Structure Optimization of Gatastatin for the Development of γ-Tubulin-Specific Inhibitor

Article abstract of DOI:10.1021/acsmedchemlett.9b00526

Gatastatin (O7-benzyl glaziovianin A) is a γ-tubulin-specific inhibitor that is used to investigate γ-tubulin function in cells. We have previously reported that the unsubstituted phenyl ring of the O7-benzyl group in gatastatin is important for γ-tubulin

Full text of DOI:10.1021/acsmedchemlett.9b00526

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Structure Optimization of Gatastatin for the Development of
γ‑Tubulin-Specific Inhibitor
Kana Shintani,‡ Haruna Ebisu,‡ Minagi Mukaiyama, Taisei Hatanaka, Takumi Chinen, Daisuke Takao,
Yoko Nagumo, Akira Sakakura, Ichiro Hayakawa,* and Takeo Usui*
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ABSTRACT: Gatastatin (O7-benzyl glaziovianin A) is a γ-tubulin-specific
inhibitor that is used to investigate γ-tubulin function in cells. We have
previously reported that the unsubstituted phenyl ring of the O7-benzyl group
in gatastatin is important for γ-tubulin inhibition. To obtain further structural
information regarding γ-tubulin inhibition, we synthesized several gatastatin
derivatives containing a fixed O7-benzyl moiety. Modifications of the B-ring
resulted in drastic decrease in cytotoxicity, abnormal spindle formation
activity, and inhibition of microtubule (MT) nucleation. In contrast, various
O6-alkylated gatastatin derivatives showed potent cytotoxicity, induced
abnormal spindle formation, and inhibited MT nucleation. We had previously
reported that O6-benzyl glaziovianin A is a potent α/β-tubulin inhibitor; thus, these new results suggest that the O6-position restricts
affinity for α/β- and γ-tubulin. Considering that an O7-benzyl group increases specificity for γ-tubulin, more potent and specific γ-
tubulin inhibitors can be generated through O6-modifications of gatastatin.
KEYWORDS: γ-Tubulin, structure−activity relationships, microtubule nucleation, spindle
progression of anaphase.11 The cytotoxicity of gatastatin (1) is
icrotubules (MTs) are dynamic polymers consisting of α-
relatively weak compared to that of conventional MT inhibitors,
Mand β-tubulin heterodimers that are involved in cell
motility, vesicle trafficking, and chromosome segregation.1,2 In
for example paclitaxel and vinblastine, although γ-tubulin is
essential for cell viability. It is therefore important to identify
more potent γ-tubulin inhibitors.
cells, polymerization of MTs starts from the MT nucleator γ-
tubulin ring complex (γTuRC).3−5 γ-Tubulin is an essential
component of γTuRC; however, the molecular mechanisms of
MT nucleation and spatiotemporal regulation of γTuRC still
remain to be revealed. One approach for revealing such
mechanisms is the application of γ-tubulin-specific inhibitors.6
In addition, γ-tubulin is an attractive target molecule for both
basic biological studies and antitumor medicines, because γ-
tubulin is activated during mitosis7,8 and is overexpressed in
certain types of glioblastomas and nonsmall cell lung cancers.9,10
Using this approach, we screened γ-tubulin-specific inhibitors
and identified gatastatin (Figure 1, 1),11 which is a derivative of
the MT dynamics inhibitor glaziovianin A (AG1) (Figure 1,
2).12,13 Gatastatin (1) has a higher affinity for γ-tubulin than
toward α- and β-tubulin heterodimers and inhibits the
Previously, we focused on the O7-benzyl group in gatastatin
(1) in a structure−activity relationship study and systematically
optimized the aromatic ring at the O7-benzyl group in
accordance with an operational Topliss tree.14,15 Some
derivatives showed stronger cytotoxicity in HeLa cells than
gatastatin (1), but the cytotoxicity of these derivatives was
independent of their inhibition of α/β- and γ-tubulin, suggesting
strong recognition of the O7-benzyl group by γ-tubulin. We also
revealed that B-ring-modified AG1 does not inhibit α/β-tubulin
polymerization activity13 and that benzylation of the O6-position
of AG1 increases cytotoxicity in HeLa cells.16 We thus assume
that modifications of the B-ring portion and O6-position increase
specificity for γ-tubulin (Figure 2). Consequently, we prepared
B-ring-modified gatastatin derivatives (3−5), O6-modified
gatastatin derivatives (6−10), and O6-modified 3′,4′-acetonide
gatastatin derivatives (11−15) (Figure 3).17,18
Received: November 17, 2019
Accepted: March 30, 2020
Published: March 30, 2020
Figure 1. Structures of gatastatin (1) and glaziovianin A (2).
https://dx.doi.org/10.1021/acsmedchemlett.9b00526
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Table 1. Cytotoxicity of Gatastatin Derivatives in HeLa
a
Cells
Compound
IC50 (μM)
Growth (%) at 100 μM
b
Gatastatin (1)
3
4
5
6
7
8
9
10
11
12
13
14
15
10.5 2.5
>100
>100
>100
>100
N.T.
100.9 11.4
77.3 13.3
100.4 7.1
99.1 3.1
N.T.
5.8 0.4
9.9 0.9
0.8 0.2
1.1 0.2
>100
N.T.
N.T.
N.T.
Figure 2. Summary of results from our previous structure−activity
relationship study of gatastatin (1) and AG1 (2).
100.8 7.9
N.T.
107.4 7.9
102.5 7.7
101.9 5.6
c
>13
>100
>100
>100
We first evaluated HeLa cell growth inhibition by gatastatin
derivatives 3−15 (Table 1). All B-ring-modified derivatives (3−
5) showed drastically reduced cytotoxicity, with IC50 values over
100 μM. Only derivative 4 showed weak cytotoxicity at 100 μM.
In contrast, O6-modified gatastatin derivatives (7−10) showed
more potent cytotoxicity than gatastatin (1). This potent
cytotoxicity was abolished by increasing the steric hindrance of
C3′ and C4’ in the B-ring portion (derivatives 13−15). This
result is similar to results of the structure−cytotoxicity
relationship study of AG1.19,20
Next, we investigated the effects of the derivatives on MT
polymerization in vitro (Figure 4) and in cells (Figure 5). For in
vitro MT polymerization, a polymerization inducer, such as 1 M
glutamate, is required for efficient MT polymerization. In these
experiments, we used 0.8 M glutamate because the high
polymerization activity in the case of 1 M glutamate prevents
examination of the inhibitory effects of derivatives (Figure 4A).
Under these conditions, the percentages of polymerized MTs
with vehicle control and colchicine, a MT polymerization
inhibitor, were 84.8 9.9% and 43.0 18.0%, respectively. MT
polymerization in the presence of 30 μM gatastatin derivative
ranged from 69−92%, with no significant differences compared
to the vehicle control.
a
HeLa cells were treated with various concentrations of each
derivative for 48 h. Cell growth was determined using a WST-8 cell
counting kit (Dojindo), and the resulting IC50 values were calculated.
b
c
Not tested. Derivative 12 precipitated at concentrations above 13
μM.
results suggest that all derivatives have minimal effects on MT
polymerization.
This conclusion is supported by MT network structures
observed in interphase HeLa cells (Figure 5). MT networks in
cells treated with 100 nM colchicine or paclitaxel were
depolymerized or bundled, respectively. In contrast, networks
treated with 30 μM of a derivative were identical to those in
DMSO-treated cells. Together with the results of in vitro
polymerization assays, we conclude that all derivatives have little
impact on α/β-tubulin heterodimers.
We next investigated the effects of derivatives on spindle
morphology in mitotic cells, as γ-tubulin activity is required for
bipolar spindle formation during mitosis. As shown in Figure 6A,
most DMSO-treated cells show normal bipolar spindles;
however, gatastatin (1) induces misaligned chromosomes and
multipole spindles, as previously reported.11 At 30 μM, most B-
ring-modified derivatives (3, 5, 11−15) did not induce
abnormal spindles. To our surprise, 3′,4′-dimethoxy gatastatin
We also examined MT polymerization in the presence of 0.2
M glutamate (Figure 4B), in which MT polymerization is not
stimulated without addition of an assembly inducer like
paclitaxel. Polymerized MT levels (%) in the presence of vehicle
control and paclitaxel were 36.3
8.1% and 91.6
5.9%,
(4) induced misaligned chromosomes (65
2.3%) despite
respectively, while the addition of 30 μM gatastatin derivative
led to polymerization levels of 31−41%, with no significant
difference from with vehicle control. Taken together, these
showing only weak cytotoxicity (Table 1, Figures 6A and 6B).
These results suggest that while the B-ring portion is important
for γ-tubulin interaction, derivative 4 stills interacts with γ-
Figure 3. Structures of B-ring-modified gatastatin derivatives (3−5), O6-modified gatastatin derivatives (6−10), and O6-modified 3′,4′-acetonide
gatastatin derivatives (11−15).
B
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Figure 4. Effects of gatastatin derivatives on MT polymerization in vitro. Porcine brain tubulin (1 mg/mL) was incubated with 10 μM colchicine, 10
μM paclitaxel, and 30 μM gatastatin derivative in the presence of either (A) 0.8 M or (B) 0.2 M glutamate for 30 min at 37 °C. Polymerized and
unpolymerized tubulin were separated by ultracentrifugation (200,000g, 10 min, 25 °C). Samples were then separated using SDS-PAGE using a 10%
gel and the amount of tubulin quantified by ImageJ. Values represent mean SD of three independent experiments.
dependent-induction of multipolar spindle formation (Figure
6C), suggesting that chromosome misalignment leads to enough
cytotoxicity and multipolar spindle formation may reflect the
degree of γ-tubulin inhibition. In contrast, O6-benzyl gatastatin
(10) induced formation of abnormal spindles containing
misaligned chromosomes in a dose-independent manner.
These results suggest that derivative 10 may be efficiently
excluded from cells compared to derivative 9 and that the
concentration of derivative 10 inside cells remains constant,
although further investigation of this possibility is required.
Taken together, these results suggest that modification of the
O6-position potentiates interaction with γ-tubulin.
We have previously reported that 30 μM gatastatin (1)
inhibits γ-tubulin-dependent MT nucleation of centrosomes in
human cells without affecting γ-tubulin localization.11 We
therefore evaluated the inhibition of MT nucleation in mitotic
cells by gatastatin derivatives. Gatastatin (1) significantly
inhibits MT nucleation from centrosomes in mitotic cells at
30 μM but not at 10 μM (Figure 7A). B-ring-modified
derivatives 3 and 5 and O6-tetrahydropyranyl gatastatin (6)
show no inhibitory activity, even at 100 μM (Supporting
Information Figure 1). 3′,4′-Dimethoxy gatastatin (4) inhibits
MT nucleation at 30 μM but not at 10 μM, indicating that the
inhibitory activity of 4 is similar to that of gatastatin (1)
(Supporting Information Figure 2). In contrast, O6-modified
gatastatin derivatives 7−10 inhibited the MT nucleation at 30
μM (Supporting Information Figure 3A). Among them,
derivative 9 showed the most potent activity and inhibited
Figure 5. None of the tested gatastatin derivatives affected the MT
MT nucleation at 0.3 μM (Figure 7B). Furthermore, we
confirmed derivative 9 inhibited GTP-binding on γ-tubulin in
vitro more potently than gatastatin (Supporting Information
Figure 4). Taken together with the results from in vitro MT
polymerization (Figure 4) and MT network in interphase cells
(Figure 5), these suggest that derivatives 7−10 are potent
inhibitors of γ-tubulin-dependent MT nucleation of centro-
somes.
In this study, we investigated the structure−activity relation-
ships of gatastatin (1). B-ring modification drastically decreases
cytotoxicity of gatastatin, although 3′,4′-dimethoxy gatastatin
(4) still possesses weak cytotoxicity (Table 1) and induces
abnormal spindle structure (Figures 6A and 6B). In contrast,
some modifications of the O6-position in gatastatin (1) produce
derivatives that greatly increase cytotoxicity (Table 1), induce
abnormal spindle formation (Figures 6A and 6B), and inhibit γ-
tubulin-dependent nucleation (Supporting Information Figure
3). In particular, our results suggest that O6-propargyl gatastatin
networks in interphase cells. Cells were treated with 100 nM colchicine
(Col) and paclitaxel (Tax) for 6 h. Cells were treated with 30 μM
gatastatin (1) and gatastatin derivatives (3−15) for 24 h. Scale bar = 25
μm.
tubulin. Furthermore, these results indicate that derivative 4 can
prevent proper spindle formation but is not enough to
constitutively activate the spindle checkpoint. Alternatively,
derivative 4 may be relatively less stable in cells, thus decreasing
its inhibitory activity. Further investigation is required to resolve
this controversy.
In contrast to B-ring-modified derivatives, all O6-modified
derivatives except O6-tetrahydropyran gatastatin (6) induced
abnormal spindles at 30 μM (Figure 6A). We then chose to
focus on derivatives 9 and 10, since these derivatives show
potent cytotoxicity (Table 1). As shown in Figure 6C, both
derivatives induced abnormal spindles at concentrations as low
as 1 μM. O6-Propargyl gatastatin (9) showed dramatic dose-
C
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Figure 6. Gatastatin derivatives (4, 7−10) induce abnormal spindle formation. (A) Effects of gatastatin derivatives on spindle formation in cells.
Exponentially growing HeLa cells are treated with 30 μM of each for 24 h and the resulting spindle morphology is classified. Values represent mean
SD of three independent experiments. Greater than 100 cells are examined for each experiment. (B) Spindle morphology of cells treated with 30 μM
gatastatin (1) or a gatastatin derivative (3−15) for 24 h. Abnormal bipolar spindles with misaligned chromosomes are observed in cells treated with
gatastatin (1) and derivatives 4 and 7−10. Red arrows indicate misaligned chromosomes. Scale bar = 10 μm. (C) Compound 9, but not 10, dose-
dependently induces multipolar spindle formation.
Figure 7. Derivative 9 inhibits MT nucleation from mitotic centrioles more potently than gatastatin. The MT areas nucleated from centrosomes (n =
20−25) were measured. (A) Gatastatin (1) inhibits MT nucleation at 30 μM. The MT polymerization inhibitor colchicine (Col) is the positive
control. (B) Derivative 9 inhibited MT nucleation at concentrations above 0.3 μM. We have used GraphPad Prism 6 (GraphPad Software, San Diego,
CA) to perform ANOVA. ****, p < 0.0001; **, p < 0.01; ns, no significance.
(derivative 9) is a more potent γ-tubulin-specific inhibitor than
gatastatin (1); therefore, we named this compound as gatastatin
G2.21 This compound would be a useful tool for investigating γ-
tubulin function in cells.
Experimental details for synthesis and analysis of
compounds 3−15; conditions for biological assays
(PDF)
AUTHOR INFORMATION
Corresponding Authors
■
ASSOCIATED CONTENT
Ichiro Hayakawa − Division of Applied Chemistry, Graduate
School of Natural Science and Technology, Okayama University,
Okayama 700-8530, Japan; orcid.org/0000-0003-2749-
9078; Phone: +81 3 5317 9363; Email: hayakawa.ichirou@
nihon-u.ac.jp
■
sı
* Supporting Information
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acsmedchemlett.9b00526.
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Takeo Usui − Faculty of Life and Environmental Sciences and
Microbiology Research Center for Sustainability, University of
Tsukuba, Tsukuba 305-8571, Japan; orcid.org/0000-0003-
2377-7499; Phone: +81 29 853 6629; Email: usui.takeo.kb@
u.tsukuba.ac.jp
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Kana Shintani − Graduate School, University of Tsukuba,
Tsukuba 305-8571, Japan
Haruna Ebisu − Graduate School, University of Tsukuba,
Tsukuba 305-8571, Japan
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Tsukuba 305-8571, Japan
Taisei Hatanaka − Division of Applied Chemistry, Graduate
School of Natural Science and Technology, Okayama University,
Okayama 700-8530, Japan
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A.; Giordano, A.; Draber, P.; Katsetos, C. D. Overexpression of γ-
tubulin in non-small cell lung cancer. Histol Histopathol 2012, 27,
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Takumi Chinen − Graduate School of Pharmaceutical Sciences,
The University of Tokyo, Bunkyo, Tokyo 113-0033, Japan
Daisuke Takao − Graduate School of Pharmaceutical Sciences,
The University of Tokyo, Bunkyo, Tokyo 113-0033, Japan
Yoko Nagumo − Faculty of Life and Environmental Sciences,
University of Tsukuba, Tsukuba 305-8571, Japan
Akira Sakakura − Division of Applied Chemistry, Graduate School
of Natural Science and Technology, Okayama University,
Okayama 700-8530, Japan; orcid.org/0000-0002-2995-
1251
(11) Chinen, T.; Liu, P.; Shioda, S.; Pagel, J.; Cerikan, B.; Lin, T.;
Gruss, O.; Hayashi, Y.; Takeno, H.; Shima, T.; Okada, T.; Hayakawa, I.;
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Hayashi, Y.; Kigoshi, H.; Usui, T.; Schiebel, E. The γ-tubulin specific
inhibitor gatastatin reveals temporal requirements of microtubule
nucleation during the cell cycle. Nat. Commun. 2015, 6, 8722.
(12) Yokosuka, A.; Haraguchi, M.; Usui, T.; Kazami, S.; Osada, H.;
Yamori, T.; Mimaki, Y. Glaziovianin A, a new isoflavone, from the leaves
of Ateleia glazioviana and its cytotoxic activity against human cancer
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(13) Chinen, T.; Kazami, S.; Nagumo, Y.; Hayakawa, I.; Ikedo, A.;
Takagi, M.; Yokosuka, A.; Imamoto, N.; Mimaki, Y.; Kigoshi, H.;
Osada, H.; Usui, T. Glaziovianin A Prevents Endosome Maturation via
Inhibiting Microtubule Dynamics. ACS Chem. Biol. 2013, 8, 884−889.
(14) Hayakawa, I.; Shioda, S.; Chinen, T.; Usui, T.; Kigoshi, H.
Structure−Activity Relationship Study of Gatastatin Based on the
Topliss Tree Approach. Heterocycles 2019, 99, 238−247.
Complete contact information is available at:
https://pubs.acs.org/10.1021/acsmedchemlett.9b00526
Author Contributions
‡K.S. and H.E. contributed equally.
(15) Topliss, J. G. Utilization of operational schemes for analog
synthesis in drug design. J. Med. Chem. 1972, 15, 1006−1011.
(16) Hayakawa, I.; Shioda, S.; Chinen, T.; Hatanaka, T.; Ebisu, H.;
Sakakura, A.; Usui, T.; Kigoshi, H. Discovery of O6-benzyl glaziovianin
A, a potent cytotoxic substance and a potent inhibitor of α,β-tubulin
polymerization. Bioorg. Med. Chem. 2016, 24, 5639−5645.
(17) See the Supporting Information.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
This work was supported by JSPS KAKENHI Grant Numbers
JP16K07710 and JP17K01949. I.H. thanks the Okayama
Foundation for Science and Technology for financial support.
The authors gratefully thank the Division of Instrumental
Analysis, Depaertment of Instrument Analysis & Cryogenics,
Advanced Science Research Center, Okayama University, for
the HR-ESIMS measurements.
(18) In this paper, we name O6-demethyl-O6-“functional group”
gatastatin derivatives as O6-“functional group” gatastatins, respectively,
for simplification.
(19) Ikedo, A.; Hayakawa, I.; Usui, T.; Kazami, S.; Osada, H.; Kigoshi,
H. Structure−activity relationship study of glaziovianin A against cell
cycle progression and spindle formation of HeLa S3 cells. Bioorg. Med.
Chem. Lett. 2010, 20, 5402−5404.
(20) Hayakawa, I.; Ikedo, A.; Chinen, T.; Usui, T.; Kigoshi, H. Design,
synthesis, and biological evaluation of the analogues of glaziovianin A, a
potent antitumor isoflavone. Bioorg. Med. Chem. 2012, 20, 5745−5756.
(21) Gatastatin G2 (second generation).
ABBREVIATIONS
■
AG1, glaziovianin A; MT, microtubule; γTuRC, γ-tubulin ring
complex; THP, tetrahydropyran
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