Design, synthesis and activity of Mnk1 and Mnk2 selective inhibitors containing thieno[2,3-d]pyrimidine scaffold

Article abstract of DOI:10.1016/j.ejmech.2018.10.070

The mitogen-activated protein kinase-interacting kinases 1 and 2 (MNK1 and MNK2) phosphorylate eukaryotic initiation factor 4E (eIF4E) and play important roles in promoting tumorigenesis and metabolic disease. Thus, inhibiting these enzymes might be valua

Full text of DOI:10.1016/j.ejmech.2018.10.070

Accepted Manuscript
Design, synthesis and activity of Mnk1 and Mnk2 selective inhibitors containing
thieno[2,3-d]pyrimidine scaffold
Xin Jin, James Merrett, Sheng Tong, Bartholomew Flower, Jianling Xie, Rilei Yu,
Shuye Tian, Ling Gao, Jiajun Zhao, Xuemin Wang, Tao Jiang, Christopher G. Proud
PII:
S0223-5234(18)30947-4
DOI:
https://doi.org/10.1016/j.ejmech.2018.10.070
EJMECH 10856
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 22 May 2018
Revised Date: 25 October 2018
Accepted Date: 31 October 2018
Please cite this article as: X. Jin, J. Merrett, S. Tong, B. Flower, J. Xie, R. Yu, S. Tian, L. Gao, J. Zhao,
X. Wang, T. Jiang, C.G. Proud, Design, synthesis and activity of Mnk1 and Mnk2 selective inhibitors
containing thieno[2,3-d]pyrimidine scaffold, European Journal of Medicinal Chemistry (2018), doi: https://
doi.org/10.1016/j.ejmech.2018.10.070.
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Graphical Abstracts

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Design, synthesis and activity of Mnk1 and Mnk2
selective inhibitors containing thieno[2,3-
d]pyrimidine scaffold
1
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2#
1
2
2
1
2
Xin Jin , James Merrett, Sheng Tong, Bartholomew Flower, Jianling Xie, Rilei Yu, Shuye
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2,3
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Tian, Ling Gao, Jiajun Zhao, Xuemin Wang, Tao Jiang* and Christopher G. Proud*
1
School of Medicine and Pharmacy, Ocean University of China, and Laboratory for Marine
Drugs and Bioproducts, Qingdao National Laboratory for Marine Science and Technology,
2
Qingdao, China; Nutrition & Metabolism, South Australian Health & Medical Research
3
Institute, North Terrace, Adelaide, SA5000, Australia; School of Biological Sciences,
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University of Adelaide, Adelaide SA5005, Australia, Department of Endocrinology, Shandong
Provincial Hospital Affiliated to Shandong University, Shandong, China.
#
These authors contributed equally to this study.
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ABSTRACT
The mitogen-activated protein kinase-interacting kinases 1 and 2 (MNK1 and MNK2)
phosphorylate eukaryotic initiation factor 4E (eIF4E) and play important roles in promoting
tumorigenesis and metabolic disease. Thus, inhibiting these enzymes might be valuable in the
treatment of such conditions. We designed and synthesized a series of 4-((4-fluoro-2-
isopropoxyphenyl)amino)-5-methylthieno[2,3-d]pyrimidine derivatives, and evaluated their
inhibitory activity against the MNKs. We found 15 compounds that were active as MNK
inhibitors and that one in particular, designated MNK-7g, which was potent against MNK1 and
substantially more potent against MNK2. The compound MNK-7g did not affect other signaling
pathways tested and had no adverse effects on cell viability. As expected from earlier studies,
MNK-7g also inhibited cell migration. Therefore, the compound MNK-7g, which forms an ionic
bond with Asp226 in MNK2 and possesses a substituted aniline in a thieno[2,3-d] pyrimidine
structure, is a promising starting point for the future development of novel drugs for treating or
managing cancer and metabolic disease.
KEYWORDS ：Mnk; eIF4E Inhibition; Thieno[2,3-d]pyrimidine; Selective inhibitor
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1
. INTRODUCTION:
The MAP kinase-interacting kinases (also termed MAP kinase signal-integrating kinases,
MNKs) are activated by phosphorylation of their activation loops by certain members of the
MAP kinase (MAPK) superfamily [1-5]. The different isoforms of MNKs (MNK1 and MNK2)
each phosphorylate the translation initiation factor eIF4E [3-5]. eIF4E binds to the 5’-cap
structure which is found on all cytoplasmic mRNAs in eukaryotes. It also interacts with other
initiation factors and thereby plays a crucial role in recruiting ribosomes to mRNAs, thereby
promoting the initiation of their translation [6].
eIF4E is implicated in tumorigenesis and cancer progression (for a review, see [7, 8]), and
several lines of data suggest that phosphorylation of eIF4E is important in solid tumors and in
specific settings in leukemia (see, for example, [9-11] and discussion in [7]). MNKs also
promote the migration of cancer cells and may therefore play a role in tumor metastasis [12-14].
It remains unclear how eIF4E phosphorylation promotes tumor formation and progression,
although a number of mechanisms have been proposed [7]. eIF4E phosphorylation and/or the
MNKs also play roles in other processes such as innate immunity and macrophage activation [15,
1
6].
We recently showed that mice lacking MNK1 or MNK2 are protected against the adverse effects
of a high-fat diet [17]. For example, they show lower weight gain, improved glucose tolerance
and better sensitivity to insulin than high fat-fed wild-type mice. Interestingly, MNK1-KO and
MNK2-KO mice are protected in distinct ways; on a high fat diet, MNK2-KO mice show lower
weight gain and much less adipose tissue inflammation than wild-type animals, while MNK1-
KO mice show similar levels of these parameters to high fat-fed wild-type mice, but still show
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improved insulin signaling and glucose tolerance [17]. Thus, MNKs may be valid targets for the
management of metabolic disorders associated with excessive caloric intake and obesity.
Inhibiting the MNKs has therapeutic potential for cancers or metabolic syndrome, especially
since MNKs are not essential in normal cells or indeed in mice under standard vivarium
conditions [18]. Specific inhibitors of the MNKs are therefore expected to show low, if any,
toxicity.
Although several compounds that inhibit MNKs have been reported, agents such as
cercosporamide or CGP57380 [19] (Fig. 1) exert off-target effects on other kinases, inhibiting
several of them with greater potency than the MNKs [20]. This made it important to identify
novel selective MNK inhibitors. Recently, two MNK inhibitors have entered the clinical research
stage, in different settings, BAY1143269 (chemical structure not published) [21] and eFT508 [22]
(
Fig. 1). Meanwhile, merestinib (Fig. 1), which is in clinical testing in an ongoing phase 1 study,
was reported as an orally bioavailable small-molecule multi-kinase inhibitor exerting good
inhibitory effect on the MNKs [23]. This compound has been studied in acute myeloid leukemia
(AML) [23]. In addition, a new inhibitor, SEL-201 (Fig. 1 [13]), was found to exert potent anti-
melanoma effects by blocking MNK1/2 [13]. Lastly, a further MNK inhibitor was reported; it is
being evaluated for the treatment of blast crisis leukemia [24].
Some recent patent applications have revealed the structures of thienopyrimidine compounds
with low nanomolar efficacy in inhibiting the MNKs. However, detailed selectivity and efficacy
data have not yet been presented. Previously, we reported a novel compound, MNK-I1 [12] (Fig.
1
) as a more potent and specific inhibitor of the MNKs than either CGP57380 or
cercosporamide. In cells, it inhibits the activities of MNK1 and MNK2 at low micromolar
concentrations [12]. While it is the best MNK inhibitor that is readily available, it has poor
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stability and requires a complicated synthetic process, making it less than ideal. Studies from
Wang’s group have revealed some new chemical skeletons of MNK inhibitors [25-28] and
indicate that substitution at the ortho-position of the fluoroaniline ring of this structure and
substituent at the C6-position of thienopyrimidine may significantly enhance the potency and
selectivity of these compounds [25].
In developing further potential inhibitors, we had three aims: (i) to obtain compounds with
enhanced potency against the MNKs; (ii) to identify compounds with selectivity for MNK2 over
MNK1 and (iii) to simplify the synthetic route. As initial steps in that direction, we set out to
determine which modifications to MNK-I1 were compatible with retention of inhibitory activity
against MNKs and, through computer modeling, gain insights into the structure-activity
relationship (SAR) of these compounds as inhibitors of MNK2. Here, we describe the synthesis
and biological evaluation of the new compounds.
Figure 1. Structures of recently-reported MNK inhibitors.
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2
. RESULTS & DISCUSSION:
.1 Identifying active analogs of MNK-I1: initial set of compounds
2
MNK1 and MNK2 share ~80% sequence identity within their catalytic domains [3]. Based on
2
+
their sequences, the MNKs belong to the Ca /calmodulin-dependent kinase group, but are not
2
+
regulated by Ca /calmodulin. Instead, the MNKs are activated by MAPK signaling pathways
[29]. Although targeting the MNKs holds the potential for treating cancer [7], little success has
been achieved in this area so far, partly due to the lack of small molecule inhibitors for them
which are sufficiently potent or specific and are also suitable for use in vivo. The protein
databank (PDB) contains crystal structures for MNK1 (PDB ID: 2hw6) and MNK2 (PDB ID:
2
ac3, PDB ID: 2hw7), albeit in inactive states where key residues in their activation loops are not
phosphorylated and their conformations are consequently not those of the active enzymes. The
MNK proteins possess a unique Asp-Phe-Asp (DFD) motif, which replaces the Asp-Phe-Gly
(DFG) motif found in all other protein kinases [30, 31]. Moreover, this DFD motif adopts an
unusual ‘DFD-out’ conformation, in which the Phe residue flips into the ATP binding pocket,
thereby blocking access for ATP and exposing an additional hydrophobic/allosteric site [32]. In
the crystal structure of MNK2 (PDB ID: 2hw7) the kinase inhibitor staurosporine binds in the
ATP-binding site promoting the kinase-active conformation to move toward the ‘DFG/D in’
configuration.
Due to the unique DFD motif, the MNKs possess a small hydrophobic pocket near the
gatekeeper which is formed by the Phe159 gatekeeper and Cys225 secondary gatekeeper
residues. The MNKs also have a larger cavity near the lower hinge region than many kinases;
this lies in a C-shaped loop owing to a hydrogen bond between Met162 and Gly165 [33]. These
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features could in principle be exploited to develop specific MNK inhibitors. We used the
docking software, MOE, to examine the binding mode of staurosporine to MNK2. The
interaction map between staurosporine and MNK2 provides important information about their
interactions: two hydrogen bonds with Glu160 and Met162 from the hinge region and the third
one with Glu92 (Fig. 2A). MNK-I1 inhibits MNKs potently with good selectivity, i.e., with little
effect on other protein kinases, and blocks the migration of cancer cells [12]. We docked
compound MNK-I1 into the binding site of MNK2, as shown in Fig. 2B, to assess its mode of
interaction at this binding site. Molecular docking revealed several key interactions, including
two hydrogen bonds involving (i) the N atom of the pyrimidine and (ii) the N atom of the C6-
position amide bond of MNK-I1 with Met162 from the hinge region, and one ionic bond
between the alkaline group at the C6-position of the thienopyrimidine of MNK-I1 and Asp226
from DFD motif. Fig. 2B indicated that the fluorine atom at the ortho-position of fluoroaniline
did not interact with any residues from the surface of MNK2. Since the fluorine at this position
complicates the synthesis, we decided to alter it, while keeping other groups the same.
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Figure 2. View of inhibitors (staurosporine and MNK-I1) in the pocket of MNK2. (A)
Stereoview of staurosporine in ATP binding site of MNK2, showing the required key
interactions with MNK2. (B) The binding mode of MNK-I1 in the pocket of MNK2. Compound
stick representation: carbon in black, nitrogen in blue, oxygen in red, sulfur in yellow, and
fluorine in green. Light blue shows the amino acid residues in MNK2.
With this result in mind, at the first stage of the compound design process, we synthesized five
compounds according to reference [25]. They included 7w with an isopropyl ether group, and 7v
with methoxyl group instead of the fluorinated isopropyl ether group, respectively; two
compounds 7x and 6d derived from 7w and one further compound 3b (see Fig. 3 and Scheme 1).
The activity of these compounds against MNKs was tested by treating mouse 3T3-L1 cells to
assess the level of phosphorylation of eIF4E. eIF4E is the best-characterized and only in vivo-
validated substrate for the MNKs [17]. It is phosphorylated by MNK1 or MNK2, but by no other
kinases, as shown by the complete loss of eIF4E phosphorylation in cells and tissues from mice
in which the genes for MNK1 and MNK2 (Mknk1 and Mknk2) have been disrupted [18]. MNKs
phosphorylate eIF4E exclusively on Ser209 [34]. Thus, phosphorylation of eIF4E provides a
reliable and diagnostic ‘read-out’ of MNK activity within cells.
Apart from MNK-I1, the compounds were inactive (7v, 3b) or only weakly active (6d, 7x)
against MNKs in 3T3-L1 cells (data were summarized in Fig. 3). 7w was the most effective, but
was less potent than MNK-I1 (did not inhibit P-eIF4E completely, unlike MNK-I1). Although
we did not obtain better compounds than MNK-I1, this indicated that altering the fluorinated
isopropyl ether group in the compound (MNK-I1) allowed activity to be retained. We also
suspected that the substituent group at the C6-position of thienopyrimidine itself had a
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substantial influence on the ability of the compound to inhibit the MNKs when other substituents
were kept the same.
We therefore focused on the C6-position. At the second stage of the compound design process,
we changed the substituent at C6-position of the thienopyrimidine to obtain a series of
compounds, 7f, 7g, 7a and 6c as shown in Fig. 3. We chose some polar and bulkier groups and
changed the lengths of the carbon chains of the substituent (7a-u in Scheme 2). We also used an
ester bond instead of an amide bond in the substituent (6a-c in Scheme 2).
Figure 3. Structural modification of MNK-I1 and the activity against MNKs of the compounds
(3b, 6d, 7v, 7w and 7x). In the first stage, we modified the ortho-position of the substituted
aniline (7v, 7w) and the C6-position (7x, 6d). Subsequently, based on the first stage, we focused
on the C6-position to obtain a series of derivatives. Their activity was tested in 3T3-L1 cells
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which were treated with the indicated compounds for 2 h. Cells were then lysed, and lysate
supernatants analysed by SDS-PAGE and western blot using the indicated antibodies.
2
.2 Synthesis of compounds
The reported synthetic routes for derivatives of the thieno[2,3-d]pyrimidine moiety of MNK-I1
25, 26] suffer from several drawbacks. We have improved the routes to obtain a series of
[
derivatives of N-(3-(dimethylamino)propyl)-4-((4-fluoro-2-((1-fluoropropan-2-yl)oxy)phenyl)
amino)-5-methylthieno[2,3-d]pyrimidine-6-carboxamide. 5-Fluoro-2-nitrophenol was converted
to 4-fluoro-2-isopropoxy-1-nitrobenzene by reacting with 2-bromopropane in the presence of
2 3
potassium carbonate (K CO ) in dimethylformamide (DMF) resulting in a yield of 70%. 4-
Fluoro-2-isopropoxy-1-nitrobenzene and commercially available 4-fluoro-2-methoxy-1-
nitrobenzene were hydrogenated over Pb/C under hydrogen gas to generate 2a and 2b (Scheme
1
), followed by coupling with 1 which was synthesized as previously reported [26] using N, N-
diisopropylethylamine (DIPEA) in isopropyl alcohol to obtain 3a, 3b respectively. Basic
hydrolysis of the methyl ester group of 3a, 3b was carried out using LiOH H O in a (1:1)
·
2
mixture of tetrahydrofuran and water to produce 4a, 4b respectively (Scheme 2). Subsequent
treatment of the carboxylic acids 4a, 4b with N-hydroxysuccinimide (NHS) and 1-ethyl-3-(3-
dimethylaminopropyl)carbodiimide (EDCI) in DMF yielded 5a, 5b.
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Scheme 1. Synthesis of Compounds 4a and 4b. Reagents and conditions: (a) DIPEA, 85°C,
IPA, N , 10 h, 63% (3a), 85% (3b). (b) THF/H O; LiOH·H O, 2 M HCl, 84% (4a), 90% (4b).
2
2
2
The intermediates 5a and 5b can be stored stably at room temperature. A series of derivatives of
MNK-I1 were obtained by reacting 5a or 5b with corresponding amine in DMF, followed by the
addition of water to collect the precipitate, which was recrystallized in tetrahydrofuran (THF)
and ether to yield 7a-t or 7u (Scheme 2) respectively. This synthetic method simplified the
purification and preparation of the compound. We converted 4a and 4b to their corresponding
esters (6a, 6b and 6c), by reacting with 2-phenylethyl bromide or 3-bromo-1-propanol or 2-
bromoethanol in the presence of potassium carbonate (K CO ) in DMF, respectively (Scheme 2).
2
3
1
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Scheme 2. Synthesis of Compounds 6a-c, 7a-u. Reagents and conditions: (a) 2-bromoethanol,
K
2
CO
3 2 3
, DMF, 12 h, rt, 48% (6a); or 2-phenylethylbromide, K CO , DMF, 12 h, rt, 48% (6b); or
3
-bromo-1-propanol, DMF, K CO , 12 h, rt, 48% (6c); (b) DMF, EDCI, NHS, rt, 61% (5a), 74%
2
3
(
5b); (c) DMF, related amine; H O.
2
2
.3 Biological activity of the new compounds against MNKs
Our initial analysis tested the full set of compounds at 3 µM, at which concentration MNK-I1
completely inhibits eIF4E phosphorylation in cells. Several compounds proved to be inactive or
much less active at inhibiting MNK function than MNK-I1 (compounds 6b, 7b-d, 7p, 7r and 7t)
(
Fig. 4A). Compounds 6c, 7e, 7q, 7s and 7u showed only partial inhibition (Fig. 4A). The
strongest inhibition was observed for 7a, 7f-i, 7k-7o (Fig. 4A). None of the compounds affected
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the total amount of eIF4E. We then tested a subset of the stronger inhibitors, 7g, 7i, 7k, 7m and
7
o, at lower concentrations, down to final concentrations of 0.1 µM. Only MNK-7g showed a
level of potency similar to MNK-I1 (Fig. 4B).
Figure 4. Evaluation of potential MNK inhibitors in 3T3-L1 cells. Mouse 3T3-L1 cells were
treated with the indicated compounds at 3 µM (A) or at the concentrations shown (B), in each
case for 2 h. As appropriate, the vehicle, DMSO, was used at a final concentration of 0.03-0.05%.
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Cells were then lysed, and supernatants analysed by SDS-PAGE and western blot using the
indicated antibodies.
The compound MNK-7g was docked in silico into the ATP binding site of MNK2 to explore the
binding mode between the compound of MNK-7g and MNK2 (see Fig. 5A, B). From the
interaction map, we observed some key features: two hydrogen bonds, with Met162 and Asn210,
one ionic bond with Asp226, and a π
−H stacking interaction with Leu90. The pattern of binding
was similar to the interaction between staurosporine and MNK2, but with differences.
Comparing the binding patterns of MNK-7g and MNK-I1 (Fig. 2B and Fig. 5B), we noticed that
the location of the two compounds in the pocket was the same, with the distinction that they can
form different hydrogen bonds to increase the stability of the binding to MNK2. The binding
modes of the other two compounds (7i and 7o) were similar to that of MNK-7g (see
supplementary material Figure 1). We conclude that a bulky basic group at the C6-position might
play a major role in the interaction with the MNKs and thus enhance the inhibition of MNKs and
also improve the solubility to some extent. In contrast, when we changed the carbon chain, as in
7
m or 7u, the inhibition became weaker than observed for MNK-I1 (Fig. 4A).
Compound 7s clearly inhibited P-eIF4E less potently than MNK-7g (Fig. 4A). Therefore, 7s was
docked into the ATP binding site to assess differences in binding mode (see Fig. 5C, D). The
binding map of 7s showed that an ionic bond was formed between protonated N atom of
pyrrolidine and negatively charged Asp226, but the hydrogen bond with Met162 was lost
compared with the binding mode of MNK-7g (Fig. 5A, C). This indicated that a carbon chain of
the appropriate length (two to three carbon atoms) can retain the hydrogen bond that the N atom
of thienopyrimidine forms with Met162, which appeared important for the inhibitory activity of
MNK2. When the amide bond (7x Fig. 3) was changed into an ester bond (6c), the compound’s
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inhibitory ability was almost unchanged. We speculated that the ester bond or amide bond had no
obvious influence on the ability to inhibit the MNKs. However, when a polar substituent at the
C6-position was replaced by a nonpolar group (compared 6d and 6b, seeing Fig. 3 and Scheme
2
), inhibition of the MNKs was slightly weaker (Fig.3 and Fig. 4A), telling us that a polar
substituent at C6-position was better. Thus, in terms of the compounds’ structures, the N atom of
the pyrimidine and a polar substituent group in the C6-position of thienopyrimidine appeared
important for binding MNK2 and a substituent group at the C6-position of appropriate carbon
chain length can enhance inhibition of the MNKs.
Figure 5. Comparison of the results between compounds 7g and 7s in the docking studies. (A)
Interaction map, showing the binding between 7g and MNK2. (B) The 3D combination mode of
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7
g and MNK2. (C) Interaction map, showing the binding pattern between 7s and MNK2. (D)
The 3D combination mode of 7s and MNK2. Compounds are shown in stick representation:
carbon in black, nitrogen in blue, oxygen in red, sulfur in yellow, and fluorine in green. Light
blue depicts the amino acid residues in MNK2.
The compounds 7g, 7i, 7k, 7m and 7o were more potent than 7f, 7h, 7j, 7l and 7n in inhibiting
MNK activity when the substituent group in the C6-position of thienopyrimidine was the same.
This indicated that a bulky group in the ortho-position of the substituted aniline may increase the
potency of MNK inhibition. Additionally, we found, by analysing the docking results of all
compounds (Supplementary Material Figure 2), that the different substituent at the ortho-position
of the substituted aniline can generate different interaction modes. Hence, we can infer that the
group at the ortho-position of the substituted aniline group plays an important role in the ability
of compounds to inhibit MNK activity.
2
.4 Biological activity of compound MNK-7g
It was important to assess whether MNK-7g had affected any other major signaling pathways
such as the ERK MAP kinase pathway (which is upstream of the MNKs), protein kinase B (also
termed AKT) which mediates the effects of many stimuli, including insulin, and the mammalian
target of rapamycin complex 1 (mTORC1) pathways, which exert manifold effects on gene
expression, metabolism and cell growth.
In 3T3-L1 cells, MNK-I1 did not affect the phosphorylation (activation) of ERK, its downstream
effector, the protein kinase RSK, or the phosphorylation of PKB, at Ser473, a substrate for
mTOR complex 2 (Fig. 6A, quantified in Fig. 6B). MNK-I1 also did not affect the
phosphorylation of 4E-BP1, a direct substrate for mTOR complex 1, or ribosomal protein (rp)
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S6, an indirect target for mTORC1 signaling (Fig. 6A, B). These data are in close agreement
with our earlier finding that MNK-I1 does not affect these pathways in human MDA-MB-231
cells [12]. However, they differ from the conclusions of Brown & Gromeier [35] who reported
that MNKs promote mTORC1 activity under a specific condition (in response to insulin-like
growth factor 1, IGF1). Our data suggested this is not a general effect as MNK inhibition clearly
does not alter mTORC1 signaling here.
MNK-7g also did not affect phosphorylation of PKB, rpS6 or 4E-BP1 (Fig. 6A, B). However,
interestingly, it did cause a slight increase in the phosphorylation of ERK and RSK perhaps by
impairing a (so far hypothetical) inhibitory ‘loop’ linking the MNKs to their upstream activator,
ERK.
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Figure 6. Comparison of the effects of MNK-I1 and MNK-7g on signaling pathways in 3T3-L1
cells. (A) 3T3-L1 cells were treated with the indicated compounds at the concentrations shown,
in each case for 2 h. Cells were then lysed, and supernatants were analysed by SDS-PAGE and
western blot using the indicated antibodies; for 4E-BP1, arrows show the differentially
phosphorylated species, the top one (PPP) being the most highly phosphorylated one. (B)
Quantification of data from three independent experiments for the indicated proteins. Error bars
are SEM. $ p < 0.001; # p < 0.01.
To assess the relative efficacy of MNK-I1 and MNK-7g against MNK1 and MNK2, we made
use of cells (MEFs) from mice in which one or the other had been knocked out [10]. In wild-type
MEFs, both MNK-I1 and MNK-7g each inhibited the phosphorylation of eIF4E completely at 5
µM and by 50% between 0.1 and 0.3 µM (Fig. 7A, B). In MNK1-KO cells, eIF4E
phosphorylation reflects the activity of MNK2, and vice versa for MNK2-KO cells. Both
compounds showed similar inhibition of P-eIF4E levels in MNK1-KO cells, with almost
complete inhibition already seen at 0.1 µM (Fig. 7A). In contrast, at 0.3 µM, either compound
only partially decreased P-eIF4E in MNK2-KO cells (Fig. 7A), 5 µM being needed to see
inhibition similar to that observed with 0.1 µM in MNK1-KO cells. This indicated that both
compounds inhibit MNK2 considerably more strongly than MNK1. To study their activities
against MNK2 further, we treated MNK1-KO cells with even lower concentrations of MNK-I1
and MNK-7g (Fig. 7C; data quantified in 7D). MNK-7g showed slightly (roughly two-fold), and
consistently, better potency against MNK2 than MNK-I1 (in MNK1-KO cells; Fig. 7D). Thus,
both compounds are selective MNK2 inhibitors, with MNK-7g showing greater selectivity, i.e.,
slightly less inhibition of MNK1 and rather stronger inhibition of MNK2.
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Figure 7. Comparison of the effects of MNK-I1 and MNK-7g on signaling pathways in wild-
type and MNK knockout MEFs. (A) MEFs (wild-type, MNK1-KO or MNK2-KO, as shown)
were treated with the indicated concentrations of MNK-I1 or 7g, in each case for 2 h. CGP57380
was used at 50 µM. Cells were then lysed, and supernatants analysed by SDS-PAGE and western
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blot using the indicated antibodies. (B) Quantification of data from three independent
experiments for the indicated proteins. Error bars are SEM. (C, D) As (A), but using lower
inhibitor concentrations; (D) shows quantification of data from 3 independent experiments; ***,
p < 0.005.
To further assess the relative efficiencies of MNK-I1 and MNK-7g against MNK1 and MNK2,
we performed in vitro kinase assays using recombinant eIF4E as substrate, and MNK1 or MNK2
expressed in human embryonic kidney (HEK) 293 cells. Extensive pilot experiments were
conducted to ensure that assays were within the linear range by adjusting the time of incubation,
the amount of MNK1 or MNK2 and the quantity of eIF4E in the assays. The data clearly showed
that MNK-7g and MNK-I1 had similar potencies against the two MNKs while, in agreement
with the data for MNK-KO MEFs, MNK-7g was more effective than MNK-I1 against MNK2
(
Fig. 8).
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Figure 8. Inhibition of MNK-catalysed phosphorylation of eIF4E by MNK-I1 and MNK-7g in
vitro. MNK assays were performed using recombinant GST-MNK1a and GST-MNK2a, with
eIF4E as substrate, in the absence or presence of MNK-7g (in A) or MNK-I1 (in B) at the
indicated concentrations. Reaction products were immunoblotted against for the indicated
phospho- (P-) or total proteins. (C) Quantification of data from A and B. Similar data were
obtained in a replicate experiment.
We extended the analysis of MNK-7g to a quite different cell type, (human) MDA-MB-231
breast cancer cells (Fig. 9A). The data showed that both MNK-7g and MNK-I1 decreased P-
eIF4E levels in these cells, and caused almost complete inhibition at 1 µM, confirming that they
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are active against human MNKs. MNK-7g tended to be slightly less effective than MNK-I1 in
these cells.
We have previously shown that MNK-I1 impairs the migration of MDA-MB-231 cells and other
cancer cells [12]. As the migration assays need to be performed over a longer time-scale than the
assays reported above (here, at least 48 h), it was important to assess whether the compounds
were stable over this time period, i.e., they continued to block P-eIF4E. As shown in Fig. 9B,
both MNK-I1 and MNK-7g still inhibited eIF4E phosphorylation at times beyond the length of
the migration assay (up to 72 h), with only a slight tendency for P-eIF4E levels to rise at later
times, especially in cells that received MNK-I1. This may suggest that MNK-I1 is less stable in
aqueous solution at 37 ºC than MNK-7g.
The effect of MNK-7g on cell migration was assessed using a 3-dimensional ‘Transwell’ assay,
where it proved to be at least as effective as MNK-I1 in blocking the migration of MDA-MB-231
cells (Fig. 9C). MNK-7g did not effect on the distribution of cells in different phases of the cell
cycle, as assessed by staining cells with propidium iodide followed by FACS analysis (Fig. 9D).
There was also no change in the proportion of ‘sub G0’ cells indicating MNK-7g did not
adversely affect cell viability. Since MNK inhibition does not affect the proliferation or cell
cycle distribution of MDA-MB-231 cells (Fig. 9D and ref. [12]), the effects on migration cannot
be a secondary consequence of reduced cell number (proliferation).
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Figure 9. Effects of MNK-I1 and MNK-7g on the migration and proliferation of MDA-MB-231
breast cancer cells. (A) MDA-MB-231 cells were treated with the indicated concentrations of
MNK-I1 or 7g, in each case for 2 h. Cells were then lysed, and supernatants analysed by SDS-
PAGE and western blot using the indicated antibodies. CGP57380 was used at 30 µM. (B)
MDA-MB-231 cells were treated with MNK-I1 or MNK-7g (3 µM) for the indicated times, and
samples were analysed as in (A). (C) Transwell migration assays were performed with collagen
as the attractant. After 24 h, the cells that had migrated were counted. Assays were performed in
triplicate and data are shown ± SEM; ***, p < 0.005; ****, p < 0.001. (D) Cell cycle analysis of
MDA-MB-231 cells treated with 3µM MNK-I1 or the indicated concentrations of MNK-7g.
Proportion (%) of cells at different stages of the cell cycle are displayed. Data are mean ± SEM
(n=5).
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3
. CONCLUSIONS
Using molecular docking and a structure-based design approach, we have achieved several
improvements to the structure of inhibitors containing a thieno[2,3-d]pyrimidine scaffold that
enhance MNK inhibition and simplify the synthetic route.
We designed and synthesized a series of MNK1 and/or MNK2 inhibitors containing a thieno[2,3-
d]pyrimidine scaffold. Our studies revealed that polar substituent groups at the C6-position of
thienopyrimidine with chain lengths of two to three carbons increase the binding efficiency and
thus inhibition of the MNKs, whilst bulky groups in the ortho-position of the substituted aniline
can increase the potency of MNK inhibition. Our docking studies pointed to two binding
interactions that are important for MNK inhibition, i.e., one hydrogen bond with Met162 from
the hinge region and another hydrogen bond with Asp226 from the DFD motif.
Our study also showed that the fluorinated isopropyl ether group at the ortho-position of the
fluoroaniline group can be substituted with an isopropyl ether group without diminishing
inhibitory activity. This modification is a practical way to simplify the synthetic route.
Another of our aims was to design and test compounds with greater selectivity towards MNK2.
We found that one such compound, MNK-7g, possessed similar potency and somewhat greater
selectivity to MNK2 when compared to MNK-I1. Importantly, MNK-7g did not affect any major
signaling pathways other than a slight increase in the phosphorylation of ERK and RSK.
Given the simplified synthetic route, high yield and good stability, compound MNK-7g has been
selected as a starting-point to develop further compounds suitable for probing the roles of MNK2
in cancer and especially in metabolic disease. This is the focus of ongoing efforts.
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4
. EXPERIMENTAL SECTION
.1 Materials and chemicals
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All cell culture solutions and supplements were purchased from Life Technologies unless
indicated otherwise. Reagents for SDS-PAGE were purchased from Bio-Rad and Sigma. The
Mnk inhibitor CGP57380 was obtained from Abcam.
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.2 Cell culture and treatment
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T3-L1 pre-adipocytes were maintained in Dulbecco's Modified Eagle's Medium (DMEM) high
glucose with pyruvate (Cat. 11995-065) supplemented with 10% (v/v) fetal bovine serum (FBS)
Ausgenex; Lot. FBS00211-1; heat-inactivated at 55°C for 30 min) and 1% penicillin-
(
streptomycin (P/S). Cells were strictly sub-cultured at 75-80% confluence every 2-3 days.
Mouse embryonic fibroblasts (MEFs) were prepared from E13.5 embryos of C57BL/6J mice in
which the genes for MNK1 or MNK2 had been homozygously knocked out [17]. MEFs were
maintained in DMEM (11995-065) supplemented with 10% (v/v) FBS and 1% P/S.
MDA-MB-231 cells were propagated as described previously [12].
All cells were maintained at 37°C in humidified air with 5% CO . Chemical treatments were
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added to the medium in DMSO vehicle at the appropriate concentrations for the indicated times
(always <0.05% v/v DMSO).
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.3 SDS-PAGE and Western blot
Cell monolayers were harvested in RIPA lysis buffer containing 50 mM Tris-HCl, pH 7.5, 150
mM NaCl, 1% NP-40 (IGEPAL CA-630, 1% sodium deoxycholate, 0.1% sodium dodecyl
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sulfate (SDS), 1 mM ethylenediaminetetraacetic acid (EDTA), 50 mM β-glycerophosphate, 0.5
mM NaVO , 0.1% 2-mercaptoethanol and protease inhibitors (Roche). After lysis, insoluble
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material was removed by centrifugation at >12,000 g for 10 min at 4°C. Protein content was
determined by the Bradford protein assay (Bio-Rad) [17]. Cell lysates were heated in sample
buffer [250 mM Tris–HCl, 10% (v/v) sodium dodecyl sulfate (SDS), 20% (v/v) glycerol, 0.12%
(w/v) bromophenol blue] at 95°C for 5 min and equal amounts of protein were then subjected to
polyacrylamide gel electrophoresis (PAGE) and electrophoretic transfer to nitrocellulose
membranes. Membranes were blocked in phosphate-buffered saline (PBS)-0.05% Tween20
containing 5% (w/v) skim milk powder for 30 min at room temperature. Membranes were
probed with the indicated primary antibody overnight at 4°C. After incubation with fluorescently
tagged secondary antibody, blots were visualised using a LI-COR Odyssey Quantitative Imaging
System. Primary antibodies were from Cell Signaling Technology, except: P-eIF4E (Life
Technologies), rpS6 (Santa Cruz) and actin (Sigma). Secondary antibodies were obtained from
Fisher Scientific and used at 1:20,000 dilution.
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.4 In vitro MNK assays
MNK assays were performed as previously described [36]. Briefly, recombinant glutathione S-
transferase (GST)-MNK fusion proteins GST-MNK1a and GST-MNK2a were purified by
glutathione pull-down from lysates of transfected HEK293 cells. Recombinant GST-MNK were
incubated in kinase assay buffer (25 mM Tris-HCl, pH7.5, 50 mM KCl and 2 mM MgCl
2
) with
20 µM ATP and 65 ng eIF4E (expressed in E. coli) as substrate at 30°C for 30 min in the
absence or presence of MNK inhibitors at indicated concentrations. Reactions were stopped by
the addition of the above sample buffer, products were analysed by SDS-PAGE/immunoblotting
using antisera against phosphorylated (P-) eIF4E Ser209, eIF4E and GST.
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4
.5 Cell migration assays
As previously described [12], for migration assays, Transwells (8 µm pore size, BD Biosciences)
were pre-coated with 10
µ
g/ml collagen (Millipore). Cells were pre-treated with inhibitors for 60
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min before seeding at 3 x 10 into the Transwell inserts. Cells that had migrated into the bottom
well after 24h were stained with DAPI (4’6-diamidino-2-phenylindole, 1:20,000) and visualized
with a Nikon Eclipse Ni microscope (
quantified using ImageJ.
×10 objective lens). DAPI-stained cell numbers were
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.6 Cell cycle analysis
Cell cycle analysis utilised the propidium iodide staining method [37]. Briefly, MDA-MB-231
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cells were seeded at 5 x 10 cells/well (6-well plate), given 24 h to attach and then treated with
indicated amounts of inhibitor or vehicle. Cells were harvested at ~70% confluence and fixed in
methanol for at least 2 h. Cells were centrifuged, resuspended in PBS and stained with 1µg/ml
propidium iodide (PI). Roughly 20,000 PI-stained cells were analysed using a BD FACSCanto II
flow cytometer. The data was analysed using BD FACSDiva software (BD Biosciences).
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.7 Statistics
Data were analysed by one-way ANOVA with Dunnett's multiple comparisons test for
significance [12]. For the MNK1-KO MEF low dosage experiment, Sidak’s multiple
comparisons test for significance was used to compare the means of pairs of columns. For the
cell cycle analysis, a two-way ANOVA with Tukey's multiple comparisons test was used. All
statistical analyses were performed using GraphPad Prism 7 software.
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.8 Molecular docking
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