells were prestimulated with 20
µ
M forskolin for 20 min. The experiment was
0
30s
performed in triplicate. Images were taken before (t ) and after (t ) the addition of NaI. Percentages
of YFP-positive cells before (
analyzed by GraphPad (n = 3) and expressed as mean values
Data were analyzed comparing treated FRT cells versus untreated FRT cells by GraphPad Prism 6
−
dark green bars) and after NaI addition (+ light green bars); data were
standard error of the mean (SEM) ( ).
±
D
software (n = 3). ***, p < 0.001 (ANOVA with Dunnett’s posthoc test).
3
. Materials and Methods
3
.1. D-QSAR Pharmacophore Modeling and Virtual Screening
The procedure has been reported in the literature [54,66–70].
3
.2. Chemistry
All reagents and solvents were purchased from commercial sources. All synthesized compounds
were first purified by silica flash chromatography and analyzed by IR, NMR, and HPLC/MS, assessing
1
13
a purity grade (>95%). Melting points were determined by Kofler. H NMR and C NMR spectra
were registered on a Bruker 300 Avance (300 MHz) spectrometer, with TMS as an internal standard. IR
spectra were recorded by a Shimadzu FTIR-8300 instrument (Shimadzu Corporation, Kyoto, Japan).
Chromatography for purification of the samples was realized by flash silica gel (Merck, 0.040–0.063 mm
)
◦
and mixtures of petroleum ether (fraction boiling at 40–60 C) and ethyl acetate as eluents. HRMS
spectra were recorded by analyzing a 10 ppm solution of each sample in a 6540 UHD Accurate-Mass
Q-TOF LC/MS (Agilent Technologies, Inc., Santa Clara, CA, USA) equipped with a Dual AJS ESI source.

Int. J. Mol. Sci. 2020, 21, 6420
11 of 18
3
.2.1. Synthesis of NV848 (N-(5-methyl-1,2,4-oxadiazol-3-yl)acetamide)
The precursor 3-amino-5-methyl-1,2,4-oxadiazole was prepared as follows: 9.5 g (0.22 mmol) of
cyanamide (previously purified with diethyl ether) was added to 1.47 g (0.22 mmol) of hydroxylamine
hydrochloride solubilized in EtOH absolute. The reaction was refluxed for 8 h. After this time, the
solvent was removed under vacuum, and 30 g (0.31 mol) of potassium acetate and 50 mL of acetic
anhydride (0.53 mol) were added in three portions to the product, cooling the bottom flask with an ice
bath. Then, the white solution was refluxed for 30 min. After cooling, the reaction was decomposed
◦
with 300 mL of ice and water. The solution was alkalinized with NaOH (45 g) and maintained at 80 C
(
bain-marie) for 45 min. In the end, the reaction was extracted with diethyl ether (6 × 100 mL).
◦
−1
1
Reaction yield 36%, MP 119–121 C. IR (cm ): 3379, 3316, 3183. H NMR (DMSO-d ): 2.43 (s, 3H),
6
6
.16 (s, 2H).
Additionally, 600 mg of 3-amino-5-methyl-1,2,4-oxadiazole was solubilized into 1 mL of pyridine
and 3 mL of acetic anhydride. The reaction was allowed to sit at room temperature overnight. After
this time, the reaction was treated with water and extracted with ethyl acetate. The product was
purified by chromatography.
◦
−1
1
NV848: Reaction yield 50%, MP 162–163 C. IR (cm ): 3254, 3205, 3120, 1695. H NMR (DMSO-d6)
δ (ppm): 2.15 (s, 3H), 2.60 (s, 3H), 10.86 (s, 1H).
3
.2.2. Synthesis of NV930 (ethyl 5-phenyl-1,2,4-oxadiazole-3-carboxylate)
NV930 was prepared by the classic amidoxime route: The amidoxime
1 (0.3 g) [71] in toluene
(
50 mL) was placed in a 250 mL round-bottomed flask. After the addition of 1.2 eq. of benzoyl chloride
and 1.2 eq. of pyridine, the reaction mixture was refluxed for 6–8 h, monitoring the consumption
of starting material by TLC. The solvent was removed under reduced pressure, followed by water
addition and extraction with ethyl acetate. The extracted reaction mixture was chromatographed on
silica gel, using eluent mixtures of petroleum ether and ethyl acetate. The obtained sample of the
oxadiazole NV930 was further purified by crystallization in ethanol.
◦
1
NV930: Reaction yield 80%, MP 50–52 C, from ethanol. H NMR (300 MHz, CDCl )
δ
(ppm):
3
8
.22 (d, J = 7.2 Hz, 2H), 7.64 (t, J = 7.4 Hz, 1H), 7.55 (t, J = 7.4 Hz, 2H), 4.55 (q, J = 7.1 Hz, 2H),
−
.47 (t, J = 7.1 Hz, 3H). FT-IR (cm ), 1720. HRMS for C H N O found 219.0699 [M + H] (Calcd.
11 10 2 3
1
+
1
2
19.0691).
3
.2.3. Synthesis of NV914 (2,3,4,5,6-pentafluoro-N-(5-(perfluorophenyl)-1,2,4-
oxadiazol-3-yl)benzamide)
NV914 was prepared by acylation with perfluorobenzoyl chloride of the amine 2 [72–74].
1.2 eq. of perfluorobenzoyl chloride and 1.2 eq. of pyridine were added to 1.0 eq of the amine
2
dissolved in toluene, and the reaction mixture was refluxed for 4 h, monitoring the consumption
of the starting material by TLC. Solvent was removed under reduced pressure followed by water
addition and extraction with ethyl acetate. The extracted reaction mixture was chromatographed on
silica gel using eluent mixtures of petroleum ether and ethyl acetate. The sample was further purified
by crystallization in ethanol.
◦
1
NV914: Reaction yield 90%, MP 208–210 C, from ethanol. H NMR (300 MHz, CDCl )
δ
(ppm):
3
−
.09 (s, 1H), FT-IR (cm ), 3290, 3180, 1750. HRMS for C HF N O found 445.9917 [M + H]
15 10 3 2
1
+
9
(Calcd. 445.9909).
3
.3. Cell Culture Conditions and Transfection of Reporter Plasmid
HeLa cells were cultured in a humidified atmosphere of 5% CO in air at 37 C in DMEM medium
◦
2
supplemented with FBS 10% (GIBCO).

Int. J. Mol. Sci. 2020, 21, 6420
12 of 18
Fisher rat thyroid (FRT) cells were cultured in 6-well plates with Coon’s modified F12 medium and
the addition of fetal bovine serum (10% FBS), 2.68 g/L sodium bicarbonate, 2 mM glutamine, 100 U/mL
penicillin, and 100 µg/mL streptomycin. Antibiotics were removed before the addition of TRIDs.
3
.4. Measurement of Luciferase Activity by Luminescence
HeLa cells were transfected with Fluc-WT and Fluc-opal plasmids combined with lipofectamine
5
2
000 (Invitrogen) in a 6-well plate at a density of 2
×
10 /ml Cells were incubated for 24 h, then PTC124
and the three selected molecules (12
cells were incubated with the detection mix Steady-Glo luciferase reagent (Promega, Madison, WI,
USA). Then, 200 L of cell suspension was plated in triplicate in a 96-well plate. Luciferase activity
was finally measured on a luminometer (Promega, Madison, WI, USA).
µM) were added for an additional 24 h. After washing with PBS,
µ
3
.5. Immunofluorescence Microscopy
Cells grown on rounded glass coverslips were fixed with methanol for 2 min. The wheat germ
agglutinin (WGA) Alexa 594 (Life Technologies, Carlsbad, CA, USA) was used to stain the cell
membrane and Golgi apparatus, while nuclei were visualized with DAPI. CFTR was detected by
◦
incubation with a mouse monoclonal antibody (ab570-mouse 1:500) overnight at 4 C, followed by a
goat polyclonal to mouse Alexa-Fluor-488 (Abcam, 1:1000, Cambridge, UK) secondary antibody for 1 h
◦
at 37 C. Cells were observed under a Zeiss Axioskop microscope (Oberkochen, Baden-Württemberg,
Germany) equipped for fluorescence.
3
.6. Western Blotting
Proteins (30 g) separated by 10% SDS-PAGE and 3–8% SDS-PAGE (0.1% SDS) were transferred
µ
to Hybond-C nitrocellulose membranes (Life Technologies, Carlsbad, CA, USA by electroblotting. The
blot was incubated with a mouse antibody anti–CFTR (Ab570 1:500), and HRP-conjugated anti-mouse
(
PIERCE, 1:5000). The target protein was detected by ECL reagents (Pierce -Thermo Fisher Scientific,
Rodano, Italy)). Anti- -tubulin antibody (mouse; Sigma-Aldrich, St. Louis, MO, USA, 1:10,000)
β
confirmed the presence of an equal amount of proteins per well. Gel bands were quantified by
ImageJ software.
3
.7. Microscope Fluorescence YFP Assay
FRT cells expressing the halide-sensitive YFP and the human WT-CFTR, G542X-CFTR, or
W1282X-CFTR cDNA were plated in a 96-well plate. After 24 h, the cells were treated with the
test compounds for 24 h. Cells were then washed with phosphate-buffered saline (PBS: 137 mM
NaCl, 2.7 mM KCl, 8.1 mM Na HPO , 1.5 mM KH PO , 1 mM CaCl , and 0.5 mM MgCl ) and
2
4
2
4
2
2
prestimulated for 20 min with forskolin (20 µM, acute dose; Selleckem, Houston, TX, USA) in a final
volume of 60
µ
L. Microplates were then transferred to a fluorescence microscope (ZEISS, Oberkochen,
0
Baden-Württemberg, Germany). YFP fluorescence images were captured by Axiocam ZEISS before (t )
and 30 s (t3 ) after the addition of 165
0s
µL (100 mM/well) of PBS-NaI, an iodide-containing solution.
All images were captured with exposition time fixed at 800 ms.
3
.8. Microplate Reader Fluorescence YFP Assay
FRT cells expressing the human WT-CFTR, G542X-CFTR cDNA, or W1282X-CFTR cDNA, together
with the halide-sensitive YFP, were plated in a-96 well plate and, after 24 h, treated with the
test compounds. After 24 h of treatment, the cells were washed with phosphate-buffered saline
(
PBS: 137 mM NaCl, 2.7 mM KCl, 8.1 mM Na HPO , 1.5 mM KH PO , 1 mM CaCl , and 0.5 mM
2 4 2 4 2
MgCl ) and prestimulated for 20 min with forskolin (20
µM, acute dose; Selleckem) in a final volume of
2
60 µL in PBS1X. Microplates were then transferred to a microplate reader to measure the fluorescence

Int. J. Mol. Sci. 2020, 21, 6420
13 of 18
before (t ) and 30 s (t30s) after the addition of 165
0
µL (100 mM/well) of PBS-NaI, an iodide-containing
solution. The assay was performed in triplicate.
3
.9. Data Analysis
All data are expressed as mean values
±
standard error of the mean. Statistical analysis was
performed by Student’s t-test and one-way ANOVA followed by Dunnet’s posthoc test, when
appropriate (by GraphPad Prism Software version 8.0.0 for Windows, San Diego, CA, USA).
A probability value (p) of less than 0.05 was regarded as significant and indicated in relevant graphs as
one symbol (*) for p < 0.05, two symbols (**) for p < 0.01, and three symbols (***) for p < 0.001.
4
. Conclusions
The readthrough approach to nonsense is worth being considered as therapy for genetic disorders
caused by the presence of a PTC in the mRNA.
The identification of molecules displaying readthrough activity of the PTCs is a very hot topic in
the challenge posed by nonsense mutations to find a valuable therapeutic treatment for the second
major cause of CF.
Some pitfalls in the readthrough process, when known, can be faced. One issue is that the NMD
pathway may lower the efficacy of TRIDs and, in this case, NMD inhibition associated with TRID
treatment can improve the amount of rescued protein.
In some cases, TRIDs can revert a nonsense mutation to a missense mutation, thus producing
a nonfunctional protein. In these situations, associating appropriate drugs to correct the missense
mutation effect should be considered as a possible solution. For this reason, in the search for new
TRIDs, besides quantitatively testing the amount of protein expressed by the induced readthrough,
it is of paramount importance to also test the functional recovery.
In this project, we designed, synthesized, and biologically tested three new 1,2,4-oxadiazoles
TRIDs showing a high readthrough activity with the production of a full length, and most importantly,
functional CFTR protein from mutant CFTR cDNAs carrying opal stop mutations.
All three molecules evidenced higher activity than ataluren when tested at 12
µM, the concentration
at which ataluren shows its best response, and even higher activity at 24
dose–response analysis.
µM, as evidenced in the
These molecules show high readthrough efficacy, both in terms of protein recovery, as confirmed by
WB analysis, and in terms of protein functionality. Following the treatment with the new compounds,
we observed Fluc-opal luminescence recovery in HeLa cells. Then, by immunofluorescence, in FRT
cells engineered with mutant CFTR cDNAs, we observed that CFTR rescued protein was properly
localized on the cell membrane. Interestingly, by halide-sensitive YFP-based assay, we demonstrated
that TRIDs restored the function of the CFTR channel encoded by PTCs harboring mRNAs. Moreover,
the three molecules showed lower cytotoxic effects when compared to ataluren and the previously
reported NV2445.
Notwithstanding their limits, pharmacological approaches to nonsense suppression are a
fascinating field in view of a wider application to different genetic diseases whose common
denominator is a nonsense mutation. In this context, the various processes involved in the readthrough
pathway require future research on synergistic effects of NMD inhibition, readthrough induction,
and postreadthrough correction.
5
. Patents
I. Pibiri, A. Pace, M. Tutone, L. Lentini, R. Melfi, A. Di Leonardo, Oxadiazole Derivatives For The
Treatment Of Genetic Diseases Due To Nonsense Mutations, PCT Int. Appl. (2019), WO 2019/101709
A1 20190531.
Supplementary Materials: The following are available online at http://www.mdpi.com/1422-0067/21/17/6420/s1.

Int. J. Mol. Sci. 2020, 21, 6420
14 of 18
Author Contributions: The manuscript was written through the contributions of all authors. Conceptualization,
I.P. and L.L.; Data curation, I.P., R.M., M.T. and L.L.; Formal analysis, R.M. and M.T.; Funding acquisition, I.P.
and L.L.; Investigation, I.P. and R.M.; Methodology, I.P., A.D.L., A.P. and L.L.; Project administration, I.P. and
L.L.; Software, M.T.; Supervision, I.P. and L.L.; Validation, I.P. and A.P.; Visualization, M.T., A.D.L. and A.P.;
Writing—original draft, I.P., R.M. and L.L.; Writing—review & editing, I.P. and L.L. All authors have read and
agree to the published version of the manuscript.
Funding: This research was funded by the Italian Cystic Fibrosis Research Foundation, grant FFC#3/2017, with the
contribution of Delegazione di Palermo, Delegazione di Vittoria (Ragusa) e Siracusa, and Delegazione FFC di
Catania Mascalucia to Laura Lentini and Ivana Pibiri.
Acknowledgments: We thank J. Inglese, NIH Chemical Genomics Center, National Institutes of Health, Bethesda,
for providing us with the FLuc190UGA plasmid, Louis Galietta (TIGEM, Napoli, Italy) and Dott.ssa Loretta Ferrera
(Gaslini Hospital, Genova, Italy) for kindly providing us with the FRT cells and the pTracer CFTRWT vector.
Conflicts of Interest: The authors declare no conflict of interest.
Abbreviations
TRID
CFTR
CF
Translational Readthrough Inducing Drugs
Cystic Fibrosis Transmembrane Regulator
Cystic Fibrosis
Ns
Nonsense
NMD
PTC
YFP
DMD
FRT
Nonsense Mediated Decay
Premature Termination Codon
Yellow Fluorescent Protein
Duchenne Muscular Dystrophy
Fischer Rat Thyroid
0
DAPI
WB
4 ,6-diamidino-2-phenylindole
Western Blot
References
1.
2.
3.
4.
5.
Zhang, Z.; Miteva, M.A.; Wang, L.; Alexov, E. Analyzing Effects of Naturally Occurring Missense Mutations.
Comput. Math. Methods Med. 2012, 2012, 805827. [CrossRef]
Chang, Y.-F.; Saadi Imam, J.; Wilkinson, M.F. The Nonsense-Mediated Decay RNA Surveillance Pathway.
Annu. Rev. Biochem. 2007, 76, 51–74. [CrossRef]
Dabrowski, M.; Bukowy-Bieryllo, Z.; Zietkiewicz, E. Translational readthrough potential of natural
termination codons in eucaryotes—The impact of RNA sequence. RNA Biol. 2015, 12, 950–958. [CrossRef]
Kellermayer, R. Translational readthrough induction of pathogenic nonsense mutations. Eur. J. Med. Genet.
2
006, 49, 445–450. [CrossRef] [PubMed]
Blanchet, S.; Cornu, D.; Argentini, M.; Namy, O. New insights into the incorporation of natural suppressor
tRNAs at stop codons in Saccharomyces cerevisiae. Nucleic Acids Res. 2014, 42, 10061–10072. [CrossRef]
[PubMed]
6
7
.
.
Blanchet, S.; Cornu, D.; Hatina, I.; Grosjeana, H.; Bertina, P.; Namy, O. Deciphering the reading of the genetic
code by near-cognate tRNA. Proc. Natl. Acad. Sci. USA 2018, 115, 3018–3023. [CrossRef] [PubMed]
Roy, B.; Friesen, W.J.; Tomizawa, Y.; Leszyk, J.D.; Zhuo, J.; Johnson, B.; Dakka, J.; Trotta, C.R.; Xue, X.;
Mutyam, V.; et al. Ataluren stimulates ribosomal selection of near-cognate tRNAs to promote nonsense
suppression. Proc. Natl. Acad. Sci. USA 2016, 113, 12508–12513. [CrossRef] [PubMed]
8
9
1
.
Dabrowski, M.; Bukowy-Bieryllo, Z.; Zietkiewicz, E. Advances in therapeutic use of a drug stimulated
translational readthrough of premature termination codons. Mol. Med. 2018, 24, 25. [CrossRef]
Tutone, M.; Pibiri, I.; Lentini, L.; Pace, A.; Almerico, A.M. Deciphering the Nonsense Readthrough Mechanism
of Action of Ataluren: An in Silico Compared Study. ACS Med. Chem. Lett. 2019, 10, 522–527. [CrossRef]
.
0. Lentini, L.; Melfi, R.; Cancemi, P.; Pibiri, I.; Di Leonardo, A. Caffeine boosts Ataluren’s readthrough activity.
Heliyon 2019, 5, e01963. [CrossRef]

Int. J. Mol. Sci. 2020, 21, 6420
15 of 18
1
1. Linde, L.; Boelz, S.; Nissim-Rafinia, M.; Oren, Y.S.; Wilschanski, M.; Yaacov, Y.; Virgilis, D.; Neu-Yilik, G.;
Kulozik, A.E.; Kerem, E.; et al. Nonsense-mediated mRNA decay affects nonsense transcript levels and
governs response of cystic fibrosis patients to gentamicin. J. Clin. Investig. 2007, 117, 683–692. [CrossRef]
[PubMed]
1
1
1
1
1
2. Keeling, K.M.; Wang, D.; Dai, Y.; Murugesan, S.; Chenna, B.; Clark, J.; Belakhov, V.; Kandasamy, J.; Velu, S.E.;
Baasov, T.; et al. Attenuation of nonsense-mediated mRNA decay enhances in vivo nonsense suppression.
PLoS ONE 2013, 8, e60478. [CrossRef] [PubMed]
3. Usuki, F.; Yamashita, A.; Higuchi, I.; Ohnishi, T.; Shiraishi, T.; Osame, M.; Ohno, S. Inhibition of
nonsense-mediated mRNA decay rescues the phenotype in Ullrich’s disease. Ann. Neurol. 2004, 55,
7
40–744. [CrossRef] [PubMed]
4. Usuki, F.; Yamashita, A.; Kashima, I.; Higuchi, I.; Osame, M.; Ohno, S. Specific inhibition of nonsense-mediated
mRNA decay components, SMG-1 or UPF1, rescues the phenotype of Ullrich disease fibroblasts. Mol. Ther.
2
006, 14, 351–360. [CrossRef]
5. Branchini, A.; Ferrarese, M.; Campioni, M.; Castaman, G.; Mari, R.; Bernardi, F.; Pinotti, M. Specific factor IX
mRNA and protein features favor drug-induced readthrough over recurrent nonsense mutations. Blood 2019
29, 2303–2307. [CrossRef]
,
1
6. Lombardi, S.; Ferrarese, M.; Marchi, S.; Pinton, P.; Pinotti, M.; Bernardi, F.; Branchini, A. Translational
readthrough of GLA nonsense mutations suggests dominant-negative effects exerted by the interaction of
wild-type and missense variants. RNA Biol. 2020, 17, 254–263. [CrossRef]
1
1
1
7. Peltz, S.W.; Morsy, M.; Welch, E.M.; Jacobson, A. Ataluren as an Agent for Therapeutic Nonsense Suppression.
Annu. Rev. Med. 2013, 64, 407–425. [CrossRef]
8. Ardicli, D.; Haliloglu, G.; Alikasifoglu, M.; Topaloglu, H. Diagnostic Pathway to Nonsense Mutation
Dystrophinopathy: A Tertiary-Center, Retrospective Experience. Neuropediatrics 2019, 50, 41–45. [CrossRef]
9. Sossi, V.; Giuli, A.; Vitali, T.; Tiziano, F.; Mirabella, M.; Antonelli, A.; Neri, G.; Brahe, C. Premature termination
mutations in exon 3 of the SMN1 gene are associated with exon skipping and a relatively mild SMA
phenotype. Eur. J. Hum. Genet. 2001, 9, 113–120. [CrossRef]
2
0. Li, N.; Wang, L.; Sun, X.; Lu, Z.; Suo, X.; Li, J.; Peng, J.; Peng, R. A novel mutation in VRK1 associated with
distal spinal muscular atrophy. J. Hum. Genet. 2019, 64, 215–219. [CrossRef]
2
1. Li, K.; Turner, A.N.; Chen, M.; Brosius, S.N.; Schoeb, T.R.; Messiaen, L.M.; Bedwell, D.M.; Zinn, K.R.;
Anastasaki, C.; Gutmann, D.H.; et al. Mice with missense and nonsense NF1 mutations display divergent
phenotypes compared with human neurofibromatosis type I. Dis. Model. Mech. 2016, 9, 759–767. [CrossRef]
[PubMed]
2
2. Xie, Y.A.; Lee, W.; Cai, C.; Gambin, T.; Nõupuu, K.; Sujirakul, T.; Ayuso, C.; Jhangiani, S.; Muzny, D.;
Boerwinkle, E.; et al. New syndrome with retinitis pigmentosa is caused by nonsense mutations in retinol
dehydrogenase RDH11. Hum. Mol. Genet. 2014, 23, 5774–5780. [CrossRef] [PubMed]
23. Kiser, K.; Webb-Jones, K.D.; Bowne, S.J.; Sullivan, S.L.; Daiger, S.P.; Birch, D.G. Time Course of Disease
Progression of PRPF31-mediated Retinitis Pigmentosa. Am. J. Ophthalmol. 2019, 200, 76–84. [CrossRef]
24. Samanta, A.; Stingl, K.; Kohl, S.; Ries, J.; Linnert, J.; Nagel-Wolfrum, K. Ataluren for the Treatment of Usher
Syndrome 2A Caused by Nonsense Mutations Int. J. Mol. Sci. 2019, 20, 6274. [CrossRef] [PubMed]
25. Banning, A.; Schiff, M.; Tikkanen, R. Amlexanox provides a potential therapy for nonsense mutations in
the lysosomal storage disorder Aspartylglucosaminuria. Biochim. Biophys. Acta Mol. Basis Dis. 2018, 1864,
6
68–675. [CrossRef]
6. Keeling, K.M. Nonsense Suppression as an Approach to Treat Lysosomal Storage Diseases. Diseases 2016, 4,
2. [CrossRef]
7. Chernushyn, S.; Gulkovskyi, R.; Livshits, L. Novel Mutation in the MECP2 Gene Identified in a Group of
Rett Syndrome Patients from Ukraine. Cytol. Genet. 2018, 52, 294–298. [CrossRef]
8. Bezzerri, V.; Bardelli, D.; Morini, J.; Vella, A.; Cesaro, S.; Sorio, C.; Biondi, A.; Danesino, C.; Farruggia, P.;
Assael, B.M.; et al. Ataluren-driven restoration of Shwachman-Bodian-Diamond syndrome protein function
in Shwachman-Diamond syndrome bone marrow cells. Am. J. Hematol. 2017, 93, 527–536. [CrossRef]
9. Bezzerri, V.; Api, M.; Allegri, M.; Fabrizzi, B.; Corey, S.J.; Cipolli, M. Nonsense Suppression Therapy:
New Hypothesis for the Treatment of Inherited Bone Marrow Failure Syndromes. Int. J. Mol. Sci. 2020, 21,
2
2
2
3
2
4
672. [CrossRef]

Int. J. Mol. Sci. 2020, 21, 6420
16 of 18
3
3
3
3
3
0. Goldmann, T.; Overlack, N.; Wolfrum, U.; Nagel-Wolfrum, K. PTC124-Mediated Translational Readthrough
of a Nonsense Mutation Causing Usher Syndrome Type 1C. Hum. Gene Ther. 2011, 22, 537–547. [CrossRef]
1. Mirtajani, S.B.; Farnia, P.; Hassanzad, M.; Ghanavi, J.; Farnia, P.; Velayati, A.A. Geographical distribution of
cystic fibrosis; The past 70 years of data analysis. Biomed. Biotechnol. Res. J. 2017, 1, 105–112.
2. Farinha, C.M.; Canato, S. From the endoplasmic reticulum to the plasma membrane: Mechanisms of CFTR
folding and trafficking Cell. Mol. Life Sci. 2017, 74, 39–55. [CrossRef] [PubMed]
3. Rowe, S.M.; Miller, S.; Sorscher, E.J. Cystic fibrosis. N. Engl. J. Med. 2005, 352, 1992–2001. [CrossRef]
[PubMed]
4. Son, J.-H.; Zhu, J.S.; Phuan, P.-W.; Cil, O.; Teuthorn, A.P.; Ku, C.K.; Lee, S.; Verkman, A.S.; Kurth, M.J.
High-Potency Phenylquinoxalinone Cystic Fibrosis Transmembrane Conductance Regulator (CFTR)
Activators. J. Med. Chem. 2017, 60, 2401–2410. [CrossRef]
3
3
3
3
5. Ye, L.; Hu, B.; El-Badri, F.; Hudson, B.M.; Phuan, P.-W.; Verkman, A.S.; Tantillo, D.J.; Kurth, M.J.
∆
f508-CFTR correctors: Synthesis and evaluation of thiazole-tethered imidazolones, oxazoles, oxadiazoles,
and thiadiazoles. Bioorg. Med. Chem. Lett. 2014, 24, 5840–5844. [CrossRef]
6. Coffman, K.C.; Nguyen, H.H.; Phuan, P.-W.; Hudson, B.M.; Yu, G.J.; Bagdasarian, A.L.; Montgomery, D.;
Lodewyk, M.W.; Yang, B.; Yoo, C.L.; et al. Constrained bithiazoles: Small molecule correctors of defective
δf508-CFTR protein trafficking. J. Med. Chem. 2014, 57, 6729–6738. [CrossRef]
7. Ponzano, S.; Nigrelli, G.; Fregonese, L.; Eichler, I.; Bertozzi, F.; Bandiera, T.; Galietta, L.J.V.; Papaluca, M. A
European regulatory perspective on cystic fibrosis: Current treatments, trends in drug development and
translational challenges for CFTR modulators. Eur. Resp. Rev. 2018, 27, 170124–170134. [CrossRef]
8. Liessi, N.; Cichero, E.; Pesce, E.; Arkel, M.; Salis, A.; Tomati, V.; Paccagnella, M.; Damonte, G.; Tasso, B.;
Galietta, L.J.V.; et al. Synthesis and biological evaluation of novel thiazole-VX-809 hybrid derivatives as
F508del correctors by QSAR-based filtering tools. Eur. J. Med. Chem. 2018, 144, 179–200. [CrossRef]
9. Fanen, P.; Wohlhuter-Haddad, A.; Hinzpeter, A. Genetics of cystic fibrosis: CFTR mutation classifications
toward genotype-based CF therapies: Genetics of CF. Int. J. Biochem. Cell Biol. 2014, 52, 94–102. [CrossRef]
0. Burke, J.F.; Mogg, A.E. Suppression of a nonsense mutation in mammalian cells in vivo by the aminoglycoside
antibiotics G-418 and paromomycin. Nucleic Acids Res. 1985, 13, 6265–6272. [CrossRef]
3
4
4
1. Manuvakhova, M.; Keeling, K.; Bedwell, D.M. Aminoglycoside antibiotics mediate context-dependent
suppression of termination codons in a mammalian translation system. RNA 2000, 6, 1044–1055. [CrossRef]
[PubMed]
4
2. Roy, B.; Leszyk, J.D.; Mangus, D.A.; Jacobson, A. Nonsense suppression by near-cognate tRNAs employs
alternative base pairing at codon positions 1 and 3. Proc. Natl. Acad. Sci. USA 2015, 112, 3038–3043.
[CrossRef] [PubMed]
4
4
3. Prayle, A.; Smyth, A.R. Aminoglycoside use in cystic fibrosis: Therapeutic strategies and toxicity. Curr. Opin.
Pulm. Med. 2010, 16, 604–610. [CrossRef] [PubMed]
4. Leubitz, A.; Frydman-Marom, A.; Sharpe, N.; van Duzer, J.; Campbell, K.C.M.; Vanhoutte, F. Safety,
Tolerability, and Pharmacokinetics of Single Ascending Doses of ELX-02, a Potential Treatment for Genetic
Disorders Caused by Nonsense Mutations, in Healthy Volunteers. Clin. Pharm. Drug Dev. 2019, 8, 984–994.
[CrossRef]
4
4
4
4
5. Arakawa, M.; Shiozuka, M.; Nakayama, Y.; Hara, T.; Hamada, M.; Kondo, S.; Ikeda, D.; Takahashi, Y.;
Sawa, R.; Nonomura, Y.; et al. Negamycin Restores Dystrophin Expression in Skeletal and Cardiac Muscles
of mdx Mice. J. Biochem. 2003, 134, 751–758. [CrossRef]
6. Friesen, W.J.; Trotta, C.R.; Tomizawa, Y.; Zhuo, J.; Johnson, B.; Sierra, J.; Roy, B.; Weetall, M.; Hedrick, J.;
Sheedy, J.; et al. The nucleoside analog clitocine is a potent and efficacious readthrough agent. RNA 2017, 23,
5
67–577.
7. Welch, E.M.; Barton, E.R.; Zhuo, J.; Tomizawa, Y.; Friesen, W.J.; Trifillis, P.; Paushkin, S.; Patel, M.; Trotta, C.R.;
Hwang, S.; et al. PTC124 targets genetic disorders caused by nonsense mutations. Nature 2007, 447, 87–91.
[CrossRef]
8. Kerem, E.; Hirawat, S.; Armoni, S.; Yaakov, Y.; Shoseyov, D.; Cohen, M.; Nissim-Rafinia, M.; Blau, H.;
Rivlin, J.; Aviram, M.; et al. Effectiveness of PTC124 treatment of cystic fibrosis caused by nonsense mutations:
A prospective phase II trial. Lancet 2008, 372, 719–727. [CrossRef]

Int. J. Mol. Sci. 2020, 21, 6420
17 of 18
4
9. Kerem, E.; Konstan, M.W.; De Boeck, K.; Accurso, F.J.; Sermet-Gaudelus, I.; Wilschanski, M.; Elborn, J.S.;
Melotti, P.; Bronsveld, I.; Fajac, I.; et al. A randomized placebo-controlled trial of Ataluren for the treatment
of nonsense mutation cystic fibrosis. Lancet Respir. Med. 2014, 2, 539–547. [CrossRef]
5
0. Aslam, A.A.; Higgins, C.; Sinha, I.P.; Southern, K.W. Ataluren and similar compounds (specific therapies for
premature termination codon class I mutations) for cystic fibrosis. Paediatr. Respir. Rev. 2017, 24, 32–34.
1. Lentini, L.; Melfi, R.; Di Leonardo, A.; Spinello, A.; Barone, G.; Pace, A.; Palumbo Piccionello, A.;
Pibiri, I. Towards a rationale for the PTC124 (Ataluren) promoted read-through of premature stop codons:
A computational approach and GFP- reporter cell-based assay. Mol. Pharm. 2014, 11, 653–664. [CrossRef]
5
[PubMed]
5
5
5
5
2. Pibiri, I.; Lentini, L.; Tutone, M.; Melfi, R.; Pace, A.; Di Leonardo, A. Exploring the Readthrough of Nonsense
Mutations by Non-Acidic Ataluren Analogues Selected by Ligand-Based Virtual Screening. Eur. J. Med.
Chem. 2016, 122, 429–435. [CrossRef] [PubMed]
3. Pibiri, I.; Lentini, L.; Melfi, R.; Gallucci, G.; Pace, A.; Spinello, A.; Barone, G.; Di Leonardo, A. Enhancement
of Premature Stop Codon Readthrough in the CFTR Gene by Ataluren (PTC124) Derivatives. Eur. J. Med.
Chem. 2015, 101, 236–244. [CrossRef] [PubMed]
4. Pibiri, I.; Lentini, L.; Melfi, R.; Tutone, M.; Baldassano, S.; Ricco Galluzzo, P.; Di Leonardo, A.; Pace, A.
Rescuing the CFTR Protein Function: Introducing 1,3,4-Oxadiazoles as Translational Readthrough Inducing
Drugs. Eur. J. Med. Chem. 2018, 159, 126–142. [CrossRef]
5. Campofelice, A.; Lentini, L.; Di Leonardo, A.; Melfi, R.; Tutone, M.; Pace, A.; Pibiri, I. Strategies against
Nonsense: Oxadiazoles as Translational Readthrough-Inducing Drugs (TRIDs). Int. J. Mol. Sci. 2019, 20,
3
329. [CrossRef]
5
5
6. Pace, A.; Buscemi, S.; Palumbo Piccionello, A.; Pibiri, I. Recent Advances in the Chemistry of 1,2,4-Oxadiazoles.
Adv. Heterocycl. Chem. 2016, 116, 85–136.
7. Rubino, S.; Pibiri, I.; Minacori, C.; Alduina, R.; Di Stefano, V.; Orecchio, S.; Buscemi, S.; Girasolo, M.A.;
Tesoriere, L.; Attanzio, A. Synthesis, structural characterization, anti-proliferative and antimicrobial T activity
of binuclear and mononuclear Pt(II) complexes with perfluoroalkyl- heterocyclic ligands. Inorg. Chim. Acta
2
018, 483, 180–190. [CrossRef]
5
8. Rubino, S.; Pibiri, I.; Costantino, C.; Buscemi, S.; Girasolo, M.A.; Attanzio, A.; Tesoriere, L. Synthesis of
platinum complexes with 2-(5-perfluoroalkyl-1,2,4-oxadiazol-3yl)-pyridine and 2-(3-perfluoroalkyl-1-methyl-
1
,2,4-triazole-5yl)-pyridine ligands and their in vitro antitumor activity. J. Inorg. Biochem. 2016, 155, 92–100.
CrossRef]
9. Tutone, M.; Pecoraro, B.; Almerico, A.M. Investigation on Quantitative Structure-Activity Relationships of
,3,4-Oxadiazole Derivatives as Potential Telomerase Inhibitors. Curr. Drug Discov. Technol. 2020, 17, 79–86.
CrossRef]
[
5
6
6
1
[
0. Vivona, N.; Ruccia, M.; Cusmano, G.; Marino, M.L.; Spinelli, D. The thermally degenerate mononuclear
rearrangement of 3-acetylamino-5-methyl-1,2,4-oxadiazole. J. Heterocycl. Chem. 1975, 12, 1327–1328.
[
CrossRef]
1. Mugnoli, A.; Barone, G.; Buscemi, S.; Lanza, C.Z.; Pace, A.; Pani, M.; Spinelli, D. On the structure of
-acetylamino-5-methyl-1,2,4-oxadiazole and on the fully degenerate rearrangements (FDR) of its anion:
3
A stimulating comparison between the results of in-silicon chemistry and laboratory chemistry. J. Phys. Org.
Chem. 2009, 22, 1086–1093. [CrossRef]
6
2. Pace, A.; Pibiri, I.; Palumbo Piccionello, A.; Buscemi, S.; Vivona, N.; Barone, G. Experimental and DFT
Studies on Competitive Heterocyclic Rearrangements. Part 2:1 A One-Atom Side-Chain versus the Classic
Three-Atom Side-Chain (Boulton-Katritzky) Ring Rearrangement of 3-Acylamino-1,2,4-oxadiazoles. J. Org.
Chem. 2007, 72, 7656–7666. [CrossRef] [PubMed]
6
6
6
3. Auld, D.S.; Lovell, S.; Thorne, N.; Lea, W.A.; Maloney, D.J.; Shen, M.; Rai, G.; Battaile, K.P.; Thomas, C.J.;
Simeonov, A.; et al. Molecular basis for the high-affinity binding and stabilization of firefly luciferase by
PTC124. Proc. Natl. Acad. Sci. USA 2010, 107, 4878–4883. [CrossRef] [PubMed]
4. Sheppard, D.N.; Rich, D.P.; Ostedgaard, L.S.; Gregory, R.J.; Smith, A.E.; Welsh, M.J. Mutations in CFTR
associated with mild-disease from Cl-channels with altered pore properties. Nature 1993, 362, 160–164.
[CrossRef] [PubMed]
5. Pedemonte, N.; Tomati, V.; Sondo, E.; Galietta, L.J.V. Influence of cell background on pharmacological rescue
of mutant CFTR. Am. J. Physiol. Cell Physiol. 2010, 298, 866–874. [CrossRef]

Int. J. Mol. Sci. 2020, 21, 6420
18 of 18
6
6
6. Almerico, A.M.; Tutone, M.; Lauria, A. 3D-QSAR pharmacophore modeling and in silico screening of new
Bcl-xl inhibitors. Eur. J. Med. Chem. 2010, 45, 4774–4782. [CrossRef]
7. Lauria, A.; Ippolito, M.; Fazzari, M.; Tutone, M.; Di Blasi, F.; Mingoia, F.; Almerico, A.M. IKK-b inhibitors:
An analysis of drug-receptor interaction by using Molecular Docking and Pharmacophore 3D-QSAR
approaches. J. Mol. Graph. Model. 2010, 29, 72–81. [CrossRef]
6
6
7
8. Almerico, A.M.; Tutone, M.; Lauria, A. Receptor-guided 3D-QSAR approach for the discovery of c-kit
tyrosine kinase inhibitors. J. Mol. Model. 2012, 18, 2885–2895. [CrossRef]
9. Almerico, A.M.; Tutone, M.; Pantano, L.; Lauria, A. A3 adenosine receptor: Homology modeling and
3
D-QSAR studies. J. Mol. Graph. Model. 2013, 42, 60–72. [CrossRef]
0. Perricone, U.; Wieder, M.; Seidel, T.; Langer, T.; Padova, A.; Almerico, A.M.; Tutone, M. A molecular
dynamics-shared pharmacophore approach to boost early-enrichment virtual screening: A case study on
peroxisome proliferator- activated receptora. Chem. Med. Chem. 2017, 12, 1399–1407. [CrossRef]
1. Branco, P.S.; Prabhakar, S.; Lobo, A.M.; Williams, D.J. Reactions of hydroxylamines with ethyl cyanoformate.
preparation of aminonitrones and their synthetic applications. Tetrahedron 1992, 48, 6335–6360. [CrossRef]
2. Buscemi, S.; Pace, A.; Pibiri, I.; Vivona, N.; Caronna, T. Fluorinated heterocyclic compounds: An assay on the
photochemistry of some fluorinated 1-oxa-2-azoles: An expedient route to fluorinated heterocycles. J. Fluor.
Chem. 2004, 125, 165–173. [CrossRef]
7
7
7
3. Buscemi, S.; Pace, A.; Frenna, V.; Vivona, N. A generalized synthesis of 3-amino-5-aryl-, 3-amino-5-
polyfluorophenyl-, and 3-amino-5-alkyl-1,2,4-oxadiazoles through ring-degenerate rearrangements.
Heterocycles 2002, 57, 811–823. [CrossRef]
7
4. Buscemi, S.; Pace, A.; Calabrese, R.; Vivona, N.; Metrangolo, P. Fluorinated heterocyclic compounds.
A photochemical synthesis of 3-amino-5-perfluoroaryl-1,2,4-oxadiazoles. Tetrahedron 2001, 57, 5865–5871.
[CrossRef]
©
2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access
article distributed under the terms and conditions of the Creative Commons Attribution
CC BY) license (http://creativecommons.org/licenses/by/4.0/).
(