Discovery of type II inhibitors of tgfβ-activated kinase 1 (TAK1) and mitogen-activated protein kinase kinase kinase kinase 2 (MAP4K2)

Article abstract of DOI:10.1021/jm500480k

We developed a pharmacophore model for type II inhibitors that was used to guide the construction of a library of kinase inhibitors. Kinome-wide selectivity profiling of the library resulted in the identification of a series of 4-substituted 1H-pyrrolo[2,

Full text of DOI:10.1021/jm500480k

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Article
Discovery of Type II inhibitors of TGF-beta-activated kinase 1 (TAK1)
and Mitogen-activated protein kinase kinase kinase kinase 2 (MAP4K2)
Li Tan, Tyzoon K. Nomanbhoy, Deepak Gurbani, Matthew Patricelli, John Hunter, Jiefei Geng, Lina Herhaus,
Jianming Zhang, Eduardo Pauls, Youngjin Ham, Hwan Geun Choi, Ting Xie, Xianming Deng, Sara J
Buhrlage, Taebo Sim, Philip Cohen, Gopal Sapkota, Kenneth Dale Westover, and Nathanael S Gray
J. Med. Chem., Just Accepted Manuscript • Publication Date (Web): 17 Jul 2014
Downloaded from http://pubs.acs.org on July 18, 2014
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Discovery of Type II inhibitors of TGFꢀbetaꢀactivated kinase 1 (TAK1)
and Mitogenꢀactivated protein kinase kinase kinase kinase 2 (MAP4K2)
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Li Tan , Tyzoon Nomanbhoy , Deepak Gurbani , Matthew Patricelli ,
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John Hunter , Jiefei Geng , Lina Herhaus , Jianming Zhang , Eduardo
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Pauls , Youngjin Ham , Hwan Geun Choi , Ting Xie , Xianming
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Deng , Sara J. Buhrlage , Taebo Sim , Philip Cohen , Gopal Sapkota ,
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Kenneth D. Westover , Nathanael S. Gray *
1. Dana Farber Cancer Institute, Harvard Medical School, Boston, MA,
0
2115, USA
2. Department of Biological Chemistry and Molecular Pharmacology,
Harvard Medical School, Boston, Massachusetts, 02115, USA
3. ActivX Biosciences, La Jolla, CA 92037, USA
4
. Departments of Biochemistry and Radiation Oncology, The University
of Texas, Southwestern Medical Center, 5323 Harry Hines Blvd., Dallas,
TX 75390, USA
5. Department of Cell Biology, Harvard Medical School, Boston,
Massachusetts, 02115, USA
6. MRC Protein Phosphorylation and Ubiquitylation Unit, College of Life
Sciences, University of Dundee, Dow Street, Dundee DD1 5EH, UK
7. Chemical Kinomics Research Center, Korea Institute of Science and
Technology, 39ꢀ1, Hawolgokꢀdong, Seongbukꢀgu, Seoul, 136ꢀ791, Korea
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*
Correspondence: nathanael_gray@dfci.harvard.edu (NSG)
Abstract
We developed a pharmacophore model for type II inhibitors that was
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used to guide the construction of a library of kinase inhibitors.
Kinomeꢀwide selectivity profiling of the library resulted in the
identification of a series of 4ꢀsubstitutedꢀ1Hꢀpyrrolo[2,3ꢀb]pyridines that
exhibited potent inhibitory activity against two mitogenꢀactivated protein
kinases (MAPKs), TAK1 (MAP3K7) and MAP4K2, as well as
pharmacologically well interrogated kinases such as p38α (MAPK14) and
ABL. Further investigation of the structure activity relationship (SAR)
resulted in the identification of potent dual TAK1 and MAP4K2
inhibitors such as 1 (NG25) and 2 as well as MAP4K2 selective inhibitors
such as 16 and 17. Some of these inhibitors possess good
pharmacokinetic properties that will enable their use in pharmacological
studies in vivo. A 2.4ꢀÅ coꢀcrystal structure of TAK1 in complex with 1
confirms that the activation loop of TAK1 assumes the DFGꢀout
conformation characteristic of type II inhibitors.
Introduction
There are approximately 518 kinases encoded in the human genome,
and they have been demonstrated to play pivotal roles in virtually all
aspects of cellular physiology. Dysregulation of kinase activity through
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numerous mechanisms results in many pathologies, but therapeutically
relevant kinases have been extensively investigated as antiꢀcancer agents.
Clinically the most successful kinase inhibitors directly target
mutationally activated kinases such as BCRꢀABL, EML4ꢀALK, mutant
EGFR, BꢀRAF and cꢀKIT that are bonafide oncogenes in the context of
tumors that harbor these lesions. Unfortunately, although genome wide
cancer sequencing efforts have uncovered a large number of kinases that
are mutated very few have been biologically validated as being singularly
important to tumor function. One challenge to efficient pharmacological
validation of new potential kinase targets is the absence of selective
inhibitors for the majority of kinases. We sought to develop efficient
kinase inhibitor discovery strategies that will enable the identification of
inhibitors with sufficient potency and selectivity that they can be used to
interrogate the functional and therapeutic consequences of inhibiting less
well characterized kinases.
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The large majority of known small molecule kinase inhibitors target
the ATP binding site with the kinase assuming a conformation otherwise
conducive to catalysis (type I inhibitors). A second, broad class of kinase
inhibitors (type II) binds to the ATP binding pocket in addition to an
adjacent hydrophobic pocket that is created when the activation loop,
which contains the conserved DFG motif, is in an ‘out’ conformation. A
number of clinically approved inhibitors such as imatinib, nilotinib and
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sorafinib have been crystallographically proven to be type II inhibitors of
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kinases such as ABL, cꢀKIT, BꢀRAF and p38 kinases. Based upon these
crystal structures we developed a pharmacophore model that defined the
structural features that are needed to access this type II binding
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conformation (Figure 1b). The model posits the need for a ‘head’
heterocyclic motif that occupies the adenosine binding pocket typically
making one to three hydrogen bonds to the kinase hinge segment, a linker
moiety that traverses the region occupied by the ‘gatekeeper’ residue, a
hydrogen bond donor/acceptor motif and a hydrophobic ‘tail’ that
occupies the pocket created by the flip of the ‘DFG’ motif of the kinase
activation loop (Figure 1b). We constructed a library of potential type II
inhibitors based upon this model and performed kinomeꢀwide selectivity
profiling in an effort to identify new inhibitors and the kinases that might
6, 7
be susceptible to inhibition by type II inhibitors.
A library of
approximately 100 potential type II inhibitors was screened against a
TM
3a
panel of over 420 kinases using the KinomeScan approach. Two
compounds to emerge from this effort are 1 (reported as “NG25” in
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references)
and 2 which possess binding affinity for TAK1 and
MAP4K2 (also known as GCK) (Figure 2). These compounds possess a
ꢀsubstitutedꢀ1Hꢀpyrrolo[2,3ꢀb]pyridine as the hingeꢀinteracting “head”
motif, a 1,3ꢀbenzoic acid linker motif inspired by imatinib and a
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ꢀtrifluormethylbenzamide ‘tail’ motif inspired by sorafinib/nilotinib.
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Herein, we report the syntheses and target identification of two
novel type II kinase inhibitors 1 and 2 of two mitogen activated protein
kinases: TAK1 (MAP3K7) and MAP4K2 (GCK). While there have been
extensive efforts to target a limited number of MAPKs such as
A,B,CꢀRAF (MAP3Ks), MEK1 (MAP2K1), ERK2 (MAPK1), p38α,β,δ,γ
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(
MAPK14,11,13,12) and JNK1,2,3 (MAPK8,9,10), the majority of other
MAPKs have not been subjected to significant inhibitor development
efforts. The mitogen activated kinases are typically classified as MAP4Ks
(6 members), MAP3Ks (21 members), MAP2Ks (7 members) and
MAPKs (14 members) for a total of 49 kinases. TAK1 mediates signaling
downstream of multiple cytokine receptors and is functionally important
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in mitogen, immune and inflammatory signaling pathways.
Recently
several inhibitors of TAK1 have been reported and characterized with
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respect to their antiꢀinflammatory and antiꢀcancer activity.
The
biological functions of MAP4K2 are less well elucidated but it may be a
regulator of NFꢀκB signaling contributing to cancer development for a
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subset of malignancies,
and its pharmacological inhibition has been
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reported to reduce the viability of colon cancer cells. MAP4K2 has also
been reported to be required to transduce signals from TGFβ receptor to
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p38. Clearly more selective inhibitors of MAP4K2 would be very useful
to further elucidate the functions of this kinase. We demonstrate that 1
and 2 and other lead compounds (Figure 2) can inhibit phosphorylation of
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proteins predicted to be downstream of TAK1 and MAP4K2 such as IKK,
p38 and JNK at concentrations of less than 100 nM. Further modification
of 1 and 2 resulted in the identification of compounds such as 16 and 17
that exhibit selectivity for MAP4K2 relative to TAK1. Broad kinase
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selectivity profiling using KiNativ , revealed that a subset of the
inhibitors could also target additional kinases such as ABL, ARG, p38,
SRC, CSK, FER, FES and EPHꢀfamily kinases suggesting that these
kinases are also susceptible to type II inhibition providing a wealth of
potential starting points for further elaborating inhibitors of these kinases.
Results
The synthesis of lead compound 1 is outlined in Scheme 1. First,
pyrrolopyridine 27 was coupled with 3ꢀhydroxyꢀ4ꢀmethylbenzoic acid
using K CO as base. The resulting intermediate was subjected to
2
3
hydrogenolysis using Pd/C to afford the dechlorinated acid 28 which was
then reacted with aniline 29 using HATU/DIEA to provide the desired
amide 30. The SEM group of 30 was deprotected under sequential acid
and basic conditions to furnish compound 1. This four step sequence
proceeded with high overall yield.
The preparation of compound 2 is outlined in Scheme 2. The iodide
intermediate 32 was prepared by reacting aniline 29 and acid 31.
Meanwhile, a methoxy group was introduced into aldehyde 33 using
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sodium methoxide followed by Wittig olefination to afford the terminal
olefin 34. The SEM protecting group was replaced with a Boc protecting
group to provide compound 35 which was coupled with iodide 32
through Heck reaction to furnish compound 36 in good yield and high
trans/cis ratio (>20:1). Deprotection of the Boc protecting group using
mildly acidic conditions provided compound 2 with retention of the high
E/Z ratio in good overall yield.
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Compounds 1 and 2 were screened against a diverse panel of over 420
kinases (DiscoveRX, KinomeScan) using an in vitro ATPꢀsite
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competition binding assay at a concentration of 10 or 1 ꢁM. These two
compounds exhibited relatively selective profiles, and for some kinases
known to accommodate a typeꢀII binding mode, e.g., p38α and ABL, both
compounds exhibited potent inhibition. In addition, they also exhibited
promising inhibition against several other kinases known to play
important roles in cancerꢀrelated signaling pathways, but whose functions
remain uncharacterized due to a lack of highly selective inhibitors, e.g.,
TAK1, MAP4K2 and EPHA2. To complement the KinomeScan profiling,
both compounds were further profiled at 5 and 0.5 ꢁM concentrations on
A375 melanoma cells utilizing the KiNativ technology (ActiveX
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Biosciences).
This liveꢀcellꢀtreatment approach measures the ability
of the compounds to protect a subset of kinases in lysates from labeling
with a lysine reactive ATP or ADP ꢀbiotin probe. Kinases that exhibited
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moderate or high inhibition of labeling by compounds 1 and 2 are listed
in Table 1 (see the Supporting Information for full profiling data).
Consistent with the KinomeScan data, at a concentration of 0.5 ꢁM, both
compounds inhibited TAK1, MAP4K2, ZAK, p38α, SRC and LYN while
compound 1 also inhibits FER and compound 2 inhibits CSK and
EPHꢀfamily kinases respectively. To confirm the observed binding of 1
and 2 to TAK1 and MAP4K2 by the KiNativ approach, we determined
the IC50 values using biochemical enzyme assays (Invitrogen,
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TM 21
SelectScreen ). Compounds 1 and 2 inhibited TAK1 with IC s of 149
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nM and 41 nM respectively, and inhibited MAP4K2 with IC s of 22 nM
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and 98 nM respectively. The in vitro IC s of most other targets from
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KiNativ profiling were also determined and some of them were further
confirmed while others were not (Table 1). Compound 1 was also
screened at 0.1 ꢁM against another panel of over 100 protein kinases
(International
Centre
for
Kinase
Profiling
http://www.kinaseꢀscreen.mrc.ac.uk/) and gave similar results (Figure S1);
the IC s against TAK1 and MAP4K2 were 15 nM and 17 nM
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respectively in that assay.
To further explore the potential of 1 and 2 to be optimized for potency
and selectivity against these kinases we synthesized 24 diverse analogs
(Table 2). To approach this optimization in a systematic fashion, this
chemotype was divided into 3 sections: head, linker and tail, and each of
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these units were varied sequentially. The head unit was altered from
pyrrolopyridinꢀ4ꢀyl in 1 to pyrrolopyrimidinꢀ4ꢀyl (e.g., 3, 13) and
substituted pyrimidinꢀ4ꢀyls (e.g., 6, 7); however, analogs of lead
compound 2 contained the original head unit. The linker unit was varied
through deletion of the methyl group (e.g., 10, 24), reversing the amide
orientation (11, 22) and altering the alkene in compound 2 via saturation
(14) or replacement with an ether linkage (15). The tail unit contained
various hydrophilic groups instead of the 4ꢀethylpiperazinꢀ1ꢀyl moiety,
along with variations of the substitution site from the 4ꢀposition to the
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ꢀposition. These changes resulted in minor variation to the syntheses
with most analogs being prepared via similar routes and in high yields.
All of the prepared analogs were subjected to KiNativ profiling using
HUH7 cell lysates which typically detects approximately 150 protein and
lipid kinases of interest. This profiling effort revealed that this class of
compounds in general provide good multiꢀtargeted scaffolds that can bind
to TAK1, MAP4K2, p38α, Abl, ZAK, CSK, FER, FES, and EPHA2 with
varying degrees of potency and selectivity. Herein we primarily focus our
discussion on how the structural changes affected the ability of the
compounds to inhibit TAK1 and MAP4K2 kinases (Table 3). With
compound
1,
we
first
changed
the
head
from
1Hꢀpyrrolo[2,3ꢀb]pyridineꢀ4ꢀyl to 7Hꢀpyrrolo[2,3ꢀd]pyrimidinꢀ4ꢀyl (3)
and 1Hꢀpyrazolo[3,4ꢀd]pyrimidinꢀyl (4) and found that the activities
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against MAP4K2 were maintained, while those against TAK1 were
clearly improved. However, the addition of a methyl group at the
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ꢀposition of the 7Hꢀpyrrolo[2,3ꢀd]pyrimidinꢀ4ꢀyl moiety (5) led to more
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potent inhibition of MAP4K2 and a decreased potency towards TAK1.
We next introduced the monocyclic head units 6ꢀ(methylamino)ꢀ
pyrimidinꢀ4ꢀyl (6) and 2ꢀ(methylamino)ꢀpyrimidinꢀ4ꢀyl (7); 6 exhibited
more potent inhibition of MAP4K2 and decreased potency against TAK1,
and 7 showed slightly decreased activities. To our surprise, the
introduction of a 6,7ꢀdimethoxyquinazolinꢀ4ꢀyl head unit (8) yielded
potent activity against both kinases while introduction of 4ꢀsubstituted
Nꢀmethylpicolinamide (9) eliminated both TAK1 and MAP4K2 potency.
We then varied the linker region and found that deletion of the methyl
group (10) had little impact on activity; while other modifications only
led to loss of activity (11). We next investigated the effect of a variety of
tail units, and found that all changes led to a decrease in activity against
both kinases. However, substitution at the 3ꢀposition of the aromatic ring
in the tail unit with 4ꢀmethylꢀ1Hꢀimidazolꢀ1ꢀyl (12) and
4
ꢀmethylpiperazinꢀ1ꢀyl (13) led to only a slight decrease in activity
against MAP4K2 but almost complete elimination of TAK1 activity. All
analogs of compound 2 generally showed reduced activity against both
kinases, with the exception of 14, which showed similar activity, and 15,
which exhibited a slight decrease in activity against TAK1.
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These results indicated that modification of the head unit could
improve activity against both kinases, although in most cases more
dramatically for MAP4K2 with the exception of 8. Alteration of the linker
unit had little or even negative effect against MAP4K2 and TAK1, while
changes in the tail unit always decreased activity against both kinases,
although this reduction was small for MAP4K2 but significant for TAK1.
This data prompted us to prepare compound 16, which combined the head
of 6, the tail of 12, and maintained the original linker of 1, and exhibited
similar inhibitory activity for MAP4K2 compared to 12. Further
modification of the tail of 16 led to 17 which exhibited good biochemical
selectivity for inhibiting MAP4K2 relative to TAK1 (37 and 2700 nM
IC s), along with best KiNativ score for MAP4K2 (85% at 1 µM). The
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results of KiNativ profiling using HUH7 cell lysates showed that only
MAP4K2 and ABL among 220 protein and lipid kinases of interest were
inhibited by 17 (Supplemental Table 3), and the inhibition of ABL was
rather weak compared with MAP4K2 (49% compared with 85% at 1 µM).
Furthermore, the weak inhibition of p38α and ZAK of 16 was eliminated
with 17 (Table 4). 17 was also subjected to KiNativ liveꢀcell profiling
using HUH7 cells and strong inhibition of MAP4K2 (88% at 1 µM)
labeling was observed suggesting that 17 is a potent and relatively
selective cellular MAP4K2 inhibitor (Supplemental Table 4).
To further understand the SAR of TAK1/MAP4K2 inhibitors, we
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solved coꢀcrystal xꢀray structures of TAK1 in complex with 1 (PDB
4
O91). Xꢀray diffraction extended to 2.4 Å and the structure was solved
using molecular replacement with PDB 2YIY as the initial search model.
Examination of the active site revealed strong additional electron density
into which 1 was easily modeled (Figure S2A). Final statistics for
diffraction and the model are given in Table S1. Interactions with the
active site residues are schematically depicted in Figure S2B. In summary,
the nitrogens in the pyrrolopyridine moiety form hydrogen bonds with
Ala107 in the hinge binding region. The linker amide is stabilized inside
the deeper binding pocket by hydrogen bonding with Glu77 and the
backbone amide of Asp175. These observations further explain why the
reversal of the amide (11) resulted in a loss of activity. The nitrogen of the
tertiary amine in the piperazinyl tail interacts with carbonyls of Ile153
and His154 on the activation loop (Figure 3c). Compared with the
structure of adenosine bound to the active site of TAK1 (PDB 2EVA)
which shows DFG in the active, flippedꢀin conformation (Figure 3b),
binding of 1 with TAK1 results in an inactive, DFGꢀout conformation
characteristic of typeꢀII inhibitors (Figure 3a).
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Simulated docking studies using MAP4K2 as the receptor resulted in
a similar overall inhibitor pose relative to the TAK1/1 coꢀcomplex
structure. However, the long side chain of Gln80 in the Cꢀhelix of TAK1
is located very close to the tail of 1 with only 3.6 Å distance between the
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carbonyl of Gln80 side chain and the 5ꢀposition of 3ꢀtrifluormethylphenyl
group, whereas the shorter side chain of Ile64 in MAP4K2 is not so
proximate in the MAP4K2/1 modeling. Substitutions at the 5ꢀposition of
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3
ꢀtrifluormethylphenyl group may be too bulky for the side chain of
Gln80 of TAK1, whereas they could possibly interact with the side chains
of Ile64 and Gln60 of MAP4K2. This potential interaction might
compensate for the loss of interactions with the two carbonyls of Ile133
and His134, providing a rationale for why 12, 16 and 17 exhibit a
preference for binding to MAP4K2 (Figure 3c, d).
Many of our compounds also exhibited potent inhibition of p38α, the
data from KiNativ and enzymatic assays are shown in Table S2. Analogs
of 1 that contained the same linker and tail as 1 were all active against
p38α (3ꢀ9), with 4 being the most potent, and 9 being inactive against
TAK1 and MAP4K2. These results indicated that modification of the
head unit was tolerated on this scaffold. We were also surprised to find
that reversing the amide connectivity of 1 afforded a very selective p38α
inhibitor (11). To prepare analogs of compound 2, we first explored
different tail units and found several compounds with potent activity
against p38α (18ꢀ20), with 19 being the most selective. Modifications of
the linker unit, as in compounds 14 and 15, were also tolerated, and these
changes sometimes led to potent activity in compounds with tail units that
had previously demonstrated poor activity (i.e., 21, IC < 10 nM).
5
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Similarly, the reversed amide connectivity (22) retained potent activity
when the methyl of the linker was deleted. In sharp contrast, its analogs
with the original amide connectivity (23, 24) were inactive against p38α.
Many of the compounds exhibited potent binding activity against
ABL kinase which is known to be potently inhibited by type II inhibitors
such as imatinib and nilotinib. To evaluate whether these new compounds
could inhibit ABL activity in a cellular context, they were evaluated for
their antiproliferative activity against BCRꢀABLꢀdependent Ba/F3 cells
(Table S3). To evaluate their potential for nonꢀspecific cytotoxicity they
were also evaluated against parental Ba/F3 cells grown in the presence of
interleukinꢀ3. The results demonstrated that many of our compounds
selectively inhibited the proliferation of BCRꢀABLꢀdependent Ba/F3
cells with EC values of less than 400 nM. Several compounds (e.g., 2,
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0) exhibited more than 50% cell growth inhibition at 10 nM, with
analogs of 2 generally being the most active. These compounds exhibit
comparable cellular potency to clinically approved secondꢀgeneration
BCRꢀABL inhibitors such as nilotinib and dasatinib. Replacement of the
(
4ꢀethylpiperazinꢀ1ꢀyl)methyl tail with a 4ꢀethylpiperazinꢀ1ꢀyl tail (25)
provided very selective inhibition against ABL (both enzymatic and
cellular); more surprising was that elimination of the trifluoromethyl
group afforded a very strong inhibitor with high selectivity (26).
Some of our compounds exhibited potent inhibition of a number of
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additional kinases (Table S4). In general, analogs of compound 2 were
more likely to inhibit ZAK, with 14 being the most active with >96%
inhibition at 1 ꢁM (KiNativ) and an IC of 72 nM (SelectScreen).
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Additionally, compound 9, an analog of 1, was very selective against
ZAK. Only analogs of 2 were found to inhibit CSK, with 14 being the
most potent. Compounds 2 and 14 exhibited potent activity against
EPHA2, although further modification of their scaffolds led to a severe
loss of activity. Surprisingly, we found that only analogs of compound 1
exhibited potency against FES and FER, with the activity of 8 being very
strong (93% and 95% inhibition at 1 ꢁM and IC s of 51 and 36 nM
5
0
respectively).
To further validate inhibitory activity against TAK1 and MAP4K2 by
these inhibitors, we next measured their ability to block the downstream
signals induced by different cytokines. First we checked their effect on
NFꢀκB signaling in mouse L929 cells following TNFα stimulation, where
we found that most inhibitors with strong TAK1 inhibition in previous
assays, could effectively reduce the phosphorylation level of IKKα/β at
1
00 nM concentrations. The degradation of IκBꢀα was also effectively
inhibited. The reported irreversible TAK1 inhibitor 37
5Zꢀ7ꢀoxozeaenol) was used as positive control and our lead compounds
22
(
1 and 2 exhibited comparable cellular potency (Figure 4a). The selective
MAP4K2 inhibitors (12, 16, and 17) and p38α inhibitors 11, 38
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(
SB203580) and 39 (PHꢀ797804) were also tested and demonstrated
no effect at the same concentrations (Figure 4b, Figure S3B). The most
selective MAP4K2 inhibitor 17 was not very effective even at
concentrations up to 1 µM (Figure S3A), which suggested MAP4K2 does
not contribute much to this pathway under these conditions.
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Next we checked the effect of 1 in MEFs and human 293 ILꢀ1R cells
stimulated with ILꢀ1α and found that the phosphorylation level of
downstream p105, p38 and JNK1/2 were all inhibited in a
doseꢀdependent manner (Figure 5aꢀb). Similar results were obtained in
mouse RAW cells following LPS stimulation (Figure 5c). We also
discovered that compound 1 (1 µM) blocked signaling following
stimulation of human Gen2.2 cells with the TLR7 agonist CL097; analog
S1 (Figure S4) which possessed moderate TAK1 and MAP4K2 inhibitory
activity (IC s of 292 nM and 296 nM, respectively) only partially
5
0
inhibited signaling. The selective MAP4K2 inhibitor 16 was not able to
block CL097 induced signaling (Figure 5d).
It was more challenging to validate the functional cellular
consequences of MAP4K2 inhibition since the effector pathways of this
kinase are less well characterized. TAK1 mediates much of the
10, 25
ILꢀ1ꢀinduced phosphorylation of p38
although recent reports suggest
that TAK1 does not mediate TGFβꢀinduced phosphorylation of p38 in
18
TAK1ꢀnull MEFs and HaCaT cells. A siRNAꢀbased screen targeting all
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MAP3Ks individually in HaCaT cells suggested roles for MAP3K4 and
18
MAP3K10 in mediating TGFβꢀinduced phosphorylation of p38.
A
similar siRNA screen targeting MAP4Ks in HaCaT cells demonstrated
that depletion of MAP4K2 but not MAP4Ks 1, 3 and 5 resulted in
substantial inhibition of both ILꢀ1 and TGFβꢀinduced phosphorylation of
p38, while TGFβꢀinduced phosphorylation of SMAD2 was unaffected
(Figure 6a). Cumulatively these studies suggest a possible role for
MAP4K2 in ILꢀ1 and TGFβꢀinduced phosphorylation of p38. We
therefore tested the ability of our selective MAP4K2 inhibitors 16 and 17
to inhibit ILꢀ1 and TGFβ induced phosphorylation of p38 MAPK in WT
and TAK1ꢀdeficient MEFs. Both 16 and 17 caused a doseꢀdependent
inhibition of ILꢀ1 and TGFβꢀinduced p38 MAPK phosphorylation but did
not inhibit the TGFβꢀinduced phosphorylation of SMAD2 (Figure 6bꢀc).
Consistent with the primary role of TAK1 in mediating ILꢀ1 signaling, in
WT MEFs, 16 and 17 caused ~50% inhibition in ILꢀ1ꢀinduced
phosphorylation of p38 at 500 nM (Figure 6bꢀc). However, in
TAK1ꢀdeficient MEFs, both 16 and 17 resulted in complete inhibition of
ILꢀ1ꢀinduced phosphorylation of p38 at 500 nM, suggesting that
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MAP4K2
may mediate the residual ILꢀ1ꢀdependent but
TAK1ꢀindependent phosphorylation of p38 (Figure 6bꢀc). The
doseꢀdependent inhibition of TGFβꢀinduced phosphorylation of p38 by
1
6 and 17 was similar in both WT and TAK1ꢀdeficient MEFs, consistent
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with the notion that TAK1 does not mediate TGFβꢀinduced
phosphorylation of p38 in these cells (Figure 6bꢀc). Compound 1, a dual
inhibitor of TAK1 and MAP4K2, exhibited similar potency to 16 and 17
while the covalent natural product TAK1 inhibitor 37, inhibited
phosphorylation of both p38 and SMAD2 at very low concentrations
regardless of presence of TAK1, consistent with the observation that
TAK1 mediates much of the ILꢀ1ꢀinduced phosphorylation of p38 and
that 37 has several additional kinase targets including MEK1 (Figure S5).
The pharmacokinetic properties of 1ꢀ5 were also evaluated following
intravenous and oral delivery in mice, respectively. Compounds 1 and 5
demonstrated favorable pharmacokinetic properties, with T1/2 values of
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.0 and 2.9 h, AUC values of 1369 and 2136 following a 10 mg/Kg oral
dose and %F values of 70.2 and 38.6, respectively (Table 5). This
suggests that these compounds should be useful for murine efficacy
studies.
Discussion
In conclusion, compounds 1 and 2, along with their analogs,
represent two novel ‘type II’ kinase inhibitor scaffolds discovered using a
kinomeꢀwide profiling approach and exhibit diverse activity against a
limited set of kinase targets. Among these compounds, 1 was especially
interesting as a very potent TAK1 inhibitor and has already been used in
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, 9
some studies, and 2 is also being utilized in cancer studies due to its
potent antiꢀproliferative effects against particular tumor types. Profiling
of these type II inhibitors reveals that a subset of the compounds also are
capable of potently inhibiting LCK, ABL, p38α, etc. and this needs to be
considered when using them to interrogate TAK1 and MAP4K2
dependent effects. As for application of TAK1 inhibitors, the most
commonly used probe nowadays is 37, which is not a selective inhibitor.
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3
7 strongly inhibits MAP2Ks and MAPKs such as MEK1 (MAP2K1)
and ERK1 (MAPK1), and many other targets such as KDR, PDGFR,
ZAK, etc. Moreover, the binding to several of these targets is covalent
and it is likely that other reactive cysteines can be targeted by this
26
compound intracellularly. Compound 1 provides a complementary
pharmacological probe of TAK1 relative to 37 due to its distinct chemical
structure, nonꢀoverlapping offꢀtarget pharmacology and reversible mode
of inhibition. The coꢀcrystal structure of TAK1/1 has helped in
rationalizing the SAR and will be used to design typeꢀII TAK1 inhibitors
in the future. Meanwhile 16 and 17 represent relatively selective
MAP4K2 inhibitors, especially compound 17 exhibits impressive
selectivity, and excellent potency in cellular assays. 11 is a selective
p38α inhibitor, both 25 and 26 are selective ABL inhibitors. By
modification of the tail moiety, these inhibitors (11, 17 and 26 etc.) can
specifically interact with the allosteric DFGꢀpockets, which may provide
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a means of achieving selectivity amongst otherwise highly homologous
kinases. The coꢀcrystal structure of TAK1/1 reveals that Gln80 is
proximal (3.6 Å) from the phenyl ring of the benzamide ‘tail’ moiety,
which suggests that a unique Hꢀbond may be able to be introduced in this
region to gain selectivity for TAK1. Finally, given their wide diversity
with respective kinase selectivity, enzymatic and cellular potency and
favorable pharmacokinetic parameters, 4ꢀsubstitutedꢀ1Hꢀpyrrolo [2,3ꢀb]
pyridines may represent privileged scaffolds for the development of
therapeutic agents targeting various kinases.
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Experimental Section
Chemistry. Unless otherwise noted, reagents and solvents were obtained
1
from commercial suppliers and were used without further purification. H
NMR spectra were recorded on 400 MHz (Varian 7600 AS) or 600 MHz
(Varian Inova), and chemical shifts are reported in parts per million (ppm,
δ) downfield from tetramethylsilane (TMS). Coupling constants (J) are
reported in Hz. Spin multiplicities are described as s (singlet), brs (broad
singlet), t (triplet), q (quartet), and m (multiplet). Mass spectra were
obtained on a Waters Micromass ZQ instrument. Preparative HPLC was
performed on a Waters Symmetry C18 column (19 x 50 mm, 5µM) using
a gradient of 5ꢀ95% acetonitrile in water containing 0.05% trifluoroacetic
acid (TFA) over 8 min (10 min run time) at a flow rate of 30 mL/min.
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Purification of compounds was performed with either a Teledyne ISCO
combiflash Rf system or a Waters Micromass ZQ preparative system.
Purities of assayed compounds were in all cases greater than 95%, as
determined by reverseꢀphase HPLC analysis.
10
11
12
13
14
15
16
17
18
19
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22
23
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25
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4ꢀmethylꢀ3ꢀ((1ꢀ((2ꢀ(trimethylsilyl)ethoxy)methyl)ꢀ1Hꢀpyrrolo[2,3ꢀb]p
yridinꢀ4ꢀyl)oxy)benzoic acid (28). To a stirred solution of 27 (570 mg, 2
mmol) and 3ꢀhydroxyꢀ4ꢀmethylbenzoic acid (340 mg, 2.2 mmol) in 13
mL of DMSO was added K CO (830 mg, 6 mmol). The reaction mixture
2
3
o
was allowed to stand for 5 h at 100 C, and then cooled to RT. The
mixture was acidified with 1N HCl solution and extracted with ethyl
acetate, the organic phase was washed with brine and dried over Na SO ,
2
4
then filtered and evaporated to give a pale yellow liquid (690 mg, 80%).
To a stirred solution of this gained liquid (690 mg, 1.6 mmol) in 10 mL of
methanol was added Pd/C (10 wt. % loading, 120 mg). The reaction
mixture was stirred under hydrogen atmosphere for 2 or 3 days. The
reaction was monitored by TLC, more Pd/C might be needed for
complete conversion during the period. Then the reaction solution was
filtered, concentrated, and purified with column chromatography (hexane :
ethyl acetate = 1:1) to give the title compound as a white solid (510 mg,
1
8
0%). H NMR (400 MHz, CDCl ) δ 8.30 (d, J = 5.6 Hz, 1H), 8.02 (d, J
3
= 8.0 Hz, 1H), 7.90 (s, 1H), 7.48 (d, J = 8.0 Hz, 1H), 7.33 (d, J = 3.6 Hz,
1
H), 6.52 (d, J = 3.6, 1H), 6.44 (d, J = 5.6, 1H), 5.78 (s, 2H), 3.65 (t, J =
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.0 Hz, 2H), 2.37 (s, 3H), 0.99 (t, J = 8.0 Hz, 2H), 0.00 (s, 9H). MS (ESI)
+
m/z 399 (M+H) .
Nꢀ(4ꢀ((4ꢀethylpiperazinꢀ1ꢀyl)methyl)ꢀ3ꢀ(trifluoromethyl)phenyl)ꢀ4ꢀme
thylꢀ3ꢀ((1ꢀ((2ꢀ(trimethylsilyl)ethoxy)methyl)ꢀ1Hꢀpyrrolo[2,3ꢀb]pyridi
nꢀ4ꢀyl)oxy)benzamide (30). To a stirred solution of 28 (400 mg, 1 mmol)
and aniline 29 (430 mg, 1.5 mmol) in 10 mL of dichloromethane was
added HATU (570 mg, 1.5 mmol), DMAP (185 mg, 1.5 mmol) and
DIEA (520 ꢁL, 3 mmol). The reaction mixture was allowed to stand for
24 h at room temperature, then diluted with ethyl acetate and washed with
water and brine, the organic phase was dried over Na SO , filtered, then
0
1
2
3
4
5
6
7
8
9
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2
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4
concentrated
and
purified
with
column
chromatography
(
dichloromethane : methanol = 20:1) to give the title compound as a
1
colorless oil (600 mg, 90%). H NMR (400 MHz, CDCl ) δ 8.21 (s, 1H),
3
8
.12 (d, J = 5.6 Hz, 1H), 7.91 (d, J = 8.0 Hz, 1H), 7.74ꢀ7.68 (m, 2H), 7.69
(s, 1H), 7.41 (d, J = 8.0 Hz, 1H), 7.22 (d, J = 3.6, 1H), 6.37 (d, J = 3.6,
H), 6.33 (d, J = 5.6, 1H), 5.64 (s, 2H), 3.61 (s, 2H), 3.55 (t, J = 8.0 Hz,
2H), 2.65ꢀ2.40 (m, 8H), 2.50 (q, J = 6.8 Hz, 2H), 2.28 (s, 3H), 1.24 (t, J =
1
6
.8 Hz, 3H), 0.90 (t, J = 8.0 Hz, 2H), ꢀ0.08 (s, 9H). MS (ESI) m/z 668
+
(M+H) .
3
ꢀ((1Hꢀpyrrolo[2,3ꢀb]pyridinꢀ4ꢀyl)oxy)ꢀNꢀ(4ꢀ((4ꢀethylpiperazinꢀ1ꢀyl)
methyl)ꢀ3ꢀ(trifluoromethyl)phenyl)ꢀ4ꢀmethylbenzamide (1). To a
stirred iceꢀcooled solution of 30 (330 mg, 0.5 mmol) in 5 mL of
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dichloromethane was added 1 mL of TFA. The reaction mixture was
o
stirred for 30 min at 0 C, and then warmed to RT. After 5 h the reaction
mixture was concentrated and dried under vacuum, then dissolved in 5
mL of THF, and 5 mL of 1N NaOH water solution was added. The
reaction mixture was stirred for 24 h and extracted with ethyl acetate. The
combined organic phase was washed with brine and dried with Na SO ,
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
2
4
then filtered and concentrated, and purified with column chromatography
(
dichloromethane : methanol = 10:1) to give the title compound as a
1
white solid (188 mg, 70%). H NMR (400 MHz, DMSO) δ 11.78 (bs, 1H),
1
0.44 (s, 1H), 8.16 (d, J = 2.4 Hz, 1H), 8.09 (d, J = 5.6 Hz, 1H), 8.02 (dd,
J = 8.4, 1.6 Hz, 1H), 7.88 (dd, J = 8.0, 2.0 Hz, 1H), 7.78 (d, J = 2.0 Hz,
H), 7.69 (d, J = 8.8 Hz, 1H), 7.58 (d, J = 8.0 Hz, 1H), 7.38 (dd, J = 3.2,
2.8, 1H), 6.32 (d, J = 5.6, 1H), 6.21 (dd, J = 3.2, 2.0 Hz, 1H), 3.56 (s, 2H),
.52ꢀ2.30 (m, 8H), 2.50 (q, J = 7.2 Hz, 2H), 2.24 (s, 3H), 1.00 (t, J = 7.2
1
2
13
Hz, 3H). C NMR (100 MHz, DMSO) δ 164.83, 157.26, 152.90, 151.59,
44.78, 138.50, 134.90, 134.11, 132.48, 132.21, 131.61, 125.31, 125.25,
1
124.04, 120.62, 117.80, 117.74, 110.13, 101.78, 97.24, 57.81, 52.97,
+
5
2.66, 51.94, 16.17, 12.18. MS (ESI) m/z 538 (M+H) .
Nꢀ(4ꢀ((4ꢀethylpiperazinꢀ1ꢀyl)methyl)ꢀ3ꢀ(trifluoromethyl)phenyl)ꢀ3ꢀiod
oꢀ4ꢀmethylbenzamide (32). To stirred solution of
a
3ꢀiodoꢀ4ꢀmethylbenzoic acid 31 (262 mg, 1 mmol) and aniline 29 (430
mg, 1.5 mmol) in 10 mL of dichloromethane was added HATU (570 mg,
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2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
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4
4
4
4
4
4
4
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5
5
5
5
5
5
5
5
5
6
1
.5 mmol), DMAP (185 mg, 1.5 mmol) and DIEA (520 ꢁL, 3 mmol). The
reaction mixture was allowed to stand for 24 h at room temperature, then
diluted with ethyl acetate and washed with water and brine, the organic
phase was dried over Na SO , filtered, then concentrated and purified
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
2
4
with column chromatography (dichloromethane : methanol 20:1) to give
1
the title compound as a yellow solid (480 mg, 90%). H NMR (600 MHz,
CDCl ) δ 8.35 (br, 1H), 8.33 (s, 1H), 7.91 (m, 2H), 7.80 (d, J = 8.4 Hz,
3
1
H), 7.63 (d, J = 9.0, 1H), 7.32 (d, J = 8.4 Hz, 1H), 3.66 (s, 2H),
3.00ꢀ2.58 (m, 8H), 2.71 (m, 2H), 2.48 (s, 3H), 1.26 (t, J = 7.2 Hz, 3H).
+
MS (ESI) m/z 532 (M+H) .
4ꢀmethoxyꢀ1ꢀ((2ꢀ(trimethylsilyl)ethoxy)methyl)ꢀ5ꢀvinylꢀ1Hꢀpyrrolo[2,
3
ꢀb]pyridine (34). To a stirred solution of 33 (400 mg, 1.20 mmol) in
MeOH (4 mL), was added sodium methoxide (348 mg, 6.43 mmol). The
o
mixture was stirred at 70 C for 5 h before being cooled to RT and
filtered through celite pad. The filtrate was concentrated under reduced
pressure and partitioned between ethyl acetate and water, the aqueous
layer was extracted with ethyl acetate three times. The combined organic
layer was washed with brine, dried over Na SO , and concentrated in
2
4
vacuo. The residue was used for the next step without purification. To a
stirred solution of methyltriphenylphosphonium iodide (923 mg, 2.28
mmol) in THF (10 mL) was added nꢀbutyl lithium (2.5 M in hexane,
o
0
.822 mL, 2.056 mmol) at ꢀ78 C. After 30 min, the reaction mixture was
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o
slowly treated with the obtained residue in THF (5 mL) at ꢀ78 C and
o
stirred for 2 h at ꢀ78 C before being quenched with saturated ammonium
chloride solution. The reaction mixture was extracted with ethyl acetate
three times and the combined organic layer was washed with brine, dried
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
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42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
over Na SO , filtered, then concentrated and purified with column
2
4
chromatography (hexane : ethyl acetate = 3:1) to give title compound as a
1
colorless oil (208 mg, 57% in 2 steps). H NMR (600 MHz, CDCl ) δ
3
8
.38 (s, 1H), 7.22 (m, 1H), 6.97 (m, 1H), 6.74 (m, 1H), 5.77ꢀ5.50 (m, 1H),
5.25 (m, 1H), 4.33 (s, 3H), 3.55 (m, 2H), 0.90 (m, 2H), ꢀ0.06 (s, 9H). MS
+
(
ESI) m/z 305 (M+H) .
tertꢀbutyl 4ꢀmethoxyꢀ5ꢀvinylꢀ1Hꢀpyrrolo[2,3ꢀb]pyridineꢀ1ꢀcarboxylate
35). To 34 (140 mg, 0.46 mmol) in CH Cl (5 mL) was added TFA (0.5
(
2
2
o
o
mL) at 0 C, the reaction mixture was stirred for 30 min at 0 C, and then
warmed to RT. After 5 h, the reaction mixture was concentrated in vacuo,
then dissolved in 5 mL of THF, and treated with 5 mL of 1N NaOH. The
reaction mixture was stirred for 24 h and extracted with ethyl acetate. The
combined organic phase was washed with brine and dried with Na SO ,
2
4
then filtered and concentrated in vacuo. The residue was then dissolved in
5 mL of CH Cl and treated with Boc O (200 mg, 0.92 mmol) and DIEA
2
2
2
(
240 ꢁL, 1.38 mmol). The reaction mixture was stirred for 24 h and
extracted with ethyl acetate, and the combined organic layer was washed
with brine, dried over Na SO , filtered, then concentrated and purified
2
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2
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2
2
2
2
2
2
2
3
3
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5
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6
with column chromatography (hexane : ethyl acetate = 2:1) to give the
1
title compound as a white solid (100 mg, 80% in 2 steps). H NMR (600
MHz, CDCl ) δ 8.63 (s, 1H), 7.54 (m, 1H), 6.94 (m, 1H), 6.76 (m, 1H),
3
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
5
.81 (m, 2H), 5.36 (m, 1H), 4.31 (s, 3H), 1.69 (s, 9H). MS (ESI) m/z 275
+
(
M+H) .
(
E)ꢀtertꢀbutyl
5ꢀ(5ꢀ((4ꢀ((4ꢀethylpiperazinꢀ1ꢀyl)methyl)ꢀ3ꢀ(trifluoromethyl)phe
nyl)carbamoyl)ꢀ2ꢀmethylstyryl)ꢀ4ꢀmethoxyꢀ1Hꢀpyrrolo[2,3ꢀb]pyridin
eꢀ1ꢀcarboxylate (36). To the mixture of 32 (58 mg, 0.110 mmol), 35 (32
mg, 0.116 mmol), DIEA (480 ꢁL, 0.275 mmol), and triꢀtꢀbutylphosphine
(1.0 M in toluene, 11 ꢁL, 0.022 mmol), was added Pd (dba) (10 mg,
2
3
0
.011 mmol) at RT. The reaction mixture was degassed and stirred under
o
Ar atmosphere at 80 C for 15 h before being cooled to RT, the filtered
and concentrated. The residue was purified with column chromatography
(dichloromethane:methanol 20:1) to give the titled compound as a white
1
solid (60 mg, 80%, E/Z > 20:1). H NMR (600 MHz, DMSO) δ 8.50 (s,
1H), 8.49 (s, 1H), 8.13 (s, 1H), 7.97 (s, 1H), 7.90 (d, J = 8.4, 1H), 7.71 (d,
J = 7.8, 1H), 7.64 (d, (d, J = 8.4, 1H), 7.50 (d, (d, J = 4.2, 1H), 7.30 (d, J
= 16.8, 1H), 7.26 (d, J = 16.8, 1H), 7.20 (d, J = 7.8, 1H), 6.71 (d, J = 4.2,
1
H), 4.24 (s, 3H), 3.58 (s, 2H), 2.55 (m, 4H), 2.49 (q, J = 7.2, 2H), 2.41 (s,
3H), 2.16 (m, 2H), 1.89 (m, 2H), 1.63 (s, 9H), 1.11 (t, J = 7.2, 3H). MS
+
(
ESI) m/z 678 (M+H) .
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(
ꢀ
E)ꢀNꢀ(4ꢀ((4ꢀethylpiperazinꢀ1ꢀyl)methyl)ꢀ3ꢀ(trifluoromethyl)phenyl)ꢀ3
(2ꢀ(4ꢀmethoxyꢀ1Hꢀpyrrolo[2,3ꢀb]pyridinꢀ5ꢀyl)vinyl)ꢀ4ꢀmethylbenzam
ide (2). To a solution of 36 (60 mg, 0.088 mmol) in CH Cl (2 mL) was
2
2
10
11
12
13
14
15
16
17
18
19
20
21
22
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28
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31
32
33
34
35
36
37
38
39
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46
47
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49
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52
53
54
55
56
57
58
59
60
o
o
added TFA (0.2 mL) at 0 C, the reaction mixture was stirred at 0 C for
.5 h then concentrated and purified with column chromatography
2
(
dichloromethane:methanol = 10:1) to give title compound as a white
1
solid (45 mg, 90%, E/Z > 20:1). H NMR (600 MHz, DMSO) δ 11.73 (s,
1
H), 10.52 (s, 1H), 8.50 (s, 1H), 8.23 (d, J = 14.4, 1H), 8.06 (d, J = 8.4,
1H), 7.78 (d, J = 8.4, 1H), 7.72 (d, J = 8.4, 1H), 7.44 (s, 1H), 7.43 (s, 1H),
.37 (d, J = 13.8, 1H), 7.36 (d, J = 7.2, 1H), 6.82 (m, 1H), 4.35 (s, 3H),
7
1
3
3.56 (s, 2H), 2.60ꢀ2.20 (m, 10H), 2.48 (s, 3H), 0.99 (t, J = 7.2, 3H). C
NMR (100 MHz, DMSO) δ 166.14, 157.01, 151.66, 143.64, 139.80,
137.41, 132.78, 132.40, 131.64, 130.89, 127.65, 126.59, 125.35, 124.334,
1
24.22, 124.07, 117.73, 114.16, 108.40, 100.31, 59.52, 57.91, 53.21,
+
52.80, 52.01, 20.09, 12.38. MS (ESI) m/z 578 (M+H) .
Compounds 3ꢀ26 were synthesized with same procedures as 1 and 2.
37ꢀ39 were commercial from Selleckchem.com.
TAK1ꢀTAB1 expression and purification. DNA encoding the
TAK1ꢀTAB1 fusion protein (kinase domain residues 31ꢀ303 and
cꢀterminal domain residues 468ꢀ497) was obtained from GeneScript
(GenScript USA Inc.860 Centennial Ave. Piscataway, NJ 08854, USA).
This was cloned into the pFastBac His6 TEV LIC cloning vector (4B)
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4
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5
5
5
5
5
5
5
5
5
5
6
(
Plasmid 30115) from). TAK1ꢀTAB1 fusion protein was expressed in Hi5
2
7, 28
insect cells and purified as described previously.
TAK1ꢀTAB1/1 Crystallization and Structure Determination.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
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0
1
2
3
4
5
6
7
8
9
0
TAK1ꢀTAB1 was concentrated to 10mg/ml and crystallized as reported
28
previously with minor modifications. Briefly, the crystals were obtained
using the hangingꢀdrop method at 20 ºC in 4µl drops by mixing protein
with equal volumes of reservoir solution [0.65ꢀ0.75M sodium citrate,
0
.2M NaCl, 0.1M Tris pH 7.0 and 5mM Adenosine]. The crystals were
washed three times in reservoir solution without adenosine. A 10 mM
solution of 1 was prepared and crystals were back soaked for overnight.
Crystals were frozen for data collection using 20% ethyleneglycol as
cryoprotectant. Diffraction data was collected at Argonne Advanced
29
Photon Source (beamline 19ꢀD) and processed with HKL3000. The
30
structure was solved by molecular replacement using Phaser, using
inactive TAK1ꢀTAB1 structures (PDB code 2YIY) as search model. Coot
31
was used for model building and refinement was carried out using both
3
2
18, 21
Phenix version 1.8.4 and Refmac version 5.8.0049.
Figures were
generated by PyMol (The PyMOL Molecular Graphics System, Version
1.6.0.0) and Meastro (Version 1.5.014) from Schrödinger, LLC.
Ba/F3 Cell Proliferation Assay. Compound efficacy against cell
proliferation was conducted in 96ꢀwell plates. Compounds were added in
serial dilutions to cell culture. After 48 hours coꢀcultured with
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compounds, cell viability was measured using CellTiterꢀGlo (Promega,
Wisconsin), and IC50s were determined by XLfit4.0 (IDBS).
Cell culture and stimulations: TAK1ꢀdeficient (TAK1ꢀ/ꢀ) and
corresponding wild type (WT) mouse embryonic fibroblasts (MEFs) were
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
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26
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29
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31
32
33
34
35
36
37
38
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41
42
43
44
45
46
47
48
49
50
51
52
53
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25
a generous gift from S. Akira (Osaka University, Japan). Human
keratinocyte (HaCaT) cells, WT and TAK1ꢀ/ꢀ MEFs were cultured in
10ꢀcm diameter dishes in Dulbecco's Modified Eagle's medium
supplemented
with
10%
Foetal
Bovine
Serum,
1%
penicillin/streptomycin mix and 2 mM LꢀGlutamine under a humidified
atmosphere with 5% CO at 37 °C. In order to knockdown the indicated
2
panels of human MAP4Ks, a pool of four siRNA duplexes designed
against each target were purchased from Dharmacon (sold as
SMARTpool siRNA) and transfected onto HaCaT cells as described
18
previously. Prior to stimulation with appropriate ligands, cells were
cultured in DMEM containing 0.1% FBS for 16h. Inhibitors were added 1
h prior to stimulation with TGFß (50 pM; 45 min), human ILꢀ1ß (1 µg/ml;
10 min) or mouse ILꢀ1α (5 ng/ml; 10 min). Cells were lysed in 0.5 ml
iceꢀcold complete lysis buffer (50 mM TrisꢀHCl pH7.5, 1 mM EGTA, 1
mM EDTA, 1 % Triton Xꢀ100, 1 mM sodium orthovanadate, 50 mM
sodium flouride, 5mM sodium pyrophosphate, 0.27 M sucrose, 5 mM
ßꢀglycerophosphate, 0.1 % (v/v) 2ꢀmercaptoethanol, 0.5 µM
microcystinꢀLR, 1 tablet per 25 ml of complete protease inhibitor
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