Discovery of a potent inhibitor of MELK that inhibits expression of the anti-apoptotic protein Mcl-1 and TNBC cell growth

Article abstract of DOI:10.1016/j.bmc.2017.03.018

Despite recent advances in molecularly directed therapy, triple negative breast cancer (TNBC) remains one of the most aggressive forms of breast cancer, still without a suitable target for specific inhibitors. Maternal embryonic leucine zipper kinase (MELK) is highly expressed in TNBC, where level of overexpression correlates with poor prognosis and an aggressive disease course. Herein, we describe the discovery through targeted kinase inhibitor library screening, and structure-guided design of a series of ATP-competitive indolinone derivatives with subnanomolar inhibition constants towards MELK. The most potent compound, 17, inhibits the expression of the anti-apoptotic protein Mcl-1 and proliferation of TNBC cells exhibiting selectivity for cells expressing high levels of MELK. These studies suggest that further elaboration of 17 will furnish MELK-selective inhibitors with potential for development in preclinical models of TNBC and other cancers.

Full text of DOI:10.1016/j.bmc.2017.03.018

Accepted Manuscript
Discovery of a Potent Inhibitor of MELK that Inhibits Expression of the Anti-
apoptotic Protein Mcl-1 and TNBC Cell Growth
Ramakrishna Edupuganti, Juliana M. Taliaferro, Qiantao Wang, Xuemei Xie,
Eun Jeong Cho, Fnu Vidhu, Pengyu Ren, Eric V. Anslyn, Chandra
Bartholomeusz, Kevin N. Dalby
PII:
S0968-0896(17)30192-X
http://dx.doi.org/10.1016/j.bmc.2017.03.018
BMC 13615
DOI:
Reference:
Bioorganic & Medicinal Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
2 February 2017
3 March 2017
8 March 2017
Please cite this article as: Edupuganti, R., Taliaferro, J.M., Wang, Q., Xie, X., Cho, E.J., Vidhu, F., Ren, P., Anslyn,
E.V., Bartholomeusz, C., Dalby, K.N., Discovery of a Potent Inhibitor of MELK that Inhibits Expression of the
Anti-apoptotic Protein Mcl-1 and TNBC Cell Growth, Bioorganic & Medicinal Chemistry (2017), doi: http://
dx.doi.org/10.1016/j.bmc.2017.03.018
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Discovery of a Potent Inhibitor of MELK
that Inhibits Expression of the Anti-
apoptotic Protein Mcl-1 and TNBC Cell
Growth
Leave this area blank for abstract info.
Ramakrishna Edupuganti a,b,c,‡, Juliana M. Taliaferro a,‡, Qiantao Wanga, Xuemei Xied, Eun Jeong Chob, Fnu
Vidhud, Pengyu Rene, Eric V. Anslync, Chandra Bartholomeuszd, and Kevin N. Dalby*,a,b
aDivision of Chemical Biology & Medicinal Chemistry, bThe Targeted Drug Discovery and Development Program, College of
Pharmacy, cDepartment of Chemistry, eDepartment of Biomedical Engineering, Cockrell School of Engineering, The University of Texas
at Austin, Texas 78712, USA
dSection of Translational Breast Cancer Research. Department of Breast Medical Oncology, The University of Texas MD Anderson
Cancer Center, Houston, Texas 77030, USA
O
H
H
N
N
O
O
O
R
HN
Focused
HN
structure-guided
design
N
N
N
N
N
N
O
O
Screening hit
15 Ki = 5.6 ± 0.4 nM
Compound 17 was docked in MELK
16 R = H,
17 R = CO2Me, Ki = 0.39 ± 0.09 nM
21 R = CO2H, Ki = 0.68 ± 0.07 nM
Ki = 0.47 ± 0.06 nM
binding pocket

Bioorganic & Medicinal Chemistry
Discovery of a Potent Inhibitor of MELK that Inhibits Expression of the Anti-
apoptotic Protein Mcl-1 and TNBC Cell Growth
Ramakrishna Edupuganti a,b,c,‡, Juliana M. Taliaferro a,‡, Qiantao Wanga, Xuemei Xied, Eun Jeong Chob, Fnu Vidhud, Pengyu Rene, Eric V. Anslync, Chandra
Bartholomeuszd, and Kevin N. Dalby*,a,b
aDivision of Chemical Biology & Medicinal Chemistry, bThe Targeted Drug Discovery and Development Program, College of Pharmacy, cDepartment of
Chemistry, eDepartment of Biomedical Engineering, Cockrell School of Engineering, The University of Texas at Austin, Texas 78712, USA
dSection of Translational Breast Cancer Research. Department of Breast Medical Oncology, The University of Texas MD Anderson Cancer Center, Houston,
Texas 77030, USA
* Corresponding author.
‡R. Edupuganti, J. M. Taliaferro contributed equally to this work.
ARTICLE INFO
ABSTRACT
Article history:
Received
Received in revised form
Accepted
Despite recent advances in molecularly directed therapy, triple negative breast cancer (TNBC)
remains one of the most aggressive forms of breast cancer, still without a suitable target for
specific inhibitors. Maternal embryonic leucine zipper kinase (MELK) is highly expressed in
TNBC, where level of overexpression correlates with poor prognosis and an aggressive disease
course. Herein, we describe the discovery through targeted kinase inhibitor library screening,
and structure-guided design of a series of ATP-competitive indolinone derivatives with
subnanomolar inhibition constants towards MELK. The most potent compound, 17, inhibits the
expression of the anti-apoptotic protein Mcl-1 and proliferation of TNBC cells exhibiting
selectivity for cells expressing high levels of MELK. These studies suggest that further
elaboration of 17 will furnish MELK-selective inhibitors with potential for development in
preclinical models of TNBC and other cancers.
Available online
Keywords:
Maternal embryonic leucine zipper kinase (MELK)
MELK inhibitor
Indolinone derivatives
Triple negative breast cancer (TNBC)
Screening hits
2017 Elsevier Ltd. All rights reserved.
Kinase inhibitor

1. Introduction
indolinone motifs represent a common pharmacophore in
ATP-competitive inhibitors.32-33
MELK, a member of the AMPK-RK family which
mediates cell survival under conditions of metabolic stress,1-2
is a mitotically regulated kinase implicated in the cell cycle
and the promotion of cell proliferation3-4 (see recent reviews5-
7). Its expression is elevated in the early stages of murine
embryonic development8-9 and is found to be highly expressed
in immature proliferating cells, such as hematopoietic cells in
bone marrow,4 multipotent neural progenitors,10 myoblasts,11
and mammary progenitors.12 In mice, MELK is detectable in
the thymus, lung and testis, but in general, exhibits a limited
expression profile in the adult.1, 9 MELK knockout is reported
to have no impact on the development of C57BL/6 mice,13
suggesting that its expression is not essential.
MELK is upregulated (mRNA and protein) in a wide array
of cancer cell types and clinical tumor samples,14-19 and gene
expression analysis has identified it as a possible glioma stem
cell marker.20-21 Its expression correlates with poor prognosis
in several cancers, including glioblastoma multiforme
(GBM),21 breast cancer,22 especially triple negative breast
23-24
cancer
(TNBC),13,
prostate
cancer,25
lung
adenocarcinoma,17 acute myeloid leukemia,26 and gastric
cancer.27 siRNA-mediated knockdown of MELK impedes the
Figure 1. Top 3 screening hits with key predicted hydrogen-bonding
interactions with MELK binding site and their IC50's
renewal of glioma stem cells10 and tumor growth.14,
21
Furthermore, MELK is also reported to induce apoptosis in
glioma stem cells21, 28 as well as in basal breast cancers,13 but
not normal neural stem cells or luminal breast cancers.13 It is
also implicated in the resistance of cancer cells to radiation.29-
The most potent of the top hits was nintedanib (BIBF-1120,
Vargatef®/Ofev®), which displayed an IC50 of 43 nM (Figure
1). Nintedanib is already FDA-approved for the treatment of
idiopathic pulmonary fibrosis and is currently undergoing
clinical trials for treatment of non-small cell lung cancer,
metastatic colorectal cancer, and ovarian cancer. However, it
is a multi-kinase inhibitor whose primary mechanism of action
is inhibition of the growth factor receptors VEGFR, PDGFR,
and FGFR. Given its favorable drug-like properties, we
decided to improve upon nintedanib’s potency and selectivity
towards MELK.
30
Taken together, these data suggest that MELK may have
value as a therapeutic target for treating various cancers,
especially those where it might serve a potentially critical
survival function (e.g. glioblastoma,21 medulloblastoma21 and
triple negative breast cancers, TNBCs13).
To date, no inhibitors developed with MELK as a primary
target are FDA-approved, and no selective inhibitor of MELK
with good oral bioavailability has been reported. A primary
goal of this study was to identify scaffolds from which to
develop new inhibitors of MELK. To this end, we describe a
highly potent indolinone inhibitor of MELK. Furthermore, we
report on its selectivity, relative to several related kinases, and
show that it preferentially inhibits the proliferation of TNBC
cell lines expressing high levels of MELK. MELK contributes
to the survival of TNBC cells by inducing the expression of
the Bcl-2-family anti-apoptotic protein Mcl-1 (induced
myeloid leukemia cell differentiation protein).31 We
demonstrate suppression of Mcl-1 expression by the inhibitor
in HCC70 cells in a dose-dependent manner. The inhibitor
also shows no activity against non-invasive, non-tumorigenic
MCF-10A human breast epithelial cells. This inhibitor (17)
therefore represents a useful starting point from which to
develop a new class of MELK inhibitor.
Figure 2. A) Docking of nintedanib (IC50 ~ 43 nM) into the ATP-binding
site of the MELK conformation PDB 4BKY. B) Crystal structure of a
benzodipyrazole inhibitor (Cpd2) in complex with the MELK catalytic
domain (PDB: 4BKZ).
While nintedanib was clearly the most potent molecule,
(IC50 = 43 nM), additional observations from our screen
directed our medicinal chemistry design strategy. The top
three most potent inhibitors (Figure 1) all exhibit a putative
alternating donor/acceptor hydrogen-bonding pattern that has
also been observed with previously published inhibitors.
Heteroatoms and substituents of the bicyclic core are spatially
oriented to interact with both the hinge region and the
conserved catalytic lysine, respectively. In addition to
nintedanib, an indolinone scaffold forms the core of three
other candidate compounds (Table S1, entries 2, 8, and 9 in
supporting data), while others contained similar heterocyclic
motifs. Notably, several of the indolines contain substituents
at the 5th or 6th position, which are predicted through modeling
2. Results and discussion
We initiated an off-target/cross-screening assay of an in-
house library of approximately 800 known kinase inhibitors to
identify inhibitors of MELK. Compounds were ranked
according to average percent inhibition at both 10 and 1 µM,
revealing 18 compounds with ≥ 50% inhibition at 1 µM. The
ten most potent compounds were further characterized by
determining each compound’s IC50 (Table S1 in supporting
data). We found that the indolinone scaffold or a similar
bicyclic core structure populated the top hits. Indeed,

studies to interact via hydrogen bonding with K40 of the
binding pocket.
amide coupling reactions using N-methyl amine and N, N-
dimethylamine after TBTU or HBTU activation (Scheme 2).
The 5 or 6-substituted indolinone derivatives 15–25 in Table 1
were evaluated for their ability to inhibit MELK.
Molecular modeling studies using Gold 5.1 (Cambridge
Crystallographic Data Center) reinforce the similarities in
binding pose and electrostatic contacts between nintedanib and
other published inhibitors (Figure 2). In the hinge region, the
backbone of C89 is predicted to interact with the enamine
nitrogen and the carbonyl of the indolinone ring of nintedanib
(Figure 2A), mirroring the hydrogen bonding pattern seen in
the crystal structure of MELK with Cpd2 (Figure 2B, PDB
4BKY).34 With both nintedanib and Cpd2, K40 is positioned
to interact with bicyclic core substituents, either directly or via
a water molecule. Interaction with K40 has been previously
described as an “activity cliff” with other MELK inhibitors, in
that loss of this interaction results in loss of compound activity
towards MELK.35-36 Taken together, the screening and
modeling data suggest that MELK not only accommodates
various substituents at the 5th and 6th positions of the
indolinone ring, but also it may be a critical element for
maintaining inhibitor potency. Thus, we elected to explore 5-
and 6-substituted indolinones.
First, we focused our efforts on 6-substituted indolinone
derivatives. After synthesizing nintedanib (hereafter called
compound 15), we hydrolyzed the methyl ester to the
carboxylic acid 20. We then made longer amine containing
amides 22 and 23 (similar to the amine motif in the known
MELK inhibitor Cpd1).34 Unfortunately, all the 6-substituted
indolinone derivatives were found to be less potent compared
to the lead compound 15 (Table 1). We subsequently shifted
our attention to the 5th position of the indolinone ring. We
made 5-substituted methyl ester, methyl ketone and fluoro
indolinones 17–19. Gratifyingly, both 5-CO2Me 17 (Ki = 0.39
nM) and the 5-F derivative 19 (Ki = 3.1 nM) were found to be
more potent compared to the lead compound 15 (Ki = 5.6 nM).
The unsubstituted indolinone 16 was also found to be active
(Ki = 0.47 nM), challenging the notion that the substituents at
the 5th or 6th position are critical for binding MELK.
Hydrolysis of the methyl ester produced inhibitor 21, which
retained MELK activity (Ki = 0.68 nM). 5-substituted amide
analogs 24 (Ki = 2.3 nM) and 25 (Ki = 8.8 nM) showed lower
activities compared to the 5-methyl ester analog 17 (Ki = 0.39
nM).
2.1. Synthesis and biological evaluation of indolinone
derivatives
The general synthetic routes are outlined in Schemes 1 and
2. The key intermediates 6–10 were prepared by acylation of
indolinones 1–5 and subsequent condensation with ortho-
benzoic acid triethylester.37 Aromatic amine intermediate 14
was prepared by literature procedures as illustrated in Scheme
1.37 Acylation of N-methyl-4-nitroaniline 11 gave chloroactyl
amide 12, which was then treated with N-methylpiperazine to
displace chloride; followed by catalytic reduction of the nitro
group which gave the key aromatic amine intermediate 14.
Reagents and conditions: (a) Ac2O, 130 °C, 8 h; (b) PhC(OEt)3, Ac2O,
120 °C, 6 h, 46–51% (for two steps a and b); (c) AcCl, AlCl3, 1,2-
dichloroethane, below 10 °C, 1 h then RT, 12 h, 72%; (d) chloroacetyl
chloride, ethyl acetate, 70 °C, 1 h, 90%; (e) N-methylpiperazine, toluene,
55 °C, 2 h; (f) Pd/C, H2, iPr-OH, RT, 12 h, 76% (for two steps e and f).
Scheme 1. Synthesis of intermediates 6–10 and 14.
Final indolinone analogs 15–19 were prepared by addition
of 14 to substituted indolinones 6–10 and subsequent
elimination of ethanol, followed by acetyl cleavage using
piperidine in one-pot (Scheme 2). Additional 5 or 6-
substituted amide analogs 22–25 were synthesized from the
corresponding 5 or 6-substituted methyl ester derivatives by
hydrolysis using aqueous 1 N NaOH and subsequent standard
Reagents and conditions: (g) DMF, 80–100 °C, 1 h; (h) piperidine, RT,
63–73% (for two steps g and h); (i) 1N aq. NaOH, dioxane:methanol (1:1),
80 °C, 5 h; (j) an amine reactant, TBTU or HBTU, HOBT, DIPEA, DMF,
RT. For yields, see experimental section.
Scheme 2. Synthesis of final compounds 15–25

2.2. Computer modeling
Compound 16, which contains no substituent at either the 5- or
6-position of the indolinone, was the least selective inhibitor
of the three. Addition of a methyl ester at the 5-position
(compound 17) increased selectivity without compromising
potency towards MELK and the hydrolysis of the methyl ester
To further understand the increase in potency by shifting
the methyl ester from the 6th to the 5th position, both
compound 15 (5-CO2Me) and compound 17 (6-CO2Me) were
docked with MELK (PDB: 4BKY) and analyzed using
Gold5.2 with the ChemPLP scoring function. The methyl
ester substituents of both compounds are within direct or
water-bridged hydrogen bonding distance of K40 (Figure 3).
A methyl ester in the 5th position, however is predicted to
force 17 deeper into the narrow groove leading to K40. This
would possibly shorten the H-bond distance and strengthen the
interaction.
to the carboxylate (compound 21) yielded
a further
improvement in selectivity, displaying > 100-fold selectivity
towards CHK1, CAMKK2, and ERK2. It is possible that the
methyl ester of 17 is hydrolyzed in vivo due to the prevalence
of esterases at the cellular and whole organism level, thus 17
may serve as a metabolic precursor for 21 in vivo. While 17
and 21 do not yet have the selectivity desired, our modeling
data suggests that further elaboration of the indolinone
scaffold will afford lead compounds exhibiting both higher
potency and selectivity.
Figure 3. Top-down view of the ATP binding pocket of MELK (PDB:
4BKY) and superposition of predicted binding poses of compounds 15
(orange sticks) and 17 (magenta sticks). Compound 15 forms hydrogen
bonds with C89 (2.7 Å), E87 (2.9 Å), and K40 (3.7 Å) of the ATP binding
pocket. 6th position substitution sterically prevents 15 from achieving
optimal complementarity with the curvature of the binding site. 5th
position methyl ester substitution in 17 forms the same hydrogen bonds,
but allows deeper penetration of the compound into the binding site,
shortening hydrogen bond lengths between MELK and the compound by
over an angstrom in some cases (C89 = 3.0 Å, E87 =1.6 Å, K40 = 3.0 Å).
Measurements were generated using Pymol software. Inhibitor-protein H-
bonds are shown as dashed gray lines for compound 15 and dashed black
lines for compound 17. Other H-bonds are shown as yellow dashes. ATP
binding pocket is shaded in light purple. Ribbon backbones of some
residues are excluded for clarity.
Figure 4. Compound 17 suppresses cell proliferation of TNBC cells in
vitro. A. Expression levels of MELK. B. 17 suppresses expression of Mcl-
1. C. 17 suppressing cell proliferation, and cells expressing high levels of
MELK are more sensitive to 17 than cells expressing low levels of MELK.
Cells were seeded in 96-well plates and the next day treated with
inhibitors at the indicated concentrations. Seventy-two hours later, cell
viability was determined using the CellTiter-Blue Cell Viability Assay.
Moreover, this modification is predicted to enable 17 to
adopt a conformation more complementary to the shape of the
binding pocket, facilitating the interaction of the indolinone
core with E87 through an additional hydrogen bond. The
potency of 16 (unsubstituted indolinone) suggests that
suboptimal substituent position significantly affects inhibitor
binding, or alternatively, that an unsubstituted indolinone can
adopt a greater number of binding modes.
2.4. Cellular selectivity
As 17 is a potent inhibitor of the MELK catalytic domain,
we sought evidence that it engages MELK in TNBC cells.
Figure 4A shows that MELK is expressed in various TNBC
subtypes,38 HCC70 (BL2 subtype), BT-549 (mesenchymal)
and SUM-159 (mesenchymal stem-like), as well as the
immortalized mammary epithelial cell line MCF10A
(classified as Basal B by molecular profiling39). 17 induces a
decrease in Mcl-1 expression in HCC70 cells, consistent with
the notion that it inhibits the MELK-eIF4B signaling axis to
abrogate Mcl-1 expression (Figure 4B)).31 As inhibition of
MELK was found to induce apoptosis in basal-like breast
cancer cells,13, 35 we used the CellTiter-Blue assay to compare
the ability of 17 to inhibit the proliferation of cell lines with
varied levels of MELK expression. While proliferation of the
HCC70, BT-549, and SUM-159 cells were impacted by
treatment with 17, the MCF10A cells remained largely
2.3. Selectivity
As a preliminary assessment of selectivity, we chose to
evaluate our inhibitors against MELK and five other kinases.
These included MELK’s most closely related family members
AMPK and NUAK1, as well as the cell cycle regulated kinase
CHK1, the AMPK family activator CAMKKII, and ERK2.
We subjected the three most potent MELK inhibitor
derivatives (16, 17, and 21) to inhibition assays to determine
their IC50’s against the six kinases. Using apparent Km 's of
ATP,
we calculated a selectivity
based on the calculated Ki for each kinase (Table 2).

unaffected at 10 µM (Figure 4C). Furthermore, TNBC cell
lines expressing higher levels of MELK protein (HCC70 and
BT549) were more sensitive to 17 than the SUM159 cells
which express low levels of MELK. These data support the
notion that 17 inhibits MELK in TNBC cells to suppress Mcl-
1 expression and reduce TNBC cell viability.
MHz, DMSO-d6): δ 8.75 (s, 1H), 8.10 (d, J = 8.0 Hz, 1H),
7.89 (d, J = 8.0 Hz, 1H), 7.49–7.58 (m, 5H), 4.01 (q, J = 7.2
Hz, 2H), 3.87 (s, 3H), 2.44 (s, 3H), 1.35 (t, J = 8.0 Hz, 3H).
HRMS m/z found 365.1260, calcd for C21H19NO5 [M]+
365.1263.
(Z)-1-acetyl-3-(ethoxy(phenyl)methylene)indolin-2-one
(7). 37 The title compound was synthesized in 46% yield using
similar procedure as described for the synthesis of compound
6 by substituting indolinone 2 for indolinone 1. Major
3. Conclusions
We have identified 5-substituted indolinones as inhibitors
of MELK and identified three tight binding inhibitors (16, 17,
1
conformer H NMR (400 MHz, DMSO-d6): δ 8.17–8.13 (m,
and 21) with
< 1nM. The most potent compound was
1H), 8.02–7.99 (m, 1H), 7.56–7.46 (m, 5H), 7.30–7.22 (m,
2H), 3.94 (q, J = 7.2 Hz, 2H), 2.43 (s, 3H), 1.33 (t, J = 7.4 Hz,
3H). LCMS m/z found 308.1, calcd for C19H18NO3 [M+H]+
308.1.
shown to inhibit Mcl-1 expression in the MELK-expressing
HCC70 TNBC cell line, and shown to preferentially decrease
the proliferation of TNBC cells expressing high levels of
MELK. It also shows little effect on the immortalized breast
epithelial cell line MCF-10A, which also expresses lower
levels of MELK. There are currently no FDA-approved
inhibitors of MELK and no selective, orally bioavailable
inhibitors have been reported. These studies suggest that
further elaboration of 17 will furnish MELK-selective
inhibitors with potential for development in preclinical models
of TNBC and other cancers.
Methyl
(Z)-1-acetyl-3-(ethoxy(phenyl)methylene)-2-
oxoindoline-5-carboxylate (8).41 The title compound was
synthesized in 48% yield using similar procedure as described
for the synthesis of compound 6 by substituting indolinone 3
for indolinone 1. 1H NMR (400 MHz, DMSO-d6): δ 8.61 (d, J
= 1.6 Hz, 1H), 8.23 (d, J = 8.4 Hz, 1H), 7.92 (dd, J = 8.4 Hz, J
= 1.6 Hz, 1H), 7.58–7.49 (m, 5H), 3.99 (q, J = 7.0 Hz, 2H),
3.87 (s, 3H), 2.45 (s, 3H), 1.37 (t, J = 7.0 Hz, 3H). HRMS m/z
found 346.1058, calcd for C19H17NNaO4 [M-Ac+Na]+
346.1055.
4. Experimental section
4.1. Chemistry
(Z)-1,1'-(3-(Ethoxy(phenyl)methylene)-2-oxoindoline-
1,5-diyl)bis(ethan-1-one) (9).37 The title compound was
synthesized in 47% yield using similar procedure as described
for the synthesis of compound 6 by substituting indolinone 4
for indolinone 1. 1H NMR (400 MHz, DMSO-d6): δ 8.58 (d, J
= 2.0 Hz, 1H), 8.23 (d, J = 8.4 Hz, 1H), 7.94 (dd, J = 8.4 Hz, J
= 2.0 Hz, 1H), 7.58–7.49 (m, 5H), 3.99 (q, J = 7.0 Hz, 2H),
2.61 (s, 3H), 2.45 (s, 3H), 1.39 (t, J = 8.0 Hz, 3H). LCMS m/z
found 350.1, calcd for C21H20NO4 [M+H]+ 350.1.
General Information. Reagents and starting materials
including indolinones 1–3, and 5, and N-Methyl-4-nitroaniline
11 were purchased from various commercial sources including
Sigma-Aldrich or Matrix Scientific and used without further
purification unless otherwise stated. 5-Acetylindolinone 4 and
aromatic amine intermediate 14 were prepared by literature
37
procedures.40,
flame-dried
All reactions were carried out in oven- or
glassware under argon. Thin-layer
chromatography (TLC) was performed using pre-coated TLC
plates with silica gel 60 F254 (EMD) or with aluminum oxide
60 F254 neutral. Flash column chromatography was performed
using 40–63 μm (230–400 mesh ASTM) silica gel (EMD).
Melting points were recorded on a Thomas Hoover capillary
melting point apparatus. NMR spectra were recorded on a
Varian NMR spectrometer. High-resolution mass and liquid
chromatography mass spectral data were obtained at the
University of Texas at Austin. Compounds were characterized
by NMR and HRMS or LCMS.
(Z)-1-acetyl-3-(Ethoxy(phenyl)methylene)-5-
fluoroindolin-2-one (10). The title compound was
synthesized in 48% yield using similar procedure as described
for the synthesis of compound 6 by substituting indolinone 5
for indolinone 1. Major conformer 1H NMR (400 MHz,
DMSO-d6): δ 8.16 (m, 1H), 7.71(m, 1H), 7.57–7.47 (m, 5H),
7.12 (m, 1H), 3.98 (q, J = 7.0 Hz, 2H), 2.41 (s, 3H), 1.33 (t, J
= 8.0 Hz, 3H). 19F NMR (376 MHz, DMSO-d6): δ - 117.6 (m).
LCMS m/z found 326.1, calcd for C19H17FNO3 [M+H]+ 326.1.
5-Acetylindolin-2-one (4). By following acylation
procedure reported in a patent,40 5-acetylindolin-2-one was
prepared from indolin-2-one and AlCl3. The crude was
recrystallized from ethyl acetate to obtain 72% of the title
compound. 1H NMR (400 MHz, DMSO-d6): δ 10.76 (br s, 1H,
NH), 7.85 (m, 1H, Ar-H), 7.80 (m, 1H, Ar-H), 6.90 (d, J = 8.0
Hz, 1H, Ar-H), 3.55 (s, 2H), 2.50 (s, 3H, Ac).
N-(4-Aminophenyl)-N-methyl-2-(4-methylpiperazin-1-
yl)acetamide (14). The title acetamide compound was
prepared from N-methyl-4-nitroaniline 11 using similar
procedure as described in literature.41 1H NMR (400 MHz,
DMSO-d6): δ 2.10 (s, 3H), 2.14–2.43 (m, 8H), 2.79 (s, 2H),
3.03 (s, 3H), 5.23 (s, 2H), 6.52–6.57 (m, 2H), 6.88–6.92 (m,
2H). HRMS m/z found 263.1866, calcd for C14H23N4O
[M+H]+ 263.1872.
General procedure for the synthesis of N-acetyl-3-
(ethoxy(phenyl)methylene)-2-oxoindolines (Compounds 6–
10). Methyl (Z)-1-acetyl-3-(ethoxy(phenyl)methylene)-2-
oxoindoline-6-carboxylate (6).37 Indolinone 1 (1000 mg, 52.3
mmol) was suspended in acetic anhydride (10 mL) and
General procedure for the synthesis of final compounds
15–19. Methyl (Z)-3-(((4-(N-methyl-2-(4-methylpiperazin-
1-yl)acetamido)phenyl)amino)(phenyl)methylene)-2-
oxoindoline-6-carboxylate (15).37 To a suspension of methyl
(Z)-1-acetyl-3-(ethoxy(phenyl)methylene)-2-oxoindoline-6-
carboxylate (6) (500 mg, 1.368 mmol) in DMF (3.5 mL) was
added N-(4-aminophenyl)-N-methyl-2-(4-methylpiperazin-1-
yl)acetamide (14) (395 mg, 1.505 mmol, 1.1 equiv.) at RT.
o
refluxed at 130 C for 8 h. The reaction mixture was allowed
o
to cool to 50 C and (triethoxymethyl)benzene (2930 mg, 131
mmol) was added. The resulting reaction mixture was stirred
o
at 120 C for 6 h. Then, volatiles were removed in vacuo and
petroleum ether was added to the obtained residue. After
triturating for 15 minutes, the separated solids were filtered
and washed with petroleum ether and then dried under vacuum
o
After heating the reaction mixture at 80 C for 1 h, it was
allowed to cool to RT. Piperidine (297 μL, 3.010 mmol, 2.2
equiv.) was then added and stirred for 2 h. Volatiles were
removed in vacuo and water was added to the obtained residue
1
to afford 974 mg (51%) of title compound. H NMR (400

and stirred for 15 min. The precipitate was then filtered under
suction and cake was washed with water, then with minimum
amount of cold methanol, and then ether. The obtained
product was purified by column chromatography (neutral
Al2O3, 0–10% methanol in CH2Cl2) to afford 532 mg (72%) of
General procedure for the synthesis of final compounds
22–25. (Z)-N-(3-(Dimethylamino)propyl)-3-(((4-(N-methyl-
2-(4-methylpiperazin-1-
yl)acetamido)phenyl)amino)(phenyl)methylene)-2-
oxoindoline-6-carboxamide (22).
1
target molecule 15. Major conformer H NMR (400 MHz,
6-carboxylic acid methyl ester 15 (250 mg, 0.46 mmol) was
DMSO-d6): δ 12.22 (s, 1H), 10.98 (s, 1H), 7.66–7.47 (m, 5H),
7.42 (s, 1H), 7.24–7.09 (m, 3H), 6.89 (d, J = 8.0 Hz, 2H), 5.83
(d, J = 8.0 Hz, 1H), 3.77 (s, 3H), 3.06 (s, 3H), 2.69 (s, 2H),
2.34–2.06 (brs, 8H), 2.10 (s, 3H). HRMS m/z found 540.2606,
calcd for C31H34N5O4 [M+H]+ 540.2605.
added to 1:1 mixture of methanol and dioxane (8 mL). The
°
resulting suspension was heated to 50 C and then 1N aqueous
NaOH solution (2.5 mL) was added. The solution was stirred
for 5 h at 80 °C and then allowed to cool to RT. Volatiles were
removed in vacuo and water was added to the obtained
residue. After stirred for 10 minutes, the separated solids were
filtered and washed with water and diethyl ether, and then
dried under vacuum to afford crude (Z)-3-(((4-(N-Methyl-2-(4-
methylpiperazin-1-
yl)acetamido)phenyl)amino)(phenyl)methylene)-2-
oxoindoline-6-carboxylic acid (20). Major conformer 1H NMR
(400 MHz, DMSO-d6): δ 12.20 (s, 1H), 10.96 (s, 1H), 7.63–
7.48 (m, 5H), 7.42 (s, 1H), 7.17 (dd, J = 8.0 Hz, 1.2 Hz, 1H),
7.13 (d, J = 8.4 Hz, 2H), 6.88 (d, J = 8.0 Hz, 2H), 5.81 (d, J =
8.4 Hz, 1H), 3.06 (s, 3H), 2.67 (s, 2H), 2.20 (brs, 8H), 2.12 (s,
3H). HRMS m/z found 525.2371, calcd for C30H31N5O4 [M]+
525.2376.
(Z)-N-Methyl-2-(4-methylpiperazin-1-yl)-N-(4-(((2-
oxoindolin-3-
ylidene)(phenyl)methyl)amino)phenyl)acetamide (16). The
title compound was synthesized in 64% yield using similar
procedure as described for the synthesis of compound 15 by
substituting (Z)-1-acetyl-3-(ethoxy(phenyl)methylene)indolin-
2-one (7) for indolinone derivative 6. A 58:42 mixture of
conformers 1H NMR (400 MHz, DMSO-d6): δ 12.19 and 12.02
(s, 1H), 10.74 and 10.59 (s, 1H), 7.61–5.75 (m, 13H), 3.17–
2.64 (m, 5H), 2.36–2.07 (m, 11H). HRMS m/z found
482.2408, calcd for C29H32N5O2 [M+H]+ 482.2400.
Methyl
(Z)-3-(((4-(N-methyl-2-(4-methylpiperazin-1-
yl)acetamido)phenyl)amino)(phenyl)methylene)-2-
oxoindoline-5-carboxylate (17). The title compound was
synthesized in 73% yield using similar procedure as described
for the synthesis of compound 15 by substituting Methyl (Z)-
1-acetyl-3-(ethoxy(phenyl)methylene)-2-oxoindoline-5-
carboxylate (8) for indolinone derivative 6. Major conformer
1H NMR (400 MHz, DMSO-d6): δ 11.97 (s, 1H), 11.13 (s,
1H), 7.63–7.47 (m, 6H), 7.13 (d, J = 8.4 Hz, 2H), 6.94 (d, J =
8.4 Hz, 1H), 6.88 (d, J = 8.4 Hz, 2H), 6.51 (s, 1H), 3.63 (s,
3H), 3.06 (s, 3H), 2.69 (s, 2H), 2.19 (brs, 8H), 2.10 (s, 3H).
HRMS m/z found 540.2613, calcd for C31H34N5O4 [M+H]+
540.2605.
To a suspension of crude 6-carboxylic acid 20 (1.0 equiv.),
HBTU or TBTU (1.2 equiv.), HOBt (1.2 equiv.) in
dimethylformamide was added DIPEA (16 equiv.) at RT.
N,N-dimethylpropane-1,3-diamine (1.5 equiv.) was then added
and continued stirred for 2h. Volatiles were removed in vacuo
and the obtained product was purified by column
chromatography (neutral Al2O3, 0–10% methanol in CH2Cl2)
or HPLC to afford the target molecule 2-oxoindoline-6-
carboxamide 22. Major conformer 1H NMR (400 MHz,
DMSO-d6): δ 12.10 (s, 1H), 10.93 (s, 1H), 8.24 (t, J = 5.2 Hz,
1H), 7.64–7.49 (m, 5H), 7.33 (d, J = 1.6 Hz, 1H), 7.12 (d, J =
8.4 Hz, 2H), 7.05 (dd, J = 8.4 Hz, 1.6 Hz, 1H), 6.87 (d, J = 8.4
Hz, 2H), 5.74 (d, J = 8.0 Hz, 1H), 3.20 (q, J = 8.0 Hz, 2H),
3.05 (s, 3H), 2.69 (s, 2H), 2.33–2.05 (m, 19H), 1.59 (m, 2H).
HRMS m/z found 610.3494, calcd for C35H44N7O3 [M+H]+
610.3500.
(Z)-N-(4-(((5-Acetyl-2-oxoindolin-3-
ylidene)(phenyl)methyl)amino)phenyl)-N-methyl-2-(4-
methylpiperazin-1-yl)acetamide (18). The title compound
was synthesized in 63% yield using similar procedure as
described for the synthesis of compound 15 by substituting
(Z)-1,1'-(3-(Ethoxy(phenyl)methylene)-2-oxoindoline-1,5-
diyl)bis(ethan-1-one) (9) for indolinone derivative 6. Major
(Z)-N-(3-Aminopropyl)-3-(((4-(N-methyl-2-(4-
methylpiperazin-1-
yl)acetamido)phenyl)amino)(phenyl)methylene)-2-
oxoindoline-6-carboxamide (23). The title compound was
synthesized using similar procedure as described for the
synthesis of compound 22 by substituting tert-butyl (3-
aminopropyl)carbamate for dimethylpropane-1,3-diamine
followed by removal of Boc protecting group with treatment
of 20% TFA in dichloromethane at RT for 3 hr. Major
1
conformer H NMR (400 MHz, DMSO-d6): δ 11.94 (s, 1H),
11.14 (s, 1H), 7.63–7.50 (m, 6H), 7.13 (d, J = 8.4 Hz, 2H),
6.94 (d, J = 8.4 Hz, 1H), 6.90 (d, J = 8.4 Hz, 2H), 6.39 (s, 1H),
3.06 (s, 3H), 2.71 (s, 2H), 2.32–2.16 (brs, 8H), 2.14 (s, 6H).
HRMS m/z found 523.2585, calcd for C31H33N5O3 [M]+
523.2583.
1
conformer H NMR (400 MHz, DMSO-d6): δ 12.15 (s, 1H),
(Z)-N-(4-(((5-Fluoro-2-oxoindolin-3-
10.98 (s, 1H), 8.37 (t, J = 5.2 Hz, 1H), 7.72–7.50 (m, 7H),
7.35 (s, 1H), 7.16 (d, J = 8.4 Hz, 2H), 7.08 (dd, J = 8.0 Hz, 1.6
Hz, 1H), 6.89 (d, J = 8.4 Hz, 2H), 5.74 (d, J = 8.4 Hz, 1H),
3.48–2.69 (m, 20H), 1.74 (m, 2H). HRMS m/z found
582.3186, calcd for C33H40N7O3 [M+H]+ 582.3187.
ylidene)(phenyl)methyl)amino)phenyl)-N-methyl-2-(4-
methylpiperazin-1-yl)acetamide (19). The title compound
was synthesized in 69% yield using similar procedure as
described for the synthesis of compound 15 by substituting
(Z)-1-acetyl-3-(Ethoxy(phenyl)methylene)-5-fluoroindolin-2-
1
one (10) for indolinone derivative 6. H NMR (400 MHz,
(Z)-N-Methyl-3-(((4-(N-methyl-2-(4-methylpiperazin-1-
yl)acetamido)phenyl)amino)(phenyl)methylene)-2-
oxoindoline-5-carboxamide (24). (Z)-3-(((4-(N-Methyl-2-(4-
methylpiperazin-1-
DMSO-d6): δ 12.09 (s, 1H), 10.76 (s, 1H), 7.63–7.48 (m, 5H),
7.12 (d, J = 8.4 Hz, 2H), 6.90–6.79 (m, 3H), 6.72 (m, 1H),
5.37 (dd, J = 10.4 Hz, 2.4 Hz, 1H), 3.05 (s, 3H), 2.67 (s, 2H),
2.18 (brs, 8H), 2.10 (s, 3H). 19F NMR (376 MHz, DMSO-d6):
δ - 123.3 (m). HRMS m/z found 500.2455, calcd for
C29H31FN5O2 [M+H]+ 500.2456.
yl)acetamido)phenyl)amino)(phenyl)methylene)-2-
oxoindoline-5-carboxylic acid (21) was synthesized using
similar procedure as described for the synthesis of compound
20 by substituting indolinone 17 for indolinone derivative 15.
1
Major conformer H NMR (400 MHz, DMSO-d6): δ 12.00 (s,

1H), 11.09 (s, 1H), 7.60–7.47 (m, 6H), 7.12 (d, J = 8.4 Hz,
2H), 6.92 (d, J = 8.0 Hz, 1H), 6.85 (d, J = 8.8 Hz, 2H), 6.59 (s,
1H), 3.05 (s, 3H), 2.69 (s, 2H), 2.19 (brs, 8H), 2.11 (s, 3H).
HRMS m/z found 526.2453, calcd for C30H32N5O4 [M+H]+
526.2449.
from American Type Culture Collection. HCC70 and BT-549
cells were maintained in RPMI 1640 medium (Life
Technologies Inc., Grand Island, NY, USA) supplemented
with FBS (10%) and antibiotic/antimycotic (1%). SUM159
cells were maintained in Ham's F-12 medium (Life
Technologies Inc.) supplemented with FBS (5%),
antibiotic/antimycotic (1%), insulin (5 µg/mL), and
hydrocortisone (1 µg/mL). MCF10A cells were maintained in
Dulbecco’s modified Eagle’s medium/F12 medium (Life
Technologies Inc.) supplemented with horse serum (10%),
antibiotic/antimycotic (1%), insulin (10 µg/mL), EGF (20
ng/mL), cholera toxin (100 ng/mL), and hydrocortisone (500
µg/mL). All cell lines used in this study were validated in
September 2016 by the Characterized Cell Line Core Facility
at MD Anderson Cancer Center by using a short tandem repeat
method based on primer extension to detect single base
deviations.
The title compound 24 was synthesized using similar
procedure as described for the synthesis of compound 22 by
substituting methylamine hydrochloride for dimethylpropane-
1
1,3-diamine. Major conformer H NMR (400 MHz, DMSO-
d6): δ 12.03 (s, 1H), 10.97 (s, 1H), 7.93 (m, 1H), 7.62–7.46 (m,
5H), 7.39 (dd, J = 8.4 Hz, 1.6 Hz, 1H), 7.11 (d, J = 8.4 Hz,
2H), 6.85 (d, J = 8.4 Hz, 1H), 6.83 (d, J = 8.8 Hz, 2H), 6.53 (s,
1H), 3.05 (s, 3H), 2.69 (s, 2H), 2.63 (d, J = 4.8 Hz, 3H), 2.19
(brs, 8H), 2.11 (s, 3H). HRMS m/z found 539.2782, calcd for
C31H35N6O3 [M+H]+ 539.2765.
(Z)-N,N-Dimethyl-3-(((4-(N-methyl-2-(4-
methylpiperazin-1-
4.3.1. Western blotting
yl)acetamido)phenyl)amino)(phenyl)methylene)-2-
oxoindoline-5-carboxamide (25). The title compound was
synthesized using similar procedure as described for the
synthesis of compound 22 by substituting dimethylamine
hydrochloride for dimethylpropane-1,3-diamine. Major
For the analysis of MELK expression, cells (1 x 106) were
seeded in 10-cm plates and incubated at 37oC with 5% CO2 for
48 h. The cell lysates were then prepared and used for Western
blotting analysis as described previously.42 For the analysis of
impact of compound 17 treatment on expression of Mcl-1,
HCC70 cells (1 x 106) were seeded in 10-cm plates overnight
and the next day serum-starved for 6 h and then treated with
compound 17 (0, 1, and 2.5 µM) for 18 h under serum
starvation condition. Following compound 17 treatment, the
cells were stimulated with TGF- (5 ng/mL) for 1 h, and then
the cell lysates were prepared and used for Western blotting
analysis as described previously.42 Proteins of interest were
probed using the following primary antibodies: anti-MELK
(1:750 dilution; R&D Systems, Inc., Minneapolis, MN, USA),
anti-Mcl-1 (1:1000 dilution; Santa Cruz Biotechnology, Inc.,
Dallas, TX, USA), and anti-β-actin (1:8000 dilution; Sigma-
Aldrich, St. Louis, MO, USA). Secondary antibodies were
horseradish peroxidase-conjugated IgG (1:10,000 dilution;
Life Technologies Inc.) for chemiluminescent signal detection
and the corresponding Alexa Fluor-conjugated IgG (1:5000
dilution; Life Technologies Inc.) for fluorescence signal
detection.
1
conformer H NMR (400 MHz, DMSO-d6): δ 12.03 (s, 1H),
10.94 (s, 1H), 7.62–7.48 (m, 5H), 7.11 (d, J = 8.4 Hz, 2H),
7.03 (dd, J = 8.0 Hz, 1.6 Hz, 1H), 6.88 (d, J = 8.0 Hz, 1H),
6.84 (d, J = 8.0 Hz, 2H), 5.86 (s, 1H), 3.05 (s, 3H), 2.76 (s,
6H), 2.69 (s, 2H), 2.18 (brs, 8H), 2.10 (s, 3H). HRMS m/z
found 553.2930, calcd for C32H37N6O3 [M+H]+ 553.2922.
4.2. Inhibitor library screening
MELK and its substrate, Bcl-GL were both recombinantly
expressed and purified for use in screening assays as detailed
in supplementary data. 752 compounds from an in-house
curated inhibitor library were subjected to a p81-based kinase
assay. 10 nM MELK 340 and 10 µM Bcl-GL in kinase assay
buffer (50 mM HEPES pH 7.5, 100 mM KCl, 0.1 mM EDTA,
0.1 mM EGTA, 10 mM MgCl2, 10 µg/mL BSA) with 10 mM
DTT were added to either 10 µM or 1 µM inhibitor aliquoted
into 96 well plates (final 1% DMSO). The mixture was
incubated at RT for 30 minutes prior to initiation of the assay
with 40 µM γ-32P-ATP (100 – 1000 CPM/pmol). 40 µL
aliquots were spotted onto a p81 96 well filter plate (Unifilter,
Whatman), quenched, and washed with 75 mM O-phosphoric
acid 8 times, followed by a final wash with acetone for drying.
Wells were then filled with scintillation fluid, sealed, and
quantified using a MicroBeta TriLux liquid scintillation
counter (PerkinElmer). Each inhibitor plate was assayed in
duplicate, with at least 4 wells without MELK to establish
background and at least 4 wells without inhibitor as a negative
control. All readings were corrected for background signal
based on the average counts from the wells without enzyme.
Percent inhibition, defined as [(1 – (CPM + inhibitor/average
CPM of negative controls))*100], was determined first at 10
µM inhibitor. The top 50 inhibitors were then re-screened in
duplicate at 1 µM. Additional methods for inhibitor screening
and selectivity assays are in supplementary data.
4.3.2. Cell proliferation assay
Cell proliferation was determined using the CellTiter-Blue
viability assay as described previously.43 Cells were seeded in
96-well plates and treated the next day with compound 17 (0-
20 µM). At 72 h after treatment, optical density at 595 nm was
determined.
Acknowledgments
The authors are grateful to CPRIT RP110532 & RP160657,
the Welch Foundation (F-1390), and startup from MD
Anderson Cancer Center (111411) to CB for financial support
of this study.
Supplementary data
Additional details of assays, experimental procedures, 1H &
19F NMR spectra, and either LCMS or HRMS are included in
the Supplementary data.
4.3. Cell culture and reagents
HCC70, BT-549, and SUM159 human TNBC cell lines and
MCF10A human breast epithelial cell line were purchased

Table 1. MELK inhibitory activity of indolinone derivatives 15–25
Compound
R1
R2
IC50 (nM)
43 ± 3.4
3.6 ± 0.4
3 ± 0.8
Ki a± SE (nM)
5.6 ± 0.4
15
16
17
18
19
20
21
22
23
24
25
CO2Me
H
H
H
0.47 ± 0.06
0.39 ± 0.09
46 ± 4.6
3.1 ± 0.6
149 ± 22
0.68 ± 0.07
358 ± 71
H
CO2Me
COMe
F
H
354 ± 35
24 ± 4.7
1145 ± 168
5.2 ± 0.5
2700 ± 540
> 5000
H
CO2H
H
H
CO2H
H
CONH(CH2)3NMe2
CONH(CH2)3NH2
H
> 650
H
H
CONHMe
CONMe2
18 ± 3.8
67 ± 11
2.3 ± 0.5
8.8 ± 1.4
aKi calculated using Equation 4, where [ATP] = 40 µM,
= 6 µM
Table 2. Selectivity of MELK inhibitor derivatives against selected kinases
Kinase
Compound 16
Ki (nM)
Compound 17
Ki (nM)
Compound 21
Ki (nM)
φa
φa
φa
MELK
0.47 ± 0.06
1.00
0.39 ± 0.09
1.00
0.68 ± 0.07
1.00
AMPK
NUAK1
CHK1
4.2 ± 0.3
1.7 ± 0.17
10 ± 0.5
8.9 ± 0.2
3.6 ± 0.2
18 ± 0.2
9.2 ± 1.7
11 ± 0.86
7 ± 0.17
24 ± 0.3
28 ± 0.2
18 ± 0.2
4.9 ± 0.6
8.6 ± 1.6
81 ± 5.6
7.2 ± 0.2
13 ± 0.2
120 ± 0.1
CAMKK2
15 ± 1.8
32 ± 0.2
31 ± 11
80 ± 0.4
85 ± 9.2
125 ± 0.2
ERK2
1160 ± 320
2500 ± 0.3
3000 ± 1300
7700 ± 0.5
19000 ± 3800
28000 ± 0.2
a

23. Komatsu, M.; Yoshimaru, T.; Matsuo, T.; Kiyotani, K.; Miyoshi, Y.;
Tanahashi, T.; Rokutan, K.; Yamaguchi, R.; Saito, A.; Imoto, S.;
Miyano, S.; Nakamura, Y.; Sasa, M.; Shimada, M.; Katagiri, T. Int. J.
Oncol. 2013, 42, 478–506.
24. Al-Ejeh, F.; Simpson, P. T.; Sanus, J. M.; Klein, K.; Kalimutho, M.; Shi,
W.; Miranda, M.; Kutasovic, J.; Raghavendra, A.; Madore, J.; Reid, L.;
Krause, L.; Chenevix-Trench, G.; Lakhani, S. R.; Khanna, K. K.
Oncogenesis 2014, 3, e100.
25. Kuner, R.; Falth, M.; Pressinotti, N. C.; Brase, J. C.; Puig, S. B.;
Metzger, J.; Gade, S.; Schafer, G.; Bartsch, G.; Steiner, E.; Klocker, H.;
Sultmann, H. J. Mol. Med. 2013, 91, 237–248.
26. Alachkar, H.; Mutonga, M. B.; Metzeler, K. H.; Fulton, N.; Malnassy,
G.; Herold, T.; Spiekermann, K.; Bohlander, S. K.; Hiddemann, W.;
Matsuo, Y.; Stock, W.; Nakamura, Y. Oncotarget 2014, 5, 12371–12382.
27. Li, S.; Li, Z.; Guo, T.; Xing, X. F.; Cheng, X.; Du, H.; Wen, X. Z.; Ji, J.
F. Oncotarget 2016, 7, 6266–6280.
28. Nakano, I.; Joshi, K.; Visnyei, K.; Hu, B.; Watanabe, M.; Lam, D.;
Wexler, E.; Saigusa, K.; Nakamura, Y.; Laks, D. R.; Mischel, P. S.;
Viapiano, M.; Kornblum, H. I. Neuro-oncology 2011, 13, 622–634.
29. Choi, S.; Ku, J. L. Biochem. Biophys. Res. Commun. 2011, 412, 207–
213.
30. Kim, S. H.; Joshi, K.; Ezhilarasan, R.; Myers, T. R.; Siu, J.; Gu, C.;
Nakano-Okuno, M.; Taylor, D.; Minata, M.; Sulman, E. P.; Lee, J.; Bhat,
K. P.; Salcini, A. E.; Nakano, I. Stem Cell Reports 2015, 4, 226–238.
31. Wang, Y.; Begley, M.; Li, Q.; Huang, H. T.; Lako, A.; Eck, M. J.; Gray,
N. S.; Mitchison, T. J.; Cantley, L. C.; Zhao, J. J. Proc. Natl. Acad. Sci.
USA 2016, 113, 9810–9815.
32. Prakash, C. R.; Raja, S. Mini Rev. Med. Chem. 2012, 12, 98–119.
33. Aronov, A. M.; McClain, B.; Moody, C. S.; Murcko, M. A. J. Med.
Chem. 2008, 51, 1214–1222.
34. Canevari, G.; Re Depaolini, S.; Cucchi, U.; Bertrand, J. A.; Casale, E.;
Perrera, C.; Forte, B.; Carpinelli, P.; Felder, E. R. Biochemistry 2013, 52,
6380–6387.
References and notes
1. Gil, M.; Yang, Y.; Lee, Y.; Choi, I.; Ha, H. Gene 1997, 195, 295–301.
2. Beullens, M.; Vancauwenbergh, S.; Morrice, N.; Derua, R.; Ceulemans,
H.; Waelkens, E.; Bollen, M. J. Biol. Chem. 2005, 280, 40003–40011.
3. Joshi, K.; Banasavadi-Siddegowda, Y.; Mo, X.; Kim, S. H.; Mao, P.;
Kig, C.; Nardini, D.; Sobol, R. W.; Chow, L. M.; Kornblum, H. I.;
Waclaw, R.; Beullens, M.; Nakano, I. Stem Cells 2013, 31, 1051–1063.
4. Saito, R.; Nakauchi, H.; Watanabe, S. Cancer Sci. 2012, 103, 42–49.
5. Jiang, P.; Zhang, D. Int. J. Mol. Sci. 2013, 14, 21551–21560.
6. Ganguly, R.; Hong, C. S.; Smith, L. G.; Kornblum, H. I.; Nakano, I. Mol.
Cancer Ther. 2014, 13, 1393–1398.
7. Ganguly, R.; Mohyeldin, A.; Thiel, J.; Kornblum, H. I.; Beullens, M.;
Nakano, I. Clin. Transl. Med. 2015, 4, 11.
8. Heyer, B. S.; Warsowe, J.; Solter, D.; Knowles, B. B.; Ackerman, S. L.
Mol. Reprod. Dev. 1997, 47, 148–156.
9. Heyer, B. S.; Kochanowski, H.; Solter, D. Dev. Dyn. 1999, 215, 344–
351.
10. Nakano, I.; Paucar, A. A.; Bajpai, R.; Dougherty, J. D.; Zewail, A.;
Kelly, T. K.; Kim, K. J.; Ou, J.; Groszer, M.; Imura, T.; Freije, W. A.;
Nelson, S. F.; Sofroniew, M. V.; Wu, H.; Liu, X.; Terskikh, A. V.;
Geschwind, D. H.; Kornblum, H. I. J. Cell Biol. 2005, 170, 413–427.
11. Niesler, C. U.; Myburgh, K. H.; Moore, F. Exp. Physiol. 2007, 92, 207–
217.
12. Hebbard, L. W.; Maurer, J.; Miller, A.; Lesperance, J.; Hassell, J.;
Oshima, R. G.; Terskikh, A. V. Cancer Res. 2010, 70, 8863–8873.
13. Wang, Y.; Lee, Y. M.; Baitsch, L.; Huang, A.; Xiang, Y.; Tong, H.;
Lako, A.; Von, T.; Choi, C.; Lim, E.; Min, J.; Li, L.; Stegmeier, F.;
Schlegel, R.; Eck, M. J.; Gray, N. S.; Mitchison, T. J.; Zhao, J. J. eLife
2014, e01763.
14. Gray, D.; Jubb, A. M.; Hogue, D.; Dowd, P.; Kljavin, N.; Yi, S.; Bai, W.;
Frantz, G.; Zhang, Z.; Koeppen, H.; de Sauvage, F. J.; Davis, D. P.
Cancer Res. 2005, 65, 9751–9761.
15. Ryu, B.; Kim, D. S.; Deluca, A. M.; Alani, R. M. PloS one 2007, 2, e594.
16. Marie, S. K.; Okamoto, O. K.; Uno, M.; Hasegawa, A. P.; Oba-Shinjo, S.
M.; Cohen, T.; Camargo, A. A.; Kosoy, A.; Carlotti, C. G., Jr.; Toledo,
S.; Moreira-Filho, C. A.; Zago, M. A.; Simpson, A. J.; Caballero, O. L.
Int. J. Cancer 2008, 122, 807–815.
35. Toure, B. B.; Giraldes, J.; Smith, T.; Sprague, E. R.; Wang, Y.; Mathieu,
S.; Chen, Z.; Mishina, Y.; Feng, Y.; Yan-Neale, Y.; Shakya, S.; Chen,
D.; Meyer, M.; Puleo, D.; Brazell, J. T.; Straub, C.; Sage, D.; Wright, K.;
Yuan, Y.; Chen, X.; Duca, J.; Kim, S.; Tian, L.; Martin, E.; Hurov, K.;
Shao, W. J. Med. Chem. 2016, 59, 4711–4723.
36. Furtmann, N.; Hu, Y.; Bajorath, J. J. Med. Chem. 2015, 58, 252–264.
37. Roth, G. J.; Heckel, A.; Colbatzky, F.; Handschuh, S.; Kley, J.;
Lehmann-Lintz, T.; Lotz, R.; Tontsch-Grunt, U.; Walter, R.; Hilberg, F.
J. Med. Chem. 2009, 52, 4466–4480.
17. Li, Y.; Tang, H.; Sun, Z.; Bungum, A. O.; Edell, E. S.; Lingle, W. L.;
Stoddard, S. M.; Zhang, M.; Jen, J.; Yang, P.; Wang, L. Lung Cancer
2013, 80, 91–98.
38. Lehmann, B. D.; Bauer, J. A.; Chen, X.; Sanders, M. E.; Chakravarthy,
A. B.; Shyr, Y.; Pietenpol, J. A. J. Clin. Invest. 2011, 121, 2750–2767.
39. Kao, J.; Salari, K.; Bocanegra, M.; Choi, Y. L.; Girard, L.; Gandhi, J.;
Kwei, K. A.; Hernandez-Boussard, T.; Wang, P.; Gazdar, A. F.; Minna,
J. D.; Pollack, J. R. PLoS One 2009, 4, e6146.
40. Heckel, A; Roth, G. J.; Kley, J.; Hoerer, S.; Uphues, I. U.S. Patent Appl.
Publ. US 20050203104 A1 Sep 15, 2005.
41. Roth, G. J.; Heckel, A.; Brandl, T.; Grauert, M.; Hoerer, S.; Kley, J. T.;
Schnapp, G.; Baum, P.; Mennerich, D.; Schnapp, A.; Park, J. E. J. Med.
Chem. 2010, 53, 7287–7295.
42. Bartholomeusz, C.; Gonzalez-Angulo, A. M.; Kazansky, A.;
Krishnamurthy, S.; Liu, P.; Yuan, L. X.; Yamasaki, F.; Liu, S.; Hayashi,
N.; Zhang, D.; Esteva, F. J.; Hortobagyi, G. N.; Ueno, N. T. Clin. Cancer
Res. 2010, 16, 1802–1811.
43. Gloeckner, H.; Jonuleit, T.; Lemke, H. D. J. Immunol. Methods 2001,
252, 131–138.
18. Rajkumar, T.; Sabitha, K.; Vijayalakshmi, N.; Shirley, S.; Bose, M. V.;
Gopal, G.; Selvaluxmy, G. BMC cancer 2011, 11, 80.
19. Risinger, J. I.; Allard, J.; Chandran, U.; Day, R.; Chandramouli, G. V.;
Miller, C.; Zahn, C.; Oliver, J.; Litzi, T.; Marcus, C.; Dubil, E.; Byrd, K.;
Cassablanca, Y.; Becich, M.; Berchuck, A.; Darcy, K. M.; Hamilton, C.
A.; Conrads, T. P.; Maxwell, G. L. Front. Oncol. 2013, 3, 139.
20. Hemmati, H. D.; Nakano, I.; Lazareff, J. A.; Masterman-Smith, M.;
Geschwind, D. H.; Bronner-Fraser, M.; Kornblum, H. I. Proc. Natl.
Acad. Sci. USA 2003, 100, 15178–15183.
21. Nakano, I.; Masterman-Smith, M.; Saigusa, K.; Paucar, A. A.; Horvath,
S.; Shoemaker, L.; Watanabe, M.; Negro, A.; Bajpai, R.; Howes, A.;
Lelievre, V.; Waschek, J. A.; Lazareff, J. A.; Freije, W. A.; Liau, L. M.;
Gilbertson, R. J.; Cloughesy, T. F.; Geschwind, D. H.; Nelson, S. F.;
Mischel, P. S.; Terskikh, A. V.; Kornblum, H. I. J. Neurosci. Res. 2008,
86, 48–60.
22. Pickard, M. R.; Green, A. R.; Ellis, I. O.; Caldas, C.; Hedge, V. L.;
Mourtada-Maarabouni, M.; Williams, G. T. Breast Cancer Res. 2009, 11,
R60.