Identification of Quinoline-Based RIP2 Kinase Inhibitors with an Improved Therapeutic Index to the hERG Ion Channel

Article abstract of DOI:10.1021/acsmedchemlett.8b00344

RIP2 kinase was recently identified as a therapeutic target for a variety of autoimmune diseases. We have reported previously a selective 4-aminoquinoline-based RIP2 inhibitor GSK583 and demonstrated its effectiveness in blocking downstream NOD2 signaling in cellular models, rodent in vivo models, and human ex vivo disease models. While this tool compound was valuable in validating the biological pathway, it suffered from activity at the hERG ion channel and a poor PK/PD profile thereby limiting progression of this analog. Herein, we detail our efforts to improve both this off-target liability as well as the PK/PD profile of this series of inhibitors through modulation of lipophilicity and strengthening hinge binding ability. These efforts have led to inhibitor 7, which possesses high binding affinity for the ATP pocket of RIP2 (IC₅₀ = 1 nM) and inhibition of downstream cytokine production in human whole blood (IC₅₀ = 10 nM) with reduced hERG activity (14 μM).

Full text of DOI:10.1021/acsmedchemlett.8b00344

Letter
pubs.acs.org/acsmedchemlett
Cite This: ACS Med. Chem. Lett. XXXX, XXX, XXX−XXX
Identification of Quinoline-Based RIP2 Kinase Inhibitors with an
Improved Therapeutic Index to the hERG Ion Channel
Pamela A. Haile,*,† Linda N. Casillas,† Michael J. Bury,† John F. Mehlmann,† Robert Singhaus, Jr.,†
Adam K. Charnley,† Terry V. Hughes,† Michael P. DeMartino,† Gren Z. Wang,† Joseph J. Romano,†
Xiaoyang Dong,† Nikolay V. Plotnikov,† Ami S. Lakdawala,† Maire A. Convery,‡ Bartholomew J. Votta,†
David B. Lipshutz,† Biva M. Desai,† Barbara Swift,† Carol A. Capriotti,† Scott B. Berger,†
Mukesh K. Mahajan,† Michael A. Reilly,† Elizabeth J. Rivera,† Helen H. Sun,† Rakesh Nagilla,†
Carol LePage,† Michael T. Ouellette,† Rachel D. Totoritis,† Brian T. Donovan,† Barry S. Brown,†
Khuram W. Chaudhary,† Peter J. Gough,† John Bertin,† and Robert W. Marquis†
†GlaxoSmithKline, Collegeville Road, Collegeville, Pennsylvania 19426, United States
‡GlaxoSmithKline, Gunnels Wood Road, Stevenage, Hertfordshire SG1 2NY, United Kingdom
S
* Supporting Information
ABSTRACT: RIP2 kinase was recently identified as a
therapeutic target for a variety of autoimmune diseases. We
have reported previously a selective 4-aminoquinoline-based
RIP2 inhibitor GSK583 and demonstrated its effectiveness in
blocking downstream NOD2 signaling in cellular models,
rodent in vivo models, and human ex vivo disease models.
While this tool compound was valuable in validating the
biological pathway, it suffered from activity at the hERG ion
channel and a poor PK/PD profile thereby limiting
progression of this analog. Herein, we detail our efforts to
improve both this off-target liability as well as the PK/PD profile of this series of inhibitors through modulation of lipophilicity
and strengthening hinge binding ability. These efforts have led to inhibitor 7, which possesses high binding affinity for the ATP
pocket of RIP2 (IC50 = 1 nM) and inhibition of downstream cytokine production in human whole blood (IC50 = 10 nM) with
reduced hERG activity (14 μM).
KEYWORDS: RIP2, NOD1, NOD2, kinase inhibitor, hERG
crosstalk or synergy.12,13 These data suggest that targeting the
attern recognition receptors (PRRs) are the key sentinels
Pthat drive the innate immune system’s response to invading NOD1/NOD2 pathway with a small molecule inhibitor may
have broad application in the treatment of a wide variety of
inflammatory diseases.
pathogens. While NOD-like receptors NOD1 and NOD2 play a
central role in identifying and mounting a response to bacterial
invasion, dysregulated signaling of these receptors have been
implicated in a variety of inflammatory diseases.1−5 Proximal to
NOD1 and 2 is receptor-interacting-protein 2 kinase (RIP2
kinase), which drives downstream signal transduction following
the activation of either NOD1 or NOD2. We and others have
demonstrated that a RIP2 kinase inhibitor is effective in blocking
signaling downstream of NOD1 and NOD2.6−11 We’ve also
demonstrated that RIP2 kinase inhibition is effective in
modulating spontaneous inflammatory responses in human
disease biopsy tissues obtained from both Crohn’s and ulcerative
colitispatientsillustratingthepotentialforRIP2kinaseinhibitors
in modulating human inflammatory diseases. It is also interesting
to note that stimulation of either NOD1 or NOD2 with their
respectiveagonists, i.e., DAP orMDP, canactsynergisticallywith
TLR agonists to produce enhanced cytokine production,
suggesting that a RIP2 kinase inhibitor may have far broader
therapeutic utility than anticipated based on this receptor
Because of the well-recognized role that both NOD1 and
NOD2 play in innate immune signaling, proinflammatory
cytokine production, and pathogenesis of inflammation, a variety
of approaches to inhibit this pathway have been explored. Early
attempts focusing on the direct inhibition of NOD1 and/or
NOD2 have not been successful in producing viable drug
candidates for a variety of reasons, most notably selectivity,
overall compound developability, and an inability to assay a
compound’s direct binding to either NOD1 or NOD2.14
Recently a variety of groups have explored the inhibition of the
more tractable downstream kinase RIP2, a protein at which both
NOD1 and NOD2 signaling converge. Not unexpectedly, it was
recognized that early kinase inhibitors such as the epidermal
Received: July 27, 2018
Accepted: September 11, 2018
© XXXX American Chemical Society
A
DOI: 10.1021/acsmedchemlett.8b00344
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Letter
growth factor tyrosine kinase inhibitor gefitinib as well as the p38
inhibitor SB203580 were also potent inhibitors of RIP2 kinase
activity.15,16 Recent reports have detailed structure-based
approaches toward the identification of RIP2 kinase inhibitors.9
These compounds are generally somewhat more selective than
those detailed above but still lack the requisite level of kinase
activity that would permit accurate interpretation of biological
phenotypes as well as the ability to progress these compounds on
to more detailed biological characterization, safety assessment
investigations, and, ultimately, man.
basicityoftheN1nitrogenofthe4-aminoquinolinecorewerenot
well tolerated as this modification weakened the critical hinge
bindinginteractionbetweenMet98andthequinazolinetemplate
thereby reducing overall inhibitor affinity. Based on this data a
strategy to reduce both the lipophilicity and aromaticity of
GSK583 was pursued. As a surrogate ahead of assessing our
targeted drug levels against QT prolongation in more complex
models, we targeted compounds with a > 100-fold window
between potencyinhumanwholebloodandactivityinthe hERG
Q-patch electrophysiology assay. In addition to improving the
window to hERG activity in vitro, it was anticipated that this
strategywouldalsohavethebeneficialeffectofreducingpotential
metabolic liabilities associated with GSK583, thereby improving
its overall pharmacokinetic profile and efficacy further widening
the therapeutic window in vivo.
We have reported recently the identification of GSK583, a
potent and selective inhibitor of RIP2 kinase (Figure 1) that was
As previously disclosed, the X-ray co-crystal structure of
GSK583 bound within the ATP binding pocket of RIP2 kinase
revealed a critical hydrogen bonding interaction between N1 of
the indazole and Asp164 of the protein (Figure 2). Additionally,
Figure 1. RIP2 kinase screening hit 1 and the selective inhibitor
GSK583.
derived from high-throughput screening hit 1.6 Analog 1 was
shown to be a potent inhibitor of RIP2 but lacked the requisite
kinase selectivity that would allow for progression into in vivo
models. Structure−activity relationship (SAR) studies aimed at
modifications of the back pocket binding group of 1 led to the
identification of GSK583. GSK583 exhibited an excellent kinase
selectivity profile as well as the ability to modulate inflammatory
responses in both murine in vivo models and ex vivo human
disease biopsy tissues obtained from both Crohn’s and ulcerative
colitis patients. Despite these promising properties GSK583 was
relegated to atool compound as further development was limited
due to a combination of potency versus the hERG ion channel
and a less than optimal pharmacokinetic profile. This
combination led to a PK/PD profile that afforded, at best, a
modest therapeutic window between this off-target interaction
relative to the human whole blood assay IC50 (237 nM). Ideally a
progressable drug candidate would possess a greater than 100-
fold window between the free Cmax at a targeted IC90 of RIP2
kinase inhibition at 24 h versus hERG related QT prolongations
in a rabbit ventricular wedge model.17 This Letter details efforts
to increase overall inhibitor efficacy and therapeutic windows by
simultaneously reducing the off-target interactions with the
hERG ion channel, increasing activity in the human whole blood
assay, and optimizing the overall pharmacokinetic profile of this
inhibitor template.
The deleterious effects of excess lipophilicity in compound
design has been well documented. With this in mind, it was
rationalized that a significant portion of the lipophilic nature of
GSK583 was imparted by the indazole back pocket moiety. This,
in combination with the basicity of the quinoline N1 nitrogen,
were likely responsible for both the interaction of GSK583 with
the hERG channel as well as the suboptimal pharmacokinetic
profile. Indeed, general strategies for reducing hERG activity
include reducing lipophilicity, lowering the pKa of basic
nitrogens, as well as the elimination of aromatic groups.18,19
Extensive SAR studies within the 4-aminoquinoline series of
RIP2 kinase inhibitors revealed that attempts to reduce the
Figure 2. X-ray co-crystal structure of RIP2 kinase inhibitor GSK583.
several weaker interactions with the indazole were observed:
proton-π interactions with Lys47 and a nontraditional hydrogen
bond of the hydrogen at the C7 position with Glu66 (2.5 Å).6,20
These data suggested that modifications that maintained the key
hydrogen bonding interaction with Asp164 while attempting to
remove the lipophilic aromatic moiety of the indazole may
reduce overall inhibitor binding affinity and potency. Azainda-
zole 2 (Table 1) in which a polar nitrogen has been incorporated
into the indazole back pocket served the purpose of reducing
overall lipophilicity relative to GSK583 but at the expense of
inhibitor potency in both the fluorescence polarization (FP) and
whole blood assay formats. Despite the reduction in lipophilicity,
this change did not resultin any appreciable reduction in potency
of 2 against the hERG ion channel relative to GSK583. The
reduction in potency of 2 is likely due to disruption of the
interaction with Glu66 in the back pocket of the RIP2 protein.
Building back lipophilicity without the aromaticity provided the
tetrahydroindazole 3, which also led to reduced activity at RIP2
kinase while maintaining activity against the hERG ion channel.
Complete removal of the aromatic ring of the indazole gave
pyrazole 4, which maintained the energetically favorable
hydrogen bonding interaction with Asp164 of RIP2 while
significantly reducing the overall lipophilicity of the molecule.
However, the reduced potency of 4 could again be attributed to a
loss of the stabilizing interaction with Glu66. Surprisingly, this
modification also maintained potency versus the hERG channel
despite the loss of aromatic character and a significant reduction
in overall lipophilicity. Incorporation of a methyl group on
position5ofthepyrazolebackpocketmoietyof5resultedinonly
B
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determining inhibitor potency. To improve upon the potency of
the 4-aminoquinoline series of RIP2 kinase inhibitors, a strategy
to further strengthen this critical interaction was employed. As
shown in Figure 1, the X-ray co-crystal structure of GSK583
bound within the ATP binding pocket of RIP2 kinase revealed
the single point hinge binding interaction between N1 of the 4-
aminoquinoline core and Met98 of the protein. In order to
strengthen this interaction, substitution of the 4-aminoquinoline
core with electron donating groups that would serve to increase
the hinge binding ability were explored.22 Examination of the X-
ray co-crystal structure suggested that the incorporation of
electron donating groups at the C7 position of the 4-
aminoquinoline template would be best tolerated as this position
is oriented away from the protein toward the solvent front.6 As
shown in Table 3, inhibitor 7, which incorporates the C7
methoxy group, resulted in a 9-fold increase in whole blood
potencyrelativeto6.23 Whilethismodificationresultedinasmall
increase in activity versus the hERG ion channel, which can be
attributed to the increased basicity of the N1 quinoline nitrogen,
the increased potency versus RIP2 kinase led to the desired
greater than 100-fold window between the human whole blood
potency and inhibition of the hERG channel. A variety of
modifications at the C7 position of the 4-aminoquinoline core
was tolerated in terms of binding activity in the fluorescence
polarization assay format (Table 3). However, small groups that
had the ability to donate electron density through resonance had
improvements in cellular activity relative to 6. Compounds with
electron withdrawing substituents (11) or with substituents that
are moderately electron donating through inductive effects (12)
did not give improved cell based activity as observed in
compounds 7−10. While compounds 9 and 10 were potent in
the human whole blood assay and lacked activity against the
hERG ion channel, the added polarity introduced by these
groups were seen to limit the oral pharmacokinetics (data not
shown) of these inhibitors thereby limiting their further
progression.
Patch clamp electrophysiology (QPatch; Sophion Bioscience)
has been regarded as the gold standard assay for assessing ion
channel modulation and the propensity of new chemical entities
to elicit QT prolongation in humans. Although the increase in
selectivity versus the hERG channel in this assay format was
encouraging, further evaluation of the proarrhythmic potential
beyond the QPatch analysis of 7 was warranted. It has been well
established that the hERG patch clamp assay, in isolation, does
notprovidethelevelofpredictivityrequiredtofullydischargethe
risk of a torsade de pointes (TdP) arrhythmia. In order to more
accurately assess these risks in a preclinical in vitro assay, the
proarrhythmic potential of inhibitor 7 was evaluated in the
arterially perfused rabbit left ventricular wedge model.24 In this
assay, four major readouts, QT prolongation (a measure of
ventricular repolarization), transmural dispersion of repolariza-
tion (Tp-e interval), impulse conduction (QRS interval), and
isometric contractile force (ICF), are measured to allow for a
more in depth, nuanced understanding of the proarrhythmic
potential of new chemical entities. Further, a TdP score derived
from QT, Tp-e, and observations of early afterdepolarizations
Table 1. Focused Back Pocket Modifications of GSK583
asmallincrease in potencyin the FP binding assayformatrelative
to 4. While small alkyl substituents at position 5 were unlikely to
maintain the interaction with Glu66 as the aliphatic C−H is less
polar and further removed from Glu66 than that of GSK583,
small alkyl substituents at the 4-position of the pyrazole were
expected to occupy hydrophobic space in the back pocket.
Gratifyingly, dimethylpyrazole 6 possessed good potency in both
the FP and whole blood assays as well as decreased activity at the
hERG channel.21 The steric interactions imparted by the methyl
groups in combination with the overall reduction in the
lipophilicity of 6 is likely responsible for the reduced affinity for
the hERG channel. In combination, this structural modification
has increased the window to hERG activity approximately 5-fold
relative to GSK583 with a 155-fold overall window between the
human whole blood potency and hERG IC50. The increased
activity in the whole blood assay format combined with the
reduced lipophilicity also resulted in an overall increase in ligand
binding efficiency as seen with an increased LipE relative to
GSK583.
The iv and po pharmacokinetics of dimethylpyrazole 6 in the
rat are summarized in Table 2. Following i.v. administration, 6
exhibited low clearance in the rat with a moderate volume of
distribution and a half-life of approximately 4 h. Upon oral
administration, 6 had an oral bioavailability of 82%. In
comparison with GSK583, the dimethylpyrazole 6 showed an
overall improved pharmacokinetic profile with a higher plasma
free fraction, both key components for improved inhibitor
efficacy and lower predicted human dose.
Although the overall pharmacokinetic profile and selectivity
versus the hERG channel of dimethylpyrazole 6 was improved
versus GSK583, a further increase of inhibitor potency would
serve to potentially widen this critical window, which was
required for further progression of this series of inhibitors. As
with most ATP-competitive kinase inhibitors, the hinge binding
interactionoftheinhibitorwiththeproteinisakeycomponentin
Table 2. Pharmacokinetic Profiles of GSK583 and 6 in the Sprague−Dawley Rat
Cmpd
IV/oral dose (mg/kg) Cl (mL/min/kg) Vdss (L/kg) IV T1/2 (h) oral Cmax (ng/mL) oral AUC (ng·h/mL)
%F
free fraction (%)
GSK583
6
1.0/2.0
2.0/2.0
15
12
2.0
2.1
7.0
4.2
260
990
980
3800
39
82
2.6
11.2
C
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Table 3. Modification of the C7 Position of Inhibitor 6
Cmpd
R
RIPK2 FP IC50 (nM)
RIPK2 hWB IC50 (nM)
cLogP
LipE
hERG IC50 (nM)
hERG/hWB fold shift
6
7
8
9
10
11
12
H
OCH3
OCH2CH3
OCH2CH2OH
OCH2CH2OMe
OCF3
6.3
1.3
1.6
2.5
2.5
6.3
2.5
100
11
41
3.3
3.0
3.5
2.1
2.8
4.1
4.3
4.9
5.9
5.3
6.5
5.8
4.1
4.3
15500
14500
>30000
>30000
30000
155
1318
>700
>2000
6000
>100
156
15
5.0
446
154
>30000
23990
Et
Table 4. Summary Data for Inhibitor 7 in Rabbit Cardiac Wedge Preparation (Mean SEM)
control
0.3 μM
1 μM
3 μM
10 μM
desired range
QT (ms)
ΔQT %
Tp-e (ms)
QRS (ms)
ICF (% change)
TdP score
353.6 10.5
360.6 10.8
2.0 1.0
64.2 5.3
34.9 2.8
−9.9 2.1
0.25
366.3 12.2
3.6 1.5
67.9 6.6
35.4 2.8
−17.3 2.8
0.75
376.1 10.2
6.4 1.7
73.3 8.0
35.2 2.4
−22.6 3.4
1.25
386.6 14.5
9.3 1.7
77.0 10.8
36.3 1.6
−28.0 4.5
1.5
Δ < 10%
59.2 4.8
33.9 2.7
0
0
NA
Δ < 10%
Δ < 50%
<2.5
Table 5. PK Parameters for Compound 7 in Rat and Dog, Discrete iv/po Studies, and Predicted Human PK
species
IV/oral dose (mg/kg) Cl (mL/min/kg) Vdss (L/kg) IV T1/2 (h) oral Cmax (ng/mL) oral AUC (ng·h/mL) %F free fraction (%)
rat
dog
1.1/2.0
1.1/2.0
1.5
12
20
6.8
1.1
3.5
1.9
3.1
2.5
6.2
740
330
259
2400
1900
2510
85
96
74
5.5
16.3
9.6
a
human (predicted)
a
Predicted using average human weight of 70 kg.
(ectopic beating) is calculated to quantify the torsadogenic
potential of drugs. The overall effects of 7 with respect to these
cardiac parameters over a concentration range of 0.3 to 10 μM
relative to control are summarized in Table 4. Here, inhibitor 7
produced a concentration-dependent prolongation of QT
interval up to 9.3% at a concentration of 10 μM. While Tp-e
also increased in a concentration-dependent manner, TdP scores
generated from the QT, Tp-e, and the absence of early
afterdepolarizations did not exceed 1.5 at the top dose of 10
μM. A TdP score of ≥2.5 has been observed for compounds
associated with clinical torsadogenic potential.24 In addition, the
effect on QRS was <10%, suggesting a lack of issues related to
impulseconduction, whiletheobserveddecreasesincontractility
fall in line with the expected inherent decrease in contractility
over time in the rabbit ventricular wedge preparation. The
industry accepted standard for a safety window between hERG
IC50 and therapeutic free Cmax plasma concentrations has been
previously reported by Redfern.25 A 30−100-fold window is
regarded as acceptable when taking disease severity and medical
need into account. Given the observation of a 9.3% QT
prolongation at 10 μM in a rabbit left ventricular wedge model
for inhibitor 7 as well as an IC50 of 14.5 μM in the hERG QPatch
assay, a maximum free Cmax of 100 nM (38.8 ng/mL) would be
required to provide a 100-fold window between free drug
concentration and hERG related activities that could lead to QT
prolongation and increased potential for torsades de pointes
arrhythmias.
Evaluationofthepharmacokineticsofanalog7(Table5)showed
that C7 substitution had little effect on the pharmacokinetic
profile relative to 6 (Table 2). Analog 7 demonstrated low
clearance and volume of distribution in the rat with an oral
bioavailability of 85% following solution dosing. In the dog, 7 is a
moderately cleared molecule with a moderately large volume of
distribution and an oral bioavailability of 96%. These
pharmacokinetic parameters were utilized in an in silico model
to predict the human pharmacokinetic profile of inhibitor 7
(Table 5). Based upon the predicted human PK profile and the
potency of inhibitor 7 in the human whole blood assay (IC50 = 10
nM; Table 3), it was determined that a 54 mg twice daily dose
(b.i.d.) would be required to inhibit RIP2 kinase at an average
90% level for a period of 24 h. At this dosing interval, a predicted
plasma Cmax of 259 ng/mL (666 nM) was estimated. Taking into
account the human free fraction of 9.6% (Table 5), the free
plasma Cmax concentration would be predicted to be 24.8 ng/mL
(64 nM). This is well below the 10 μM free Cmax determined in
the rabbit left ventricular wedge required to trigger a
prolongation of the QT interval and potentially lethal
arrhythmias (156-fold shift) and the 14.5 μM activity at the
hERG ion channel (227-fold shift).
In addition to selectivity versus the hERG ion channel
inhibitor, 7 also possessed broad kinase selectivity when tested
against a panel of 299 kinases (see Supporting Information). In
total, 15 of the kinases in the panel were inhibited >70% at a
concentration of 1 μMof inhibitor 7. Not unexpectedly off-target
kinaseinteractionswerepredominantlyobservedtobewithinthe
tyrosinekinasesubfamilyofkinasesstemmingfromeithertheSrc
family kinase or Ephrin receptor subfamily with the exceptions of
To place this improvement in selectivity versus the hERG
channel in the proper therapeutically relevant context, a
prediction of the human pharmacokinetics was conducted.
D
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ABL2 and ZAK kinases, which are inhibited at levels of 81% and
89% inhibition, respectively. BRK, FRK, c-SRC, EPHB4, and
TXK are all inhibited at levels >90% inhibition.
The X-ray co-crystal structure of 7 bound within the ATP
bindingpocketofRIP2kinase(Figure3)revealedittobindinthe
more suitable clinical candidate for a novel target where levels of
inhibitionneededtohaveaneffectoninflammatorydiseaseshave
not yet been defined.
We have detailed previously the discovery of the potent and
kinome-selective RIP2 kinase inhibitor GSK583. Despite
GSK583’s positive attributes, it was not progressed to more
advanced safety studies due to a combination of limiting
properties: potent interaction with the hERG ion channel, less
than optimal pharmacokinetics, as well as subpar efficacy in an in
vivo model of inflammation. In this Letter we have detailed
efforts to modify the GSK583 template to a progressable
compound to be evaluated in advanced safety studies. The
cornerstone of the strategy to identify a more suitable inhibitor
for advancement was to reduce the overall lipophilicity of
GSK583, which was identified as a key contributor to the lack of
selectivity versus the hERG channel as well as the poor
pharmacokinetics and overall in vivo efficacy. Toward this end,
structure-enabled design suggested elimination of the aromatic
ring of the back pocket benzimidazole and introduction of a C7
methoxy group leading to the potent and selective RIP2 kinase
inhibitor 7. These efforts have resulted in an overall increase in
potency in the human whole blood assay, reduced affinity for the
hERG channel, significantly improved pharmacokinetics in
preclinical species, greater efficacy in an in vivo model of
inflammation, and a reasonable predicted human dose. Taken
together, these improvements in both the physical properties of
inhibitor 7 as well as this inhibitor’s overall efficacy now warrant
its evaluation in more advanced safety studies.
Figure 3. X-ray co-crystal structure of 7 bound within the ATP binding
pocket of RIP2 kinase (2.53 Å).
mannerofatypeIkinaseinhibitorwiththehingeinteractionwith
Met98 through the quinoline nitrogen, sulfone interaction with
the Ser25 in the glycine rich loop, and interaction with the
Asp164 of the DFG loop through two water molecules. Each of
these interactions is important for activity as determined through
our previous SAR studies.6
To establish a PK/PD relationship of inhibitor 7 and compare
it with the efficacy of GSK583, dimethylpyrazole 7 was evaluated
in vivo in the acute MDP-driven rat peritonitis model, which has
been previously detailed.6 Following oral administration of
inhibitor 7, the reduction of cytokine levels at 2 h for each dose
group was used to generate an in vivo IC50 value of 19 nM (7.5
ng/mL) (Figure 4). This value is in good agreement with the
ASSOCIATED CONTENT
* Supporting Information
■
S
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acsmedchem-
lett.8b00344.
Experimental details and characterization of compounds
2−12; biological assay details; kinome selectivity data for
compound 7; data collection and refinement statistics and
exemplars of the density for compound 7 (PDF)
Accession Codes
The PDB codes are as follows: 6HMX for RIP2/7 co-crystal
structure.
AUTHOR INFORMATION
Corresponding Author
*E-mail: pamela.a.haile@gsk.com.
■
Figure 4. (a) Concentration−response plot for inhibitor 7 at a single
time point (2 h) over ten doses. (b) Comparison of human whole blood
IC50 values and drug levels of GSK583 and compound 7.
ORCID
Pamela A. Haile: 0000-0003-3950-4890
Notes
measured rat in vitro whole blood IC50 value of 13 nM (5.0 ng/
mL), which in turn is in close agreement with the human whole
blood value of 11 nM (4.3 ng/mL), indicative of coherent
pharmacology across species in vitro and a good correlation
between in vitro and in vivo assays in rats. Putting the human
whole blood IC50 value into context with the drug levels in rat
following a 2 mg/kg oral dose allows for a direct comparison of
tool GSK583 and compound 7. Observed blood levels of
compound 7 are above its human whole blood IC50 over a period
of 24 h while GSK583 blood concentrations fall below its
corresponding human whole blood IC50 after only 4 h. This
comparisonatcomparabledosesindicatesthatmuchlowerdoses
of compound 7 would be needed to achieve targeted inhibition
levels over a 24 h period as compared to GSK583, and thus, a
The authors declare the following competing financial
interest(s): All GSK authors are/were employees and stock-
holders of GlaxoSmithKline when this work was completed.
ACKNOWLEDGMENTS
■
The authors would like to express appreciation to Emma Jones,
Phil Hardwicke, and Robert Leydon for protein production, Julie
Cox for screening support, Ashley Leister and Amy Gibble for
purification support, and Allison Beal for review.
ABBREVIATIONS
NOD, nucleotide-binding oligomerization domain protein;
PRR, pattern recognition receptors; RIP2, receptor interacting
■
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ACS Medicinal Chemistry Letters
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Vucic, D. Disruption of XIAP-RIP2 Association Blocks NOD2-
Mediated Inflammatory Signaling. Mol. Cell 2018, 69, 551−565.
(12) van Heel, D. A.; Ghosh, S.; Butler, M.; Hunt, K.; Foxwell, B. M.;
Mengin-Lecreulx, D.; Playford, R. J. Synergistic enhancement of Toll-
like receptor responses by NOD1 activation. Eur. J. Immunol. 2005, 35,
2471−2476.
protein 2; IBD, inflammatory bowel disease; FP, fluorescence
polarization; clogP, calculated partition coefficient; MW,
molecular weight; LipE, lipophilic ligand efficiency; ie-DAP, G-
D-glutamyl-meso-diaminopimelic acid;MDP, muramyldipetide;
TLR, toll-like receptor; hERG, human ether-a-go-go-related
gene
(13) Fritz, J. H.; Girardin, S. E.; Fitting, C.; Werts, C.; Mengin-
Lecreulx, D.; Caroff, M.; Cavaillon, J. M.; Philpott, D. J.; Adib-Conquy,
M. Synergistic stimulation of human monocytes and dendritic cells by
Toll-like receptor 4 and NOD1- and NOD2-activating agonists. Eur. J.
Immunol. 2005, 35, 2459−2470.
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DOI: 10.1021/acsmedchemlett.8b00344
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