Pharmacophore-Based Design of New Chemical Scaffolds as Translational Readthrough-Inducing Drugs (TRIDs)

Article abstract of DOI:10.1021/acsmedchemlett.9b00609

Translational readthrough-inducing drugs (TRIDs) rescue the functional full-length protein expression in genetic diseases, such as cystic fibrosis, caused by premature termination codons (PTCs). Small molecules have been developed as TRIDs to trick the ribosomal machinery during recognition of the PTC. Herein we report a computational study to identify new TRID scaffolds. A pharmacophore approach was carried out on compounds that showed readthrough activity. The pharmacophore model applied to screen different libraries containing more than 87000 compounds identified four hit-compounds presenting scaffolds with diversity from the oxadiazole lead. These compounds have been synthesized and tested using the Fluc reporter harboring the UGA PTC. Moreover, the cytotoxic effect and the expression of the CFTR protein were evaluated. These compounds, a benzimidazole derivative (NV2899), a benzoxazole derivative (NV2913), a thiazole derivative (NV2909), and a benzene-1,3-disulfonate derivative (NV2907), were shown to be potential new lead compounds as TRIDs, boosting further efforts to address the optimization of the chemical scaffolds.

Full text of DOI:10.1021/acsmedchemlett.9b00609

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Letter
Pharmacophore-Based Design of New Chemical Scaffolds as
Translational Readthrough-Inducing Drugs (TRIDs)
Marco Tutone,* Ivana Pibiri,* Riccardo Perriera, Ambra Campofelice, Giulia Culletta, Raffaella Melfi,
Andrea Pace, Anna Maria Almerico, and Laura Lentini
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ABSTRACT: Translational readthrough-inducing drugs (TRIDs)
rescue the functional full-length protein expression in genetic
diseases, such as cystic fibrosis, caused by premature termination
codons (PTCs). Small molecules have been developed as TRIDs to
trick the ribosomal machinery during recognition of the PTC. Herein
we report a computational study to identify new TRID scaffolds. A
pharmacophore approach was carried out on compounds that
showed readthrough activity. The pharmacophore model applied to
screen different libraries containing more than 87000 compounds
identified four hit-compounds presenting scaffolds with diversity
from the oxadiazole lead. These compounds have been synthesized
and tested using the Fluc reporter harboring the UGA PTC.
Moreover, the cytotoxic effect and the expression of the CFTR
protein were evaluated. These compounds, a benzimidazole
derivative (NV2899), a benzoxazole derivative (NV2913), a thiazole derivative (NV2909), and a benzene-1,3-disulfonate derivative
(NV2907), were shown to be potential new lead compounds as TRIDs, boosting further efforts to address the optimization of the
chemical scaffolds.
KEYWORDS: Pharmacophore modeling, cystic fibrosis, premature termination codons, nonsense mutation, HTVS
aminoglycosides including renal, auditory, and vestibular
toxicities, that have limited their widespread clinical use as
translational readthrough-inducing drugs (TRIDs). In this
context, recently a new aminoglycoside ELX-2, showing
potential activity as a TRID and less side effects, has been
launched in phase II clinical trials.10
Previously, in 2007 Ataluren (aka PTC124) was proposed
by PTC Therapeutics as able to promote the readthrough of
premature but not normal termination codons.11 The small
molecule Ataluren is a diaryl-1,2,4-oxadiazole, is less toxic than
aminoglycosides, and has been suggested as a potential
treatment of genetic disorders caused by nonsense mutations,
particularly those involving the UGA premature codon.12
Results of the phase II and III trials showed improvement in
markers of CFTR function but no improvements in sweat
chloride levels or nasal potential difference.13 PTC Ther-
apeutics concluded phase III clinical trials in 2014 for CF and
n recent years many efforts have been dedicated to
Ipersonalized medicinal approaches to genetic disease. In
this context, with cystic fibrosis being a largely diffused genetic
pathology, researchers have focused on the therapy of the basis
genetic defect. Approximately 10%−15% of the cystic fibrosis
(CF) cases are due to nonsense-mutations in the cystic fibrosis
transmembrane conductance regulator gene (CFTR).1 The
presence of a nonsense mutation, giving rise to a premature
termination codon (PTC) in the mRNA, produces a truncated
protein that is rapidly degraded, so the patients lack the
functional protein and suffer a hardest form of the disease.
Therapies targeting this specific genetic defect have been
addressed and, besides aminoglycosides, heterocyclic scaffolds
play the main roles.2−6
Concerning patients with nonsense mutations, in the last ten
years, the only pharmaceutical option was the translational
readthrough of the PTC in order to bypass the PTC and
restore to a sufficient extent the expression of a functional
protein. Aminoglycosides (e.g., gentamicin, tobramycin,
paromomycin) had been previously studied to this aim. They
suppress the normal proof-reading function of the ribosome
allowing the translation and lead to the insertion of a near-
cognate amino acid at the PTC site.7,8 However, severe side
effects have been reported9 by prolonged treatments with
Special Issue: In Memory of Maurizio Botta: His
Vision of Medicinal Chemistry
Received: December 16, 2019
Accepted: February 18, 2020
Published: February 18, 2020
https://dx.doi.org/10.1021/acsmedchemlett.9b00609
© XXXX American Chemical Society
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Figure 1. Pharmacophore features for the actives data set: HBA, H-bond acceptor; AR, aromatic; H, hydrophobic; NI, negative ionic.
Duchenne Muscular Dystrophy (DMD), in order to evaluate
the long-term safety of Ataluren. At the conclusion of this
study, Ataluren has been approved for DMD while for CF it
was evidenced that, although cystic fibrosis patients who
received this treatment had beneficial effects, patients taking
chronic inhaled tobramycin did not show the same benefits,
allowing the researchers to hypothesize that the two drugs
were competing at the level of the ribosome.
Additional confirmation phase III clinical study resulted in
the approval of the drug under the trade name Translarna for
DMD patients, while the trial for CF patients was suspended in
April 2017 due to conflicting results.13,14
available experimental data on our previous identified
compounds.16−18,20 As starting point of our analysis, we
considered the 24 active compounds in FLuc assay over the 61
synthesized compounds. Nine of the most promising
compounds have a carboxylic group (Ataluren and NV2445,
included); for this reason, we decided to identify these nine
compounds as the active data set (Figure 1).
Pharmacophore models have been generated using Li-
gandScout 4.3 by Inte:Ligand GmbH.21,22 The data set
compounds have been randomly split into a training and a
test set (80%−20%). Before models generation, conformers’
generation and alignment have been performed. The
pharmacophore features have been identified in a ligand-
based mode, and ten different pharmacophore models have
been identified. The better one (Model 1) showed a score of
0.90 out of 1 and for each compound matched 7 to 10 features.
Model 1 consists of three aromatic features, two hydrophobic
features, 5 H-bond acceptor features, and one negative ionic
feature (Figure 2).
In order to refine and validate the model we employed the
data set of active compounds previously identified by us and a
data set of 450 decoys generated using the DUD-E tool.23,24
For the validation of the pharmacophore model related to its
insight power, we considered the enrichment factor (EF) and
the area under the curve (AUC) of the receiver operating
characteristic (ROC) curve. The validation test identified 29
hits, all the 24 actives and five decoys. Figure 2 (bottom)
displays the ROC plot, the AUC, and the EF factor values at
Considering Ataluren’s failure to be the prospective lead
compound as a TRID and its biological target still not being
clear15 prompted us to further studies on the topic in order to
find a valuable substitute to Ataluren.
In this context, we identified a set of small molecules
containing an oxadiazole heterocyclic core by means of virtual
screening on public and in-house libraries and cell-based
experimental studies.16,17 The compound NV2445 (Figure 1)
showed the most promising activity both in FLuc assays and in
cells expressing a nonsense-CFTR-mRNA.18,19 Notwithstand-
ing these efforts with the aim to identify an effective alternative
to Ataluren, new and more potent TRIDs are necessary for the
treatment of the disease. In this paper, we performed an in
silico design focusing on the identification of new chemical
scaffolds not related with 1,2,4- or 1,3,4-oxadiazoles. We
carried out a pharmacophore-based modeling study exploiting
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Figure 3.
1. Thus, the benzimidazole derivative NV2899 was prepared
by cyclocondensation in DMF between 3,4-diaminobenzoic
Scheme 1
Figure 2. (top) 3D representation of the best model (Model 1
legend: red sphere, H-bond acceptor; red arrows, negative ionizable
area; yellow sphere, hydrophobic interactions; blue donuts, aromatic
ring); (bottom) ROC curve for the Model 1 (legend: AUC, area
under the curve; EF, enrichment factor).
1%, 5%, 10%, and 100% of the best pharmacophore model. As
shown in Figure 2, the early enrichment (EF 1%) is equal to
19.8 with an AUC value of 1.00 demonstrating that our
pharmacophore model was able to discriminate between active
and inactive compounds. Overall, the model has a preference
for active compounds with an AUC value of 1 in the first 10%
and AUC = 0.72 at 100%, and an EF value of 7.5 in the first
10% until 100% showing a good accuracy for new hits
identification.
In the end, this model was used as a query to screen 5
different databases: (1) an OTAVA commercial library
containing 2775 potential mRNA binders; (2) the Drugbank
library updated to January 2018 containing 8721 compounds;
(3) the Maybridge hit discover library containing 52146
compounds; (4) an in-house library of 1829 small molecules
designed by the Almerico group;25−28 (5) an in-house library of
about 26000 small molecules, consisting of aromatic
pentatomic heterocycles with two heteroatoms or more, their
benzo-condensed analogs, and their open chain precursors,
designed by the Pace and Pibiri group.18 The overall used
library contained about 87000 compounds. The search yielded
23 hits compounds. Among these, four hits were chosen
among the most synthetically accessible. They do not contain
the oxadiazole core (Figure 3).
acid and 2, obtained in turn from chlorobenzaldehyde 1 and
metabisulfite (Scheme 1A).
NV2909, a 2-(3,5-dichlorophenoxymethyl)-thiazole deriva-
tive, was synthesized in one step from the corresponding
chloromethyl-thiazole 3 and 3,5-dichlorophenol 4 in acetoni-
trile in the presence of a base (Scheme 1B).
Also, the 2-(3,5-dichlorophenyl)benzoxazole NV2913 was
prepared from 3,5-dichlorophenyl acyl chloride 5 and the aryl
ester 6, likely through a not isolated amido intermediate
(Scheme 1C).
The hit compounds were easily obtained in one or two steps
from commercially available compounds according to Scheme
C
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As shown in Scheme 1D, NV2907 was obtained from 2,4-
difluorophenol 7 and 1,3-disulfonyl chloride 8, in DCM and
potassium carbonate as a base.
compounds, we observed an increase of luciferase activity
(Figure 4).
FRT CFTR wild type cells were used to determine the
possible cytotoxic effects of NV2907, NV2909, NV2899, and
NV2913 and to visualize the impact on cell proliferation. In the
graphs in Figure 5 are reported the percentages of dead cells
and proliferating cells after treatment with 12 μM NV2907,
NV2909, NV2899, and NV2913. PTC124 at 12 μM was used
as control. Our results showed similar increase in dead cells at
24−48−72 h in all samples analyzed (Figure 5 top). We also
evaluated the expression of the CFTR protein after treatment
with the selected compounds. FRT cells stably transfected with
the pTracer vectors containing either the WT-CFTR(CFTR-
WT) or the G542X-CFTR human cDNA were used. The
expression of the CFTR WT and G542X-CFTR after
treatment with 12 μM of NV2899, NV2907, NV2909, and
NV2913 was detected by immunofluorescence microscopy.
After 24 h of treatment with the two compounds NV2899 and
NV2909, we observed CFTR expression in G542X-CFTR
FRT cells. In contrast CFTR expression was not revealed in
G542X-CFTR FRT untreated cells (negative control) (Figure
6).
To evaluate the activity of the newly synthesized small
molecules in promoting the readthrough of PTCs, we used the
FLuc cell-based assay.16,17 To this aim HeLa cells were
transfected transiently with the plasmids pFLuc-WT (control)
and pFLuc-opal (UGA stop mutation).29 After transfection,
Hela cells were treated for 24 h with the compounds (NV2907,
NV2909, NV2899, NV2913) and subsequently the activity of
the Fluc protein was measured. HeLa cells transfected with the
pFluc-WT plasmid were used as positive control and showed
high levels of luciferase activity (Figure 4). As negative control
In conclusion, our studies, with the aim to identify a valuable
substitute of Ataluren for the treatment of nonsense mutation
in CFTR gene, allowed us to find out new promising chemical
scaffolds as TRIDs. Following a ligand-based pharmacophore
approach together to a virtual screening protocol, several small
molecules capable to allow the in vitro expression of the gene
pFLuc-opal (UGA stop mutation) were discovered. These
molecules have different scaffolds from the known 1,2,4- and
1,3,4-oxadiazoles. In particular, the identified compounds are a
benzimidazole derivative (NV2899), a benzoxazole derivative
(NV2913), a thiazole derivative (NV2909), and a benzene-1,3-
disulfonate derivative (NV2907). These compounds showed
low cytotoxic effect and displayed the expression CFTR
protein in transfected cells. We envision that these molecules
could to be potential lead compounds as TRIDs, and further
studies will be performed in order to optimize these scaffolds.
Figure 4. Histogram showing luciferase (FLuc) activity after 24 h of
exposition to PTC124 and the new identified compounds NV2899,
NV2907, NV2909, NV2913 (all at the concentration of 12 μM) in
HeLa FLuc-opal transfected cells.
pFluc-opal transfected HeLa cells were used, and these cells
did not show any activity. Moreover, when these pFluc-opal
cells were treated with Ataluren and the four different
Figure 5. (top) Histograms showing live and dead cells at 24−48−72 h post treatment with the indicated compounds. (bottom) Graphs showing
cell proliferation at the same time intervals (24−72 h).
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Figure 6. Immunofluorescence analysis to visualize CFTR (green-Ab570) expression in G542X-CFTR FRT cells treated with the indicated
compounds for 24 h. Cell membrane was stained in red and nuclei in blue with DAPI.
degli Studi di Palermo, 90123 Palermo, Italy; orcid.org/
0000-0001-5059-8686; Email: marco.tutone@unipa.it
Ivana Pibiri − Dipartimento di Scienze e Tecnologie Biologiche,
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* Supporting Information
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acsmedchemlett.9b00609.
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Chimiche e Farmaceutiche (STEBICEF), Universita degli Studi
di Palermo, 90123 Palermo, Italy; Email: ivana.pibiri@
unipa.it
Materials and methods (PDF)
Authors
AUTHOR INFORMATION
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Corresponding Authors
Riccardo Perriera − Dipartimento di Scienze e Tecnologie
Biologiche, Chimiche e Farmaceutiche (STEBICEF), Universita
degli Studi di Palermo, 90123 Palermo, Italy
̀
Marco Tutone − Dipartimento di Scienze e Tecnologie
Biologiche, Chimiche e Farmaceutiche (STEBICEF), Universita
̀
E
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(4) 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 (15), 6729−6738.
(5) 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
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Synthesis and Biological Evaluation of Novel Thiazole- VX-809
Hybrid Derivatives as F508del Correctors by QSAR-Based Filtering
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Mammalian Cells in Vivo by the Aminoglycoside Anthiotics G-418
and Paromomycin. Nucleic Acids Res. 1985, 13 (17), 6265−6272.
(8) Manuvakhova, M.; Keeling, K.; Bedwell, D. M. Aminoglycoside
Antibiotics Mediate Context-Dependent Suppression of Termination
Codons in a Mammalian Translation System. RNA 2000, 6 (7),
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(9) Prayle, A.; Smyth, A. R. Aminoglycoside Use in Cystic Fibrosis:
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(6), 604−610.
Ambra Campofelice − Dipartimento di Scienze e Tecnologie
Biologiche, Chimiche e Farmaceutiche (STEBICEF), Universita
degli Studi di Palermo, 90123 Palermo, Italy
Giulia Culletta − Dipartimento di Scienze e Tecnologie
Biologiche, Chimiche e Farmaceutiche (STEBICEF), Universita
degli Studi di Palermo, 90123 Palermo, Italy; Dipartimento di
Scienze Chimiche, Biologiche, Farmaceutiche ed Ambientali,
̀
̀
̀
Universita di Messina, 98168 Messina, Italy
Raffaella Melfi − Dipartimento di Scienze e Tecnologie
Biologiche, Chimiche e Farmaceutiche (STEBICEF), Universita
degli Studi di Palermo, 90123 Palermo, Italy
̀
Andrea Pace − Dipartimento di Scienze e Tecnologie Biologiche,
̀
Chimiche e Farmaceutiche (STEBICEF), Universita degli Studi
di Palermo, 90123 Palermo, Italy
Anna Maria Almerico − Dipartimento di Scienze e Tecnologie
Biologiche, Chimiche e Farmaceutiche (STEBICEF), Universita
degli Studi di Palermo, 90123 Palermo, Italy
Laura Lentini − Dipartimento di Scienze e Tecnologie
Biologiche, Chimiche e Farmaceutiche (STEBICEF), Universita
degli Studi di Palermo, 90123 Palermo, Italy
̀
̀
Complete contact information is available at:
https://pubs.acs.org/10.1021/acsmedchemlett.9b00609
(10) Rowe, S. M.; Mutyam, V.; Alroy, I.; Huertas, P. WS01.3
Translational Read-through of CFTR Nonsense Mutations and
Inducement of Cystic Fibrosis Transmembrane Conductance
Regulator (CFTR) Function by ELX-02 Treatment. J. Cystic Fibrosis
2018, 17, S2.
Author Contributions
The manuscript was written through contributions of all
authors. All authors have given approval to the final version of
the manuscript.
(11) 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 (7140), 87−91.
(12) 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
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Funding
We are grateful for funding of this work from Fondazione
Ricerca Fibrosi Cistica−Onlus (FFC), Italy grant FFC#3/2017
with the contribution of Delegazione di Palermo, Delegazione
di Vittoria (Ragusa) e Siracusa and Delegazione di Catania
Mascalucia and Fondazione Terzo Pilastro International.
Notes
The authors declare no competing financial interest.
(13) 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. Ataluren for the Treatment of Nonsense-
Mutation Cystic Fibrosis: A Randomised, Double-Blind, Placebo-
Controlled Phase 3 Trial. Lancet Respir. Med. 2014, 2 (7), 539−547.
(14) Aslam, A.; Jahnke, N.; Remmington, T.; 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.
ACKNOWLEDGMENTS
■
We thank J. Inglese, NIH Chemical Genomics Center,
National Institute of Health, Bethesda, for providing us with
FLuc190UGA plasmid and Louis Galietta (TIGEM, Napoli,
Italy) and Loretta Ferrera (Gaslini Hospital, Genova, Italy) for
kindly providing us the FRT cells and the pTracer CFTR-WT
vector.
(15) Raymer, B.; Bhattacharya, S. K. Lead-like Drugs: A Perspective.
J. Med. Chem. 2018, 61, 10375−10384.
ABBREVIATIONS
■
(16) 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.
(17) 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.
(18) 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.
(19) 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 (4), 522−527.
(20) Campofelice, A.; Lentini, L.; Di Leonardo, A.; Melfi, R.;
Tutone, M.; Pace, A.; Pibiri, I. Strategies against Nonsense:
TRIDs,translational readthrough inducing drugs; PTC,prema-
ture termination codon; CF,cystic fibrosis; CFTR,cystic
fibrosis transmembrane conductance regulator; FLuc,firefly
luciferase
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