Targeting the MKK7-JNK (Mitogen-Activated Protein Kinase Kinase 7-c-Jun N-Terminal Kinase) Pathway with Covalent Inhibitors

Article abstract of DOI:10.1021/acs.jmedchem.9b00102

The protein kinase MKK7 is linked to neuronal development and the onset of cancer. The field, however, lacks high-quality functional probes that would allow for the dissection of its detailed functions. Against this background, we describe an effective co

Full text of DOI:10.1021/acs.jmedchem.9b00102

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Brief Article
Targeting the MKK7–JNK (Mitogen-Activated Protein Kinase Kinase
7–c Jun N-Terminal Kinase) Pathway with Covalent Inhibitors
Patrik Wolle, Julia Hardick, Shane J.F. Cronin, Julian Engel, Matthias
Baumann, Jonas Lategahn, Josef Penninger, and Daniel Rauh
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.9b00102 • Publication Date (Web): 15 Feb 2019
Downloaded from http://pubs.acs.org on February 16, 2019
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Journal of Medicinal Chemistry
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Targeting the MKK7–JNK (Mitogen-Activated Protein Kinase Kinase 7–
c-Jun N-Terminal Kinase) Pathway with Covalent Inhibitors
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Patrik Wolle,1,2,‡ Julia Hardick,1,2,‡ Shane J.F. Cronin,3 Julian Engel,1,† Matthias Baumann,4
Jonas Lategahn,1,2 Josef M. Penninger,3 and Daniel Rauh1,2,*
1Faculty of Chemistry and Chemical Biology, TU Dortmund University, Otto-Hahn-Strasse 4a, 44227 Dortmund (Germany).
2Drug Discovery Hub Dortmund (DDHD) am Zentrum für integrierte Wirkstoffforschung (ZIW), 44227 Dortmund
(Germany).
3Institute of Molecular Biotechnology, Austrian Academy of Sciences, Dr. Bohr Gasse 3, AT-1030 Vienna (Austria).
4Lead Discovery Center GmbH, Otto-Hahn-Strasse 15, 44227 Dortmund (Germany).
KEYWORDS: mitogen-activated protein kinase kinase 7 (MKK7), click chemistry, drug discovery, covalent inhibitor.
ABSTRACT: The protein kinase MKK7 is linked to neuronal development and the onset of cancer. The field, however, lacks high-
quality functional probes that would allow for the dissection of its detailed functions. Against this background, we describe an
effective covalent inhibitor of MKK7 based on the pyrazolopyrimidine scaffold.
disease, rheumatoid arthritis and inflammatory diseases.16
INTRODUCTION
MKK7 is activated by several MAP3 kinases and in turn
The development of highly potent and selective chemical
phosphorylates JNK together with MKK4.17 In contrast to
probes to determine the function of proteins in cells is
MKK4, which can also phosphorylate p38 MAPK, MKK7
absolutely necessary to study their role in cellular signaling.
phosphorylates only the different isoforms of JNK.18 The exact
Many other ways to explore the function of a protein are
role of MKK7 in different cellular processes is still a topic of
possible, but most of them are less efficient or not specific.1
current research. For example, it seems to be an important part
Therefore small molecule modulators, with a specific target
of neuronal development and is involved in the generation and
and high potency can be used to look deeper into the function
growth of dendrites.19 It was also found to be involved in some
of single proteins, for example protein kinases, which are
special cases of cancer development.20 So far only few
highly involved in cellular signaling.2
inhibitors are published to target MKK7, and most of them
The human kinome comprises about 500 protein kinases,3
inhibit MKK7 as an off-target. Sogabe et al. showed a
which renders the design of selective kinase inhibitors a major
derivative of hypothemycin – a known natural product
challenge due to the highly conserved ATP pocket.4 One
addressing a cysteine right before the DFG-motif in several
promising approach is to target cysteines with reactive groups
that form a covalent linkage.5 The covalent bond formation
leads to higher residence time and therefore an increase in
potency and selectivity.5, 6 Although covalent inhibitors have
been avoided in medicinal chemistry in the past, given
concerns regarding their potential for off-target reactivity and
immune responses,7 this approach was recently successfully
exemplified in covalently targeting protein kinases such as
EGFR (epidermal growth factor receptor) and BTK (Bruton’s
tyrosine kinase) with clinically approved drugs, osimertinib,8-
kinases,21 among them MEK1 (Cys276) – to bind covalently to
Cys218 in MKK7 and provokes an IC50 of 1.3 µM.22 More
recently, Shraga et al. identified small fragments through
docking endeavors, that covalently bind to MKK7 and inhibit
cellular JNK activity with EC50 values in the micromolar
range.23 Therefore, further research requires the development
of highly potent and selective probes to gain further insight in
to MKK7 biology.
We recently reported pyrazolopyrimidine-based inhibitors
that target gatekeeper-mutant (T790M) drug resistance in
EGFR,24, 25 and were able to solve their crystal structure.24 The
structure of apo MKK7 is known, and the alignment of MKK7
apo with the ligand bound EGFR structure shows high
similarity regarding the ATP binding pocket. Both kinases
carry a methionine gatekeeper, a reactive cysteine at the end of
the hinge region as well as the conserved catalytic lysine
10
afatinib,11, 12 and ibrutinib (1)13, 14. These address a cysteine
at the end of the hinge region connecting the N-terminal and
C-terminal part of the kinase domain. In the entire kinome
only 11 kinases bear a cysteine at this position (Figure S1).
One of these kinases is MKK7 (Figure 1a), a member of the
mitogen-activated protein kinase family.15 It is part of the
c-Jun N-terminal kinase (JNK) pathway, which is involved in
stress signaling, for example. JNK’s were identified in
numerous human diseases, like Parkinson’s and Alzheimer’s
(Figure 1
and
Figure S1).
Furthermore,
the
pyrazolopyrimidine-based inhibitor of BTK, ibrutinib (1), was
reported to exhibit MKK7 off-target activity.26 We therefore
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considered pyrazolopyrimidines well-suited as ligands of (Figure 2a) and therefore, we synthesized the derivatives 4–6
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8
MKK7 that could alkylate the analogous cysteine with a
Michael acceptor system.
that were believed to occupy the observed pocket. This
focused series of triazole-containing compounds showed
mixed results when they were tested in the biochemical assay
(Table 1). The para-keto benzene derivative 4a set the lead
with an IC50 value of 10 nM against MKK7, provoking a 4-
times increased potency as compared to the initial compound
3a. It is worth noting that 4a markedly improved the
selectivity towards EGFR and its mutant variant. The
derivatives 5 and 6, which showed moderate inhibition (788
and 669 nM, respectively), seemed not to fit properly into the
backpocket. As a reference, we tested ibrutinib, an approved
inhibitor of BTK, which inhibited MKK7 with an IC50 of
78 nM. We synthesized the reversible counterpart 4b of the
most potent inhibitor 4a. Subsequent testing revealed a
complete loss of activity against MKK7 which led to the
conclusion that these compounds form a covalent bond to
Cys218, that is inevitable for inhibitory activity.
Here, we present the structure-based design, synthesis and
evaluation of a pyrazolopyrimidine-based covalent inhibitor to
specifically target MKK7. Mass spectrometry demonstrated
the covalent bond formation and subsequent X-ray structures
yielded insight into the binding mode, as well as the final
evidence for targeting Cys218 in MKK7. Biochemical and
cellular characterization revealed the inhibitory potency and
kinome profiling as well as pharmacokinetic analysis
substantiate our approach.
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Table 1. Half-maximal inhibitory concentrations (IC50)
determined for 4-aminopyrazolopyrimidine-based inhibitors
1–6 against MKK7 and EGFR.a
Figure 1. Similarity between the binding site of MKK7 and
EGFR-T790M. (A) Sequence overlay of MKK7-wt and EGFR-
T790M which share the same cysteine in the ATP binding site.
(B) Deeper insight into the ATP binding pocket to illustrate the
high similarity between the EGFR structure bound
pyrazolopyrimidine based compound 2 (PDB ID: 5J9Z; left) and
MKK7 apo protein (PDB ID: 2DYL; right). Important amino acid
sidechains are highlighted – catalytic lysine (purple), methionine
gatekeeper (cyan) and the reactive cysteine (red).
IC50 [nM]
Compound
EGFR-
MKK7b
EGFR-WTc
RESULTS
L858R/T790Mc
Rational Design and Biochemical Characterization of
Pyrazolopyrimidine-Based MKK7-Inhibitors. We took
advantage
302  104
40  8
35  14d
1489  659d
8006  3453
649  129
6  1
3  1d
693  336d
>10000
677  52
28 ± 9
2
of
the
previously
described
3a
4-aminopyrazolopyrimidine EGFR inhibitor 2,24 which
showed a high inhibitory effect on EGFR and its mutants and
moderate potency on MKK7 (Table 1). Structural alignment of
2 in complex with EGFR and MKK7 showed the already
mentioned high similarity of the two binding sites as well as
some space in the backpocket for further compound design
and optimization (Figure 1). Therefore, compound 3a served
as a suitable starting point since it provides an alkyne as the
derivatization point for copper-catalyzed click-chemistry.25
Furthermore, compound 3a showed a significant inhibitory
effect against MKK7 in biochemical assays. We were able to
gain a co-crystal structure of the kinase in complex with 3a
and verified the additional space in the backpocket of MKK7
between the Met212-gatekeeper and the catalytic Lys165
4a
10  3
4b
>10000
788  172
669  161
78  21
5
6
85  6
104  16
40  2
ibrutinib (1)
<1
aValues are the mean ± SD of three independent measurements
in duplicates. bDetermined utilizing an ADP-Glo assay.
cDetermined utilizing an HTRF assay. dPreviously reported
results24, 25
.
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Kinetik and MS-Based Characterization of Covalent
inhibition against only seven off-target kinases (BLK, BMX,
BTK, ITK, JAK3, mTOR and S6K) which contain the reactive
cysteine residue isostructural to Cys218 in MKK7, with S6K
being the only exception.
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2
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4
5
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7
8
Bond Formation and X-Ray Co-Crystal Structure
Determination. To further characterize the covalent character
of the bond formation, we determined the binding kinetics of
the two-step process of (i) reversible alignment and (ii)
subsequent alkylation for compound 4a. We noticed that the
inhibitory potency was dependent on the reaction time and that
at least 10 min were required to gain full potency against
MKK7. Although we were not able to derive the kinetic values
Ki and kinact due to high fluctuations at a short preincubation
time, a qualitative assessment of the bond formation was
gained (Figure S2). In order to define the covalent binding
mode and to obtain a deeper insight into the structural
organization within the kinase domain we performed mass
spectrometry experiments, followed by MS/MS studies, and
finally crystallization of 4a in complex with MKK7.
Incubation of 3a and 4a with the MKK7 kinase domain
resulted in a mass increase corresponding to complete labeling
with the covalent inhibitors (Figure S3). MS/MS studies
confirmed the covalent modification of compounds 3a and 1 at
the intended Cys218 (Figure S4). Furthermore, the obtained
crystal structures verified that both inhibitors 3a and 4a
formed a covalent bond to Cys218 at the end of the hinge
region (Figure 2). The acrylamide provided good orientation
and geometry toward the reactive cysteine, forming a
hydrogen bond to Lys221 for inhibitor 3a, which was not seen
in the structure of 4a, because of missing electron density. The
4-aminopyrazolopyrimidine core also formed two direct
hydrogen bonds with the peptide backbone of the hinge
region.
Moreover, we analyzed compounds 4a and 4b in cellular
studies to evaluate their influence on the phosphorylation of
JNK and its downstream signaling (Figure 3, Figure S6). 4a
revealed potent inhibition of cellular JNK at a concentration of
1 µM and completely inhibited its downstream effector cJun.
Compound 4b showed no effect on the JNK-pathway and
thereby demonstrated the already described assumption that
covalent bond formation is necessary for activity.
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Figure 3. Western blot analysis of the inhibitory effect of
compound 4a and 4b against the JNK pathway.
Physicochemical and Pharmacokinetic Characterization.
We
also
characterized
the
with
most
respect
potent
to
4-aminopyrazolopyrimidine
4a
physicochemical and in vitro ADME parameters and
compared it to the structurally-related reference 1 (Table 2).
The inhibitor 4a revealed a low lipophilicity with a cLogP
value of 1.78. The determination of the ligand-lipophilicity
efficiency (LLE) allows the estimation of the potency of a
compound with consideration of its lipophilicity (Figure S7).
To increase potency without simultaneously increasing
lipophilicity is a major challenge in compound optimization.
Compound 4a is related to a LLE of 6.22, which is an
acceptable range and is superior to ibrutinib (LLE = 3.48).28
Despite the low lipophilicity of the most active compound, 4a
exhibited a low aqueous kinetic solubility (SolRank) with a
value of 8 µM. This low solubility ranking may explain the
undetectable flux over an artificial membrane (PAMPA
assay). 4a and 1 were further determined in the Caco-2 cell
permeability assay as a cellular model system to investigate
intestinal drug absorption. Both compounds showed a high
cellular apical (A) to basolateral (B) permeability with 20 and
4010-6 cm/s, respectively. Significant lower efflux values of
13 and 0.910-6 cm/s for 4a and 1 predicting good oral
absorption. From the MDCKII-MDR1 cellular permeability
assay, an assay for predicting brain permeation, we concluded
a CNS penetration with an efflux ratio of 0.4. We observed a
good stability in both human and mouse plasma (>70%) and
Figure 2. Crystal structure of MKK7 in complex with (A)
compound 3a and (B) compound 4a. Shown is the electron
density (2FO-FC map contoured at an RMSD of 1) of MKK7/3a
(PDB ID: 6IB0) at a resolution of 2.6 Å and of MKK7/4a (PDB
ID: 6IB2) at a resolution of 2.1 Å. Shown are also the amino acid
sidechains of the hinge region (white) and the α helix C (green).
The QR-codes provide an augmented reality view of 3D models
of the binding sites.27
Kinase Selectivity Profiling and Analysis of Cellular
Potency. These promising results observed with the potent
inhibitor 4a encouraged us to determine its kinase selectivity
profile (ProQinase) at a concentration of 1 µM against a panel
of 320 kinases (Figure S5). Compound 4a displayed an
excellent selectivity profile and showed more than 50%
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also a high plasma protein binding (>93%), features that are in the JNK-pathway. Besides the high clearance in human and
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frequently observed for covalent inhibitors. The potent
inhibitor 4a displayed a moderate microsomal stability in liver
microsomes (154 and 124 µL/min/mg) but obviously
improved as compared to the reference ibrutinib, which
revealed a clearance of 234 and >1000 µL/min/mg in human
and mouse liver microsomes, respectively (Figure S8). Despite
the high clearance in microsomes, the overall favorable profile
of 4a encourages us to investigate its in vivo pharmacokinetic
properties in the future.
mouse liver microsomes, inhibitor 4a demonstrated an overall
good pharmacokinetic profile, thus rendering it a valuable
probe for further investigation to elucidate the function of
MKK7 in cellular studies and in vivo.
EXPERIMENTAL SECTION
Protein Purification. For the crystallization a construct of MKK7
was used containing amino acids 117-423 with a non-cleavable N-
terminal His6-Tag. For the Assay the construct contained amino acids
1-462 with a TEV-cleavable N-Terminal His6-Tag, containing the
activation mutations S273D and T275D. The protein for
crystallization was expressed in BL21 DE3 E. coli at 18 °C for 20 h,
while the protein for the assay was expressed in SF9 insect cells. The
cells were lysed using French press and centrifuged. The supernatant
was then loaded on a Ni-Affinity Chromatography (Qiagen Ni-NTA
Superflow 5mL). Washed with buffer A (50 mM Tris, 500 mM NaCl,
25 mM Imidazole, 5% glycerol, pH 8) and eluted with a gradient of
buffer B (50 mM Tris, 500 mM NaCl, 500 mM Imidazole, 5%
glycerol, pH 8). The protein was concentrated, and then applied to a
gel filtration chromatography (GE HiLoad 16/600 75pg) using buffer
C (25 mM Tris, 100 mM NaCl, 10% glycerol, pH 7.4). The eluted
protein fractions were combined and concentrated to 10 mg/mL and
then used for crystallization.
Protein crystallization and Data collection. Crystallization of
MKK7 with Inhibitor 3a and 4a was performed by incubating
10 mg/mL MKK7 with a threefold molar excess of inhibitor (10 mM
DMSO stock) for 1 h at 4 °C. Crystals were grown using the hanging
drop method at 20 °C after mixing 1 μL protein-inhibitor solution
with 1 μL reservoir solution (180-220 mM Sodium Citrate, 15-25%
PEG3350). All crystals were frozen with further addition of 20% (v/v)
glycerol. The data sets were collected at the PX10SA beamline of the
Swiss Light Source (PSI,Villingen, Switzerland). All data sets were
processed with XDS29 and scaled using XSCALE29. Structure
Determination and Refinement of the complex crystal structures were
solved by molecular replacement with PHASER30 using PDB ID:
2DYL as template. The MKK7 molecule in the asymmetric unit was
manually adjusted using the program COOT31. The refinement was
performed with REFMAC532. Inhibitor topology files were generated
using the Dundee PRODRG2 server33. Refined structures were
validated with PROCHECK33 and the PDB validation server. Data
collection, structure refinement statistics, PDB ID codes, further
details for data collection are provided in Table S1. PyMOL34 was
used for generating the figures.
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Table 2. Physicochemical and in vitro pharmacokinetic
parameters for 4a and 1 as a reference compound.
4a
1.78
6.22
8
ibrutinib (1)
cLogPa
LLEb
3.63
3.48
36
SolRank [µM]
PAMPA [% Flux]
n.d.
38
Caco-2 Papp (A–B)
20
40
0.9
[10-6 cm/s]
Caco-2 Papp (B–A)
[10-6 cm/s]
13
Caco-2 ratio Papp
(B–A)/(A–B)
0.6
0.02
MDCKII-MDR1 Papp (A–B)
9
4
22
[10-6 cm/s]
MDCKII-MDR1 Papp (B–A)
[10-6 cm/s]
3
MDCKII-MDR1 ratio Papp
(B–A)/(A–B)
0.40
0.13
Plasma Stability
human / mouse [% Remain]
>99 / 70.4
96.0 / 92.7
79.7 / 97.5
97.6 / 98.9
Plasma Protein Binding
human / mouse [% Bound]
Microsomal Stability
(Phase I) CLint
154 / 124
234 / >1000
human / mouse [µL/min/mg]
ADP-Glo Assay. The ADP-Glo Assay was performed according to
the instructions provided by the supplier with 20 nM Kinase (MKK7
beta 1, S273D T275D) and 5 µM Substrate (JNK1 K55M).
The dilution of the compound was done with the Echo 520
(LabCyte) acoustic pipetting robot and the liquid handling was
performed with a multidrop (Thermo Scientific). The readout was
performed with a Tecan M1000 infinite plate reader.
acLogP was calculated with Seurat (consensus model using
ChemAxon and Klopman’s models and the PhysProp database).
bLigand lipophilicity efficiency (LLE) was calculated from
pIC50(MKK7) – cLogP. n.d. = not detectable.
Mass spectrometry. The MS experiments were performed on a
VelosPro IonTrap (Thermo Scientific). The protein was diluted to a
concentration of 1 mg/mL and combined with 2-fold molar excess of
the compound and incubated for 30 min on ice. Then the solution was
centrifuged, and the supernatant was then used for the measurements.
The measurement was performed with an EC 50/3 Nucleodur C18
1.8µm column (Macherey and Nagel) and gradient of the mobile
phase A (0.1% formic acid in water) to B (0.1% formic acid in
acetonitrile).
For the spectra’s visualization, XCalibur and MagTran were used.
For the MS/MS experiments the protein was incubated with the
compound as described before. Then denaturized using SDS-sample
buffer (50 mM Tris-HCl) pH 6.8, 2% SDS, 10% glycerol, 1% beta-
mercaptoethanol, 12.5 mM EDTA and 0.02% bromphenol blue) and
then separated via SDS-Gel electrophoresis with a 15% SDS-gel.
The corresponding band was cut out and washed with 200 µL
DISCUSSION AND CONCLUSIONS
In summary, we have developed a potent, selective, and
covalent inhibitor of MKK7, which shows an excellent
biochemical inhibitory effect and selectivity over EGFR and
its mutant variant. A kinase profiling study displayed high
selectivity for only seven off-target enzymes because of the
isostructural cysteine residues in six of these kinases. Mass
experiments revealed the covalent bond formation and further
MS/MS studies confirmed the intended cysteine had been
targeted. X-ray analysis confirmed the predicted binding mode
and provided insight into the binding site for further
compound optimization. Additionally, western blot analysis
showed the high cellular inhibitory effect of 4a on the
phosphorylation of JNK as well as the downstream signaling
solution
A (3:1 25 mM ammonium hydrogen carbonate and
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acetonitrile) for 15 min at 37 °C. The solution was replaced with
concentration of 5 µM after incubation in plasma obtained from
different species for 1 h at 37 °C.
200 µL solution B (1:1 25 mM ammonium hydrogen carbonate and
acetonitrile) and incubated again for 15 min at 37 °C. After removing
the solution, the samples were treated with 100 µL reductive solution
C (50 mM DTT, 25 mM ammonium hydrogen carbonate) for 1 hour
at 37 °C. For the following alkylation the solution was replaced with
1
2
3
4
Plasma protein binding. Assessment of plasma protein binding
was measured by equilibrium dialysis by incubating plasma with the
compound of interest at a concentration of 5 µM for 6 h at 37 °C
followed by LC/MS-based determination of final compound
concentrations.
100 µL solution
D (55 mM Iodacetamide, 25 mM ammonium
5
6
7
8
hydrogen carbonate) and incubated for one hour at 20 °C.
Microsomal stability assay (phase I). Metabolic stability under
oxidative conditions was measured in liver microsomes from different
species by LC/MS-based measuring of depletion of compound at a
concentration of 3 µM over time up to 60 minutes for HLM and
MLM at 37 °C. Based on the compound half-life t1/2, the in vitro
intrinsic clearance CLint was calculated.
Chemistry. All final compounds were purified to ≥95% purity
confirmed by NMR analysis as well as LC/MS analysis. Detailed
synthetic procedures are described in the supporting information.
The alkylation solution was replaced by solution B and washed two
times for 15 min at 37 °C. The solution was removed, and the samples
were dehydrated with 10 µL acetonitrile for 10 min. The acetonitrile
was removed and 40 µL of the digest solution (0.1 mg/mL trypsin,
25 mM ammonium hydrogen carbonate) was added and incubated for
20 hours at 30 °C.
After incubation, 3.5 µL of a 10% TFA solution was added and
incubated for 30 min at 0 °C. The solution was then used for further
experiments.
Cell culture, western blotting and antibodies. DRGs were
collected and dissociated as previously described.35 DRGs were
cultured in laminin-coated 12-well plates for three hours. Media was
replaced, and compounds were then added for a further 2 hours unless
indicated otherwise. The neurons were lysed, and proteins extracted
using RIPA buffer supplemented with phosphatase inhibitors and
Western blots were performed for phosphor-cJun (cell signaling,
3270), cJun (cell signaling, 9165), phospho-JNK (cell signaling,
4668) and JNK (cell signaling, 9252).
SolRank. For determining the kinetic solubility, the compound
was diluted from a 10 mM stock in DMSO to a final concentration of
500 μM in 50 mM Hepes buffer, pH 7.4. Following an incubation of
90 min at room temperature on a shaker, the aqueous dilution was
filtered through a 0.2 μm PVDF filter, and the optical density between
250 and 500 nm was measured at intervals of 10 nm. The kinetic
solubility was calculated from the area under the curve (AUC)
between 250 and 500 nm and normalized to absorption of a dilution
of the compound in acetonitrile.
PAMPA (Parallel artificial membrane permeability assay). The
compound was diluted from a 10 mM stock in DMSO to a final
concentration of 500 µM in 50 mM Hepes buffer pH 7.4 and
transferred onto a transwell membrane covered with a membrane-
forming solution of 10% 1,2-Dioleyl-sn-glycero-3-phosphocholine
(Sigma Aldrich) and 0.5% (w/v) cholesterol (Sigma Aldrich) in
dodecane. Following an incubation of 16 hours at room temperature
in a wet chamber, the optical density of the solution in the receiver
well was measured between 250 and 500 nm in intervals of 10 nm.
The percent flux was calculated from the AUC between 250 and
500 nm and normalized to the absorption of the compound following
a 16 hours incubation in a parallel transwell containing a membrane
covered with 50% MeOH in 50 mM Hepes buffer pH 7.4.36
Caco-2 assay. For Caco-2 cell assay, a 10 mM DMSO stock of the
compound was diluted to a final concentration of 5 μM in HBSS
buffer pH 7.4 and incubated for 2 hours at 37 °C and 5% CO2 on a
monolayer of Caco-2 cells (ATCC) that had been grown on a
transwell membrane (Millipore, Schwalbach, Germany) for 21 days.
The compound concentration was measured in the receiver as well as
the donor well. Apparent permeability (Papp) from either the apical to
basolateral direction or vice versa was calculated by the equation: Papp
= 1/AC0 (dQ/dt), where A is the membrane surface area, C0 is the
donor drug concentration at t = 0, and dQ/dt is the amount of drug
transported within the given time period of 2 hours.
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ASSOCIATED CONTENT
Supporting Information
The Supporting Information is available free of charge on the
ACS Publications website.
Supplementary Tables and Figures, and Experimental Section –
Chemistry (PDF)
Molecular formula strings, MFS (CSV)
Accession Codes
PDB codes for MKK7 bound with 3a and 4a are 6IB0 and 6IB2,
respectively.
AUTHOR INFORMATION
Corresponding Author
*Prof. Dr. Daniel Rauh, Phone: +49 (0)231/755-7080, eMail:
daniel.rauh@tu-dortmund.de, Twitter: @DDHDortmund, Web:
DDHDortmund.de.
ORCID
Patrik Wolle: 0000-0001-6309-6773
Julia Hardick: 0000-0002-0062-1849
Jonas Lategahn: 0000-0001-5993-7082
Daniel Rauh: 0000-0002-1970-7642
Present Addresses
†Lead Discovery Center GmbH, Otto-Hahn-Strasse 15, 44227
Dortmund (Germany).
Author Contributions
‡P.W. and J.H. contributed equally to this work.
ACKNOWLEDGMENT
This work was co-funded by the German Federal Ministry for
Education and Research (NGFNPlus and e:Med) (Grant No.
MDCKII-MDR1. To measure cellular permeability, compounds
were applied at a concentration of 10 µM in HBSS to either the apical
(A) or basolateral (B) side of a MDCKII-MDR cell monolayer and
incubated for 2 hours at 37 °C. Compound concentrations on each
side of the monolayer were determined by LC-MS/MS and the
apparent permeability (Papp) was calculated in the apical to basolateral
(A–B) and basolateral to apical (B–A) directions according to the
following equation: Papp (A–B) = (CB  VB  0.001) / (t  A 
Ct0,A).
BMBF
01GS08104,
01ZX1303C),
the
Deutsche
Forschungsgemeinschaft (DFG), the German federal state North
Rhine-Westphalia (NRW) and the European Union (European
Regional Development Fund: Investing In Your Future) (EFRE-
800400), NEGECA (PerMed NRW), EMODI and Drug
Discovery Hub Dortmund (DDHD).
ABBREVIATIONS
ADME, absorption, distribution, metabolism, and excretion; BTK,
Bruton’s tyrosine kinase; CNS, central nervous system; EGFR,
epidermal growth factor receptor; JNK, c-Jun N-terminal kinase;
Plasma stability. Plasma stability was measured by LC/MS-based
determination of the percentage of remaining compounds at a
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(14) Byrd, J. C.; Furman, R. R.; Coutre, S. E.; Flinn, I. W.;
LLE, ligand-lipophilicity efficiency; MKK7, mitogen-activated
protein kinase kinase 7; PAMPA, parallel artificial membrane
permeability assay.
Burger, J. A.; Blum, K. A.; Grant, B.; Sharman, J. P.; Coleman, M.;
Wierda, W. G.; Jones, J. A.; Zhao, W.; Heerema, N. A.; Johnson, A.
J.; Sukbuntherng, J.; Chang, B. Y.; Clow, F.; Hedrick, E.; Buggy, J.
J.; James, D. F.; O'Brien, S. Targeting BTK with ibrutinib in relapsed
chronic lymphocytic leukemia. N. Engl. J. Med. 2013, 369, 32-42.
(15) Tournier, C.; Whitmarsh, A. J.; Cavanagh, J.; Barrett, T.;
Davis, R. J. The MKK7 gene encodes a group of c-Jun NH2-terminal
kinase kinases. Mol. Cell. Biol. 1999, 19, 1569-1581.
(16) Sabapathy, K. Role of the JNK pathway in human diseases.
Prog. Mol. Biol. Transl. Sci. 2012, 106, 145-169.
(17) Yamauchi, J.; Kaziro, Y.; Itoh, H. Differential regulation of
mitogen-activated protein kinase kinase 4 (MKK4) and 7 (MKK7) by
signaling from G protein beta gamma subunit in human embryonal
kidney 293 cells. J. Biol. Chem. 1999, 274, 1957-1965.
(18) Wang, X.; Destrument, A.; Tournier, C. Physiological roles
of MKK4 and MKK7: insights from animal models. Biochim.
Biophys. Acta 2007, 1773, 1349-1357.
(19) Yamasaki, T.; Kawasaki, H.; Arakawa, S.; Shimizu, K.;
Shimizu, S.; Reiner, O.; Okano, H.; Nishina, S.; Azuma, N.;
Penninger, J. M.; Katada, T.; Nishina, H. Stress-activated protein
kinase MKK7 regulates axon elongation in the developing cerebral
cortex. J. Neurosci. 2011, 31, 16872-16883.
(20) Tornatore, L.; Sandomenico, A.; Raimondo, D.; Low, C.;
Rocci, A.; Tralau-Stewart, C.; Capece, D.; D'Andrea, D.; Bua, M.;
Boyle, E.; van Duin, M.; Zoppoli, P.; Jaxa-Chamiec, A.; Thotakura,
A. K.; Dyson, J.; Walker, B. A.; Leonardi, A.; Chambery, A.;
Driessen, C.; Sonneveld, P.; Morgan, G.; Palumbo, A.; Tramontano,
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Figure 1. Similarity between the binding site of MKK7 and EGFR-T790M. (A) Sequence overlay of MKK7-wt
and EGFR-T790M which share the same cysteine in the ATP binding site. (B) Deeper insight into the ATP
binding pocket to illustrate the high similarity between the EGFR structure bound pyra-zolopyrimidine based
compound 2 (PDB ID: 5J9Z; left) and MKK7 apo protein (PDB ID: 2DYL; right). Important amino acid
sidechains are highlighted – catalytic lysine (purple), methionine gatekeeper (cyan) and the reactive
cysteine (red).
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Figure 2. Crystal structure of MKK7 in complex with (A) compound 3a and (B) compound 4a. Shown is the
electron density (2FO-FC map contoured at an RMSD of 1) of MKK7/3a (PDB ID: 6IB0) at a resolution of 2.6
Å and of MKK7/4a (PDB ID: 6IB2) at a resolution of 2.1 Å. Shown are also the amino acid sidechains of the
hinge region (white) and the α helix C (green). The QR-codes provide an augmented reality view of 3D
models of the binding sites.24
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Figure 3. Western blot analysis of the inhibitory effect of compound 4a and 4b against the JNK pathway.
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