Synthesis and biological evaluation of optimized inhibitors of the mitotic kinesin Kif18A

Article abstract of DOI:10.1021/cb500789h

The mitotic spindle, a highly dynamic structure composed of microtubules, mediates the segregation of the previously duplicated genome into the two nascent daughter cells. Errors in this process contribute to pathology including tumor formation. Key for the shape and function of the mitotic spindle are kinesins, molecular motor proteins that convert chemical energy into mechanical work. Due to their fast mode of action, small molecules are valuable tools to dissect the dynamic functions of kinesins during mitosis. In this study, we report the identification of optimized small molecule inhibitors of the mitotic kinesin Kif18A. Using BTB-1, the first identified Kif18A inhibitor, as a lead compound, we synthesized a collection of derivatives. We demonstrate that some of the synthesized derivatives potently inhibited the ATPase activity of Kif18A with a half maximal inhibitory concentration (IC₅₀) value in the low micromolar range. In vitro analysis of a panel of Kif18A-related kinesins revealed that the two most potent compounds show improved selectivity compared to BTB-1. Structure-activity relationship studies identified substituents mediating undesired inhibitory effects on microtubule polymerization. In summary, our study provides key insights into the mechanism of action of BTB-1 and its analogs, which will have a great impact on the further development of highly selective and bioactive Kif18A inhibitors. Since Kif18A is frequently overexpressed in solid tumors, such compounds are not only of great interest for basic research but also have the potential to open up new strategies for the treatment of human diseases.

Full text of DOI:10.1021/cb500789h

Articles
pubs.acs.org/acschemicalbiology
Synthesis and Biological Evaluation of Optimized Inhibitors of the
Mitotic Kinesin Kif18A
Joachim Braun,†,§ Martin M. Mockel,‡,§ Tobias Strittmatter,† Andreas Marx,† Ulrich Groth,†
̈
and Thomas U. Mayer*,‡
†Department of Chemistry and Konstanz Research School Chemical-Biology (KoRS-CB), University of Konstanz, Universitatsstr. 10,
̈
78467 Konstanz, Germany
‡Department of Biology and Konstanz Research School Chemical-Biology (KoRS-CB), University of Konstanz, Universitatsstr. 10,
̈
78467 Konstanz, Germany
S
* Supporting Information
ABSTRACT: The mitotic spindle, a highly dynamic structure
composed of microtubules, mediates the segregation of the
previously duplicated genome into the two nascent daughter
cells. Errors in this process contribute to pathology including
tumor formation. Key for the shape and function of the mitotic
spindle are kinesins, molecular motor proteins that convert
chemical energy into mechanical work. Due to their fast mode
of action, small molecules are valuable tools to dissect the
dynamic functions of kinesins during mitosis. In this study, we
report the identification of optimized small molecule inhibitors
of the mitotic kinesin Kif18A. Using BTB-1, the first identified
Kif18A inhibitor, as a lead compound, we synthesized a
collection of derivatives. We demonstrate that some of the
synthesized derivatives potently inhibited the ATPase activity of Kif18A with a half maximal inhibitory concentration (IC50) value
in the low micromolar range. In vitro analysis of a panel of Kif18A-related kinesins revealed that the two most potent compounds
show improved selectivity compared to BTB-1. Structure−activity relationship studies identified substituents mediating
undesired inhibitory effects on microtubule polymerization. In summary, our study provides key insights into the mechanism of
action of BTB-1 and its analogs, which will have a great impact on the further development of highly selective and bioactive
Kif18A inhibitors. Since Kif18A is frequently overexpressed in solid tumors, such compounds are not only of great interest for
basic research but also have the potential to open up new strategies for the treatment of human diseases.
he genetic integrity of each organism is intimately tied to
the majority of chromosomes scattered throughout the
Tthe faithful segregation of its genome during mitosis. In spindle.4,8 Notably, multiple cancer types display elevated
Kif18A levels, suggesting that its function might be beneficial
for the survival and development of these cells.10−12 Previously,
we reported the identification of BTB-1 (1, Figure 1A), the first
small molecule inhibitor of Kif18A.13 1 reversibly inhibited the
microtubule-stimulated ATPase activity of Kif18A with a half
maximal inhibitory concentration (IC50) of 1.7 μM. Treatment
of HeLa-cells with 1 caused an increase in the percentage of
cells that were in mitosis indicative of 1 being bioactive in tissue
culture cells. However, in contrast to the depletion of Kif18A,
treatment with high concentrations of 1 did not result in
elongated spindles. On the basis of this phenotype, we
speculated that Kif18A might not be the relevant binding
partner of 1 in cells.
eukaryotes, this challenging task is mastered by the mitotic
spindle, a bipolar structure composed of microtubule
filaments.1 The organization of the complex network of
dynamic microtubules into the bipolar spindle shape and the
process of chromosome segregation depends on the coordi-
nated activities of kinesins.2 Kinesins are molecular motor
proteins that hydrolyze ATP and convert the released energy
into mechanical work. Depending on the molecular architec-
ture, kinesins typically perform the mechanical work in either of
the three following forms: they move towards the highly
dynamic plus end of microtubules, move towards the less
dynamic minus end, or modulate microtubule dynamics.3 The
mitotic kinesin Kif18A is unique in that it integrates plus-end
directed motility with the ability to affect microtubule
dynamics.4−9 Previous studies revealed that the function of
Kif18A is essential for the accurate architecture of the mitotic
spindle and proper alignment of chromosomes in mammalian
cells, i.e. cells depleted of Kif18A by RNA interference (RNAi)
display elongated spindles with hyperstable microtubules and
To better understand the mode of action of 1, we
synthesized a collection of 20 BTB-1 derivatives and analyzed
Received: October 1, 2014
Accepted: November 17, 2014
© XXXX American Chemical Society
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Figure 1. (A) Structure of BTB-1 (1). (B) Synthesis of BTB-1 analogs 2−21.
these compounds in respect of their inhibitory activity against
Kif18A, selectivity, and effect on cells. We identified novel
inhibitors of Kif18A with an IC50 value in the low micromolar
range. Structure−activity relationship (SAR) studies depicted
substituents mediating undesired side effects on microtubule
polymerization. Notably, one of the most potent Kif18A
inhibitors identified in this study had no detectable inhibitory
effect on microtubule polymerization. These insights into the
mechanism of action of 1 and its derivatives provide essential
guidelines for the future development and evaluation of highly
Finally, the sulfone linker was changed to an ether or sulfoxide
to analyze the effect of the geometry of the linker on the
inhibition of Kif18A. The main reaction (Figure 1B) utilized to
synthesize this series of BTB-1 derivatives was a nucleophilic
aromatic substitution (SNAr) under basic conditions to displace
activated chloride at the aromatic core by aromatic thiols or
thiolates as described for BTB-1.13−15 The resulting sulfides
were oxidized to the corresponding sulfons using H2O2 (Figure
1B, method A, Table 1) or to the racemic sulfoxides by meta-
chloroperoxybenzoic acid (mCPBA; Figure 1B, method B,
Table 1).
potent and bioactive Kif18A inhibitors.
Compound 2 was synthesized according to Moore et al. by
the AlCl3 mediated reaction of benzol and the corresponding
sulfonyl chloride in 46% yield (Figure 1B, method C).16 The
biphenyl ether 21 was synthesized using a SNAr under basic
conditions in DMSO in 96% yield (Figure 1B, method D).17 All
synthesized compounds were purified by column chromatog-
raphy and/or successive recrystallization. The chemical identity
of the molecules was confirmed by elementary and NMR
analysis validating the described synthesis routes as robust and
cost-efficient methods to assemble a small scale compound
collection in a fast manner. For detailed synthetic procedures,
see the Supporting Information.
Biochemical Evaluation of the Newly Synthesized
BTB-1 Analogs. Kinesins hydrolyze ATP to walk along
microtubules, and this activity can be easily quantified by an
enzyme coupled assay (ECA) where ATP hydrolysis results in
the oxidation of NADH and, thus, in a decrease in the
absorbance at 340 nm (Figure 2A).18 Using the recombinant
motor domain of Kif18A fused to a His-Tag (Kif18AMD), the
conditions of the ECA were adjusted to obtain 50% inhibition
RESULTS AND DISCUSSION
■
Design of BTB-1 Derivatives and Their Synthesis. In
order to get a better understanding of which structural motifs of
1 are necessary for its inhibitory activity against its target
Kif18A and to potentially find more effective inhibitors, a
subset of closely related analogs was synthesized (2−21). To
this end, the unsymmetrically substituted diarylsulphone 1 was
divided into three scaffolds: the two phenyl moieties I and II
and a linker between them (Figure 1A). At position R1, electron
withdrawing substituents like fluorine, trifluoromethyl, and
nitro groups were introduced to elucidate how altered
electronegativity and sterical demand at this position impact
the inhibitory activity and compound lipophilicity.
In addition, the requirement of the NO2 substituent in the
ortho position to the linker for the inhibitory activity was
analyzed by shifting it in meta position. The second phenyl ring
R2 was exchanged to thiophene, and the aromatic system was
expanded to naphthalene. Furthermore, various substituents
(Cl, MeO, Me) were introduced in para position to the linker.
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Figure 2B) showed an inhibition of greater than 25% at a
concentration of 5 μM, and these compounds were selected for
further analysis.
Table 1. BTB-1 Analogs and Yields
no.
R1 (scaffold I)
R2 (scaffold II)
Ph
method
yield
74%13
1
2
Cl
A
Next, the IC50 values of these five compounds were
determined using the ECA (Figure 2C, Supporting Informa-
tion). While all compounds showed a dose dependent
inhibition in the low micromolar range, none of them was
more potent than 1 (1.7 0.2 μM). 13 and 5 were the most
potent inhibitors with IC50 values of 3.0 0.2 μM and 4.8
0.4 μM, respectively, indicating that the replacement of the
halogene atom is tolerated. The introduction of more bulky
substituents like NO2 (3) or CF3 (6) as well as the exchange of
the phenyl moiety R2 to a thiophene (7) resulted in reduced
potency (IC50 values: (3) 10.2 2.0 μM, (6) 10.3 1.9 μM,
and (7) 6.4 0.9 μM).
1-chloro-2-nitro-4-(phenylsulfonyl)
benzene
C
46%
3
NO2
NO2
F
Ph
A
B
A
A
A
A
B
A
A
B
A
B
A
B
A
B
A
A
D
25%
38%
12%
26%
68%
6%
4
Ph
5
Ph
6
CF3
Cl
Ph
7
2-thiophene
2-naphthalene
2-naphthalene
4-methylbenzene
4-methoxybenzene
4-methoxybenzene
Ph
8
Cl
9
Cl
66%
7%
10
11
12
13
14
15
16
17
18
19
20
21
Cl
Cl
12%
72%
3%
Cl
ECA analysis of Kif18AMD activity in the absence of
microtubules revealed that 13 and 5like 1had no
inhibitory effect on the basal, microtubule-independent ATPase
activity of Kif18A (Figures S1 and S2). Furthermore, ATP
titration experiments indicate that 13 acts in an ATP-
competitive manner (Figure S3). Thus, these data suggest
that the identified derivativeslike 1 itself13are ATP-
competitive inhibitors that can only inhibit Kif18A when it is
bound to microtubules. To analyze compound selectivity, the
effects of the two most potent compounds 5 and 13 on the
ATPase activity of other kinesins highly related to Kif18A were
tested in vitro. To this end, ECA was performed in the presence
of 100 μM 5 or 13 and His-tagged motor domains of the
H
H
Ph
13%
5%
H
4-methoxybenzene
4-methoxybenzene
4-methylbenzene
4-methylbenzene
4-chlorobenzene
4-chlorobenzene
H
2%
NO2
NO2
Cl
18%
20%
55%
77%
96%
NO2
4-chloro-2-nitro-1-phenoxybenzene
at 5 μM 1 (Supporting Information). ATPase activity of
Kif18AMD in the presence of DMSO was normalized to 100%.
Of the tested 20 derivatives, five compounds (3, 5, 6, 7, and 13,
Figure 2. (A) Scheme of ECA used to quantify the ATPase activity of Kif18AMD. (B) Quantification of the inhibitory activity of BTB-1 (1) and
derivatives tested at 5 μM. Average of three independent experiments and standard deviations are shown. (C) Dose−response curves of 1, 3, 5, 6, 7,
and 13. Average of three independent experiments and standard deviations are shown. (D) Quantification of the inhibitory activity of 100 μM 1, 5,
and 13 on the ATPase activity of His-tagged motor-domain of the indicated mitotic kinesins. Average of three independent experiments and
standard deviations are shown. For further details, see the Supporting Information.
C
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mitotic kinesins Kif3A, Kif4A, Kif5A, Mklp1, Eg5, MPP1, or
MCAK. Compared to 1, 5 and 13 showed reduced inhibitory
activity towards Kif3A and Kif5A and comparable inhibitory
activity towards the remaining tested kinesins, suggesting that
the substitution of chloride with hydrogen and fluoride atoms
increased compound selectivity.
Cellular Evaluation. Due to their fast and often reversible
mode of action, small molecules are versatile tools to dissect
dynamic cellular processes. To analyze if the identified novel
Kif18A inhibitors are active in cells, we first analyzed the five
most potent compounds 3, 5, 6, 7, and 13 for their cytotoxicity
on HeLa cells.19 Determination of the half maximal effective
concentration (EC50 values) using an alamar blue assay (Figure
3, Supporting Information) revealed that compound 3 showed
Figure 4. (A) Assay scheme for cell synchronization and compound
treatment. First, cells were synchronized in S-Phase using thymidine.
Nine hours after the release, cells were treated with DMSO, 50 nM
Nocodazole (Noc), or 50 μM compounds 1, 5, 6, and 13 followed by
formalin fixation and immunostaining. (B) Representative immuno-
fluorescence images of cells treated with DMSO, 50 nM Nocodazole
(Noc), or 50 μM compounds 1, 5, 6, and 13. For each condition,
merged images with DNA (blue), GFP-Kif18A (green), and
microtubules (red) are shown on the left. GFP-Kif18A signal in gray
is shown on the right (scale bar = 10 μm).
Figure 3. Quantification of compound cytotoxicity at indicated
concentrations. Average of three independent experiments and
standard deviations are shown. For further details, see the Supporting
Information.
the highest cytotoxicity with 1.1 0.3 μM followed by 6 with
2.6 0.4 μM. 1 and 7 were less toxic with EC50 values of 35.8
9.0 μM and 23.1 7.3 μM, respectively. For 5, the lowest
cytotoxicity was observed with EC50 values above 50 μM, and
13 did not show a significant cytotoxicity at all.
of chromosomes were observed in HeLa cells treated with 50
μM 6 (Figure 4B). Since Kif18A depletion results in elongated
spindles with hyperstable microtubules,4,8 the observed
phenotypes tempted us to speculate that Kif18A is not the
relevant binding partner of 113 or 6 in cells.
Biochemical Evaluation on Tubulin Polymerization.
The spindle phenotype induced by 6 was reminiscent of the
phenotype caused by low doses of Nocodazole, a microtubule
destabilizing compound (Figure 4B). To analyze if 6 as well as
1, 3, 5, and 13 target microtubules, a turbidity-based in vitro
microtubule polymerization assay was performed (Figure 5A,
Supporting Information).21,22 Microtubule polymerization
efficiency in the presence of the solvent control DMSO was
normalized to 100%. Notably, at 50 μM, all compounds had an
inhibitory effect on microtubule polymerization, with 3 being
the most potent molecule (Figure 5B).
Initial immunofluorescence analysis revealed that derivative 3
caused unspecific cytotoxic effects that affected both dividing
and nondividing cells, and therefore, this compound was
excluded from further microscopic studies. To analyze in detail
the effect of 1 and derivatives 5, 6, and 13 on the architecture of
the mitotic spindle, we used a previously established HeLa cell
line that stably expresses green fluorescent protein (GFP)
tagged Kif18A.20 Cells synchronized in S-Phase were released
for 9 h and then treated with the solvent control DMSO or 50
μM of 1 or derivatives 5, 6, and 13 (Figure 4A, Supporting
Information). Thirty minutes after compound addition, cells
were chemically fixed, and tubulin (red) and DNA (blue)
structures were visualized by immunostaining and treatment
with the dye Hoechst 33342, respectively. Microscopic analysis
of mitotic spindles revealed that neither 5 nor 13 significantly
affected spindle structures or alignment of chromosomes
(Figure 4B). Furthermore, the accumulation of GFP-Kif18A
at the plus ends of microtubuleswhich as shown previously
depends on the motor activity of Kif18A5−7was not affected
by the treatment with 5 or 13. This effect together with the
observed normal spindle structures suggests that treatment with
derivative 5 or 13 does not result in efficient Kif18A inhibition
in cells. Consistent with previous reports,13 analysis of HeLa
cells treated with 50 μM 1 revealed severe defects in spindle
morphology and chromosome alignment (Figure 4B). Sim-
ilarly, multiple spindle poles, highly disorganized, and
fragmented microtubule structures and no detectable alignment
Compound titration experiments revealed an IC50 value of
1.2 0.1 μM for 3, indicating that the high cytotoxicity of the
compound might be due to its potent inhibitory effect on
microtubule polymerization (Figure 5C). 1 and 6 had moderate
effects on microtubule polymerization with IC50 values of 27.5
3.3 μM and 9.2
1.0 μM, respectively (Figure 5C). 13
inhibited microtubule polymerization with an IC50 value of
209.4 37.3 μM, and 5 did not show any significant effect on
the tubulin polymerization up to a concentration of 1 mM
(Figure 5C). Thus, these studies revealed that the introduction
of bulky substituents in phenyl moiety I at the para position to
the linker drastically increased the undesired inhibitory effect
on microtubule polymerization.
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Figure 5. (A) Schematic representation of microtubule polymerization. (B) Quantification of the effect of 50 μM 1, 3, 5, 6, and 13 on microtubule
polymerization. (C) Dose−response curves of 1, 3, 6, and 13 on microtubule polymerization in vitro. Derivative 5 did not show an effect on the
tubulin polymerization up to 1 mM. For B and C, an average of three independent experiments and standard deviations are shown. For detailed
information, see the Supporting Information.
While we cannot formally exclude the possibility that 5 and 13
are not membrane permeable, the fact that the structurally
related compounds 1 and 6 induced cellular effects argues
against this idea. Possibly, post-translational modifications of
Kif18A in cells or its association with binding partners render
Kif18A resistant to the action of 5 and 13. Notably, 1 and 6
caused a cellular phenotype reminiscent of low doses of
Nocodazole and in vitro studies confirmed that these
compounds potently inhibited microtubule polymerization.
Since microtubule dynamics affect the accumulation of
Kif18A to the plus ends of microtubules,7 it is difficult to
determine if altered Kif18A localization in cells treated with 1
or 6 is a direct effect of Kif18A inhibition or indirectly caused
by the undesired effect on microtubule dynamics. Furthermore,
our in vitro microtubule polymerization assays revealed that the
high cytotoxicity of 3 is likely to be mediated by its severe effect
on microtubule polymerization. These data suggest that the
introduction of sterically demanding electron withdrawing
groups at R1 like NO2 (3) or CF3 (6) shift the inhibitory
activity towards microtubules, whereas small substituents like
hydrogen (13) or fluoride (5) influence the activity in favor of
Kif18A inhibition (Figure 6B).
DISCUSSION AND CONCLUSION
■
Using BTB-1 as a lead compound, we synthesized derivatives
2−21 in one or two steps starting from commercially available
building blocks. The described synthesis route allowed the
robust and cost-efficient assembly of a small scale compound
collection. IC50 studies identified several BTB-1 derivatives that
potently inhibited the ATPase activity of Kif18A in the low
micromolar range.
SAR studies of identically substituted sulfone (3 and 13) and
sulfoxide (4 and 14) derivatives highlight the importance of the
sulfone functionality for the inhibitory activity of the
compounds against Kif18A. Consistently, replacing the sulfone
linker of 1 with an ether linkage (21) resulted in an almost
complete loss of inhibitory activity (Figure 6A). Further SAR
studies revealed that the introduction of either electron
withdrawing (19 and 20) or donating (10−12, 15−18)
substituents in the para position to the linker at phenyl moiety
II was not tolerated. Similarly, expanding the sterical demand of
phenyl moiety II by replacing it with naphthalene (8) resulted
in a complete loss of activity (Figure 6A). Within phenyl
moiety I, shifting the NO2 group from the ortho (1) to meta (2)
position to the sulfone linker caused compound inactivity, while
replacing chloride (1) at R1 with hydrogen (13) or different
electron withdrawing groups like fluoride (5), NO2 (3), or CF3
(6) had no drastic effect on compound activity (Figure 6A).
Unfortunately, the most potent derivatives 5 and 13 did not
interfere with the localization of Kif18A to the plus ends of
microtubules in mitotic cells, suggesting that treatment with
these derivatives did not result in efficient Kif18A inhibition.
In summary, we reported herein the design, synthesis, and
biological evaluation of a BTB-1 analog library. We identified
several novel derivatives that potently inhibited the ATPase
activity of Kif18A. Most importantly, our SAR analysis enabled
us to separate the undesired effect on microtubule polymer-
ization from the aimed inhibition of Kif18A. These insights
provide an important guideline for the future design and
synthesis of specific and bioactive Kif18A inhibitors. Such
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German Research Foundation (DFG) and scientific support
by the Konstanz Research School Chemical Biology (KoRS-
CB). We thank the Bioimaging Center (BIC) and NMR facility
of the University of Konstanz for technical support.
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METHODS
■
Full experimental details are given in the Supporting Information.
ASSOCIATED CONTENT
* Supporting Information
Detailed synthetic procedures of all compounds and biological
assays are available. This material is available free of charge via
the Internet at http://pubs.acs.org.
■
S
AUTHOR INFORMATION
Corresponding Author
*E-mail: Thomas.U.Mayer@uni-konstanz.de.
■
Author Contributions
§These authors contributed equally.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
We gratefully acknowledge funding by the SFB 969 “Chemical
and Biological Principles of Cellular Proteostasis” of the
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