Photoswitchable CENP-E Inhibitor Enabling the Dynamic Control of Chromosome Movement and Mitotic Progression

Article abstract of DOI:10.1021/jacs.9b12782

Interfering with mitosis is a potential cancer therapy strategy. However, the lack of controllability of antimitotic drugs in cell growth suppression causes severe side effects and limits their clinical utility. Herein, we developed an azobenzene-based ph

Full text of DOI:10.1021/jacs.9b12782

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Communication
Photoswitchable CENP-E inhibitor enabling the dynamic
control of chromosome movement and mitotic progression
Noushaba mafy, Kazuya Matsuo, Shota Hiruma, Ryota Uehara, and Nobuyuki Tamaoki
J. Am. Chem. Soc., Just Accepted Manuscript • Publication Date (Web): 13 Jan 2020
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Photoswitchable CENP-E inhibitor enabling the dynamic control of
chromosome movement and mitotic progression
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Noushaba Nusrat Mafy†, Kazuya Matsuo†, Shota Hiruma‡, Ryota Uehara‡, #,*, Nobuyuki Tamaoki†,*
†Research Institute for Electronic Science, Hokkaido University, Kita 20, Nishi 10, Kita-ku, Sapporo, 001-0020, Japan
‡Graduate School of Life Science, Hokkaido University, Kita 21, Nishi 11, Kita-ku, Sapporo, 001-0021, Japan
#Faculty of Advanced Life Science, Hokkaido University, Kita 21, Nishi 11, Kita-ku, Sapporo, 001-0021, Japan
Supporting Information Placeholder
associated genes and determine their functions.3-5
ABSTRACT: Interfering with mitosis is a potential cancer
However, a drawback of anti-mitotic cancer therapy is its
therapy strategy. However, the lack of controllability of
potential side effects on normal proliferative cells, which
anti-mitotic drugs in cell growth suppression causes
limits their clinical application. Photopharmacological
severe side effects and limits their clinical utility. Herein,
tools such as photostatins6 to reversibly control
we developed an azobenzene-based photoswitchable
microtubule dynamics with light illumination may
inhibitor of CENP-E, a mitotic kinesin required for
overcome this limitation, but development of
chromosome transportation. The new inhibitor enabled
photoswitchable tools for a wide variety of targets
including motor proteins would further facilitate tissue-
selective mitotic inhibition.
reversible photoswitching of CENP-E activity with ~10-
fold change in IC50 between cis and trans
photoisomerization states both in vitro and in living cells.
It also enabled repeatable photoswitching of CENP-E-
dependent chromosome congression and hence mitotic
progression with UV/Vis light illumination cycles. Using
this technique, we could specify the exact process of
mitotic progression in which CENP-E plays an
indispensable role. Our data demonstrate the power of
photochemical approach for highly controllable mitotic
interference as well as for discovery of precise molecular
functions in dynamic cellular processes.
During mitosis in eukaryotes, replicated chromosomes
are equally segregated into two daughter cells to precisely
transmit genetic information.1 This process is mediated
Figure 1. (a) Authentic CENP-E inhibitor, GSK923295. (b)
by a microtubule-based bipolar structure, the mitotic
spindle. The spindle captures all chromosome pairs at
their kinetochores, transports them to cell equatorial plane
at metaphase by motor proteins, and then separates each
pair into opposite cellular poles during anaphase.
Accurate mitotic progression is secured by spindle
assembly checkpoint (SAC) that is activated in the
presence of uncaptured chromosomes and stops mitotic
progression until all chromosomes are captured and
aligned by the spindle.2 Spindle malfunctioning causes
prolonged mitotic arrest, eventually leading to cell death.
Therefore, development of inhibitors that block spindle
function has been a promising strategy for cancer therapy,
and enormous efforts have been made to discover spindle-
Chemical structures of photo-switchable CENP-E inhibitors.
(c) Reversible cis-trans photoisomerization of 4.
Different types of microtubule-associated motor
proteins make specialized contributions to force
generation in dynamic reorganization of spindle
microtubules and chromosome segregation.7 Among
them, centromere-associated protein
E
(CENP-E;
kinesin-7) binds to the kinetochores and transports
chromosomes along spindle microtubules from the
spindle poles toward the equatorial plate to complete
chromosome congression.8 GSK923295 (Figure 1a) is a
selective CENP-E inhibitor under clinical trials, which
locks CENP-E in a “rigor” microtubule-bound state by
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blocking inorganic phosphate release in its ATPase
cycle.9-11
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Figure 2. (a) Absorption spectra of 4 (33 µM) in BRB-80 buffer (pH 6.9): acetonitrile (1:1) solution at room temperature
before light illumination (BI, black), at PSS365nm (red), or at PSS510nm (blue). Inset: Absorbance changes at 338 nm under
alternating 365/510 nm illumination. (b, c) CENP-E ATPase assay (b) or cell proliferation assay (c) with GSK923295 (gray)
and 4 before light illumination (black), at PSS365nm (red), or at PSS510nm (blue). Mean ± S.E., n = 3. (d) Schematics of
completely-aligned chromosomes with active CENP-E and misaligned polar chromosomes with inhibited CENP-E. (e)
Immunofluorescence of CENP-E and -tubulin in HeLa cells treated with DMSO, GSK923295 (1.0 µM) or 4 (1.0 µM) in the
presence of MG-132 (20 µM) for 2 h. Scale bar, 5 µm. White, magenta, or green arrows indicate misaligned chromosomes,
aligned chromosomes, or CENP-E accumulation, respectively. (f) Frequency of misaligned chromosomes in DMSO-,
GSK923295- (2.0 µM) or 4- (1.0 µM) treated cells (Mean ± S.E., n = 3, >150 cells in total). Light intensities in all experiments
were described in Supporting Information.
15
Previous studies have found that GSK923295 inhibits
chromosome alignment and mitotic progression in cancer
cells and also suppresses tumor growth in xenograft
models.9-10,12-13 However, despite of its potential as a
cancer therapeutic target, there has been no report on
development of a photoswitchable CENP-E inhibitor to
date.
Our photoswitchable CENP-E inhibitor enabled
dynamic modulation of CENP-E-dependent chromosome
movement and mitotic progression in high temporal
resolution, providing a potential optotherapeutic method
and a new tool for studying precise role of CENP-E in
mitosis.
To design the photoswitchable CENP-E inhibitors, we
employed the well-established molecular scaffold of
azobenzene derivatives as a reversible photoswitch for
photopharmacology.16-19 The core imidazopyridine ring
in GSK92329510 was replaced with azobenzene
Herein, we describe a photochemical approach for
control of CENP-E functions using cis-trans
photoisomerization of azobenzene derivatives that we
previously used for photoregulation of motor proteins.14-
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derivatives (Figure 1b). We synthesized 1-3 with a
phenylazo group (azoPh) at 2-, 3-, 4-position,
respectively, and 4 with a pyrazolylazo group (azoPyr) at
4-position (Figure 1b; Supporting information Figure
S12-S59), which was previously reported to exhibit high
photoswitchability20.
light illumination in colorimetric cell proliferation assay
(Figure 2c and S6). Consistent with previous studies10,
GSK923295 exhibited strong cell growth inhibition with
IC50 30 nM. Cell viability was not affected by light
illumination either in the presence or absence of
GSK923295, ruling out possible side effects of light
illumination on essential cellular processes (Figure S6).
On the other hand, 4 had substantially higher efficacy at
non-illuminated state (IC50 0.29 µM) or PSS510nm (IC50
0.29 µM) than at PSS365nm (IC50 2.4 µM). Therefore,
although its efficacy on cell proliferation was ca. 10-fold
less than that of GSK923295, 4 showed evident
photoswitchability of cell proliferation inhibition.
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Photoswitchability of 1-4 was studied using absorption
spectroscopy with ultraviolet (UV) and visible (Vis) light
illumination in aqueous solution (Figure 2a and S1).
Before light illumination, 1-4 in pure trans isomer (Figure
S2) exhibited two absorption bands corresponding to
* (around 330 nm) and n* (around 440 nm)
electronic transitions. These intensities were dramatically
altered upon 365 nm or 436 nm light illumination to reach
photostationary state (PSS) containing > 70% cis or trans
isomer, respectively, for 1-3 (determined by HPLC in
Figure S2, Table S1). Presumably due to the well-
separated electronic transitions in arylazopyrazole20, 4
showed higher photo-switchability (93% of cis isomer at
PSS365nm and 86% of trans isomer at PSS510nm) than 1-3.
All compounds underwent the reversible cis-trans
photoisomerization for many cycles without any fatigue.
Thermal relaxation lifetimes for cis isomers in 1-4 were
> 24 h at 37 ºC (Figure S3, Table S1).
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To gain further insight into inhibitory mechanism of 4,
we investigated the effect of GSK923295 or 4 in spatial
distribution of the chromosomes and endogenous CENP-
E in HeLa cells by immunofluorescence using anti-
CENP-E antibody (Figure 2d and e). To facilitate the
analysis of chromosome dynamics in pre-anaphase,
during
which
CENP-E-dependent
chromosome
congression takes place, anaphase onset was blocked by
a proteasome inhibitor MG-132 in all conditions.22 In
control, all mitotic cells possessed completely aligned
chromosomes at the metaphase plate with evident
accumulation of CENP-E at their kinetochores (Figure
2d-f and S7). As previously reported10,23, GSK923295
induced frequent misalignment of chromosomes at
spindle poles. In this condition, CENP-E was delocalized
from the kinetochores, and instead ectopically
accumulated to the spindle poles due to microtubule
treadmilling that drives poleward movement of the rigor-
bound CENP-E (Figure 2e).10,22 Similar chromosome
misalignment was induced by 4 in > 80% cells at non-
illuminated state or PSS510nm, while it was in less than
30% cells at PSS365nm. The 4-induced chromosome
misalignment at non-illuminated state or PSS510nm was
also accompanied by evident CENP-E delocalization.
This indicates that 4 blocks CENP-E at rigor state in
living cells and perturbs chromosome congression in a
photo-switchable manner.
Next, we investigated inhibitory effects on steady-state
ATPase activity of purified CENP-E using an NADH-
coupled method21. Due to the poor water solubility of 1-
3, their inhibitory activities were tested at the maximum
concentration of 5 µM in BRB80 buffer containing 10%
DMSO (Figure S4). Only 3 bearing 4-azoPh in pure trans
form inhibited CENP-E before light illumination (46%
inhibition at 5 µM), whereas no inhibitory effect was
shown at PSS365nm. Compared to 1-3, 4 showed much
better water solubility (> 100 µM in BRB-80 buffer
containing 0.1% DMSO) and inhibited ATPase activity
in non-light illuminated condition (IC50 5.9 µM), which
was ca. 10-fold less potent than GSK923295 (IC50 0.58
µM). The efficacy of 4 dramatically decreased at
PSS365nm (IC50 120 µM) but remained equivalent to the
non-light illuminate state at PSS510nm (IC50 14 µM),
demonstrating its sharp photoswitchability of the
inhibitory property (Figure 2b). These trends are probably
because only the extended structures included in trans-3
and 4, not in these cis isomers are acceptable to the
sterically restricted binding site of CENP-E. In
microtubule co-sedimentation assay, 4 induced stable
binding between polymerized microtubules and CENP-E
with ADP in non-light illuminated condition or at
PSS510nm, but to lesser extent at PSS365 (Figure S5).
Therefore, similarly to GSK923295, 4 also locked
CENP-E in a rigor microtubule-bound state, but in a
photo-switchable manner.
To determine whether 4 effects the intracellular
function of CENP-E in a photo-switchable manner, we
compared proliferation of HeLa cells treated with
GSK923295 or 4 for 48 h with or without 12 h-interval
Figure 3. (a) Live imaging of mitotic chromosomes in a 4-
treated LLC-PK1 cell under alternating 365 nm (red) and
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510 nm (blue) light illumination. White arrows: misaligned
chromosomes. (b) A kymograph of mitotic chromosome
movement along cell division axis. The misaligned
chromosomes are highlighted and tracked by lines with
different colors. Vertical bars, 5 µm. Horizontal bar, 5 min.
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Figure 4. (a) Live imaging of 4-treated HeLa cells under alternating 365/510 nm light illumination. White or magenta arrows
indicate misaligned or aligned chromosomes, respectively. The timing of 365 nm light illumination was set as 0 min.
Chromosome dynamics were categorized into three patterns as described in the main text. Scale bar, 5 µm. (b) Frequency of
different chromosome dynamics patterns in (a). At least 38 mitotic cells containing misaligned chromosomes before light
illumination from two independent experiments were analyzed for each condition.
We further investigated 4-mediated photo-control of
chromosome congression by live imaging with
alternating 365 nm and 510 nm light illumination. To
facilitate chromosome tracking at high magnification, we
used LLC-PK1 cells that has a flat cell morphology in
mitotic phase. Chromosomes were stained with far-red
HeLa cells (Figure 4). Cells were co-treated with MG-132
and 4 at non-illuminated state to induce chromosome
misalignment, incubated for different duration (3-30 min)
at PSS365nm to induce chromosome congression to various
extent, and then further incubated at PSS510nm for 30 min.
In case cells were incubated only for 3 min at PSS365nm
,
fluorescent
dye
(SiR-DNA)
not
to
affect
misaligned chromosomes still remained in all cells at the
end of PSS365nm, and these chromosomes immediately
stopped congression or started to move poleward at the
subsequent PSS510nm (pattern I in Figure 4a). As the
duration of PSS365nm increased, larger population of cells
completed chromosome alignment by the end of
PSS365nm. This was further confirmed by clearance of a
SAC component Mad2 from the kinetochores, which
occurs only after establishment of the end-on attachment
of spindle microtubules on the kinetochore (Figure
S10).25-26 Importantly, among the cells that completed
chromosome alignment during PSS365nm, very small
population showed chromosome re-misalignment within
30 min at the subsequent PSS510nm (pattern II), whereas
vast majority of them maintained complete chromosome
alignment (pattern III). Quantification clearly
demonstrates time-dependent transition from pattern I to
III in the mode of chromosome dynamics in light
illumination cycle (Figure 4b). Further extension of
PSS510nm resulted in a gradual increase in chromosome re-
misalignment, but more than 50% of cells kept complete
alignment even after 3-h incubation at PSS510nm (Figure
S11). These data demonstrate that CENP-E activity is not
required for the maintenance of chromosome alignment
within the physiologically-relevant time frame of mitotic
progression (i.e. 30 min), whereas its inhibition impairs
the maintenance of extremely prolonged metaphase state.
photoisomerization of 4 by the excitation light (Figure
S8). Consistent with the fixed cell analysis, pretreatment
with 4 induced misaligned polar chromosomes before
light illumination. However, after 365 nm light
illumination, the misaligned polar chromosomes
gradually moved toward the equatorial plane. By the
subsequent 510 nm light illumination, the equator-ward
movement of the misaligned chromosomes immediately
ceased and substantial portion of them started to move
toward the spindle poles (Figure 3a, movie S1). This
suggests immediate inhibition of CENP-E by 4 upon
photoisomerization to PSS510nm
. Direction of the
chromosome movement changed repeatedly in the
subsequent light illumination cycle, demonstrating a
repeatable photoswitching of chromosome congression
by 4 (Figure 3b and S9).
During this experiment, we noticed that chromosomes
that had once completely aligned at the equatorial plane
at PSS365nm did not immediately come out of the
metaphase plate at the subsequent PSS510nm. This
indicates that, while CENP-E is continuously required for
congression of chromosomes, it becomes dispensable
once these chromosomes are aligned. It has been
controversial whether CENP-E plays active roles in the
maintenance of chromosome alignment.24 Since 4 is an
ideal chemical tool to investigate contribution of CENP-
E to different stages of mitosis, we further addressed this
issue using a systematic UV/Vis illumination cycle in
In summary, we successfully developed the photo-
switchable CENP-E inhibitor 4 that enabled reversible
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switching of CENP-E activity both in vitro and in the
REFERENCES
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intracellular environment. Using this technique, we could
modulate CENP-E-dependent chromosome movement
and the progression of mitosis in a reversible manner. The
fast photoswitching of 4 also enabled us to specify the
exact mitotic stage in which CENP-E plays an
indispensable role. Our data demonstrate the power of
reversible photo-regulatory tool in elucidating molecular
functions in dynamic cellular processes. Considering
potential high spatial resolution of photochemical
technics, it would be an intriguing future study to apply
our inhibitor for specific growth suppression of cancer
cells within living organisms, which may open up an
avenue of a novel scheme for cancer therapy.
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Supporting Information.
The Supporting Information is available free of charge on
the
ACS
Publications
website.
Experimental procedures and additional data (PDF)
Movie S1: Dynamic photoregulation of mitotic
chromosomes in a living cell (AVI)
AUTHOR INFORMATION
Corresponding Author
E-mail for N.T.: tamaoki@es.hokudai.ac.jp or for R.U.:
ruehara@sci.hokudai.ac.jp
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENT
We thank Dr. Daniel W. Gerlich (IMBA, Austria) for the
Mad2-GFP/H2B-mCherry HeLa cell line. We also thank the
Instrumental Analysis Division of the Global Facility Center
(Creative Research Institution, Hokkaido University) for
HR-MS measurements with Thermo Scientific Exactive,
and for providing insight and expertise. This work was
performed under the International Cooperative Research
Program of “Dynamic Alliance for Open Innovation
Bridging Human, Environment and Materials” in “Network
Joint Research Center for Materials and Devices”. This work
was supported by JSPS KAKENHI for Early-Career
Scientists (Grant # 19K15709), Takeda Science Foundation,
The Tokyo Biochemical Research Foundation, Konica
Minolta Science and Technology Foundation, Noguchi
Foundation, Japan Foundation for Applied Enzymology,
Foundation for Interaction in Science & Technology, and
Akiyama Life Science Foundation to K.M, Grants-in-Aid for
Scientific Research B (Grant # 19H03219), on Innovative
Areas “Singularity Biology (No.8007)” (19H05413) and
Fostering Joint International Research B (19KK0181) of
MEXT, the Orange Foundation, the Smoking Research
Foundation, and the Kato Memorial Bioscience Foundation
to R.U., and Grants-in-Aid for Scientific Research B (Grant
# 18H02042) and TOKYO OHKA Foundation for the
Promotion of Science and Technology to N.T.
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N
N
N
N
N
N
365 nm
O
H
O
Cl
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N
N
H
N
H
Cl
N
510 nm
N
O
H
O
O
O
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trans isomer
cis isomer
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Reversible
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photoswitch!
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DNA
CENP-E
DNA
CENP-E
inhibited CENP-E
active CENP-E
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