Target Identification of Kinase Inhibitor Alisertib (MLN8237) by Using DNA-Programmed Affinity Labeling

Article abstract of DOI:10.1002/chem.201702033

Accurate identification of the molecular targets of bioactive small molecules is a highly important yet challenging task in biomedical research. Previously, a method named DPAL (DNA-programmed affinity labeling) for labeling and identifying the cellular targets of small molecules and nucleic acids was developed. Herein, DPAL is applied for the target identification of Alisertib (MLN8237), which is a highly specific aurora kinase A (AKA) inhibitor and a drug candidate being tested in clinical trials for cancer treatment. Apart from the well-established target of AKA, several potential new targets of MLN8237 were identified. Among them, p38 mitogen-activated protein kinase (p38) and laminin receptor (LAMR) were validated to be implicated in the anticancer activities of MLN8237. Interestingly, these new targets were not identified with non-DNA-based affinity probes. This work may facilitate an understanding of the molecular basis of the efficacy and side effects of MLN8237 as a clinical drug candidate. On the other hand, this work has also demonstrated that the method of DPAL could be a useful tool for target identification of bioactive small molecules.

Full text of DOI:10.1002/chem.201702033

A Journal of
Accepted Article
Title: Target Identification of Kinase Inhibitor Alisertib (MLN8237) Using
DNA-Programmed Affinity Labeling
Authors: Dong-Yao Wang, Yan Cao, Le-Yi Zheng, Lang-Dong Chen,
Xiao-Fei Chen, Zhan-Ying Hong, Xiaoyu Li, and Yi-Feng Chai
This manuscript has been accepted after peer review and appears as an
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of the final Version of Record (VoR). This work is currently citable by
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To be cited as: Chem. Eur. J. 10.1002/chem.201702033
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Target Identification of Kinase Inhibitor Alisertib (MLN8237) Using
DNA-Programmed Affinity Labeling
Dong-Yao Wang+[a], Yan Cao+[a], Le-Yi Zheng+[a], Lang-Dong Chen , Xiao-Fei Chen , Zhan-Ying
[a]
[a]
[
a]
[a]
[b]
[a]
Hong , Zhen-Yu Zhu , Xiaoyu Li* , and Yi-Feng Chai*
Abstract: Accurate identification of the molecular targets of
bioactive small molecules is a highly important yet challenging task
in biomedical research. Previously we have developed a method
named DPAL (DNA-Programmed Affinity Labelling) for labelling and
identifying the cellular targets of small molecules and nucleic acids.
Here we applied DPAL for the target identification of Alisertib
(MLN8237), a highly specific Aurora kinase A (AKA) inhibitor and a
drug candidate being tested in clinical trials for cancer treatment.
Besides its well-established target of AKA, several potential new
targets of MLN8237 were identified. Among them, p38 mitogen-
activated protein kinase (p38) and laminin receptor (LAMR) were
validated to be implicated in the anti-cancer activities of MLN8237.
Interestingly, these new targets were not identified with non-DNA-
based affinity probes either in our experiments or in previous studies.
This work may facilitate the understanding the molecular basis of the
efficacy and side-effects of MLN8237 as a clinical drug candidate.
On the other hand, this work has also demonstrated that the method
of DPAL could be a useful tool for target identification of bioactive
small molecules.
Figure 1. a) An affinity probe usually composes the ligand (SM), a reactive
group for target capture, and an affinity tag for target isolation. b) DNA-
programmed affinity labelling (DPAL).[ DPAL has a dual-probe system: a
3]
binding probe (BP) and a capture probe (CP). Red star denotes the photo-
crosslinker.[4]
Introduction
Target identification (target ID) of bioactive small molecules is an
important yet highly challenging task in biomedical research. In
drug discovery, target ID is of great importance in understanding
the molecular mechanism underlying the efficacy, toxicity, and
side-effects of drug molecules.[1] Among the many target ID
strategies, the classic affinity purification is the most widely used
approach for identifying the direct binding targets of small
molecules.[2] Typically in affinity purification, a covalent affinity
probe is prepared composing the small molecule as the bait, an
affinity tag for imaging or target enrichment and isolation, and a
reactive group for covalent capture of the target upon ligand
binding (Figure 1a).[2b] Since these add-on structures should not
interfere with target-ligand binding, it is often necessary to
prepare multiple probes with varied configurations, test their
performances, and then choose the best one to conduct the
target ID experiments. In response, researchers have developed
elegant strategies to address this issue. For example, Hosoya,[
Suzuki,[6] Young,[7] and their respective co-workers designed
several types of highly compact “all-in-one” affinity probes for
target ID; Yao and co-workers developed a “minimalist” linker
strategy to minimize the impact of add-on structures on target
binding;[1f, 8] the Cravatt group designed a series of fully
functionalized small-molecule probes incorporating the
crosslinker as part of the binding structure. Previously, we
reported a DNA-based affinity labeling method named DPAL
(DNA-Programmed Affinity Labeling).[3-4, 10] Instead of using a
single tri-functional probe, DPAL has a dual-probe system: a
binding probe (BP) where the small molecule is conjugated with
a short DNA strand, and a capture probe (CP) in which the
5]
[9]
[
a]
D. Wang, Y. Cao, L. Zheng, L. Chen, X. Xing, Z. Hong, Z. Zhu, Prof
Y. Chai
affinity tag and the crosslinker are conjugated with a
complementary DNA strand (Figure 1b). After BP binds to the
target, CP hybridizes on BP and brings the photo-crosslinker
close to the target, and then light triggers the covalent capture of
the target. Since CP is not involved in target binding, the
potential interferences from the affinity tag and the crosslinker
are obviated. Previously, we reported the proof-of-principle
target ID studies of DPAL for small molecules[3-4] and nucleic
acids.[10] Here we report the application of DPAL in identifying
the cellular targets of Alisertib, an Aurora kinase A (AKA)
inhibitor and an anti-cancer drug candidate being tested in
School of Pharmacy
Second military medical university
No.325 Guohe Road, Shanghai 200433 (P.R.China)
E-mail: yfchai@smmu.edu.cn
Prof X. Li
[
b]
Department of Chemistry
The University of Hong Kong
Pokfulam Road, Hong Kong SAR, China.
Email: xiaoyuli@hku.hk
Supporting information for this article is given via a link at the end of
the document.
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clinical trials.[11]
AKA is a serine/threonine protein kinase and is one of the
three kinases in the Aurora kinase family,[12] which has
generated significant interests in cancer research recently,
mostly due to their high expression levels and positive
associations with the progression of many malignant cancers.[13]
On the molecular level, AKA plays essential roles in nearly every
stage of cell mitosis.[
12a, 13e, 14]
Unsurprisingly, AKA has become
a prime target in anti-cancer drug discovery and numerous AKA
inhibitors have been developed and are being tested in clinical
trials.[15] Among them, Alisertib, also called MLN8237 (1; Figure
a), is one of the most intensively pursued AKA inhibitors[
15f, 16]
2
and has exhibited superb tumor-suppressing activities in many
phase II and III clinical trials.[17] However, although MLN8237 is
a highly specific AKA inhibitor, it may also interact with other
cellular proteins, similar to the many kinase inhibitors targeting
the ATP-binding site of multiple kinases.[18] Therefore, a more
comprehensive study of its cellular targets may be beneficial for
the better understanding of its efficacy, toxicity, and side-effects
of MLN8237 as a clinical drug candidate. Indeed, the recent
termination of Alisertib Phase III trial in Peripheral T-cell
Lymphoma (PTCL) signifies the urgency and importance of this
aspect.[19] Here in this study, using the DPAL method, besides
AKA, we have identified 7 additional proteins that may bind to
MLN8237. Interestingly, none of these potential targets was
identified with non-DNA-based probes or reported in previous
target ID studies.[20] Furthermore, the biological implications of
two putative targets, p38 and laminin receptor, in the anti-cancer
activities of MLN8237, were investigated.
Figure 2. a) Structures of MLN8237, MLN8054, probe 3 and 4. b) Hep3B cells
were treated with DMSO (control), MLN8237, or probe 3 (1 µM) for 48 hours.
Cells were stained with Annexin V-FITC and propidium iodide, and apoptotic
cells was quantified by flow cytometry. c) After affinity pulldown, captured
proteins were eluted from streptavidin beads and analysed by 12% SDS-
PAGE. M: molecular weight ladder; beads: pulldown with streptavidin beads
only (no probe added); 3: pulldown with probe 3; 4: pulldown with control
probe 4. Red arrow indicates the AKA protein band.
Results and Discussion
Non-DNA-based MLN8237 affinity probes identified AKA
Design and synthesis of non-DNA-based MLN8237 affinity
probes
First, we confirmed that probe 3 has similar activity as MLN8237
in inducing apoptosis in Hep3B cells (Figure 2b). Hep3B is a
type of hepatocellular carcinoma (HCC) cells known to be
sensitive to MLN8237 inhibition.[23] Next, probes 3 and 4 (1.0
µM) were incubated with Hep3B cell lysates (5.0 mg/mL) at 4 °C
for 12 hours, respectively. After irradiation under 365 nm for 90
seconds, streptavidin beads were added to isolate captured
proteins. In order to thoroughly remove non-specific background,
a stringent washing condition of 1% SDS (x10) was used.
Captured proteins were eluted and resolved with electrophoresis.
As shown in Figure 2c, a band at ~45 kD, which matches the
molecular weight of AKA, showed specificity for probe 3 (red
arrow; Figure 2c). This band was excised, extracted, analyzed
by TOF-MS/MS, and finally confirmed to be the expected target
AKA (Table S1). Notably, many non-specific bands were also
observed, indicating significant non-specific crosslinking by the
phenylazide crosslinker.[4]
Previously, Weissleder and co-workers designed an AKA
imaging probe based on the structure of MLN8054 (2; Figure 2a),
a close analog of MLN8237.[21] MLN8054 is also a potent AKA
inhibitor but with less AKA selectivity and less favorable clinical
profiles.[16] More recently, Yao and co-workers performed a
multiplexed imaging and proteomic target profiling of MLN8237
using affinity probes designed with the “minimalist” linker
strategy.[20a] These studies, also corroborated by crystallographic
analysis,[22] have clearly shown that modifications at the
carboxylic acid position of MLN8054 and MLN8237 do not
significantly affect their AKA binding. Based on these reports, we
prepared a non-DNA-based tri-functional probe with a biotin tag
and a phenylazide photo-crosslinker by modifying the carboxylic
acid of MLN8237 (3; Figure 2a). Phenylazide has been
extensively used in protein labeling with excellent efficiency[2h]
and the biotin tag is for the enrichment of captured proteins. In
addition, a negative control probe without the MLN8237 motif (4;
Figure 2a) was also prepared to control for non-specific
crosslinking. The synthesis route of these probes and
experimental details are provided in Scheme S1.
Target identification of MLN8237 with DPAL
Our previous studies have shown that DPAL has excellent target
specificity with very low level of non-specific labeling;[ therefore,
we decided to apply DPAL to the target ID of MLN8237. First,
3]
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of MLN8237.[20a] Intriguingly, except for AKA, none of the 32
proteins overlaps with the ones identified with DPAL. This
discrepancy may be due to the differences in probe structure,
cell type (HeLa vs. Hep3B), and pulldown condition (in situ vs. in
vitro) of these two methods. Nevertheless, these results suggest
that DPAL may provide an alternative approach complementing
non-DNA-based affinity probes for target identification.
As a highly specific AKA inhibitor, the anti-cancer activity of
MLN8237 is considered to be primarily based on AKA inhibition,
which leads to cell mitotic arrest and apoptosis, the hallmark
phenotype induced by MLN8237 in cancer cells.[12c, 15c] However,
recent studies have shown that MLN8237 also inhibits cell
migration and adhesion and is implicated in cancer metastasis
and invasion.[24] Therefore, among the 7 identified proteins, we
selected p38 mitogen-activated protein kinase (p38) and laminin
receptor (LAMR) for further investigation (p3 and p2,
respectively; Figure 3b), as they both play important roles in cell
migration and adhesion. The validation of functional connections
between MLN8237 with p38 and LAMR may facilitate a better
understanding of the molecular mechanism underlying the anti-
cancer activities of MLN8237.
Figure 3. a) Structures of DNA-based binding probes and capture probe; the
wavy lines denote DNA strands. See the Supporting Information for details on
the structure, synthesis, characterization, and DNA sequences of these probes
Validation of p38 as a cellular target for MLN8237
(
Figure S3 and Table S2). b) After DPAL experiments with BP-1 + CP and BP-
[
3]
2
+ CP, respectively, captured proteins were analysed by 15% SDS-PAGE.
Seven protein bands that are specific for BP-1 (p1-p7) were excised,
extracted and characterized by MS (Table S3). Identities of proteins are as
marked. Probes: 1.0 µM each; buffer: 1x PBS and 0.1 M NaCl; Hep3B cell
lysates: 5.0 mg/mL; irradiation: 365 nm, 4 °C for 90 seconds.
p38 is well known for its diverse roles in cellular responses to
stress stimuli and involvement in cell differentiation, apoptosis,
[25]
autophagy, and migration.
Dysregulation of p38 is closely
associated with cancer progression.[
25a, 26]
Interestingly, Yao and
co-workers also identified MAPK1, a close family member of p38,
as one of the MLN8237 targets in HeLa cells.[20a] We first
determined the binding affinity between MLN8237 and p38
protein with surface plasmon resonance (SPR). As shown in
Figure 4a, MLN8237 binds p38 with a moderate affinity of 8.23
MLN8237 was conjugated to the 5’-end of a 16-nt DNA strand
as the binding probe (BP-1, Figure 3a); a complementary 26-nt
DNA modified with a 3’-phenylazide and a 5’-fluorescein (FAM)
group was prepared as the capture probe (CP). A control
binding probe without the MLN8237 motif was also prepared
μM (K
(
d
), which is significantly lower than its IC50 against AKA
1.2 nM).[27] Next, we measured the inhibition of cellular p38
(
BP-2; Figure 3a).
activity by MLN8237 with a p38 MAP kinase assay kit. In brief,
live Hep3B cells were treated with MLN8237 at different
concentrations and lysed. The catalytic activity of p38 was then
determined by detecting the phosphorylation products of a p38
substrate peptide ATF-2 (activating transcription factor 2).[ As
shown in Figure 4b, MLN8237 inhibited the cellular p38 activity
in a dose-dependent manner, demonstrating the functional effect
of MLN8237 on p38.
Following a previously developed DPAL protocol,[3] CP was
mixed with either BP-1 or BP-2, respectively (1.0 µM each); the
mixture was incubated with Hep3B cell lysates (5.0 mg/mL) at
4
°C for 12 hours. After irradiation under 365 nm UV light, two
28]
rounds of ethanol precipitation were performed to separate CP-
labelled proteins from unlabelled ones. The precipitated pellet
was re-dissolved and analysed by in-gel fluorescence. As shown
in Figure 3b, in contrast to non-DNA-based probes, significantly
less non-specific labelling was observed and multiple bands that
are specific for MLN8237 were detected. Seven of these specific
protein bands were excised (p1-p7; Figure 3b), extracted, and
characterized by mass spectrometry (Table S3). Besides AKA, 9
proteins were identified: tumour rejection antigen (gp96), TER
ATPase, serine dehydratase, heat shock protein 60 (HSP60), α-
tubulin 8 (TUBA8), p38 mitogen-activated protein kinase (p38),
laminin receptor (LAMR), and nucleoside diphosphate kinase A
and B (NME1 and NME2).
A canonical mechanism for cellular p38 activation is the
phosphorylation of the activation loop by upstream MAPK
kinases (e.g.: MKK3 or MKK6).[25b, 29] We performed docking of
MLN8237 with p38 crystal structure (PDB: 1LEZ);[30] it is shown
that MLN8237 binds p38 at a site overlapping with the MKK3-
binding site (Figure 4c and 4d). This model suggests that
MLN8237 may compete with MKK3 for p38 binding, thereby
impeding the phosphorylation/activation of p38. In order to test
this, Hep3B cells were treated with MLN8237, lysed, and then
blotted with anti-phosphorylated-p38 (p-p38) antibody. Indeed,
the. phosphorylation level of p38 decreased upon MLN8237
treatment (Figure 4e).
Recently, Yao and co-workers performed a multiplexed and
comprehensive target profiling of MLN8237 using a proteomic
approach;[8a] 32 proteins were identified as the potential targets
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Figure 4. (a) SPR sensorgram and fitting curve for measuring MLN8237-p38 binding affinity; (b) Measurement of the inhibition of p38 activity in Hep3B cells by
MLN8237; (c) Docking of MLN8237 to p38 crystal structure (PDB: 1LEZ). The most populated and lowest energy pose is shown; docking score: -69.35 kcal/mol.
MLN8237 is shown in stick, with carbons in magenta, nitrogens in blue, oxygen in orange, fluorine in cyan, and chlorine in green. H-bonds interactions are shown
with dashed black lines. Selected protein residues are highlighted as golden sticks. (d) Crystal structure of p38 in complex with its activator MKK3 peptide (PDB:
1LEZ). MKK3 peptide is shown in stick, with carbons in light blue, nitrogens in blue, and oxygen in red. (e) Hep3B cells were treated with MLN8237 at various
concentrations (37 °C, 48 hours) and then lysed. Expression levels of selected proteins in the apoptosis cascade were determined with Western blot using
respective antibodies. (f) Hep3B cells were treated with MLN8237 as same as in (e) and stained with Annexin V-FITC and propidium iodide. Cell apoptosis was
then quantified by flow cytometry. (g) Quantification of apoptotic cells in (f).
Furthermore, we have also verified that MLN8237 induced
significant cell apoptosis after 48 hours, as determined by flow
cytometry (Figure 4f). Western blots show the expression levels
of key proteins in the apoptosis signaling cascade (p53, Bcl-2,
Bax, cleaved caspase-9, and cleaved caspase-3) have been
affected by MLN8237 in accordance to apoptosis activation
(Figure 4e).[20b] This effect may partially result from p38 inhibition,
in addition to the well-validated AKA inhibition. Collectively, the
above results have demonstrated that p38 is a cellular target of
MLN8237 and the inhibition of p38 may contribute to the pro-
apoptotic activity of MLN8237.
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Figure 5. (a) SPR sensorgram and fitting curve for measuring MLN8237-LAMR binding affinity. (b) Hep3B cells were treated with MLN8237 as the
same as in Figure 4 and stained with Annexin V-FITC and propidium iodide; cell apoptosis was then analysed by flow cytometry. (c) Quantification of
apoptotic cells in (b). (d) Results of the wound-healing assay. Hep3B cells were treated with MLN8237; after 24 hours, images were captured and cell
migration rates were quantified. (e) Results of trans-well assay. Hep3B cells were treated with MLN8237; after 8 hours, images of cell penetration of
the Matrigel membrane were captured. (f) ELISA measurement of extracellular LMN concentrations after MLN8237 treatment. (g) Hep3B cells were
treated with MLN8237 at various concentrations for 24 hours and then lysed. Levels of LAMR and cleaved caspase-3 were determined using Western
blot with respective antibodies (h) Docking of MLN8237 to LAMR (PDB: 3BCH). The most populated and lowest energy pose is shown; docking score:
-
56.10 kcal/mol. MLN8237 is shown in stick, with carbons in magenta, nitrogens in blue, oxygen in orange, fluorine in cyan, and chlorine in green. H-
bonds interactions are shown with dashed black lines. Selected protein residues are highlighted in golden sticks. The “peptide G” site is highlighted in
bright red colour.
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Validation of LAMR as a cellular target for MLN8237
Conclusions
Laminins (LMNs) are a family of glycoproteins mostly found in
the extracellular matrix (ECM) and they play important and
diverse roles in regulating cell adhesion, migration, tissue
development, and intercellular communications.[31] LAMR, the
major cell membrane receptor for LMNs, is significantly
upregulated in solid tumors and functions as a promoter for
cancer metastasis and invasion.[31-32] Therefore, the identification
of LAMR as a potential target may be relevant to the relatively
less explored anti-migration activity of MLN8237.[20b, 24] The
binding affinity of MLN8237 with LAMR was determined by SPR
to be 1.84 μM (Figure 5a and Table S4), stronger than p38
binding but still weaker than AKA binding. Next, we performed
wound-healing and trans-well assays to examine the inhibitory
abilities of MLN8237 on cell migration and invasion, respectively.
However, considering MLN8237 is cytotoxic and can induce
significant cell apoptosis after 48 hours (Figure 4f and 4g), we
first verified that only a small percentage of cell apoptosis
occurred if cells were treated less than 24 hours (< 10% at 5.0
μM of MLN8237; Figure 5b and 5c). Therefore, the would-
healing and trans-well assays were performed with shortened
time durations. As shown in Figure 5d, in wound-healing assay,
MLN8237 was able to reduce the migration rate of Hep3B cells
by ~38% after 24 hours, while in the trans-well assay, significant
inhibition of cell invasion was observed after only 8 hours
In summary, using a DNA-based affinity labeling approach,
we have identified several potentially new targets for MLN8237,
a highly specific AKA inhibitor being tested as an anti-cancer
drug in clinical trials. Among the new targets identified, p38 and
LAMR were validated and we have demonstrated that binding to
these targets contribute to the anti-cancer activities of MLN8237.
The binding affinity of MLN8237 with p38 is modest and the
corresponding functional effect may probably be masked by
AKA inhibition in the cell; however, the effect of MLN8237 on
LAMR is more prominent and may underlie, at least partially, the
anti-migration and anti-invasion activities of MLN8237.[24] Overall,
this work may facilitate the better understanding of the efficacy,
toxicity, and side effects of MLN8237 as a clinical drug
candidate on the molecular level.
In this study, the targets identified with DPAL were not
clearly distinguished in our initial attempt using the more
conventional, non-DNA-based tri-functional probes. Instead,
significant non-specific labeling was observed (Figure 2c). More
intriguingly, except AKA, none of the targets identified with
DPAL overlaps with the ones identified by Yao and co-workers,
who used different probe strategy (minimalist probe design and
in situ labeling) and
a more comprehensive proteomic
approach.[20a] It is also worth noting that Yao’s approach was
able to capture indirectly binding targets in the protein complex,
a highly useful capability that has yet to be explored with the
DPAL method (e.g. varying the site of CP hybridization on the
BP DNA[3, 10]). Currently, we are studying the other potential
(
Figure 5e). These results have confirmed the anti-migration and
anti-invasion activities of MLN8237.
Next, we asked whether the attenuated cell migration and
invasion capabilities are correlated with MLN8237-LAMR binding. targets in Figure 3c to investigate their binding modes with
LAMR mediates cell migration and adhesion by forming a
complex with α -integrin and then binding to LMN on the cell
MLN8237 (direct or indirect) and functional connections with the
biological activities of MLN8237. Nevertheless, the results
obtained in these studies highlighted the importance of probe
design and the complementarity of different approaches in target
identification.
6
surface.[33] Studies have shown that peptides[34] and small
molecules[35] can disrupt LMN binding, thereby releasing LMN to
ECM and promoting proteolytic digestion of LMN by
[
36]
cathepsins, which results in lower LMN concentration and the
loss of cell adhesion and migration capabilities.[34] Using an
ELISA assay, we measured the LMN concentration in the
supernatant of Hep3B cells treated with MLN8237. Indeed,
MLN8237 decreases LMN concentration dose-dependently
Experimental Section
DNA-based probe syntheses
(
Figure 5f). Moreover, we also confirmed that the cellular level of
LAMR and cleaved caspase-3 remained mostly unchanged after
MLN8237 treatment, suggesting the decrease of LMN
concentration was not due to the change of expression level of
LAMR or cell apoptosis (Figure 5g).
Furthermore, docking of MLN8237 with LAMR indicates that
MLN8237 binds at a site partially overlapping with the so-called
For BP-1, to a solution of MLN8237 (15.54 mg, 0.03 mmol) in 120 μL
DMSO, N-hydroxysuccinimide (3.45 mg, 0.03 mmol) and EDC (3.31mg,
0.03 mmol) were added. The reaction was allowed to vortex at room
temperature for 4 hours. After centrifugation, 90 μL of the supernatant of
the mixture and 90 μL of the 5’-amine-modified DNA stock solution
2 3
(approximately 30 μM) were directly added to 60 μL saturated Na CO .
The mixture was vortexed at room temperature for another 4 hours. The
product was obtained by ethanol precipitation and then purified with
HPLC. For BP-2, the 5’-amine-modified DNA was purchased and directly
used in the affinity pulldown experiments. For CP: The 5’-FAM-, 3’-
amine-modified DNA was dissolved in a solution of azide-NHS ester (S2,
see the Supplementary Inforation; 7.8 mg, 0.03 mmol) in 120 μL DMSO
“
peptide G” site (amino acid 161-180) on LAMR, the major
binding site for LMN (Figure 5h).[37] This suggests MLN8237 may
compete for LAMR binding with LMN, in corroboration with the
results of the LMN assay in Figure 5f. Collectively, the above
results indicate that the anti-migration and anti-invasion activities
of MLN8237 may, at least partially, arise from LAMR binding.
and 30 μL saturated Na
2 3
CO . The mixture was vortexed at room
temperature for hours. The product was obtained by ethanol
4
precipitation and purified with HPLC.
DNA probe sequences
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Chemistry - A European Journal
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BP-1: 5’-MLN8237-GGACTTAAGGGATGCA-3’);
BP-2: 5’-NH2-GGACTTAAGGGATGCA-3’;
CP: 5’-FAM-CCGTCAAACCTGCATCCCTTAAGTCC-3’-azide
Solis and Wets local search was used for energy minimization on a user-
specified proportion of the population. The docked conformation of small
molecule was generated after a reasonable number of evaluations.
General procedure for affinity pulldown experiments with non-DNA-
based probes
General procedure for flow cytometry
Hep3B cells were plated in six-well cell culture plates at a density of
1×105 cells per well. After incubation overnight, the cells were
replenished with fresh medium and treated with vehicle alone (0.05%
DMSO, v/v) or MLN8237 at 0.2, 1.0, and 5.0 μM for 24 or 48 hours. The
cells were then trypsinized and centrifuged at 1,000x g for 5 minutes to
pellet the cells. Each sample was re-suspended in 1 mL of binding buffer
(10 mM HEPES/NaOH, pH 7.4, 140 mM NaCl, 2.5 mM CaCl2). An
aliquot of cell suspension (195 μL) was mixed with 5 μL of Annexin V-
FITC and incubated for 10 minutes in the dark. The cells were washed
once using 1x PBS and re-suspended in 190 μL of binding buffer and 10
μL of 20 μg/mL propidium iodide was added. The cells were then
subjected to apoptotic analysis using flow cytometry.
Hep3B cells were lysed (3 mg) and incubated with either DMSO, probe 3
or 4 (1 µM) at 4 °C for 12 hours. The mixture was irradiated at 365 nm for
1
minute, and then 20 μL streptavidin beads was added. The suspension
was vortexed at 4 °C for another 4 hours. In order to thoroughly remove
non-specifically binding proteins, stringent washing buffer (1% SDS) was
used. The beads were washed with 1% SDS for 10 times and then 1x
PBS for 3 times. 1x loading buffer was added to the beads and then
boiled at 95 °C for 5 minutes to elute streptavidin-captured proteins.
Eluted samples were directly analyzed by 12% SDS-PAGE.
General procedure for DPAL experiments and in-gel fluorescence
Analysis
General procedure for Western blot
The DPAL experiments were performed based on our previously
[
3]
reported procedure. In brief, BP-1 or BP-2 was hybridized with CP in
water, respectively. Hep3B cells were lysed (3 mg) and incubated with
pre-hybridized DNA probes (final concentration: 1 µM each probe) at
Hep3B cells were treated with MLN8237 (0, 0.2, 1.0, and 5.0 μM),
washed with re-chilled 1x PBS, lysed with radio-immunoprecipitation
assay (RIPA) buffer containing the protease inhibitor and phosphatase
inhibitor cocktails, and then centrifuged at 12,000x g for 15 minutes at
4 °C. Protein concentrations were measured using Bradford assay. Cell
lysate samples (30 µg) were dissolved in SDS sample buffer and
resolved by SDS-PAGE gel (12%). After electrophoresis, proteins were
transferred onto a nitrocellulose and the blotted following the typical
blocking, washing and visualization protocol.
4
°C for 12 hours (typical reaction volume: 200 µL). The mixture was
irradiated at 365 nm for 90 seconds. After two rounds of ethanol
precipitation, the precipitated pellet was resolved in 60 µL and 15 µL 5x
loading buffer was added and then boiled at 95 °C for 5 minutes. The
samples were resolved with 12% SDS-PAGE. The specific protein bands
shown in Figure 3b were excised and submitted for protein identification
using MALDI-TOF/TOF-MS.
Wound-healing assay
General procedure for SPR Analysis
Hep3B cells were seeded in six-well plates and cultured until confluent. A
sterile pipette tip was used to make a straight scratch would, and then
cells were washed with 500 μL PBS twice to remove cell debris. Cells
were cultured with DMEM medium (2 mL each well without FBS, 37 °C,
5% CO2) and treated with MLN8237 (0, 0.2, 1.0, 5.0 µM). In control
experiments, only the same volume of water was added. Cell images
were captured at designated time points after scratch as indicated in the
main text with a microscope. Cell migration rate was measured based on
the distance of the edges of scratch using the Image J software, following
the manufacturer’s instructions. Relative migration rate was quantified
against the water only control experiments.
Proteins were immobilized on a CM5 chip via the typical EDC/NHS-
mediated crosslinking reaction. Small molecule ligands were diluted in
PBS at concentrations ranging from 125 nM to 64 µM. The analysis was
then performed according to the protocol provided by the instrument
manufacturer. In each analysis, the middle concentration was duplicated
at the end of the wash run to confirm the stability of the sensor surface.
The parameters of SPR were set as follows: flow rate, 30 µL/min; contact
time, 120/180 s, disassociation time, 300 s. Affinity curve fitting was
performed with the Biacore T200 software using a steady-state affinity
d
model (1:1) to calculate disassociation constant (K ).
P38 cellular activity assay
Trans-well assay
Hep3B cells were treated with different concentrations of MLN8237 for 24
hours. Cells were then collected and re-suspended in lysis buffer and
subjected to repeated freeze/thaw cycles to fully lyse the cells. The p38
enzymatic activities were measured with a p38 MAP kinase assay kit
The trans-well chamber membranes were pre-coated with 50 μL 1:1
mixture of Matrigel and DMEM medium. 5 x 104 Hep3B cells in serum-
free DMEM medium were added to each upper chamber and DMEM
medium with 15% FBS was added to the lower chamber. The cells were
then treated with 0, 0.2, 1.0 or 5.0 μM MLN8237. After 8 hours’
incubation at 37 °C, cells remaining on the upper surface of membrane
were removed and cells that have migrated to the underside of
membrane were stained with 0.1% crystal violet for 30 minutes. The
migrated cells on the underside of membrane were then photographed
under a microscope at 500 magnification.
(Cell Signalling) following the manufacturer’s protocol.
Molecular modeling
The docking program Autodock 3.0.315 was used to automatically dock
MLN8237 to target proteins. The crystal structures of human laminin
receptor (PDB: 3BCH) and p38 MAP kinase in a complex with its
activator MKK3B (PDB: 1LEZ) were obtained from Protein Data Bank
Laminin concentration assay
(PDB). The missing atoms and residues were modeled in Sybyl 6.8.17.
Atomic charges were taken as Kollman united-atom for macromolecules
and Gasteiger-Marsili for MLN8237. The Lamarckian genetic algorithm
Hep3B cells were treated with different concentrations of MLN8237 for 24
hours, and then cell culture supernatants were collected and spun at
2000x g for 10 minutes at 4 °C. The concentration of laminin was
(LGA) was applied to analyze small molecule-protein interactions and the
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Chemistry - A European Journal
10.1002/chem.201702033
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measured with an ELISA kit (Cell Signaling) following the manufacturer’s
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other experimental details are provided in the Supplementary Information.
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Keywords: target identification • DNA-programmed affinity
labeling • Alisertib • kinase inhibitor • MLN8237
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Entry for the Table of Contents (Please choose one layout)
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Dong-Yao Wang, Yan Cao, Le-Yi
Zheng, Lang-Dong Chen, Xin-Rui Xing,
Zhan-Ying Hong, Zhen-Yu Zhu, Xiaoyu
Li*, and Yi-Feng Chai*
Page No. – Page No.
Target Identification of Kinase
Inhibitor Alisertib (MLN8237) Using
DNA-Programmed Affinity Labelling
The application of DPAL (DNA-Programmed Affinity Labelling) for the target
identification of Alisertib (MLN8237), an Aurora kinase A (AKA) inhibitor and an
anti-cancer drug candidate being tested in clinical trials. Besides its known target
AKA, several new targets of MLN8237 were identified and their relevant biological
implications were verified.
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