Identification of Potent and Selective RIPK2 Inhibitors for the Treatment of Inflammatory Diseases

Article abstract of DOI:10.1021/acsmedchemlett.7b00258

NOD2 (nucleotide-binding oligomerization domain-containing protein 2) is an internal pattern recognition receptor that recognizes bacterial peptidoglycan and stimulates host immune responses. Dysfunction of NOD2 pathway has been associated with a number of autoinflammatory disorders. To date, direct inhibitors of NOD2 have not been described due to technical challenges of targeting the oligomeric protein complex. Receptor interacting protein kinase 2 (RIPK2) is an intracellular serine/threonine/tyrosine kinase, a key signaling partner, and an obligate kinase for NOD2. As such, RIPK2 represents an attractive target to probe the pathological roles of NOD2 pathway. To search for selective RIPK2 inhibitors, we employed virtual library screening (VLS) and structure based design that eventually led to a potent and selective RIPK2 inhibitor 8 with excellent oral bioavailability, which was used to evaluate the effects of inhibition of RIPK2 in various in vitro assays and ex vivo and in vivo pharmacodynamic models.

Full text of DOI:10.1021/acsmedchemlett.7b00258

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Letter
Identification of Potent and Selective RIPK2
Inhibitors for the Treatment of Inflammatory Diseases
Xiaohui He, Sara Da Ros, John Nelson, Xue-Feng Zhu, Tao Jiang, Barun Okram, Songchun
Jiang, Pierre-Yves Michellys, Maya Iskandar, Sheryll Espinola, Yong Jia, Badry Bursulaya,
Andreas Kreusch, Mu-Yun Gao, Glen Spraggon, Janine E Baaten, Leah Clemmer, Shelly
Meeusen, David Huang, Robert Hill, Vân Nguyen-Tran, John Fathman, Bo Liu, Tove Tuntland,
W Perry Gordon, Thomas Hollenbeck, Kenneth Ng, Jian Shi, Laura Bordone, and Hong Liu
ACS Med. Chem. Lett., Just Accepted Manuscript • DOI: 10.1021/acsmedchemlett.7b00258 • Publication Date (Web): 27 Sep 2017
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Identification of Potent and Selective RIPK2 Inhibitors for the
Treatment of Inflammatory Diseases
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Xiaohui He,* Sara Da Ros, John Nelson, Xuefeng Zhu, Tao Jiang, Barun Okram, Songchun
Jiang, Pierre-Yves Michellys, Maya Iskandar, Sheryll Espinola, Yong Jia, Badry Bursulaya,
Andreas Kreusch, Mu-Yun Gao, Glen Spraggon, Janine Baaten, Leah Clemmer, Shelly
Meeusen, David Huang, Robert Hill, Vân Nguyen-Tran, John Fathman, Bo Liu, Tove Tuntland,
Perry Gordon, Thomas Hollenbeck, Kenneth Ng, Jian Shi, Laura Bordone* and Hong Liu
Genomics Institute of the Novartis Research Foundation, 10675 John Jay Hopkins Drive, San Diego, California
92121
KEYWORDS: RIPK2, NOD2, kinase inhibitors, structure-based drug design
ABSTRACT: NOD2 (nucleotide-binding oligomerization domain-containing protein 2) is an internal pattern recognition
receptor that recognizes bacterial peptidoglycan and stimulates host immune responses. Dysfunction of NOD2 pathway has
been associated with a number of autoinflammatory disorders. To date, direct inhibitors of NOD2 have not been described
due to technical challenges of targeting the oligomeric protein complex. Receptor interacting protein kinase 2 (RIPK2) is an
intracellular serine/threonine/tyrosine kinase, a key signaling partner and an obligate kinase for NOD2. As such, RIPK2
represents an attractive target to probe the pathological roles of NOD2 pathway. To search for selective RIPK2 inhibitors,
we employed virtual library screening (VLS) and structure based design that eventually led to a potent and selective RIPK2
inhibitor 8 with excellent oral bioavailability which was used to evaluate the effects of inhibition of RIPK2 in various in
vitro assays, ex vivo and in vivo PD models.
Activation of NOD2 in myeloid cells by pathogenic bacteria
and genetic variants in NOD21 lead to increased cytokine
secretion suggesting that a NOD2 inhibitor would be beneficial
for auto-inflammatory disorders, such as inflammatory
arthritis,2, 3 early onset sarcoidosis, Blau Syndrome,4-7 multiple
sclerosis8 and allergic inflammation9. However, NOD2 has
been an elusive target for pharmacological inhibition for many
years. Therefore, downstream targets of the pathway can
provide an alternative approach for modulating NOD2 pathway
activity. Receptor-interacting protein kinase 2 (RIPK2, RICK
or CARDIAK), a key downstream modulator of NOD2
signaling possessing serine/threonine and tyrosine kinase
activity, has been recently discovered.10-13 Upon intracellular
bacterial exposure, NOD2 binds to RIPK2 and trigger nuclear
factor κB (NF-κB) mediated cytokine responses. Thus, RIPK2
represents a critical mediator of NOD2 signaling and becomes
a promising target for developing novel chemical inhibitors. A
number of agents have been described to inhibit RIPK2,
including Gefitinib,13 SB203580,14,15 WEHI-345,16 OD36 and
OD38,17 Ponatinib18 and recently GSK58319 (Figure 1). Among
these reported RIPK2 inhibitors, the EGFR tyrosine kinase
inhibitor Gefitinib and the p38 inhibitor SB203580 were
chemical tools used to demonstrate the function of RIPK2 in
NOD pathways but lacked kinase specificity. WEHI-345,
OD36 and OD38 were reported to be more selective RIPK2
inhibitors. WEHI-345 was shown to specifically delay NF-κB
activation upon NOD stimulation and consequently prevent
cytokine production in vitro and in vivo. OD36 demonstrated
inhibition of recruitment of inflammatory cells in an in vivo
peritonitis model. In a separate study,18 three FDA approved
drugs, Ponatinib, Sorafenib, and Regorafenib, also showed
promising results in terms of inhibiting RIPK2 and reducing
inflammation. In the same study, the crystal structure of RIPK2
Figure 1. Selected small molecule RIPK2 inhibitors.
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complexed with Ponatinib revealed an allosteric site that holds atom occupying a small hydrophobic pocket. Importantly, it
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promise for the identification of more selective inhibitors.
GSK583 was reported to be a potent, selective and bioavailable
RIPK2 inhibitor. It demonstrated the role of RIPK2 in
modulation of intestinal inflammation in both murine and
human systems. Collectively, these findings identified RIPK2 a
promising target for the treatment of inflammatory diseases in
which NOD2: RIPK2 activity is aberrantly high (i.e.,
sarcoidosis, Blau syndrome, asthma, multiple sclerosis, and
Crohn’s disease when patients do not harbor the loss-of-
function mutation). Herein we describe a novel RIPK2
inhibitor, which is potent, selective and bioavailable. The
RIPK2 inhibitor identified here significantly decreased pro-
inflammatory cytokine secretion in both cellular and in vivo
models. In addition, we describe phosphorylation of ribosomal
protein S6 in monocytes as a result of NOD2 activation, an
event attenuated upon RIPK2 inhibition. Hence,
phosphorylation of S6 could be potentially used as a biomarker
for predicting the activity of the RIPK2 inhibitors in patients.
was noted that the region around the 5- and 6-positions of the
molecule was open and near the unique Ser25 of RIPK2. We
speculated that chemical modification around this region could
potentially impact potency and kinase selectivity.
Table 1. Early SAR evolution
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RIPK2 ADP
transcreener
IC50 (µM)*
Lyn/Lck IC50
(µM)*
Liver
microsomal ER
(human/mouse)
1.5
NA/9.2
0.92/0.99
<0.21/0.61
0.44/0.86
0.80/0.88
1
2
3
4
0.024
0.038
0.09
0.05/0.13
0.15/0.51
9.7/NA
*Data are the mean of three measurements, SEM < ± 30%.
Table 2. SAR exploration at the 3-position*
Figure 2. (A) VLS confirmed hit 1. (B) Co-crystal structure of
compound 1 in RIPK2 kinase domain (2.9 Å resolution). Hydrogen
bonds are indicated by dashed lines.
Compd
R
RIPK2 ADP
transcreener
IC50 (µM)**
Liver
microsomal
ER m/h
To search for novel selective RIPK2 inhibitors, a virtual
library screen (VLS) was performed with ~11 million of
commercial and Novartis proprietary compounds using a 2D
profile QSAR method developed at Novartis20 and a 3D
pharmacophore search. Hits (~100,000) were then evaluated by
flexible docking (Glide®, Schrödinger, Inc.) to an in-house
obtained RIPK2 protein structure. Approximately 10,000 hits
with best docking scores were then clustered and analyzed. A
subset from VLS were tested for RIPK2 inhibition in a RIPK2
HTRF ADP transcreener assay.21 Various scaffolds with IC50
ranging from nanomolar to micromolar were identified.
2-Cl
>10
NA
5a
4
3-Cl
0.09
0.52
0.46
0.04
0.13
>10
NA
4-Cl
NA
5b
5c
5d
5e
5f
2-MeO
NA
3-MeO
NA
4-MeO
NA
2-CONH2
3-CONH2
4-CONH2
3-OH
NA
0.43
0.16
0.035
0.016
0.18
0.07
0.011
0.005
0.045
0.009
NA
5g
5h
5i
After further evaluation of the hits, including ligand
efficiency (LE), lipophilic efficiency (LiPE), kinase selectivity,
and binding mode, compound 1 was considered for further
characterization (Figure 2A). Although it was a compound with
micromolar activity (IC50 = 1.5 µM), it exhibited attractive
ligand efficiency (LE = 0.32) and lipophilic efficiency (LiPE =
3.5). In addition, the co-crystal structure revealed compound 1
bound to the ATP binding site with DFG motif adopting a
DFG-in conformation (Figure 2B), highlighting compound 1 as
a type I inhibitor with clearly defined interactions with the
protein. As shown in Figure 2B, there is a single hinge
interaction between N1 of compound 1 and the amide from
Met98 in the hinge region of RIPK2. The gate keeper Thr95-
OH acts as a H-bond donor to form a H-bond with the backbone
carbonyl of Glu96 and a H-bond acceptor to form a H-bond with
the amide NH of compound 1. The phenyl ring is twisted out of
plane by ~50˚ and fits into the back pocket with the chlorine
NA
0.84/0.34
0.74/0.4
NA
4-OH
5j
3-CN
5k
5l
3-Cl-4-CONH2
3-MeO-4-OH
3-Cl-4-OH
3-Cl-5-MeO
3-Cl-5-OH
NA
0.82/0.48
0.83/0.56
NA
5m
5n
5o
5p
0.90/0.67
* Refer to Supporting Information for synthetic protocols; **Data
are the mean of three measurements, SEM < ± 30%.
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Early structure-activity relationship (SAR) led to the disco- As listed in Table 2, a series of aromatic derivatives at the 3-
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3
4
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6
7
8
very of compound 2. By adding a t-butyl sulfone group at 5-
position and a methoxy group at 6-position, the potency of 2
was dramatically improved. Meanwhile, the metabolic stability
(liver microsomal extraction ratio) was also improved by
changing the core from pyrazolopyridine to imidazopyridine.
Evaluation of 2 in a small in-house enzymatic kinase profiling
panel revealed activity on many other kinases, especially the
Src family of kinases, such as Lck and Lyn (Table 1).
position with different substitution patterns and electronic
properties were prepared to probe the back pocket. It was noted
that ortho substitutions consistently reduced activity (5a, 5c and
5f), presumably, the larger torsional angle resulting from the
ortho substitutions as well as its clash with the gatekeeper side
chain kept the benzene ring from fitting optimally into the back
pocket. Electron donating groups seemed more favorable than
the electron withdrawing groups (5d vs. 4, 5g and 5k). Both
meta- and para- substituted analogs in general showed more
favorable activities and the two phenol analogs (5i and 5j)
demonstrated the best RIPK2 activities. Building upon these
important findings, a series of disubstituted analogs were
generated to see if there is a synergistic effect (5l to 5p). We
were pleased to see that the disubstituted analogs were much
more potent than their mono-substituted counterparts.
Further analysis of early SAR identified compound 3, an ester
analog of 2. Although compound 3 can not act as H-bond donor
to form H-bond interaction with gate keeper Thr95, its potency
was still comparable to compound 2. This implied that whereas
this H-bond interaction may not determine compound 3 activity
on RIPK2, it did contribute to kinase target specificity. Indeed,
compound 3 activity on Lck and Lyn was significantly reduced
(~ 3 fold). Compound 4, with a simple C-C bond replacing the
amide bond, was thus designed and synthesized. As expected,
compound 4 demonstrated potent inhibitory activity on RIPK2
while its activities over Src family kinases were significantly
reduced (Table 1). A docking study of compound 4 in RIPK2
ATP binding site (Supporting Information, Figure SI-4)
indicated that the chlorobenzene moiety occupied the back
pocket region differently when compared to the hit compound
1, which might contribute to the improved kinase selectivity. A
systematic SAR study of the imidazolepyridine core was carried
out to optimize potency (Supporting Information for synthetic
protocols), as well as ADME-PK properties for in vivo investi-
gations.
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Table 4. Optimization for metabolic stability and clearance*
Compd
R
RIPK2 ADP Liver
Clearance
(ml/min/kg)
mouse***
52
transcreener
microsomal
IC50 (µM)** ER (m/r/h)
Me
Et
0.006
0.63/0.49/0.51
6h
Table 3. Bioisosteres for phenol replacement*
0.005
0.058
0.61/0.54/0.53
NA
42
7a
7b
NA
iPr
0.007
0.007
0.79/NA/0.73
0.54/0.44/0.38
0.78/0.58/0.59
42
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7c
7d
7e
CH2CF3
(CH2)2OMe 0.005
0.008
Compd
R
RIPK2 ADP hPBMC
transcreener IL8 IC50
IC50 (µM)** (µM)**
Liver
microsomal
ER (m /r /h)
0.49/NA/0.55
109
7f
0.003
0.003
0.59/0.45/0.39
0.5/0.19/<0.21
18
26
7g
8
0.20
0.02
0.12
NA
0.13
NA
NA
6a
6b
6c
* Refer to Supporting Information for synthetic protocols; **Data
are the mean of three measurements, SEM < ± 30%; *** Data
obtained from mouse in vivo PK study.
0.95/0.83/0.5
NA
Although compound 5n and 5p showed single digit
nanomolar enzymatic activity, these phenols demonstrated poor
metabolic stability (Table 2) and low oral exposure (5n, F% =
2%; 5p, F% = 0%) in mouse pharmacokinetics (PK) studies,
presumably due to their susceptibility to oxidation and phase II
metabolism.22 A bioisostere approach was then executed to
search for replacement of the phenols in these potent analogs
(Table 3, Supporting Information for synthetic protocols). It
was found that the 6-indazole (6a) and the 5-benzimidazole (6c)
analogs were much less potent compared to their phenol
counterparts (5i and 5j); 5-indazole (6b) was equally potent to
its phenol analog 5j. However, the reduced cellular activity
(MDP-stimulated human peripheral blood mononuclear cell
(hPBMC) IL8 secretion assay) and poor metabolic stability
(liver microsomal ER in mouse, rat and human = 0.95, 0.83 and
0.5 respectively) indicate that 6b is not an ideal phenol
0.021
0.029
0.06
0.032
0.029
0.04
0.5/0.47/<0.21
0.41/NA/<0.21
0.8/0.45/<0.21
6d
6e
6f
0.002
0.006
0.009
0.009
0.74/0.77/0.5
0.63/0.49/0.51
6g
6h
* Refer to Supporting Information for synthetic protocols; **Data
are the mean of three measurements, SEM < ± 30%.
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replacement either. We were encouraged to see that both the 3- contributes to the binding activity while the orientation of the
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4
5
6
7
8
N-acyl-phenyl (6d) and the 2-aminopyridin-4-yl (6e) deriva-
tives exhibited good biochemical and cellular potency as well
as metabolic stability. In addition, consistent to synergistic
effects observed from 5l to 5p, introducing meta- substitution
in the same aromatic ring (6g and 6h) further boosted the
potency by 5-10 folds. Compound 6h, with the best metabolic
stability (liver microsomal ER in mouse, rat and human =
0.63/0.49/0.51), was then chosen for PK study in mice. The
compound demonstrated good bioavailability (81%), but
relatively high clearance (52mL/min /kg).
aromatic ring in the back pocket impacts the kinase selectivity.
The piperazine side chain extends toward the edge of ATP
binding pocket and is exposed to solvent which explains
modification around this region exhibits no impacts on binding
affinity (7a to 7f).
Combining the co-crystal structure information with the
results from the metabolic identification study, we proposed
further strategies to minimize core oxidation. One strategy was
to reduce electron density of the pyridine ring while intensifying
the H-bond interaction with Asp164. This strategy was
exemplified by compound 7g. By replacing Cl with F in the 3-
pyridinyl ring of 7a, 7g improved the potency, the metabolic
stability (liver microsomal ER m/r/h = 0.59/0.45/0.39) and in
vivo clearance (18mL/min /kg).
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To further optimize the metabolic stability and pharmaco-
kinetic profile, a metabolite identification study was conducted
on compound 6h in rodent and human liver microsomes. The
study revealed that O-demethylation at position 7, as well as
oxidation of the aromatic core and at the t-butyl group were the
primary metabolic pathways. Optimization was then carried
out accordingly.
A second strategy for further optimization was to replace the
metabolically labile t-butyl sulfone group with an isopropy
sulfone group. Compound 8 was obtained from this effort
which not only retained the activity of 7g but also demonstrated
a better metabolic stability profile.
A PK study revealed that compound 8 exhibited low
clearance and good oral exposure in mice. Moreover, it
displayed low clearance and high oral bioavailability in rats and
dogs as well (Table 5). The compound also demonstrated dose
proportionality from 10 mg/kg to 300 mg/kg in rat PK studies
(data not shown). Kinase selectivity of 8 was confirmed by a
broad kinase panel profiling (Nanosyn 250 kinases kinome
profile, Supporting Information). At 5µM, 90% percent of
kinases showed <50% inhibition, while at 0.5µM, 98% of
kinases showed <50% inhibition. All of these findings
highlighted compound 8 as a promising tool compound for
mechanistically investigating the role of RIPK2 inhibition in
vivo.
Figure 3. Co-crystal structure of 7f with RIPK2 kinase domain (2.9
Å resolution). Hydrogen bonds are indicated by dashed lines.
Table 5. PK parameters of 8 in mouse, rat and dog.
As exemplified in Table 4, to overcome the O-demethylation
metabolic pathway, the methoxy group was replaced with
various alkoxy groups (Supporting information for synthetic
protocols). All analogs illustrated in Table 4 demonstrated
comparable RIPK2 enzymatic activity except the cyclopropyl
analog 7b, probably due to the unique π character of
cyclopropane C-C bond as compared to normal alkanes.
Different metabolic stabilities were observed, with compounds
7d and 7f improving the most and 7a maintaining a similar
profile to 6h. Both 7c and 7e showed reduced metabolic
stability across species. Murine PK studies of these compounds
revealed that, despite the good in vitro metabolic stability (liver
microsomal ER m/h = 0.49/0.55), 7f showed extremely high
clearance in vivo (109mL/min/kg). Moderate improvement of
clearance were observed for compounds 7a and 7d.
Parameters
Mouse
Rat
Dog
Dosage(iv/po, mg/kg) 2.0/10.0
5.0/20.0
12.6
0.5/2
7.8
CL (ml/min/kg)
Vss(L/kg)
26.1
1.1
1.6
2.0
T1/2 (h)
1.0
3.2
5.0
AUC(h*nM) iv
AUC(h*nM) po
Cmax (nM) po
Oral BA (%F)
3377
7951
3886
47.1
17539
91278
14218
130.1
2844
15391
2343
135.3
X-ray co-crystal structures of 7f complexed to the kinase
domain of RIPK2 was obtained (Figure 3), albeit with relatively
low resolution of 2.9 Å. Similar to compound 1, compound 7f
binds to ATP binding site of the kinase and forms single hinge
interaction with Met98. There is a hydrogen bond interaction
between the sulfone group and the hydroxyl side chain of the
RIPK2’s unique Ser25 residue; another visible interaction is the
hydrogen bond between the pyridyl amino group and the side
chain of catalytic residue Asp164 which is involved in the
hydrogen bond networks with Lys47 and Gly66. We speculated
that the hydrogen bond with the pyridyl amino group
Accordingly, we next evaluated the role of RIPK2 in IL-6
secretion by using mouse bone marrow derived macrophages
(mBMDM). mBMDM were pretreated with the corresponding
RIPK2 inhibitors for 30 minutes, then stimulated with 5 µg/mL
of muramyl dipeptide (MDP, a known activator of the NOD2/
RIPK2 pathway) and
a
very low concentration of
lipopolysaccharides (LPS, 1ng/mL, under this concentration,
LPS alone did not stimulate IL6 secretion, however, it was
necessary to stimulate a significant IL6 signal when combined
with MDP). Compound 7e and 8 effectively suppressed the
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secretion of IL6 in a dose dependent manner with IC50 of 6nM Assessment of the effect of 8 in a rat ex vivo PK/PD model of
1
2
3
4
5
6
7
8
and 12nM, respectively (Figure 4A). In addition, compound 8
demonstrated inhibition of TNFα secretion in both rat and
human whole blood stimulated by MDP (Figure 4B and Figure
4C).
cytokine secretion is shown in Figure 6A. Female Wistar rats
were dosed with 8 at 30 mg/kg. Blood was collected at different
time points (0, 0.5, 3, 5 and 24hr) and challenged ex vivo with
MDP. It was observed that blood samples treated with 8
significantly attenuated MDP-induced cytokines secretion at all
time points.
In an attempt to identify downstream targets for RIPK2, we
employed a mass cytometry method known as CyTOF (Figure
5, see Supporting Information for method details). First, we
treated human peripheral blood mononuclear cell (hPBMC)
with MDP or LPS and identified a number of proteins that were
phosphorylated, including ERK1/2, p38, and S6. We observed
increased phosphorylation of the above proteins specifically in
monocytes and dendritic cells. S6 was the protein that showed
In order to demonstrate pharmacodynamic activity in target
tissues to treat Inflammatory Bowel Diseases such as blood and
gut, rat in vivo challenge studies were carried out with
pretreatment of 8 at 30 mg/kg followed by intraperitoneal
injection of MDP. Blood and colon were collected 4h post MDP
treatment for measurement of cytokine secretion. As illustrated
in Figure 6, compound 8 treatment significantly reversed MDP-
induced pro-inflammatory cytokines in rat colon (Figure 6B).
In blood (Figure 6C), we observed moderate but significant
reduction in IL6 and TNFα.
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Figure 4. RIPK2 inhibitor suppresses cytokines secretion. A)
Compounds 7e and 8 suppress MDP stimulated IL6 secretion in
mouse BMDM; B) Compounds 7e and 8 suppress MDP-stimulated
TNF secretion in rat whole blood; C) Compound 8 suppresses
MDP-stimulated IL8 secretion in human whole blood.
the most profound phosphorylation upon MDP and LPS
treatment. Pre-treatment of hPMBC with the RIPK2 inhibitor
7e for 15 min. blocked S6 phosphorylation only when the cells
were stimulated with MDP but not LPS, suggesting a selective
effect of the RIPK2 inhibitor on pS6 modulation when the
NOD2 pathway is activated.
Figure 6. RIPK2 inhibitor 8 suppresses cytokine release in rat ex
vivo and in vivo models. A) Rats (n=6/group) were dosed with 8 at
30mg/kg, blood was collected at different time points (0, 0.5, 3, 5
and 24hr), plasma concentration of 8 at different time points are
shown in left panel; MDP was added in plasma samples of each
time point and incubated for 23hr to show that 8 inhibits MDP-
stimulated cytokines (IL6 and TNFα) release in all time points. B)
and C) Rats (n=6/group) were dosed with either vehicle or 8 at
30mg/kg followed by intraperitoneal injection of MDP. Colon (B)
was collected 4h post MDP stimulation for measurement of
cytokines (IL6, IL8 and TNFα) secretion; Blood (C) was collected
4h post MDP stimulation for measurement of cytokines (IL6, IL8
and TNFα) secretion. Error bars represent the standard deviation of
6 animals.
In summary, we have identified structurally distinct, potent
and selective RIPK2 inhibitors through extensive structure
based design and SAR exploration. Compound 8 was tested in
multiple in vitro assays and in vivo models to demonstrate its
activity in suppressing cytokine secretion upon activation of the
NOD2:RIPK2 pathway. In addition, we described activation of
the NOD2 pathway led to profound increase in phosphorylation
of S6 in monocytes and this event was reversed by RIPK2
inhibitor 8. These findings will definitely encourage additional
investigations for validation and could potentially provide
convenient biomarker for RIPK2 activity.
Figure 5 Human PBMC CyTOF experiments. Human PBMC were
treated with MDP and LPS respectively, increased phosphorylation
for ERK1/2, p38, and S6 were observed in monocytes and dendritic
cells. RIPK2 inhibitor 7e blocks phosphorylation of ERK1/2, p38
and S6 induced by MDP and not by LPS.
We also confirmed the inhibition of phosphorylation of S6 in
hPBMC by FACS (Supporting Information, Figure SI-1),
further supporting specificity for the NOD2:RIPK2 pathway.
We observed that both 7e and 8 significantly reduced
phosphorylation of S6 only upon MDP treatment.
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(10) Inohara, N.; del Peso, L.; Koseki, T.; Chen, S.; Nunez, G. RICK,
Supporting Information
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Lindqvist, L. M.; Fiil, B. K.; Khakham, Y.; Ko, H. J.; Sandow, J. J.;
Falk, H.; Holien, J. K.; Chau, D.; Hildebrand, J.; Vince, J. E.; Sharp, P.
P.; Webb, A. I.; Jackman, K. A.; Muhlen, S.; Kennedy, C. L.; Lowes,
K. N.; Murphy, J. M.; Gyrd-Hansen, M.; Parker, M. W.; Hartland, E.
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Production. Nat. Commun. 2015, 6, 6442.
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Experiment details are provided for the preparation of 4, 5a-p, 6a-
6h, 7a-h and 8, analytical data, kinome profile data for compound
8, X-ray crystallography experiments of compound 1 and 7f,
docking study of 4 and experimental details for in vitro assay and
in vivo animal models. This material is available free of charge via
the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
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*To whom correspondence should be addressed. Tel: 858-332-
4714/858-332-4747. E-mail: xhe@gnf.org /lbordone@gnf.org
ACKNOWLEDGMENT
The authors would like to thank Paul Calvin and his team
(compound management), David Jones (NMR), John Isbell and
Todd Groessl (DMPK), as well as Jennifer Shaffer for assistance
with crystallization experiments, Ananda Herath for flow
chemistry consultation and staff at the Advanced Light Source,
BL503 (Berkeley, CA) for beamline assistance. We thank Bryan
Laffitte, Jon Loren and Ignacio Sancho-Martinez for critical
reading of the manuscript.
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Identification of Potent and Selective RIPK2 Inhibitors for the
Treatment of Inflammatory Diseases
Xiaohui He,* Sara DaRos, John Nelson, Xuefeng Zhu, Tao Jiang, Barun Okram, Songchun
Jiang, Pierre-Yves Michellys, Maya Iskandar, Sheryll Espinola, Yong Jia, Badry Bursulaya,
Andreas Kreusch, Mu-Yun Gao, Glen Spraggon, Janine Baaten, Leah Clemmer, Shelly
Meeusen, David Huang, Robert Hill, Vân Nguyen-Tran, John Fathman, Bo Liu, Tove Tuntland,
Perry Gordon, Thomas Hollenbeck, Kenneth Ng, Jian Shi, Laura Bordone* and Hong Liu
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