Sibiriline, a new small chemical inhibitor of receptor-interacting protein kinase 1, prevents immune-dependent hepatitis

Article abstract of DOI:10.1111/febs.14176

Necroptosis is a regulated form of cell death involved in several disease models including in particular liver diseases. Receptor-interacting protein kinases, RIPK1 and RIPK3, are the main serine/threonine kinases driving this cell death pathway. We scree

Full text of DOI:10.1111/febs.14176

Sibiriline, a new small chemical inhibitor of Receptor-Interacting Protein Kinase 1,
prevents immune-dependent hepatitis
Fabienne Le Cann†,1,2, Claire Delehouzé†,3, Sabrina Penna-Leverrier†,1,2, Aveline Filliol1,2,
Arnaud Comte4, Olivier Delalande5, Nathalie Desban3, Blandine Baratte3, Isabelle Gallais1,2,
Claire Piquet-Pellorce1,2, Florence Faurez1,2, Marion Bonnet1,2,6, Yvette Mettey7, Peter
Goekjian8, Michel Samson1,2, Peter Vandenabeele9,10, Stéphane Bach,3 and Marie-Thérèse
Dimanche-Boitrel,1,2
1 INSERM UMR 1085, Institut de Recherche sur la Santé, l’Environnement et le Travail, F-
35043 Rennes, France.
2 Biosit UMS 3080, Université de Rennes 1, F-35043 Rennes, France.
3 Sorbonne Universités, UPMC Univ Paris 06, CNRS USR3151, Protein Phosphorylation
and Human Disease Laboratory, Station Biologique, F-29688 Roscoff, France.
4 CNRS UMR 5246, Chimiothèque, ICBMS, Université Claude Bernard Lyon 1, F-69622
Villeurbanne, France.
5 CNRS UMR 6290, Institut de Génétique et Développement de Rennes, Université de
Rennes 1, F-35043 Rennes, France.
6 Division of Infection & Immunity, College of Biomedical and Life Sciences, Cardiff
University, Cardiff, United Kingdom.
7 Laboratoire Chimie Organique, Faculté de Médecine-Pharmacie, Laboratoire Signalisation
et Transports Ioniques Membranaires, Université de Poitiers, CNRS, 86073 Poitiers Cedex 9,
France.
8 CNRS UMR 5246, Laboratoire Chimie Organique 2-Glycosciences, Université de Lyon,
ICBMS, Université Claude Bernard Lyon 1, F-69622 Villeurbanne, France.
9 Molecular Signaling and Cell Death Unit, VIB Inflammation Research Center, Ghent,
Belgium.
10 Department of Biomedical Molecular Biology, Ghent University, Ghent, Belgium.
†These authors contributed equally to this work
 These authors share senior authorship
Corresponding authors:
S. Bach, Protein Phosphorylation and Human Disease Laboratory, Station Biologique, Place
Georges Teissier, F-29688 Roscoff, France
Tel: +33 2 98 29 23 91 Fax: +33 2 98 29 25 26 Email: bach@sb-roscoff.fr
M-T. Dimanche-Boitrel, INSERM UMR 1085, Institut de Recherche sur la Santé, 2 avenue du
Pr Léon Bernard, F-35043 Rennes, France
Tel: +33 2 23 23 48 99 Fax: +33 2 23 23 47 94 Email: marie-therese.boitrel@univ-rennes1.fr
Running title: Sibiriline, a RIPK1 inhibitor preventing hepatitis
Article type : Original Article
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lead to differences between this version and the Version of Record. Please cite this article as
doi: 10.1111/febs.14176
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Abbreviations: ALT, alanine aminotransferase; AST, aspartate aminotransferase; ATP,
adenosine triphosphate; AU, arbitrary unit; Con A, concanavalin A; DR, death receptor; MD,
molecular docking; MEFs, mouse embryonic fibroblasts; Nec-1, necrostatin-1; RT; room
temperature; Sib, Sibiriline; Sib-i, inactive Sibiriline; TNF, tumor necrosis factor; FasL, Fas
ligand; TRAIL, TNF-related apoptosis-inducing ligand.
Keywords: necroptosis, RIPK1 inhibitor, molecular docking, Con A-induced hepatitis, kinase
inhibitor
Abstract
Necroptosis is a regulated form of cell death involved in several disease models including in
particular liver diseases. Receptor-Interacting Protein Kinases, RIPK1 and RIPK3, are the
main serine/threonine kinases driving this cell death pathway. We screened a non-commercial
kinase-focused chemical library allowed us to identify Sibiriline as new inhibitor of
necroptosis induced by TNF in Fas-Associated protein with Death Domain (FADD)-deficient
Jurkat cells. Moreover, Sib inhibits necroptotic cell death induced by various death ligands in
human or mouse cells while not protecting from caspase-dependent apoptosis. By using
competition binding assay and recombinant kinase assays, we demonstrated that Sib is a
rather specific competitive RIPK1 inhibitor. Molecular docking analysis shows that Sib is
trapped closed to human RIPK1 ATP-binding site in a relatively hydrophobic pocket locking
RIPK1 in an inactive conformation. In agreement with its RIPK1 inhibitory property, Sib
inhibits both TNF-induced RIPK1-dependent necroptosis and RIPK1-dependent apoptosis.
Finally, Sib protects mice from Concanavalin A-induced hepatitis. These results reveal the
small molecule Sib as a new RIPK1 inhibitor potentially of interest for the treatment of
immune-dependent hepatitis.
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Introduction
Excessive hepatocyte cell death has been identified as a central pathophysiological
mechanism in both acute and chronic liver injuries, involving mostly cell death by apoptosis
and necrosis (oncosis) [1]. More recently, necroptosis, a specific form of regulated necrosis,
has also been implicated in liver disease models [2,3]. Necroptosis is a caspase-independent
non-apoptotic backup cell death mechanism initiated upon blockade of apoptosis [4]. This cell
death pathway is dependent on serine-threonine Receptor-Interacting Protein Kinases, RIPK1
and RIPK3, and a pseudokinase MLKL (Mixed-Lineage Kinase domain-Like) (see for review
[5]). Moreover, it is inhibited by necrostatin-1 (Nec-1), the first small tryptophan-based
molecule inhibitor of RIPK1 kinase activity [4,6].
Necroptosis may occur in response to various stimuli [5]. The TNF-induced
necroptosis pathway has been the most intensively studied. Upon TNF stimulation when
caspase-8 is inhibited or absent [7,8], RIPK1 in complex II recruits RIPK3, resulting in
autophosphorylation and activation of the latter. Caspase-8 and FLIPL in complex II negatively
regulates RIPK1 and RIPK3 by proteolysis [9]. Then, these kinases interact with each other
via their RIP homotypic interaction motifs (RHIM) domains promoting necrosome formation
[10], recruitment and phosphorylation of MLKL [11, 12] which in turn oligomerizes [13] and
is recruited to negatively charged phosphoinositolphosphates [14], causing loss of plasma
membrane integrity [15]. Necroptosis contributes to the pathogenesis of several inflammatory
diseases including ischemic reperfusion injury, acute pancreatitis, sepsis, neurodegeneration
and viral infection [16]. Although the contribution of necroptosis in liver disease is still an
open question [17], some studies reported that necroptosis is also involved in inflammatory-
driven liver diseases. On the one hand, RIPK3-deficient mice are protected from ethanol-,
acetaminophen (APAP)-induced hepatocyte injury or non-alcoholic steatohepatitis (NASH)
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[18-20]. On the other hand, RIPK3 expression is increased in liver biopsies of chronic ethanol-
fed mice, alcoholic liver disease patients, NASH patients or patients with other chronic liver
diseases (hepatitis B and C) [18,20,21]. Furthermore, Nec-1 or Nec-1s protect against APAP-
or Concanavalin A (Con A)-induced hepatoxicity [22-25]. To date, the only possible candidate
to be used in the clinic would be the anticancer drug B-RafV600E Dabrafenib which besides
mutant Raf targeting also inhibits RIPK3 and alleviates APAP-induced liver injury [26].
Altogether these data suggest that targeting necroptosis could be a novel cellular process to be
therapeutic targeted in liver diseases.
Nec-1 (5-((1H-indol-3-yl)methyl)-3-methyl-2-thioxoimidazolidin-4-one) was identified
in a phenotype screen for small molecule inhibiting TNF-induced necroptosis in human
monocytic U937 cells [4, 27] and further, was shown to be an allosteric inhibitor of RIPK1
kinase [6]. However, Nec-1 is not specific for RIPK1 because its chemical scaffold is similar
to methyl-thiohydantoin-trytophan (MTH-Trp), an inhibitor of indoleamine 2,3-dioxygenase
(IDO), an immune regulator [28]. Structure-activity relationship (SAR) study revealed that
small substituent Cl at the 7-position of the indole ring leads to the compound necrostatin-1s
(Nec-1s) [27] having an increased selective RIPK1 inhibitory activity [29] and lacking IDO
inhibitory activity [28]. However, necrostatins have poor pharmacokinetic properties. More
recently, other classes of compounds were described such as the 1-aminoisoquinolines, 5-
phenylpyrrolo[2,3-b]pyridines, 5-arylpyrrolo[2,3-b]pyridines, furo[2,3-d]pyrimidines, analogs
of Bcr-Abl inhibitor ponatinib, PN10 (fusion of the scaffold of ponatinib and Nec-1s), the
benzo[b][1,4]oxazepin-4-ones, the compound GSK’963 and the N-benzyl-N-hydroxy-2,2-
dimethylbutanamide [30-36]. The previously described biological activities validate kinase
RIPK1 as a promising therapeutic target for discovering new compounds to treat various
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human disorders [37]. The quest for an optimized clinical candidate is still in progress with the
synthesis of the first-in-class RIP1 kinase specific inhibitor (GSK2982772) [38] and will
benefit from the description of new potent RIPK1 inhibitors.
In order to find new inhibitors of RIPK1, we screened an in-house kinase-focused
library of small chemical compounds (the ReCC, Roscoff essential Chemical Collection,
Roscoff, France) using a phenotypic screen based on the inhibition of TNF-induced
necroptosis in human FADD-deficient Jurkat T cells (in vitro model described in [39]). Among
1015 tested compounds, 4-(1H-pyrrolo[2,3-b]pyridin-2-yl)phenol (Sibiriline, Sib) was selected
as the best hit. Interestingly, we describe here the biochemical properties and both in vitro and
in vivo effects of this new pyrrolo[2,3-b]pyridine-based derivative as a potent RIPK1 inhibitor/
necroptosis inhibitor. Our results indicate that the kinase activity of RIPK1 is dose-
dependently affected by Sib. In silico molecular modeling and docking studies reveal that Sib
interacts with RIPK1 kinase domain in a hydrophobic “back” pocket [32], between the
gatekeeper and the DFG (DLG in RIPK1) motif, adjacent to the ATP-binding site, thus
locking RIPK1 in an inactive DLG out conformation. Finally, to assess the therapeutic
efficacy of Sib in liver diseases, we studied its effects in Con A-induced hepatitis in C57Bl/6
mice. This acute hepatitis is a mouse model of immune-mediated liver injury [40]. A single
intraperitoneal injection of Sib protects mice from liver injury induced by Con A, and
therefore, reveals Sib as a promising new chemical scaffold of RIPK1 inhibitor, with a
straightforward, high-yielding synthesis, which is effective in the treatment of the preclinical
model of immune-dependent hepatitis.
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Results
Sibiriline is a new necroptosis inhibitor
To identify novel necroptosis inhibitors, we screened a non-commercial kinase-focused
chemical library of 1015 compounds for their ability to block TNF-induced necroptosis in
FADD-deficient human Jurkat cells (Fig. 1A). These compounds were tested at the
concentration of 10 µM and compared to the well-characterized RIPK1 inhibitor necrostatin-1
(Nec-1). Among the tested drugs, Sibiriline (Sib) stood out as the most potent molecule at
rescuing cells from necroptotic cell death whereas Sib-i came out as its inactive analog (Fig.
1B). Similarly to Nec-1 (EC50=0.3 µM), Sib efficiently blocked necroptotic death in FADD-
deficient Jurkat cells in a concentration-dependent manner with an EC50 value of 1.2 µM (Fig.
1C). The inactive derivative Sib-i was unable to block necroptosis in FADD-deficient Jurkat
cells even at the high concentration of 50 µM (Fig. 1C). Importantly, Sib has a very low
cytotoxic effect against human peripheral blood leukocytes (PBL) (Fig. 2A) or human retinal
pigment epithelial cells (RPE-1 hTERT) (Fig. 2B).
Sib blocks necroptotic but not apoptotic cell death
To further analyze Sib inhibitory effect, we compared the ability of Sib and Nec-1 to block
necroptosis in murine and human cell lines in vitro. Necroptosis was induced by a wide range
of well-described necroptotic stimuli such as TRAIL+zVAD-fmk, TNF+zVAD-fmk or
FasL+zVAD-fmk in human U937 or mouse L929sAhFas cells. In all of these models, Sib
efficiently blocked necroptosis in a dose-dependent manner, and with an efficacy almost
similar to Nec-1 (Fig. 3A). This efficient protection from necroptosis by Sib was evidenced by
measurements of both plasma membrane permeabilization (% of propidium iodide positive
cells) and intracellular ATP levels (Fig. 3A). This latter assay highlighted the fact that Sib
inhibitory effect appeared at 2.5 µM and was almost complete at 5 µM. Notably and like Nec-
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1, Sib did not rescue from apoptosis induced by TRAIL, FasL or staurosporine in Jurkat cells
(Fig. 3B,C), showing that Sib is a selective inhibitor of necroptosis but not of apoptosis. It is to
be noted that in a different human cell line resistant to extrinsic apoptosis, the FADD-deficient
Jurkat cells, TNF-induced necroptosis can also be prevented by Sib. Conversely, Fas-induced
apoptosis in the L929AsFas mouse model is not sensitive to Sib (data not shown). Overall,
these data evidenced that Sib specificity towards inhibiting necroptosis, but not apoptosis, does
not depend upon the stimuli, the cell type, or even the species.
Sib rescues cells from necroptosis even after cell death initiation and allows cell survival
and proliferation
In the hypothetical perspective of using Sibiriline as a necroptosis inhibitor in medecine, we
tested its activity when administered after, rather than before, necroptosis initiation. In the
following experimental set up, Sib or Nec-1 was added at the non-toxic concentration of 10
µM between 1 h to 4 h after TNF-dependent necroptosis induction in FADD-deficient Jurkat
cells. When added in the first couple of hours following necroptosis induction, Sib promotes
an almost complete recovery by decreasing cell death to approximately 10%. When added 3 or
4 h after necroptosis induction, both Sib or Nec-1 treatment rescued necroptotic FADD-
deficient Jurkat cells, reducing by a minimum of 50 % the number of propidium iodide (PI)
positive cells (Fig. 4A). To further refine Sib’s potential at preventing cells from a necroptotic
wave, a clonogenic assay was performed 24 h after necroptosis induction by TNF+zVAD-fmk
in L929 cells in presence or not of Sib, Sib-i, Nec-1 or Nec-1s. Eight days later, culture plates
were stained with crystal violet and showed that treatment with Sib, Nec-1 or Nec-1s but not
with Sib-i allowed the formation of colonies (Fig. 4B), suggesting that Sib as other well-
known necroptosis inhibitors (Nec-1 or Nec-1s), not only protected cells from cell death but
allowed them to survive and proliferate.
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Sib is a potent inhibitor of RIPK1
In order to investigate the mechanism of action of Sib and its target, the selectivity of Sib and
Sib-i was tested at 10 µM against a panel of 456 kinases including eight lipid kinases in an in
vitro competition binding assay (KINOMEscan, DiscoverX, San Diego, CA, USA). RIPK1
was shown to be among the top kinases targeted by Sib as only 0.2% of the initial amount of
the kinase was still on the affinity matrix after competition with Sib (Table S1 and blue circle
on Fig. 5A). Sib-i is not a potent interactor of the tested kinases (Fig.5A). Death-Associated
Protein Kinase-3 (DAPK3) is only poorly affected by Sib-i (32% of the initial amount of
kinase is still on the affinity matrix after competition with 10 µM Sib-i, Fig. 5A). Using this
competition binding assay, the dissociation constant (Kd) of Sib for RIPK1 was calculated
with a standard dose-response curve and showed the high affinity of Sib for RIPK1 with a Kd
in nM range and equal to 218 nM (Fig. 5B, left panel). High affinity of Nec-1 or Nec-1s for
RIPK1 was also confirmed with Kd equal to 31 nM or 8 nM, respectively (Fig. 5B, right
panel).
To further confirm the inhibition of RIPK1 enzymatic activity by Sib, an in vitro auto-
phosphorylation assay using recombinant full-length RIPK1 (GST-tagged) was performed.
This assay showed an inhibition of RIPK1 auto-phosphorylation by Sib in a dose-dependent
manner, Sib-i having no effect (Fig. 6A). The catalytic activity of RIPK1 was also quantified
by monitoring the phosphorylation of Myelin-Basic Protein (MBP) a known substrate of
RIPK1. Consistently, Sib efficiently inhibits the phosphorylation of MBP in a dose-dependent
manner with an IC50 equal to 1.03 µM (Fig. 6B).
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Finally, to characterize the mechanism of RIPK1 inhibition by Sib, we analyzed the effect of
increasing the concentrations of ATP (up to 1mM of cold ATP) on the inhibition of auto-
phosphorylation by 10µM of Sib. Results obtained show that in presence of 1 mM cold ATP,
the inhibition of the RIPK1 auto-phosphorylation by Sib was decreased. It suggests an ATP-
competitive mechanism of RIPK1 inhibition by Sib (Fig. 6C). This result was previously
described for Nec-1 [6]. Very interestingly, Sib had a very low inhibitory effect on RIPK3
kinase activity with an IC50 equal to 101.9 µM (Fig. 6D). Altogether, these data demonstrated
that Sib is a specific inhibitor of RIPK1 kinase activity. In agreement with this biochemical
activity, Sib was able, with a similar efficacy to Nec-1, to inhibit RIPK1-dependent
necroptosis induced by the combination TNF+TAK1i+zVAD-fmk by reducing the
percentage of Syber Green (% SG) positive cells measured in function of time (Fig. 7A, left
panel). This necroptotic pathway was characterized by the lack of caspase-3 activity (very
low DEVD-AMC fluorescence) (Fig. 7A, right panel). Moreover, Sib like Nec-1 inhibited
RIPK1-dependent apoptosis induced by the combination of TNF+TAK1i [41,42] by
decreasing both the % SG positive cells and caspase-3 activity measured as a function of time
(Fig. 7B).
Molecular modeling of the RIPK1-Sib complex
To further understand the mechanism of inhibition by the small chemical compound, we
studied in silico the RIPK1-Sib complex to establish a possible model of RIPK1 inhibition by
Sib. Molecular docking simulations of Sib to RIPK1 kinase and molecular dynamics
trajectory computed for the best complex indicated a preferential binding mode similar to the
one described for Nec-1s on a site next to the ATP-binding site (Fig. 8 and Fig.9). Contact
residues determined included notably the seven key amino acids (Leu70, Val76, Leu78,
Leu90, Asp156, Ser161, Phe162) (Fig. 8A) involved in the interaction with necrostatins (see
[43] for details on RIPK1-Necrostatins complexes). An exhaustive list of surrounding
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residues (3.5Å cut-off) also included the amino acids Met67, Lys77, Lys79, Met92, Val134
and Ile154 (Fig. 9). Note here that Met92 is the gatekeeper residue of RIPK1 [6] and the
catalytic triad residues are Lys45, Glu63 and Asp156 as reported [43]. Sib thus binds in the
“back” pocket of RIPK1 between the DLG motif and the gatekeeper. Despite the presence of
a canonical H-bond donor / H bond acceptor motif, Sib, like Nec-1s, does not appear to bind
to the hinge region, at least in this conformation of the protein (Fig. 8B,C).
In contrast, the inactive compound Sib-i did not contact the C-lobe region of RIPK1 (Fig. 9).
Comparison between the contact residues predicted for active compounds (Sib and Nec-1s)
and inactive compound (Sib-i) did indicate the position of a good inhibitor that should
promote hydrophobic interactions with the long helix defined by residues from Pro111 to
Lys132, thus locking the conformation of the enzyme by reducing the mobility of the two
main subdomains of the kinase towards each other (Fig. 9).
Sib protects mice from Concanavalin A-induced hepatitis
The identification of Sib as a specific necroptosis and RIPK1 inhibitor as Nec-1 prompted us
to test this compound in a murine model of immune-dependent hepatitis induced by
Concanavalin A (Con A) treatment. In this model, we have previously demonstrated a
protective effect of an intravenous (i.v.) injection of Nec-1 or Nec-1s against Con A-induced
hepatitis [22, 25]. Here, we studied the protective effect of an intraperitoneal (i.p.) injection of
3 mg/kg Sib or 6.25 mg/kg Nec-1s 1 h before an i.v. injection of 12 mg/kg Con A.
Pre-treatment with either Sib or Nec-1s protected mice from Con A-induced hepatotoxicity by
significantly decreasing serum AST/ALT levels measured 24 h after treatment (Fig. 10A).
Moreover, Sib or Nec-1s significantly decreased liver damage by reducing the size of
perivascular and parenchymal zones of necrosis stained with Hematoxylin and Eosin (Fig.
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10B, see arrows) and the percentage of necrotic area (Fig. 10C). In accord with the protection
afforded by Sib or Nec-1s, the loss of mouse body weight induced by ConA treatment was
significantly reduced by Sib or Nec-1s pretreatment (Fig. 11). Altogether these data suggest
that 3 mg/kg Sib had an almost comparable efficacy to 6.25 mg/kg Nec-1s in protecting mice
against Con A-induced hepatitis.
Discussion
By using a cellular screen and a unique non-commercial kinase-focused chemical library of
1015 small molecular compounds, we identified a new inhibitor of TNF-induced necroptosis
in Jurkat Fadd-deficient cells with a micromolar EC50 concentration. Sibiriline (Sib) , 4-(1H-
pyrrolo[2,3-b]pyridin-2-yl)phenol is a new pyrrolo[2,3-b]pyridine-based derivative compound.
Our present data enlarge the number of small chemical compounds having a potent necroptosis
inhibitory activity [44]. Sib was also able to inhibit TRAIL- or FasL-induced necroptosis but
not apoptosis regardless of the human or murine cell line origin. Protection from necroptotic
cell death was obtained at 1-5 µM concentration of Sib in our diverse cellular systems,
suggesting a large therapeutic window for potential clinical application. Moreover, protection
from necroptosis has been afforded by Sib up to 4 hours after the induction of death, which
could allow a therapeutic potential in the case of cardiac infarction or stroke. Regarding the
mode of action underlying necroptosis inhibition by Sib, a KINOMEscan competition binding
assay at DiscoverX (456 kinases and activated mutant kinases) was performed and showed
that RIPK1 was the top kinase inhibited by Sib. In contrast to Nec-1 which did not bind to
RIPK2 and RIPK4 [45], Sib bound to RIPK4 to a certain extent (Table S1). We showed in this
study that the kinase activity of RIPK3 is only poorly affected by Sib (approximately 100
times less potently inhibited compared to RIPK1). The Kinome scan revealed also that JAK2,
PDGFR KIT and KIT (V559D) are other major kinases targeted by Sib. Chemical synthesis
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of Sibiriline analogues, notably driven by the molecular docking reported in this study, would
be necessary to improve the selectivity of this compound. As expected, no kinase was
efficiently targeted by the inactive counterpart, Sib-i. We have also determined a Kd binding
constant of Sib for RIPK1 of 218 nM whereas the Kd of Sib-i was greater than 30000 nM. The
in vitro competitive assay used for both Kd study and kinase profiling is based on an active-
site/ligand binding technology developed by DiscoverX (Fremont, CA, United States): it can
detect the effect of compounds that bind the kinase active site and directly (sterically) or
indirectly (allosterically) prevent kinase binding to one immobilized ligand. The majority of
kinase inhibitors developed to date target the ATP binding site. Based on the selectivity
reported for Sib, we hypothesized that Sib also targets the ATP binding site of RIPK1. We
showed here that ATP competes with Sib for the inhibition of RIPK1 autophosphorylation
(Fig. 6C). Such result was already described for Nec-1 [6], suggesting some similarities in the
mode of action between Sib and Nec-1. In silico molecular modeling and docking studies
highlighted these similarities. To understand the interaction mode of Sib with RIPK1,
molecular docking was performed based on the co-crystal structure of RIPK1 with Nec-1s
(PDB: code 4ITH). The predictive binding mode was shown on Figure 8. As was the case for
Nec-1s, Sib was located in the hydrophobic “back” [32] pocket between the N-lobe and C-lobe
of the RIPK1 kinase domain, interacting with both the DLG motif and the gatekeeper. This
pocket is formed by a number of residues including Leu70, Val76, Leu78, Leu90, Asp156,
Ser161 and Phe162 (see [43] for details on RIPK1-Necrostatins complexes). In analogy with
the inhibition of RIPK1 by Nec-1s, we hypothesized that Sib locks RIPK1 in an inactive
conformation (“DLG (Asp-Leu-Gly)-out” state of kinase) notably by interaction with Asp156
of the DLG motif and neighboring Ser161. Interactions with the C-lobe also appear to be an
important element for successful inhibition. Our results suggest that Sibiriline shares
characteristics of the type II kinase inhibitor class, which target the inactive enzymatic
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conformation, with the caveat that it does not interact with the hinge region, despite its H-bond
donor – H-bond acceptor motif. Since the description of the druggability of necroptosis [4],
numerous chemobiological studies have led to the discovery of molecules that can bind to the
catalytic site of RIPK1 [37]. As examples, compounds characterized by a GlaxoSmithKline
group include notably pyrrolo[2,3 b]pyridine-based derivatives [30]. Interestingly, and
similarly to the putative mechanism of Sibiriline described here, these small chemical
compounds share also all characteristic of the type II kinase inhibitor class. This class of
compounds is believed to perturb the Phe side chains of the catalytic site inwards to obstruct
the ATP-binding cleft.
Since Nec-1 or Nec-1s have already been shown to protect mice from Concanavalin A (Con
A)-induced hepatitis [22,24,25], we tested the effect of Sib in this model of murine hepatitis.
Pretreatment of mice with 3 mg/kg Sib has almost the same protective effect against Con A-
induced hepatitis than pretreatment of mice with 6.25 mg/kg Nec-1s, by decreasing serum
AST/ALT levels and liver injury. Sib seems to have no toxic adverse effect since this
compound allowed mice to recover some weight after Con A treatment. Altogether these data
demonstrated efficacy of Sib in vivo which make Sib to be a highly promising template for
lead optimization.
Materials and methods
Cell lines and culture conditions
SV40 large T-immortalized MEFs and L929sAhFas were kind gifts of the University of
Ghent (Belgium). These cell lines were cultured in DMEM high glucose (Invitrogen, Thermo
Fisher Scientific, St Aubin, FR) supplemented with 10 % FCS and 2 mM L-glutamine
(Invitrogen). HT29, Jurkat wild-type A3, FADD-deficient Jurkat I 2.1, L929, U937, RPE-1
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hTERT human cell lines were obtained from ATCC (American Type Culture Collection,
Rockville, MD, USA). HT29 cells were cultured in EMEM (Eurobio, Les Ulis, FR)
supplemented with 10 % FCS and 2 mM L-glutamine. Jurkat, FADD-deficient Jurkat and
U937 cells were cultured in RPMI containing Glutamax (Invitrogen) supplemented with 15
% FCS. RPE-1 hTERT cells were cultured in DMEM-F12 containing Glutamax (Invitrogen)
supplemented with 10 % FCS. All cells were cultured under a 5 % CO2 atmosphere at 37 °C.
Chemicals and biological compounds
Recombinant human Flag-tagged TRAIL (TRAIL-Flag) and z-VAD-fmk were obtained from
Enzo Life Sciences (Villeurbanne, FR). Necrostatin-1 (Nec-1) and necrostatin-1s (Nec-1s)
were from Calbiochem (VWR International, Fontenay-sous-Bois, FR). The TAK1 kinase
inhibitor, NP-009245, was from AnalytiCon Discovery GmbH (Postdam, DE). Anti-Flag M2
IgG1 antibody, cycloheximide (CHX), propidium iodide, were obtained from Sigma-Aldrich
(St Quentin-Fallavier, FR). TNFα were obtained from Invitrogen. This TNF was used in the
screen of new necroptosis inhibitors. Recombinant human TNFα, was also produced and
purified to at least 99% homogeneity at VIB Protein service facility (Ghent, BE). It has a
specific biological activity of 6.8x107 IU/mg and was used at 2000 IU/ml (67 ng/ml) in
FADD-deficient Jurkat or at 600 IU/ml (20 ng/ml) in MEFs. The multi-aggregated Ig-CD95L
(or Ig-FasL) was a kind gift of Dr P. Legembre (CLCC Eugene Marquis, Rennes, FR) and
was used at 200 ng/ml.
Synthesis of 4-(1H-pyrrolo[2,3-b]pyridin-2-yl)phenol (Sib) : following literature procedure
[46], a solution of 3-picoline (200 mg, 2.2 mmol, 1 eq) in anhydrous THF (10 ml) was added
dropwise to a solution of LDA (freshly prepared by adding nBuLi (1.5 ml, 2.5 M solution in
hexanes, 3.9 mmol) to diisopropylamine (0.54 ml, 3.9 mmol) in anhydrous THF (10 ml)) at
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0°C under argon. The orange mixture was stirred for 20 min before dropwise addition of a
solution of 4-methoxybenzonitrile (316 mg, 2.2 mmol, 1 eq) in anhydrous THF (10 ml). After
1 h at 0°C, more LDA solution in THF (10 ml, 3.9 mmol, prepared as above) was added
dropwise and the reaction was slowly warmed to RT over 1 h before being heated to reflux in
a water bath for 2 h. After cooling, the yellow solution was quenched carefully with saturated
NH4Cl (10 ml) and water (40 ml) was added. The precipitate was filtered, washed with diethyl
ether and water, dried under vacuum to afford 2-(4-methoxyphenyl)-1H-pyrrolo[2,3-
b]pyridine as a light yellow solid (327 mg, 68%). Analytical data are in accordance with the
literature.
2-(4-methoxyphenyl)-1H-pyrrolo[2,3-b]pyridine (100 mg, 0.44 mmol) was suspended in
anhydrous dichloromethane (5 ml) and cooled to -78°C. Boron tribromide (1.3 ml of 1M
solution on dichloromethane, 1.3 mmol, 3 eq) was added dropwise and the dark red mixture
was stirred for 16 h while warming to RT. The reaction was carefully quenched with water,
dichloromethane (10 ml) was added and the solid was dissolved by addition of 1M sodium
hydroxide (15 ml). After stirring for 10 min, the aqueous phase was washed with
dichloromethane (2x10 ml) and the pH was adjusted to 5 with 1M hydrochloric acid. The
resulting precipitate was filtered, washed with water and dried under vacuum to obtain a tan
solid (68 mg, 74%). 1H NMR (300 MHz, DMSO-d6) : 11.93 (bs, 1H), 9.71 (bs, 1H), 8.14 (dd,
J = 4.7, 1.3 Hz, 1H), 7.85 (dd, J = 7.8, 1.3 Hz, 1H), 7.76 (d, J = 8.7 Hz, 2H), 7.02 (dd, J = 7.8,
4.7 Hz, 1H), 6.85 (d, J = 8.7 Hz, 2H), 6.70 (d, J = 2.0 Hz, 1H). 13C NMR (75 MHz, DSMO-d6)
: 157.7, 149.6, 141.9, 139.0, 127.1, 126.9, 122.7, 121.3, 115.9, 115.8, 95.1. MS (ESI): m/z 211
[M+H]+.
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Synthesis of the inactive derivative Sib-i has been described previously [47].
Cell-based screening assay for characterization of necroptosis inhibitors
The details of the cell-based assay used to screen a library of small chemical compounds have
been described in Miao and Degterev, 2009 [39]. The Jurkat FADD-deficient I 2.1 cell line
was maintained in RPMI 1640 medium (Gibco) containing Glutamax and 15% fetal calf serum
(Eurobio). Necroptosis was induced by addition of 10 ng/ml of human recombinant TNF-α
(Invitrogen). Necrostatin-1 (Nec-1, Calbiochem) was used as model necroptosis inhibitor.
Cells were maintained in 75 cm2 flask and passed every 2 or 3 days. Chemical collections
analyzed were formatted in 96-well plates with 80 molecules per plate at 10 mM in 100%
DMSO. For each collection plate, two cell plates were seeded (one treated with TNF-α and the
other one without TNF-α). Cells were seeded at 20 000 cells/well, in 40 μl of medium, in a 96-
well clear, flat bottom plate (CytoOne, Starlab) before treatment. Then, 40 μl of medium with
or without TNF-α at 25 ng/ml were added to all wells in the corresponding plate. Immediately
after TNF-α addition, 20 μl of diluted compound at 50 μM were added to the plates. Final
concentration of each chemical compound was 10 μM at 0,1 % DMSO. Eight positive (Nec-1
at 10 μM final) and eight negative (DMSO) controls were used in each plate to validate the
assay. Cells were incubated at 37°C, 5% CO2 for 24 h before performing MTS viability assay,
described hereafter. Compounds were diluted before treating cells. Liquid handling was
performed using the Nimbus Microlab liquid handler (Hamilton Robotics, Bonaduz,
Switzerland) under microbiological safety workbench. The stock solutions of compounds at 10
mM were diluted directly in culture medium to obtain a solution at 50 µM before treating
cells.
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Cell death assays
Cell viability was measured by a water soluble tetrazolium compound MTS (3-[4,5-
dimethylthiazol-2-yl]-5-[3-carboxymethoxy-phenyl]-2-[4-sulfophenyl]-2H-tetrazolium, inner
salt) (CellTiter 96® AQueous Non-Radioactive Cell Proliferation Assay (Promega,
Fitchburg, WI, USA), according to the manufacturer's instructions. This method is based on
the reduction of MTS tetrazolium compound by viable cells to generate a colored formazan
product. Human peripheral blood lymphocytes (PBL) and human RPE-1 hTERT cells were
seeded in 96-well plates at 2.5x105 or 8x103 cells per well, respectively. After 24 h of
treatment, 20 μl of assay solution was added. After 3 h incubation at 37°C, the absorbance at
490 nm and 630 nm was measured by a microplate reader (SPECTROstar Nano, BMG
Labtech). The results were corrected by subtracting the background reading (after 2 h
incubation with blank medium) and viability was calculated with % viability = mean (OD490
OD630) sample/mean (OD490 - OD630) control x 100.
-
Detection of necrotic cells was done by using propidium iodide staining (500 ng/ml) and flow
cytometry analysis. U937, Jurkat or L929 sAhFas cell lines were seeded at 2.5x105 cells/well
in 24-well plate. Concerning necroptotic models: U937 cells were pretreated with z-VAD-
fmk (30 µM) for 30 min and then treated for 20 h with TRAIL-Flag (200 ng/ml) and 2 µg /ml
M2 anti-Flag antibody. L929 sAhFas cell line were pretreated with z-VAD-fmk (20 µM) for
30 min and then with TNF (10 ng/ml) or Ig-FasL (200 ng/ml) for 16 h. FADD-deficient
Jurkat cells were treated with 10 ng/ml TNF. As for apoptotic models, wild-type Jurkat cells
were treated either with TRAIL-Flag (20 ng/ml) and 2 µg/ml M2 anti-Flag antibody, Ig-FasL
(200 ng/ml) or staurosporine (100 nM) for 18 h. After treatment, percentage of propidium
iodide positive cells was determined by flow cytometry analysis of fluorescence (FL-2)
(FACScalibur, Becton Dickinson, Le Pont de Claix, FR) and expressed as %/NT.
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Clonogenic Assay
After treatment with TNF/zVAD-fmk in presence or absence of inhibitory compounds (Sib,
Sib-i, Nec-1 or Nec-1s) as described above, L929 cells were seeded out in an appropriate
dilution, here 1,000 cells per well (in 6-well plates), and allow for 8 days to form colonies. The
subsequent colonies were fixed with ice cold methanol and stained with crystal violet (50%
ethanol).
RIPK1 and RIPK3 kinase assays
Human RIPK1 full length GST-tagged was expressed by baculovirus in Sf9 insect cells
(SignalChem, product #R07-10G, Richmond, CA, USA). The protocol used to detect the
enzymatic activity is adapted from Degterev et al., 2008 [6]. For detection of RIPK1
autophosphorylation, kinase reaction was initiated mixing 5 l of RIPK1, 5 l of 3X kinase
reaction buffer (5 mM MOPS pH 7.2, 2.5 mM -glycerophosphate, 4 mM MgCl2, 2.5 mM
MnCl2, 1 mM EGTA, 0.4 mM EDTA, 50 µg/ml BSA, 0.05 mM DTT), 2 l H2O and 3 l of
the tested molecule. The mixture was kept on ice for 10 minutes. During the incubation, the
ATP solution was prepared by mixing 5 l of 3X kinase reaction buffer, 4 l H2O, 6 l cold
ATP at 150 M and 2 Ci of -32P ATP. This ATP solution was then mixed with RIPK1 and
the tested compound and incubate for 30 minutes at 30 °C. Reactions were stopped by boiling
in 5 l of loading buffer for 3 minutes at 95 °C. 25 l of each reaction were loaded per well in
pre-cast NuPage 12% Bis-Tris gel (Thermo Fisher Scientific) and analyzed by SDS-PAGE.
Autophosphorylated RIPK1 band was visualized by analysis in a Typhoon Phosphorimager
(GE Healthcare Life Sciences, Velizy-Villacoublay, FR). Alternatively, the kinase activity of
RIPK1 can be detected using the “gold standard” P81 phosphocellulose assay. Note here that
the P81 ion exchange chromatography paper (GE Healthcare) used in this study is no longer
provided by the manufacturer. For a final volume of 30 µl, 2 µl of kinase was assayed in
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buffer described above with 0.1 µg/µl of myelin basic protein (MBP, Sigma, #M1891) as
substrate and in the presence of 15µM [γ-33P] ATP (3,000 Ci/mmol; 10 mCi/ml). Controls
(maximal kinase activity) were performed with appropriate dilutions of dimethylsulfoxide.
After extensive washes with a solution of phosphoric acid at 1% in water using a FilterMate
Harvester (PerkinElmer, Waltham, MA), the wet filters were counted in the presence of 20µl
ACS (GE Healthcare) scintillation fluid using a TopCount® Scintillation and Luminescence
Counter (PerkinElmer, Waltham, MA).
The kinase activity of RIPK3 (human, recombinant, expressed by baculovirus in Sf9 insect
cells) was detected in RIPK1 buffer with 0.1 µg/µl of MBP as substrate following the P81
phosphocellulose assay described here before.
Kinase binding assays
Binding assays were performed by DiscoverX (Freemont, CA, USA). These assays are based
on a competition binding assay that quantitatively measures the ability of a compound to
compete with an immobilized, active-site directed ligand. See Fabian et al., 2005 for details
on this assay technology [48]. In brief, streptavidin-coated magnetic beads were mixed with
biotinylated small-molecule ligands to generate affinity resins for the assay. After blocking,
binding reactions were assembled by combining DNA tagged kinases, inhibitors and liganded
affinity beads. The ability of the tested compound to compete with the immobilized ligand is
measured via quantitative PCR of the DNA tag. The percent inhibition was calculated by
measuring the amount of kinase captured on the solid support as a function of the test
compound concentration. In this study, binding assays were used for (i) the analysis of
selectivity against a large panel of kinases (service « KINOMEscan ») and for (ii) the
determination of binding constant (Kd) of small chemical compound for RIPK1 (service
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« KdELECT »). Dissociation constants (Kds) are calculated by measuring the amount of
kinase captured on the solid support as a function of the test compound concentration with
top concentration = 30,000 nM.
Analysis of cell death and caspase-3 activation
MEFs were seeded at 7,500 cells per well in 96-well plate. The next day, cells were pre-
treated with the indicated compounds for 30 min and then stimulated with human TNF (600
IU/ml) in combination with 1 µM TAK1i with or without 20 µM z-VAD-fmk in the presence
of 5 µM SytoxGreen (Invitrogen) and 20 µM DEVD-MCA (PeptaNova GmbH, Sandhausen,
DE). SytoxGreen intensity and caspase-3 activation were measured at intervals of 1 hour
using a Fluostar Omega fluorescence plate reader (BMG Labtech GmbH, Ortenberg, DE),
with excitation/emission filters of 485/520 nm for SytoxGreen and 360/460 nm for DEVD-
MCA, gains set at 1,100, 20 flashes per well, and orbital averaging with a diameter of 3 mm.
Percentage of cell death was calculated as (induced fluorescence – background fluorescence)/
(maximal fluorescence – background fluorescence) x 100. The maximal fluorescence is
obtained by full permeabilization of the cells using Triton X-100 at a final concentration of
0.1 %. The caspase-3 activity was calculated as (induced DEVD-AMC fluorescence -
background fluorescence) x 100/ (SytoxGreen fluorescence obtained from the Triton X-100
permeabilized cells).
ATP concentration measurement
U937 and L929 sAhFas were seeded at 2.104 cells/well in white 96-well plate for 24 h. U937
cells were pretreated with z-VAD-fmk (30 µM) for 30 min and then treated for 20 h with
TRAIL-Flag (200 ng/ml) and 2 µg/ml M2 anti-Flag antibody. L929 sAhFas cell line were
pretreated with z-VAD-fmk (20 µM) for 30 min and then treated with mouse TNF (10 ng/ml)
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or Ig-FasL (200 ng/ml) for 16 h. Concentration of cellular ATP was measured using
CellTiter-Glo® Luminescent Cell viability assay (Promega) according to the manufacturer’s
instruction and as previously described [22]. ATP concentration was expressed as %/NT ±
SD.
Molecular docking
In order to complete the RIPK1 structure available from PDB structures, we performed
homology modeling of full RIPK1 protein, notably for the completion of the missing residues.
The selected template was ITHJ_B as only loop 177REVDGTAKKNGG188 is missing in this
structure. Homology modeling did not seem reliable because it did not propose any
structuration of this loop despite PSSPred results predicting that a majority of residues should
be involved in alpha-helix secondary structure. PEPFold ab-initio method allowed us to model
the loop efficiently and finally enabled us the completion of the RIPK1 model for molecular
docking computations. Next, a Normal Mode Analysis (NMA) was achieved with ElNemo
program to modulate the RIPK1 conformations available from the initial model, improving the
variety of protein structures that will be used during docking by the addition of two more
models for RIPK1 after modes selection (following ElNemo standard validation of essential
modes). The final three models of RIPK1 were used as biological target (receptor) for the
exploration of possible association modes with compounds Nec-1s, Sib and Sib-i. Docking
calculations were performed by using the standard protocol proposed in the VINA
implementation of YASARA suite (global docking, 1000 runs per target / per compound).
Results have been clusterized thanks to the g cluster command of the Gromacs program
(Jarvis-Patrick method). After ranking of the poses, the best positioning of the ligand towards
the receptor (energy criteria) was submitted to a short molecular dynamics simulation in water
for 10 nanoseconds in order to improve the model of compound-RIPK1 complex. Final models
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were obtained after energy minimization after checking that the ligand was stable during the
MD trajectory. Ligand-contacting residues were detected using a 3.5Å cut-off distance and
pharmacophores were derived from a standard analysis by using Ligand Scout program.
Concanavalin A-induced hepatitis
Seven to nine-week-old female C57Bl/6 WT were purchased from Janvier (Le Genest St Isle,
FR). Con A was provided by Sigma Aldrich and diluted at 12 mg/kg in PBS/5% DMSO
(vehicle). C57Bl/6 WT mice were pretreated for 1 hour with i.p. administration of 3 mg/kg
Sib or 6,25 mg/kg Nec-1s diluted in vehicle before retro-orbital administration of 12 mg/kg
Con A for 24 hours. At the end of treatment, mice were sacrified (vehicle, n=5; Sib, n=4;
Nec-1s, n=4; vehicle+Con A, n=10; Sib+Con A, n=13; Nec-1s+Con A, n=10). All animals
received human care and all study protocols comply with the institution’s guidelines. Authors
were authorized to lead animal experimentation by "La direction des Services Vétérinaires"
(license M Samson #A3523840), the project was authorized by the "Comité Régional
d’Ethique d'Expérimentation Animal CREAA (license given by the "Ministère de l'Education
Nationale et de la Recherche" # 7576).
At the end of the experiment, mice body weights were measured and fragments of mouse
livers were fixed in 4% paraformaldehyde and embedded in paraffin for Hematoxylin/Eosin
staining. Serum aspartate aminotransferase (AST) and alanine aminotransferase (ALT) were
measured in mouse serum according to the IFCC primary reference procedures and using the
Olympus AU2700 Autoanalyser (Olympus France, Rungis, FR). The percentage of liver
necrosis was determined using image analysis software (NDP view, Hamamatsu). The total
liver area taken into account was comprised between 18 and 51 mm2.
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Statistical analyses
Data presented were acquired from a minimum of three independent experiments. They are
expressed as means ± SD or ± SEM. Statistical analyses for in vitro studies were performed
with unpaired t tests. For in vivo studies, Mann-Whitney U test was used for comparison of
control group parameters with treatment group. All statistical analyses were performed with
GraphPad Prism6 software (GraphPad Software, San Diego, CA, USA).
Acknowledgements
Dr. Sandrine Ruchaud is warmly acknowledged for her continuing support and critical
reading of the manuscript. We would like to thank Béatrice Foll-Josselin for cell culture and
Mathieu Bertrand and Kathrin Weber for helpful scientific advices. The authors also thank
the Cancéropôle Grand-Ouest (axis: Nature sea products in cancer treatment), GIS IBiSA
(Infrastructures en Biologie Santé et Agronomie, France) and Biogenouest (Western France
life science and environment core facilty network) for supporting KISSf screening facility
(Roscoff, France). We also thank the animal house facilities and the immunohistology
platform (Biosit, Rennes). Michel Samson, Stéphane Bach and Marie-Thérèse Dimanche-
Boitrel were supported by INCA PLBIO2012 (“NECROTRAIL” Program). MT Dimanche-
Boitrel’s group was also supported by grants from Conseil Régional de Bretagne (ID2 Santé),
and the Ligue Contre le Cancer (committees Ille et Vilaine and Maine et Loire). F Faurez was
supported by INCa (post-doctoral fellow). M Bonnet was supported by the European
University of Brittany (Chaire d’excellence). Research in the group of Prof. P. Vandenabeele
is supported by grants from the Vlaams Instituut voor Biotechnologie (VIB), from Ghent
University (MRP, GROUP-ID consortium), grants from the 'Foundation against Cancer'
(2012-188 and FAF-F/2016/865), grants from the Fonds voor Wetenschappelijk Onderzoek
Vlaanderen (FWO) (FWO G.0875.11, FWO G.0A45.12N, FWO G.0787.13N, FWO
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G.0C37.14N, FWO G.0E04.16N), grants from the Flemish Government (Methusalem
BOF09/01M00709 and BOF16/MET_V/007), a grant from the Belgian science policy office
(BELSPO)(IAP 7/32). This work is part of shared PhD ship between universities of Rennes
and Ghent.
Author contributions
FLC designed, performed an analyzed in vitro and in vivo data. CD designed, performed and
analyzed in vitro data and protein kinase production and phosphorylation. SPL designed,
performed and analyzed in vitro data. AF designed, performed and analyzed in vivo data. AC
synthetized the compounds and characterized them by RMN and mass spectrometry. OD
conducted in silico molecular modeling and docking analyses. ND was in charge of cell
culture, MTS viability assay and autophosphorylation assay. BB was in charge of protein
kinase purification and phosphorylation assays. IG was in charge of cell culture and MTS
assay. CPP designed, performed and analyzed in vivo data. FF designed and performed in
vitro data. MB designed and performed in vivo data. YM was the chemist who synthetized
the compound sibiriline for the first time. PG analyzed results of chemical synthesis and
revised the manuscript. MS designed, performed and analyzed in vivo data. PV revised the
manuscript. SB and MTDB participated in the study, design and coordination. All the authors
read and approved the final manuscrit submitted for publication.
Conflict of Interest
The authors declare no conflict of interest.
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