Cerpegin-derived furo[3,4-c]pyridine-3,4(1H,5H)-diones enhance cellular response to interferons by de novo pyrimidine biosynthesis inhibition

Article abstract of DOI:10.1016/j.ejmech.2019.111855

There is an increasing interest in the field of cancer therapy for small compounds targeting pyrimidine biosynthesis, and in particular dihydroorotate dehydrogenase (DHODH), the fourth enzyme of this metabolic pathway. Three available DHODH structures, fe

Full text of DOI:10.1016/j.ejmech.2019.111855

European Journal of Medicinal Chemistry xxx (xxxx) xxx
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Research paper
Cerpegin-derived furo[3,4-c]pyridine-3,4(1H,5H)-diones enhance
cellular response to interferons by de novo pyrimidine biosynthesis
inhibition
Simon Hayek a, Nicolas Pietrancosta a 1, Anna A. Hovhannisyan b,
,
,
Rodolphe Alves de Sousa a, Nassima Bekaddour a, Laura Ermellino a c, Enzo Tramontano c,
d
d
e
f
f
ꢀ
ꢀ
Stephanie Arnould , Claude Sardet , Julien Dairou , Olivier Diaz , Vincent Lotteau ,
Sebastien Nisole , Gagik Melikyan **, Jean-Philippe Herbeuval a,
g
b,
Pierre-Olivier Vidalain a
,
*
a
ꢀ
ꢀ
Chimie et Biologie, Modelisation et Immunologie pour la Therapie (CBMIT), Laboratoire de Chimie et Biochimie Pharmacologiques et Toxicologiques,
ꢀ
Universite Paris Descartes, CNRS UMR8601, Paris, France
b Department of Organic Chemistry, Yerevan State University, Yerevan, Armenia
c Laboratory of Molecular Virology, Department of Life and Environmental Sciences, University of Cagliari, Cagliari, Italy
d
ꢀ
ꢀ
ꢀ
Institut de Recherche en Cancerologie de Montpellier, INSERM U1194, Universite de Montpellier, Institut Regional du Cancer de Montpellier, Montpellier,
France
e
ꢀ
ꢀ
ꢀ
Chimie Bio-inorganique des Derives Soufres et Pharmacochimie (CBDSP), Laboratoire de Chimie et Biochimie Pharmacologiques et Toxicologiques,
ꢀ
Universite Paris Descartes, CNRS UMR8601, Paris, France
f
ꢀ
Centre International de Recherche en Infectiologie, INSERM U1111, CNRS UMR5308, Universite Lyon 1, ENS de Lyon, Lyon, France
g
ꢀ
Institut de Recherche en Infectiologie de Montpellier, CNRS UMR9004, Universite de Montpellier, Montpellier, France
a r t i c l e i n f o
a b s t r a c t
Article history:
There is an increasing interest in the field of cancer therapy for small compounds targeting pyrimidine
biosynthesis, and in particular dihydroorotate dehydrogenase (DHODH), the fourth enzyme of this
metabolic pathway. Three available DHODH structures, featuring three different known inhibitors, were
used as templates to screen in silico an original chemical library from Erevan University. This process led
to the identification of P1788, a compound chemically related to the alkaloid cerpegin, as a new class of
pyrimidine biosynthesis inhibitors. In line with previous reports, we investigated the effect of P1788 on
the cellular innate immune response. Here we show that pyrimidine depletion by P1788 amplifies
cellular response to both type-I and type II interferons, but also induces DNA damage as assessed by
Received 9 September 2019
Received in revised form
2 November 2019
Accepted 4 November 2019
Available online xxx
Keywords:
Interferon
Pyrimidine biosynthesis
Cerpegin
DHODH
g
H2AX staining. Moreover, the addition of inhibitors of the DNA damage response led to the suppression
of the P1788 stimulatory effects on the interferon pathway. This demonstrates that components of the
DNA damage response are bridging the inhibition of pyrimidine biosynthesis by P1788 to the interferon
signaling pathway. Altogether, these results provide new insights on the mode of action of novel py-
rimidine biosynthesis inhibitors and their development for cancer therapies.
DNA damage response
© 2019 Elsevier Masson SAS. All rights reserved.
1. Introduction
* Corresponding author. Current Address: Centre International de Recherche en
DNA and RNA syntheses require large amounts of purine and
pyrimidine nucleosides as precursors. This makes quickly dividing
cells highly dependent on de novo nucleoside biosynthesis, whereas
quiescent and slowly growing cells essentially rely on intracellular
recycling of nucleosides or their uptake from extracellular fluids.
Consequently, cancer cells are particularly sensitive to nucleoside
biosynthesis inhibitors such as 5-fluorouracil, methotrexate, or 6-
ꢀ
Infectiologie (CIRI), INSERM U1111, CNRS UMR5308, Universite Lyon 1, ENS de Lyon,
21 Avenue Tony Garnier, 69365, Lyon, Cedex 07, France.
** Corresponding author.
E-mail addresses: gagik.melikyan@yahoo.com (G. Melikyan), pierre-olivier.
vidalain@inserm.fr (P.-O. Vidalain).
1
ꢀ
Current Address: Sorbonne Universite, INSERM, CNRS, Neurosciences Paris
Seine - Institut de Biologie Paris Seine (NPS - IBPS), 75005 Paris, France. Sorbonne
ꢀ
ꢀ
Universite, CNRS, Laboratoire des Biomolecules (UMR7203), 75005 Paris, France.
https://doi.org/10.1016/j.ejmech.2019.111855
0223-5234/© 2019 Elsevier Masson SAS. All rights reserved.
Please cite this article as: S. Hayek et al., Cerpegin-derived furo[3,4-c]pyridine-3,4(1H,5H)-diones enhance cellular response to interferons by de
novo pyrimidine biosynthesis inhibition, European Journal of Medicinal Chemistry, https://doi.org/10.1016/j.ejmech.2019.111855

2
S. Hayek et al. / European Journal of Medicinal Chemistry xxx (xxxx) xxx
mercaptopurine, which are extensively used in clinical practice.
Among antimetabolites that are currently investigated for their
antitumoral properties, molecules targeting dihydroorotate dehy-
drogenase (DHODH), the fourth enzyme of de novo pyrimidine
biosynthesis, are the subject of intense research [1e3]. In this
metabolic pathway, three enzymatic steps are required to convert
glutamine, aspartate, and bicarbonate into dihydroorotate (DHO),
which is the substrate for DHODH [3]. Its oxidation by DHODH leads
to orotate, which is converted into uridine monophosphate (UMP)
that serves as a precursor for cytidine and thymidine biosynthesis.
This makes DHODH a rate-limiting enzyme in the biosynthesis of
pyrimidine nucleosides, and a potential target for antimetabolite
development.
canonical and IFN-independent pathway, and (ii) enhance the
expression of IFNs and ISGs when induced by viruses or viral PAMPs
such as double-stranded RNA molecules [22,31e41]. Data from the
literature suggest that DHODH inhibitors amplify the cellular
response to type I IFNs, but this was only shown using a reporter
gene containing Interferon-Stimulated Response Elements (ISRE)
driving transcription of luciferase and never explored in detail
[32,34]. The mechanism by which the inhibition of pyrimidine
biosynthesis promotes the expression of IFNs and ISGs remains
poorly understood and thus needs to be further documented
[22,32,36,40,41]. The positive interaction between the inhibition of
pyrimidine biosynthesis and the innate immune response likely
contributes to the antiviral activity of DHODH inhibitors and it is
tempting to speculate that it could also participate to the antitumor
effect of these molecules as well.
DHODH, anchored by its N-terminal extremity to the internal
mitochondrial membrane, uses flavin mononucleotide (FMN) and
ubiquinone (or coenzyme Q10) as cofactors in a two-step redox
reaction. Ubiquinone is usually located in the mitochondrial
membrane. Close contact between DHODH and the membrane al-
lows ubiquinone to shuttle into a hydrophobic channel of this
enzyme in order to contact FMN as recently modelled [42]. A ma-
jority of DHODH inhibitors that have been described in the litera-
ture compete with ubiquinone for binding to this channel, and
interact with a limited number of well-characterized residues
[1e3]. Here, we took advantage of three high-definition structures
available in the Protein Data Bank (PDB) for human DHODH bound
to unrelated inhibitors to develop and benchmark a pipeline for in
silico screening (4OQV, 3KVJ, 4RR4). This led to the identification of
P1788, a compound featuring structural components of the plant
alkaloid cerpegin [43,44], as a new class of pyrimidine biosynthesis
inhibitors. We then used this compound to establish that cellular
response to both type-I and II IFNs is amplified in cell cultures
where pyrimidine biosynthesis is blocked. We provide evidence
that pyrimidine biosynthesis inhibition by P1788 activates the DNA
damage response to amplify the interferon response. Altogether,
our results reinforce the link between this metabolic pathway and
the innate immune response and its therapeutic potential in
various acute and chronic diseases.
In the 90s, brequinar, a potent DHODH inhibitor passed pre-
clinical studies, and was evaluated for its antitumor properties in
multiple phase-I and phase-II trials [1e3]. However, severe adverse
effects and a lack of efficacy in patients with different types of solid
tumors stopped its development. This setback also led to the
disregard of DHODH inhibition as a valuable strategy for anticancer
therapies. Concomitantly, teriflunomide, another DHODH inhibitor,
and its related prodrug (leflunomide), were evaluated successfully
and approved for the treatment of rheumatoid arthritis and later on
for multiple sclerosis despite significant side effects [1e3]. This
drug was shown to impair the proliferation of immune cells by
inhibiting pyrimidine biosynthesis. It also interferes through tyro-
sine kinase inhibition and Aryl Hydrocarbon Receptor (AhR) acti-
vation, which altogether contribute to its immunosuppressive
properties [4]. Despite the failure of brequinar in past clinical
studies, recent publications renewed interest in DHODH as a target
in cancer therapy and both old and recent series of DHODH in-
hibitors were shown to have promising antitumor properties. This
was demonstrated in various in vivo models when administered
alone or in combination with other drugs, in particular in acute
myeloid leukemia (AML), neuroblastoma, PTEN (Phosphatase and
TENsin homolog) mutant triple negative breast cancer cells, and
KRAS (V-Ki-ras2 Kirsten rat sarcoma viral oncogene homolog)
mutant pancreatic cancer cells [5e14]. Several DHODH inhibitors,
including brequinar, leflunomide and new molecules such as
BAY2402234 or PTC299, are currently being evaluated in clinical
trials for cancer therapy [15,16]. Some pyrimidine biosynthesis in-
hibitors also exhibit broad-spectrum antiviral activity in vitro
[1e3,17e19] and in vivo [20e22], opening new fields of application
for this class of antimetabolites. In this context, the identification of
novel DHODH inhibitors with original chemical and pharmaco-
logical properties has become a priority to overcome the limita-
tions of existing molecules [23e27].
2. Materials and methods
2.1. Chemical syntheses
All solvents and chemicals were purchased from the Armenian
Institute of Applied Chemistry (ARIAC). Dimethylformamide (DMF),
dimethylacetal (DMA) and xylene were dried using standard pro-
cedure. 1H NMR and 13C NMR spectra were recorded in deuterated
dimethyl sulfoxide (DMSO‑d6) at ambient temperature using a
500 MHz nuclear magnetic resonance (NMR) spectrometer equip-
ped with a 5 mm cryogenically cooled probe 1H/13C/2D optimized
for carbon-13 detection with z-gradients. The chemical shifts were
reported in ppm downfield from tetramethylsilane (TMS). For
compounds P2702, P2703, P2705 and P2706, 1H NMR and 13C NMR
spectra were recorded with a Varian Mercury 300 spectrometer
operating at 300 MHz and 75 MHz with TMS as internal standard in
DMSO‑d6/CCl4 solution at 303 K. Electrospray ionization (ESI) and
high resolution mass spectrometry (HRMS) analyses were con-
ducted using a Thermo Scientific High Resolution Exactive Orbitrap
Mass spectrometer. The measurements were performed on the
NMR platform and Mass spectrometry platform of UMR8601 CNRS
laboratory. Elemental analyses were carried out by the CNRS
microanalysis service at Gif-sur-Yvette. Melting points were
determined on a Kofler hot-stage microscope and are uncorrected.
Beyond its cytostatic effects, DHODH inhibition is known to have
a strong impact on gene expression, cell metabolism, and differ-
entiation [5,12,14,15,28]. Our team is interested in the existing re-
lationships between pyrimidine biosynthesis and the activation of
innate immunity, with a specific focus on the interferon (IFN)
response. IFN-a/b (or type I IFNs) are key antiviral and antitumoral
cytokines that are produced by both immune and non-immune
cells. They are released upon stimulation by viral PAMPs (Path-
ogen-Associated Molecular Patterns) such as double-stranded RNA
molecules, or cellular DAMPs (Damage-Associated Molecular Pat-
terns) including DNA breaks. Once bound to their target receptors,
IFN-a/b mobilize the JAK (Janus Kinase) and STAT (Signal Trans-
ducers and Activators of Transcription) signaling cascade to induce
a large panel of target genes, the ISGs (Interferon-Stimulated
Genes), which encode numerous antiviral and antitumoral factors.
IFNs and ISGs are therefore essential for the innate immune
response against viruses and for the control of carcinogenesis
[29,30]. Most interestingly, it is now well documented that py-
rimidine biosynthesis inhibitors (i) induce some ISGs by a non-
Please cite this article as: S. Hayek et al., Cerpegin-derived furo[3,4-c]pyridine-3,4(1H,5H)-diones enhance cellular response to interferons by de
novo pyrimidine biosynthesis inhibition, European Journal of Medicinal Chemistry, https://doi.org/10.1016/j.ejmech.2019.111855

S. Hayek et al. / European Journal of Medicinal Chemistry xxx (xxxx) xxx
3
2.1.1. Procedure for the synthesis of cerpegin N-Substituted
derivatives
ligand structure by high-temperature molecular dynamics using
the BEST algorithm. The different conformations of ligands were
placed into the active site and HotSpots were matched as triplets.
After a manual optimization and curation step, the ligand poses
were scored. Because ligand hydrogens were removed during the
docking process, they were added back to the ligand poses and
optimized by minimization. The ligand poses with the highest
LibDock scores were retained and clustered according to their
binding mode.
The general procedure used for the synthesis of substituted
cerpegin analogs was described in our previous reports [43,45].
Initial (E)-ethyl 4-(2-(dimethylamino)vinyl)-5,5-disubstituted-2-
oxo-2,5-dihydrofuran-3-carboxylates (II) were synthesized ac-
cording to previously described methods [43e46]. Briefly,
condensation of 2-oxo-2,5-dihydrofurans (I; 10 mmol) and DMF/
DMA (1.44 ml; 12 mmol) was achieved in 3 h in boiling anhydrous
xylene (10 ml) to give the corresponding compounds (II) in yields of
up to 80e90% (Scheme 1). In a flask fitted with a reflux, compound
(II) (10 mmol) and the corresponding amine (40 mmol) were mixed
in anhydrous xylene (7 mL). In the case of amines with low boiling
points (bp < 100 ꢀC) the reaction was undertaken without xylene
using an excess of amine. The mixture was boiled for 15 h (extra 2 h
after cessation of dimethylamine evolution), cooled to room tem-
perature, and 5 mL of light petroleum was added. The resulting
precipitates were filtered, washed with ether and dried. Yields and
physical data of the resulting compounds are provided in Supple-
mentary Materials and Methods.
2.1.4. Cells and culture conditions
Cells were cultured at 37 ꢀC and 5% CO2 in Dulbecco’s Modified
Eagle’s Medium (DMEM; Sigma-Aldrich; D6429) containing 10%
fetal calf serum (FCS; Sigma-Aldrich; F0804), penicillin and strep-
tomycin. Experiments were performed on HEK-293 cells stably
transfected with the ISRE-Luciferase reporter gene (ISRE-Luc), with
or without NanoLuc as an additional reporter gene [32,48]. ISRE-
Luc and ISG expression levels were unaffected by the presence of
this additional reporter gene, and data from cells with or without
NanoLuc were treated indifferently. Human peripheral blood
mononuclear cells (PBMC) from one healthy donor were isolated by
density centrifugation with Lymphoprep medium (StemCell Tech-
nologies) from leucocytes concentrates obtained when platelet-
pheresis was performed (Etablissement Français du Sang; Paris;
France). To activate PBMCs, cells were treated for 24 h with the
2.1.2. Synthesis of N-cyclopropyl-4-methyl-2-oxo-1-oxaspiro[4.5]
dec-3-ene-3-carboxamide (P2708)
A mixture of ethyl 4-methyl-2-oxo-1-oxaspiro[4.5]dec-3-ene-3-
carboxylate (2.38 g; 10 mmol) and cyclopropylamine (0.9 mL;
13 mmol) in ethanol (1 mL) was left at room temperature 20 h. The
resulting precipitate was filtered, washed with cold ether and
recrystallized from ethanol. Yields and physical data of the result-
ing compound are provided in Supplementary Materials and
Methods.
TLR7/8 ligand R848 at 5 mg/ml (Sigma-Aldrich) and supernatants
were harvested. Cytokine expression levels were assessed using the
LEGENDplex Human Anti-Virus Response Panel (BioLegend). Re-
sults are presented in Supplementary Table I.
2.1.5. Reagents, cytokines and small compounds
2.1.3. In silico screening
Firefly luciferase expression in culture wells was measured us-
ing the Bright-Glo (Promega) or Britelite plus reagents (Perki-
nElmer), according to the manufacturer’s recommendations.
Cellular viability was determined by quantification of adenosine
triphosphate (ATP) in culture wells using the CellTiter-Glo assay
(Promega). Bioluminescence was measured for 0.1 s with a lumin-
The in silico docking was performed with Discovery Studio
(Discovery Studio Modeling Environment, release 4.5; Dassault
Systemes BIOVIA: San Diego, 2015) as an interface to LibDock, a
docking algorithm developed by Diller and Merz [47]. LibDock uses
protein site features called HotSpots which are tagged as polar or
apolar. Prior to the docking procedure, the receptor HotSpot file
was calculated. In parallel, ligands were prepared (i.e. tautomer
generation, protonation depending of pKa, or stereoisomer gener-
ation), then random ligand conformations were generated for each
ometer (EnSpire; PerkinElmer). Recombinant IFN-
Sigma-Aldrich or PBL Assay Science. Recombinant IFN-
a
was from
was from
g
Roussel Uclaf (a kind gift of Dr. Mounira Chelbi-Alix) or Miltenyi
Biotech. Uridine, orotate, dihydroorotate, PF477736 (Checkpoint
Scheme 1. Synthesis of cerpegin derivatives.
Please cite this article as: S. Hayek et al., Cerpegin-derived furo[3,4-c]pyridine-3,4(1H,5H)-diones enhance cellular response to interferons by de
novo pyrimidine biosynthesis inhibition, European Journal of Medicinal Chemistry, https://doi.org/10.1016/j.ejmech.2019.111855

4
S. Hayek et al. / European Journal of Medicinal Chemistry xxx (xxxx) xxx
Kinase 1 inhibitor), mirin (MRE11 inhibitor), C646 (P300/CREB-
Binding Protein inhibitor), teriflunomide, brequinar, and mitoxan-
trone were all from Sigma-Aldrich. Vidofludimus was from Sell-
eckchem. AZD6738 (Ataxia telangiectasia and Rad3-related kinase
inhibitor) was from Euromedex.
resolution structures of DHODH binding chemically-unrelated in-
hibitors: 4OQV, 3KVJ and 4RR4 [49,50]. These structures were not
selected on the basis of the activity of the compounds, but because
the compounds display different binding modes to human DHODH.
Indeed, all three compounds are interacting with Arg136 and FMN
in the hydrophobic funnel but the rest of the interactions with
DHODH are highly diverse (Fig. 1B). This provided three slightly
different DHODH structures to undertake an in silico screening. To
benchmark our screening protocol, we showed that algorithm-
based docking of each inhibitor perfectly matched the corre-
sponding X-ray derived DHODH structure as assessed by a low
Root-Mean-Square Deviation (RMSD) comparison (Fig. 1B).
The virtual high-throughput screening (vHTS) was performed
on a set of 1,587 compounds from Yerevan State University, a
chemical library that encompasses a large diversity of original
structures. To ensure a time/precision ratio compatible with vHTS,
we used a protocol in which amino acid side chains of the protein
are left flexible only around the binding site, and we tested 10
random conformers for each ligand. The resulting ligand poses
were further filtered to select compounds expected to bind Arg136
and FMN, and then ranked according to their docking scores as
described in the Material and Methods section. The entire library
was screened against the three selected DHODH structures. Finally,
a total of 26, 49, and 55 compounds respectively fulfilled our
criteria when using the 4OQV, 3KVJ and 4RR4 structures (Fig. 1C).
To further increase the stringency of our screening, we selected the
11 compounds identified with at least two of the three above-
mentioned structures. Interestingly, this subset included two
molecules, P2703 and P1788 (Fig. 1D), which featured a 1,1,5-
trimethylfuro[3,4-c]pyridine-3,4(1H,5H)-dione component similar
to one seen in the alkaloid cerpegin. To our knowledge, such
double-ringed structure with two adjacent keto groups contacting
Arg136 was new for DHODH inhibitors and thus deserved further
investigations.
2.1.6. Metabolite analysis
Nucleoside/nucleotide quantification in HEK-293 cells was per-
formed by high-performance liquid chromatography (HPLC)-
coupled spectrophotometry as previously described in Ref. [38].
Experimental details are provided in the “Supplementary Materials
and Methods”.
2.1.7. Gene expression analysis by RT-qPCR
ISG expression levels were determined by Reverse Transcription
and quantitative Polymerase Chain Reaction (RT-qPCR) as follows.
Total RNAs were extracted from 2 ꢁ 105 cells using the RNeasy
Micro kit including DNase (Qiagen). Reverse transcription of
cellular RNA into cDNA was achieved using the RevertAid H Minus
first-strand cDNA synthesis kit (Thermo Scientific). Real-time PCRs
were performed in duplicate using the Takyon ROX SYBR Master-
Mix dTTP blue kit (Eurogentec) and a 7900HT Fast RT-PCR system
(Applied Biosystems). Transcripts were quantified using the
following program: 3 min at 95 ꢀC, followed by 35 cycles of 15 s at
95 ꢀC, 25 s at 60 ꢀC, and 25 s at 72 ꢀC. For each transcript, CT values
were normalized both to the expression levels of RPL13A (60S ri-
bosomal protein L13a) and unstimulated control samples using the
DDCT
2-
method. The primers used for the quantification of tran-
scripts are presented in Supplementary Table II.
2.1.8. H2AX immunostaining
g
Cells were harvested after 24 h of culture, washed with PBS,
trypsinized and fixed for 30 min in paraformaldehyde (PFA 4%).
Cells were washed and permeabilized with Perm Buffer III (BD
Biosciences) for 5 min. After one washing step, cells were stained
with a mouse monoclonal anti-
gH2AX antibody (1/400 dilution,
3.2. Functional validation of selected hits
3F2 Clone, ThermoFisher) or a matching isotypic control in
phosphate-buffered saline (PBS) þ 2% bovine serum albumin (BSA).
After 45 min at 4 ꢀC, cells were washed again and incubated with a
secondary Alexa Fluor 488-conjugated goat anti-mouse antibody
(1/500, A21121, ThermoFisher). Cells were washed after 45 min at
Since our main interest in DHODH inhibitors is their capacity to
boost the cellular innate immune response, we used a reporter cell
line expressing luciferase under the control of five interferon-
stimulated response elements (ISRE-Luc) to assess the biological
activity of the selected compounds. We and other groups have
previously shown that in such a reporter system, cellular response
4
ꢀC of incubation, and analyzed by flow cytometry using a FACS-
Canto II (BD Biosciences).
to IFN-
incubated with increasing concentrations of selected compounds in
the presence of recombinant IFN- , and luciferase activity was
a/b is amplified by DHODH inhibitors [32,34]. Cells were
3. Results
a
3.1. In silico identification of DHODH inhibitors
quantified 24 h later. Of the 11 compounds tested, 9 were inactive
but the two related compounds P2703 and P1788 efficiently
Ubiquinone is
a
rather large cofactor made of
a
1,4-
increased cellular response to IFN-a (Fig. 2A). To further confirm the
benzoquinone and
a
tail of 10 isoprenyl chemical subunits
inhibition properties of this chemical series, we tested the capacity
of P1788 to block cellular proliferation, a phenotype that is also
associated with DHODH inhibition. P1788 efficiently inhibited the
proliferation of HEK-293 cells when monitored over three days
(Fig. 2B).
(Fig. 1A). The structure of human DHODH bound to ubiquinone, or
its reduced form ubiquinol, has not been resolved yet. However,
more than 50 co-structures of human DHODH featuring chemical
inhibitors are available in the PDB. The vast majority of these in-
hibitors were found to bind a hydrophobic funnel localized at the
contact interface of DHODH with the mitochondrial membrane.
This lipophilic pocket was considered as the binding site for ubi-
quinone allowing a direct contact with FMN at its extremity as
recently modelized by Costeira-Paulo J. et al. [42]. As shown in
Fig. 1A, our molecular docking protocol confirmed that ubiquinone
perfectly fits within this binding pocket. We also validated that
Arg136, which is essential for DHODH activity and mutated in pa-
tients with Miller syndrome, interacts with one of the keto groups
of ubiquinone as reported in Ref. [42]. To perform the molecular
docking-based screening, we selected in PDB three available high-
3.3. Structure activity relationships
To determine some structure activity relationship for this
chemical series, and confront them with the docking model
determined for P2703 and P1788 (Fig. 1D), we prepared the array of
chemical analogs of cerpegin depicted in Fig. 3. Each compound
was tested for its ability to boost cellular response to IFN-
ISRE-Luc reporter cells as shown in Fig. 2A. A dose-response
ranging from 0.8 to 150 M was obtained for each molecule, and
the corresponding pEC50s were calculated to compare their
a using
m
Please cite this article as: S. Hayek et al., Cerpegin-derived furo[3,4-c]pyridine-3,4(1H,5H)-diones enhance cellular response to interferons by de
novo pyrimidine biosynthesis inhibition, European Journal of Medicinal Chemistry, https://doi.org/10.1016/j.ejmech.2019.111855

S. Hayek et al. / European Journal of Medicinal Chemistry xxx (xxxx) xxx
5
Fig. 1. In silico screening procedure for the identification of novel DHODH inhibitors. (A) DHODH structure was retrieved from PDB (3KVJ), showing orotate (red), FMN (green),
and Arg136 (blue). The DHODH inhibitor present in the ubiquinone-binding site of the structure was removed and replaced by a ubiquinone analog without the hydrophobic tail to
facilitate the in silico docking (yellow). Ubiquinone structure is shown in the upper left panel as a reference. (B) Schematic 2-D diagrams of small molecule inhibitors as bound to
DHODH in PDB structures 4OQV, 3KVJ and 4RR4. For each inhibitor, the RMSD value estimates the position overlap between structural data and results of the in silico docking. (C)
Venn diagram showing the results of the screenings performed on DHODH structures from 4OQV, 3KVJ, and 4RR4. We selected the 11 hit compounds identified in more than one
screen for further evaluation. (DeE) Schematic 2-D diagrams of P2703 and P1788 bound to DHODH as determined by in silico docking. (For interpretation of the references to colour
in this figure legend, the reader is referred to the Web version of this article.)
biological effect (Fig. 3). Norcerpegin (1,1-dimethylfuro[3,4-c]pyri-
dine-3,4(1H,5H)-dione), the original scaffold of this chemical series,
was inactive. The difference of effect seen between P1788 and
compounds P2512, P1781 or P2705 demonstrated that a cyclo-
propyl substituent on the nitrogen was essential. Furthermore,
compounds with the larger N-cyclobutyl (P2707), N-cyclopentyl
(P1792), or N-cyclohexyl (P1793) groups were also inactive. Simi-
larly, P2706 exhibiting an extended cyclopropylmethyl group
showed no activity at tested concentrations. Altogether, these re-
sults support our docking model in which this cyclopropyl group
fully occupies the DHODH hydrophobic pocket made of FMN,
Val134 and Val143 (Fig. 1D). We also found that a double hydro-
phobic substitution on position 1 of the furopyridine scaffold was
essential as seen for compounds featuring a spirocyclopentane
(P2717), a spirocyclohexane (P1788) or a phenyl and a methyl group
(P2703). This is consistent with our docking model where such
substituents interact with Ala59 or Ala55 at the entry side of the
hydrophobic pocket (Fig.1D). Finally, opening the pyridine group as
in P2708 also impaired the biological activity of the molecule. This
last result is in line with a structuring role for the furopyridine
Please cite this article as: S. Hayek et al., Cerpegin-derived furo[3,4-c]pyridine-3,4(1H,5H)-diones enhance cellular response to interferons by de
novo pyrimidine biosynthesis inhibition, European Journal of Medicinal Chemistry, https://doi.org/10.1016/j.ejmech.2019.111855

6
S. Hayek et al. / European Journal of Medicinal Chemistry xxx (xxxx) xxx
P1788 in the absence or presence of uridine. The addition of uridine
completely reverted the effect of P1788, thus demonstrating that
pyrimidine depletion is indeed responsible for the amplified
response to IFN-a as shown in Fig. 4B. In order to determine more
precisely which step of pyrimidine biosynthesis is inhibited by
P1788, the same experiment was performed using a culture me-
dium supplemented with either DHO or orotate (Fig. 4C). While
DHO showed no effect on cellular response to IFN-a, orotate
completely reverted the effect of P1788. As DHO and orotate are
respectively the substrate and product of DHODH, these results
confirmed that P1788 is inhibiting this critical step of the pyrimi-
dine biosynthesis pathway.
3.5. P1788 amplifies cellular response to both type I and type II IFNs
As stated above, it has previously been shown that DHODH in-
hibition amplifies cellular response to IFN-a/b [32,34]. However,
this was only documented using an ISRE-Luc reported gene and
never validated by the actual monitoring of cellular ISGs. We took
advantage of P1788 as a novel inhibitor to address this question.
Cells were treated with increasing concentrations of IFN-
absence or presence of P1788, and expression levels of 12 ISGs were
determined by RT-qPCR. ISGs were induced by IFN- alone, but
a in the
a
P1788 further enhanced their expression (Fig. 5A). For example, a 4
to 5-fold increase in Mx2 expression level was observed when cells
stimulated with IFN-
gether, this confirmed that P1788 can boost the cellular response to
IFN-
To our knowledge, it is still unknown if pyrimidine biosynthesis
inhibitors are also able to amplify the cellular response to IFN-
a were co-treated with P1788 (Fig. 5B). Alto-
Fig. 2. Cellular response to IFN-a is amplified by P1788 and P2703. (A) HEK-293 cells
a
.
with the ISRE-Luc reporter gene were treated with P1788, P2703 or DMSO alone in the
absence or presence of IFN- (150 IU/ml). Expression of the ISRE-Luc reporter gene was
determined 24 h later, and results were normalized using cells treated with IFN-
a
g.
a
alone as reference. (B) Cells were treated with DMSO or P1788 at different concen-
trations, and the number of viable cells was determined using the CellTiter-Glo reagent
after 24, 48 and 72 h of culture. Results were expressed as a percentage relative to
untreated cells at t ¼ 0 h. Data represent means SD of three (A) or two (B) indepen-
dent experiments. **p < 0.01; ***p < 0.001 as calculated by one-way analysis of vari-
ance (ANOVA) with Bonferroni’s post hoc test.
This is particularly interesting because this cytokine, which is
massively produced by Natural Killer (NK) cells and CD8þ T lym-
phocytes, plays a key role in antitumoral immune responses and
adaptive immunity in general. We thus investigated the effects of
P1788 on this pathway. Cells with the ISRE-Luc reporter gene were
stimulated with recombinant IFN-g in the absence or presence of
P1788. As shown in Fig. 6A, the reporter gene was induced by IFN-
g
,
scaffold. Altogether, these results validate our docking model for
P2703 and P1788 in the ubiquinone binding pocket of DHODH.
To compare the activity of P2703 and P1788 with well-
characterized inhibitors of DHODH, teriflunomide, vidofludimus
and brequinar were evaluated in the same cellular assay (Fig. 3).
Teriflunomide and vidofludimus were significantly more active
and P1788 further increased its expression. To further validate this
observation, we collected supernatants of human PBMCs stimu-
lated with R848, a synthetic ligand for TLR7. Such conditioned su-
pernatants contain IFN-g as shown in Supplementary Table I and
were used to stimulate cells with the ISRE-Luc reporter gene. P1788
strongly increased the cellular response to conditioned superna-
tants from PBMCs (Fig. 6B). Our results demonstrate that P1788 is
able to amplify cellular response to both type I and type II
interferons.
than P2703 and P1788 (EC50 ¼ 8.9 and 2.8
mM vs 27 and 44 mM,
respectively), but all four molecules showed EC50s within the
micromolar range. Only brequinar was much more active with an
EC50 equal to 56 nM. Because P2703 and P1788 were within a range
of activity close to fully optimized molecules like teriflunomide or
vidofludimus, we decided to further investigate the properties of
these molecules. Although P2703 was slightly more active, P1788
was selected as the prototype of this chemical series because a
larger batch had been produced and was readily available.
3.6. Key factors of the DNA damage response link P1788 to the IFN
signaling pathway
Pyrimidine depletion by DHODH inhibitors has been shown to
block DNA replication, leading to S phase accumulation and the
activation of DNA damage response [9] (Fig. 7A). It is also well
known that DNA damage are able to prime or induce the IFN
response to prevent infections or tumor formation, depending on
the cellular system [29,51]. The DNA damage response could
therefore functionally link the inhibition of DHODH to the innate
immunity as recently documented by Luthra P. et al. [40]. To
address this question in our cellular system, we first determined if
P1788 treatment induced the DNA damage response by measuring
3.4. P1788 targets the pyrimidine biosynthesis pathway
To demonstrate that P1788 is indeed targeting the pyrimidine
metabolism, we treated cells with this molecule and quantified
nucleoside levels by liquid chromatography coupled with spectro-
photometry. As shown in Fig. 4A, pyrimidine levels collapsed in
P1788-treated cells (U and C), whereas purine levels were unaf-
fected (G and A). This shows that P1788 interferes with pyrimidine
homeostasis as expected. We then determined if the amplification
g
H2AX, the phosphorylated form of H2AX (H2A histone family
member X) on serine 139. As shown in Fig. 7B, P1788 induced a
significant increase in H2AX staining as determined by flow
cytometry. Similar H2AX levels were achieved with the reference
DHODH inhibitor teriflunomide but the signal was much higher
of IFN-
depletion in treated cells. To address this question, cells with the
ISRE-Luc reporter gene were treated with recombinant IFN- and
a/
b
signaling by P1788 is a consequence of pyrimidine
g
g
a
Please cite this article as: S. Hayek et al., Cerpegin-derived furo[3,4-c]pyridine-3,4(1H,5H)-diones enhance cellular response to interferons by de
novo pyrimidine biosynthesis inhibition, European Journal of Medicinal Chemistry, https://doi.org/10.1016/j.ejmech.2019.111855

S. Hayek et al. / European Journal of Medicinal Chemistry xxx (xxxx) xxx
7
Fig. 3. Structure/activity relationships. Chemical analogs of P1788 and P2703 were tested for their ability to boost cellular response to IFN-
a
in HEK-293 cells with the ISRE-Luc
reporter gene. Teriflunomide, vidofludimus and brequinar were tested as reference DHODH inhibitors in the same cellular assay. pEC50s, corresponding to -log10 of the half maximal
effective molar concentrations, were computed from a four-parameter fitting curve using Prism software (GraphPad). Binding energies were also computed by in silico docking for
comparison with pEC50s. FTD (“failed to determine”) is indicated when binding energies could not be computed. The chemical structure of cerpegin is shown as a reference.
with mitoxantrone, a potent topoisomerase II inhibitor that induces
massive DNA damage. Furthermore, P1788 effect was neutralized
by the addition of uridine, thus implicating pyrimidine depletion in
this effect. The inhibition of de novo pyrimidine biosynthesis by
P1788 is therefore associated to the induction of a mild DNA
damage response which probably reflects the accumulation of
stalled DNA forks in S phase.
The exonuclease activity of the double-strand break repair
protein MRE11, which is part of the MRE11/RAD50/NBS1 (MRN)
complex, plays an important role in the stabilization, resection, and
restart of stalled DNA forks (Fig. 7A). MRE11 also exhibits some
endonuclease activity involved in the cleavage and release of
single-stranded DNA from the resection site which contributes to
IFN activation [52]. We tested the effect of mirin, an inhibitor of the
Please cite this article as: S. Hayek et al., Cerpegin-derived furo[3,4-c]pyridine-3,4(1H,5H)-diones enhance cellular response to interferons by de
novo pyrimidine biosynthesis inhibition, European Journal of Medicinal Chemistry, https://doi.org/10.1016/j.ejmech.2019.111855

8
S. Hayek et al. / European Journal of Medicinal Chemistry xxx (xxxx) xxx
DNA breaks and then phosphorylate CHK1 (Checkpoint Kinase 1)
to block cell cycle progression upon DNA damage [53]. We tested
potent inhibitors of either ATR (AZD6738) or CHK1 (PF477736) in
our system. ATR or CHK1 inhibitors abolished the effects of P1788
on the IFN-a response, further supporting a role of the DNA damage
response as shown in Fig. 7D and E. Interestingly, ATR or CHK1
inhibition also showed significant effects on the cellular response
to IFN-a in the absence of P1788. This suggests that a steady-state
activation of ATR and CHK1 in these cells somehow contributes to
the IFN response. As stated, these treatments showed limited
(AZD6738) or no effect (PF477736) on cellular viability (Supp.
Fig. 1B). Finally, we evaluated the contribution of P300 and CBP
(CREB-Binding protein), two related histone acetyltransferases that
are activated downstream of the DNA damage response but also
regulate the expression of ISGs. We thus treated cells with C636, a
small compound inhibiting both P300 and CBP, and induced the
ISRE-Luc reporter gene with IFN-
shown in Fig. 7F, the inhibition of P300/CBP slightly decreased
cellular response to IFN- , but most importantly abolished the ef-
a with or without P1788. As
a
fect of P1788. These results confirm the functional link between the
DNA damage response and the IFN-potentiating effect of P1788.
4. Discussion
De novo pyrimidine biosynthesis is essential to fulfill the
metabolic needs of quickly proliferating cells. As such, there is an
increased interest in the field of cancer for molecules blocking
critical steps of this enzymatic pathway. Here, we detailed the
identification of P1788 and related molecules as a new series of
pyrimidine biosynthesis inhibitors. These compounds were
selected by virtual screening using three structures of DHODH co-
crystallized with chemically distinct inhibitors bound to the ubi-
quinone/ubiquinol pocket. This approach was previously used by
other teams and our results confirm its efficacy [54e57]. We then
established the inhibition of pyrimidine biosynthesis pathway in
P1788-treated cells by measuring nucleoside levels and using
cellular response to IFN-a as a functional readout. The fact that
orotate but not DHO nullified the effects of P1788 in this test sup-
ports the inhibition of DHODH by this molecule. It is of interest that
P1788 and related molecules of this chemical series feature the
unique chemical scaffold found in the alkaloid cerpegin (Fig. 3)
[43,44]. Cerpegin is found in the Indian plant Ceropegia juncea,
which has been used for centuries in folk medicine as a tranquilizer,
anti-inflammatory, analgesic, and anti-ulcer therapy. Modern
studies showed that compounds with the cerpegin scaffold inhibit
the 20S subunit of the proteasome [43,45]. The results described
here show that compounds also based on the cerpegin scaffold but
with different additional chemical groups inhibit pyrimidine
biosynthesis. The two-edged fused polycyclic moieties of this
chemical series are actually reminiscent of known DHODH in-
hibitors, including brequinar, dicoumarol, and DD778 that we
recently described [38]. Brequinar interacts with Arg136 of DHODH
through its carboxylic acid group whereas in dicoumarol, the
lactone oxygen atom was suggested to play this role [58]. According
to our in silico model, the interaction of P1788 with Arg136 is
mediated by the diketone motif. To our knowledge, this mode of
interaction was not previously reporter for DHODH inhibitors and
clearly distinguishes the P1788 series from known DHODH
inhibitors.
Fig. 4. P1788 is inhibiting pyrimidine biosynthesis. (A) Cells were treated with
DMSO or increasing concentrations of P1788. 24 h later, cells were harvested and
intracellular levels of purine (G/A) and pyrimidine (C/U) were determined by HPLC-
coupled spectrophotometry. Results were normalized using DMSO-treated cells as a
reference. (B) Cells were treated with increasing concentrations of IFN-
P1788 (80 M) or P1788 (80 M) and uridine (125 M). 24 h later, expression of the
ISRE-Luc reporter gene was determined. Results were normalized using cells treated
at 250 IU/ml as a reference. (C) Same experiment as in (A), but
a with DMSO,
m
m
m
with DMSO þ IFN-
a
culture medium was supplemented with 3 mM of orotate or DHO instead of uridine.
Data represent means SD of three independent experiments. ***p < 0.001 as calcu-
lated by two-way ANOVA.
MRE11 nuclease activity, on the cellular response to IFN-a in the
absence or presence of P1788. As shown in Fig. 7C, mirin completely
abolished the booster effect of P1788 on ISRE-Luc induction by IFN-
We took advantage of our lead molecule P1788 to characterize in
details the impact of pyrimidine biosynthesis inhibition on cellular
response to IFNs. It is well known that DHODH inhibition can (i)
induce ISGs independently of IFN production or JAK/STAT signaling
and (ii) amplify the expression of both ISGs and IFN genes in
response to double-stranded RNA molecules, a viral PAMP that
a
. Cellular viability was not affected by this treatment as shown in
Supp. Fig. 1A. The Ataxia telangiectasia and Rad3-related kinase
(ATR) are co-activated with the MRN complex by single-stranded
Please cite this article as: S. Hayek et al., Cerpegin-derived furo[3,4-c]pyridine-3,4(1H,5H)-diones enhance cellular response to interferons by de
novo pyrimidine biosynthesis inhibition, European Journal of Medicinal Chemistry, https://doi.org/10.1016/j.ejmech.2019.111855

S. Hayek et al. / European Journal of Medicinal Chemistry xxx (xxxx) xxx
9
Fig. 5. ISG induction by IFN-
a
is enhanced in the presence of P1788. (A) HEK cells with the ISRE-Luc reporter gene were cultured with increasing concentrations of IFN-
a
and co-
treated with DMSO or P1788 (80
mM). 16 h later, induction levels of indicated ISGs were determined by RT-qPCR, and expressed as fold changes relative to control cells (DMSO
alone). Results highlighted in bold correspond to statistically significant values when comparing untreated to P1788-treated cells from three independent experiments (p < 0.05
determined by two-way ANOVA with Bonferroni’s post hoc test). (B) Corresponding data for Mx2 mRNA expression were displayed as a bar chart showing mean values SD.
*p < 0.05; ***p < 0.001 as calculated by two-way ANOVA with Bonferroni’s post hoc test.
DHODH is inhibited as assessed with an ISRE-Luc reporter gene
[32,34]. Here, we have analyzed a dozen of ISGs by RT-qPCR, and
found that their induction by recombinant IFN-a is, as expected,
enhanced in the presence of P1788. This illustrates that the cellular
stress associated to pyrimidine deprivation is acting at multiple
steps of the innate immune response. Most interestingly, we also
found that P1788 amplifies cellular response to IFN-
g. This was
shown using purified recombinant IFN- or conditioned superna-
g
tants from activated PBMCs. IFN-
significantly differs from IFN-
g
activates a signaling cascade that
as it binds a specific receptor
a
/
b
(IFNGR1/2) and mobilizes Jak1/Jak2 kinases d as opposed to Jak1/
Tyk2 for IFN- d to phosphorylate STAT1. STAT2 is not involved
in the signaling cascade induced by IFN- . Our results and the
literature suggest that a common mechanism triggered by pyrim-
idine deprivation amplifies cellular response to both IFN- , IFN-g,
a/b
g
a/b
and RLR ligands. IFNs rely on STAT1/2 for gene transcription,
whereas RLRs activate Interferon Regulatory Factors 3 (IRF3). It is
therefore tempting to speculate that a common costimulatory
factor associates to these transcription factors when pyrimidine
biosynthesis is impaired. Discovering this mechanism remains a
major challenge in our understanding of the mechanisms linking
pyrimidine metabolism to innate immunity.
Fig. 6. Cellular response to IFN-
ISRE-Luc reporter gene were treated with DMSO or P1788 (80
presence of recombinant IFN- (150 IU/ml). Luciferase activity in culture wells was
determined 24 h later. Results were normalized using cells treated with DMSO þ IFN-
g
is amplified by P1788. (A) HEK-293 cells with the
mM) in the absence or
g
g
as reference. **p < 0.01 as calculated by two-tailed t-test. (B) Same experiment as in
(A), but cells were stimulated with different dilutions ranging from 1/8 to 1/64 of
conditioned supernatants from activated PBMCs. Results were normalized using cells
treated with 1/8 diluted supernatants as reference. Data represent means SD of three
independent experiments. *p < 0.05; ***p < 0.001 determined by two-way ANOVA
with Bonferroni’s post hoc test.
Here, we showed that P1788 treatment was associated to the
induction of a DNA damage response as assessed by
gH2AX stain-
ing. Furthermore, the enhanced response to IFN- was dependent
a
on MRE11, ATR, CHK1, and P300/CBP. MRE11, ATR, and CHK1 are all
activated by single-stranded genomic DNA, in particular at stalled
binds and activates RIG-like receptors (RLRs). Literature also sug-
gested that cellular response to type I IFNs is amplified when
Please cite this article as: S. Hayek et al., Cerpegin-derived furo[3,4-c]pyridine-3,4(1H,5H)-diones enhance cellular response to interferons by de
novo pyrimidine biosynthesis inhibition, European Journal of Medicinal Chemistry, https://doi.org/10.1016/j.ejmech.2019.111855

10
S. Hayek et al. / European Journal of Medicinal Chemistry xxx (xxxx) xxx
Fig. 7. Immunostimulatory properties of P1788 is linked to DNA damage response. (A) Simplified model of the DNA damage response induced by pyrimidine deprivation at
stalled DNA replication forks. (B) Cells were treated with DMSO, P1788 (80 M), teriflunomide (Ter.; 50 M) or mitoxantrone (Mit.; 50 nM). 24 h
M), P1788 þ uridine (Uri.; 125
later, cells were harvested and H2AX level was determined by immunostaining and flow cytometry analysis. Data represent means SD of three independent experiments.
**p < 0.01; ***p < 0.001 as calculated by two-tailed t-test. (CeF) HEK-293 cells with the ISRE-Luc reporter gene were treated with DMSO, IFN- (150 IU/ml) or IFN-
þ P1788
(80 M), in the absence or presence of mirin (C), AZD6738 (D), PF477736 (E) or C636 (F) to inhibit MRE11, ATR, CHK1 and P300/CBP, respectively. Luciferase activity in culture wells
was determined 24 h later, and results were normalized using cells treated with IFN- alone as reference. Data represent means SD of at least three independent experiments
(BeF). *p < 0.05; **p < 0.01; ***p < 0.001 as calculated by standard Student’s t-test (B) or one-way ANOVA with Bonferroni’s post hoc test (CeF).
m
m
m
g
a
a
m
a
DNA replication forks which likely accumulate when intracellular
pyrimidine concentration drops below a certain threshold [52].
This further supports the idea that the DNA damage response
represents the functional link between the inhibition of pyrimidine
biosynthesis and the innate immune response. Most interestingly,
we and others have previously shown that Interferon Regulatory
Factor 1 (IRF1) is essential to ISG induction in cells treated with
pyrimidine biosynthesis inhibitors alone, double-stranded RNA, or
both [32,40]. IRF1 is a transcription factor that is upregulated by the
DNA damage response and promotes the expression of ISGs, either
alone or in association with STATs and IRFs [59,60]. In addition, it
was shown that IRF1-mediated transcription relies on P300/CBP,
two related histone acetyltransferases that we implicated in the
signaling cascade downstream of P1788 [61]. In a recent report by
Luthra et al., it has been shown that DNA damage associated with
the inhibition of pyrimidine biosynthesis activate ATM (Ataxia
Telangiectasia Mutated), a kinase activated by double-stranded
DNA breaks, which was essential to the induction of ISGs by py-
rimidine biosynthesis inhibitors [40]. In line with this work, we
conclude from our observations that the DNA damage response
links pyrimidine deprivation to the innate immune response and to
IRF1, and show that P300/CBP is also involved in this phenomenon.
In conclusion, we have identified and characterized a new series
of compounds inhibiting pyrimidine biosynthesis. Future work will
Please cite this article as: S. Hayek et al., Cerpegin-derived furo[3,4-c]pyridine-3,4(1H,5H)-diones enhance cellular response to interferons by de
novo pyrimidine biosynthesis inhibition, European Journal of Medicinal Chemistry, https://doi.org/10.1016/j.ejmech.2019.111855

S. Hayek et al. / European Journal of Medicinal Chemistry xxx (xxxx) xxx
11
acsmedchemlett.6b00316.
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hibitors with optimized pharmacological properties. These mole-
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showed that cellular response to IFN- is also enhanced in the
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tives in cancer therapy as IFN- contributes to the antitumoral
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that the benefit of DHODH inhibitors relies not only on their
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compounds described in this manuscript have been patented.
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I. Menguy, I. Schreiber, C. Perrault, S. Vougier, B. Benhamou, B. Zhang, T. He,
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ꢀ
We acknowledge the technical support of Stephanie Dupuy from
ꢀ
the Flow Cytometry Platform at Universite Paris Descartes. We
thank the chemical library of UMR8601 of Paris Descartes Univer-
sity for preparing and providing access to Yerevan’s complete
database for virtual and physical screening. We thank Dr. Farah
Hodeib and Dr. Yves Janin for proofreading the manuscript. This
work was supported by the Agence Nationale de la Recherche
(ChemInnate program to POV and SN), Campus France (Programme
CEDRE), SantImmune from the Fondation Paris Descartes, the
Centre National de la Recherche Scientifique (CNRS; www.cnrs.fr),
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G.J. Maghzal, S. Magalhaes Novais, M. Olsinova, L. Krobova, Y.J. An,
~
E. Davidova, Z. Nahacka, M. Sobol, T. Cunha-Oliveira, C. Sandoval-Acuna,
H. Strnad, T. Zhang, T. Huynh, T.L. Serafim, P. Hozak, V.A. Sardao,
W.J.H. Koopman, M. Ricchetti, P.J. Oliveira, F. Kolar, M. Kubista, J. Truksa,
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ꢀ
ꢀ
and the Institut National de la Sante Et de la Recherche Medicale
(INSERM). SH was supported by the National Council for Scientific
ꢀ
Research (Lebanon) and the Universite Saint-Esprit de Kaslik
(USEK).
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Appendix A. Supplementary data
Supplementary data to this article can be found online at
https://doi.org/10.1016/j.ejmech.2019.111855.
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