Interactions of the antitumor macrolide aplyronine a with actin and actin-related proteins established by its versatile photoaffinity derivatives

Article abstract of DOI:10.1021/ja310495p

The antitumor and apoptogenic macrolide aplyronine A (ApA) is a potent actin-depolymerizing agent. We developed an ApA acetylene analog that bears the aryldiazirine group at the C34 terminus, which formed a covalent bond with actin. With the use of the ph

Full text of DOI:10.1021/ja310495p

Communication
pubs.acs.org/JACS
Interactions of the Antitumor Macrolide Aplyronine A with Actin and
Actin-Related Proteins Established by Its Versatile Photoaffinity
Derivatives
Masaki Kita,* Yuichiro Hirayama, Kota Yamagishi, Kozo Yoneda, Ryosuke Fujisawa, and Hideo Kigoshi*
Graduate School of Pure and Applied Sciences, University of Tsukuba, 1-1-1 Tennodai, Tsukuba, 305-8571, Japan
S
* Supporting Information
Recent progress in chemical biology research has accelerated
ABSTRACT: The antitumor and apoptogenic macrolide
aplyronine A (ApA) is a potent actin-depolymerizing
agent. We developed an ApA acetylene analog that bears
the aryldiazirine group at the C34 terminus, which formed
a covalent bond with actin. With the use of the
photoaffinity biotin derivatives of aplyronines A and C,
Arp2 and Arp3 (actin-related proteins) were specifically
purified as binding proteins along with actin from tumor
cell lysate. However, Arp2 and Arp3 did not covalently
bind to aplyronine photoaffinity derivatives. Thus, actin-
related proteins might indirectly bind to ApA as the
ternary adducts of the actin/ApA complex or through the
oligomeric actin.
identification of the targets of bioactive natural products as well
as observations of their dynamic behaviors in living systems.8 In
addition, photoaffinity labeling combined with mass spectro-
metric approaches has been developed as a useful proteomics
tool.9 To develop novel actin-targeting antitumor agents, we
performed chemical and biological studies on ApA and related
actin-targeting macrolides. With the use of fluorescent
derivatives of aplyronines, we have shown that 1 causes rapid
disassembly of the actin cytoskeleton and the dephosphor-
ylation of focal adhesion kinase in tumor cells with caspase-
dependent apoptosis.7 Furthermore, with the use of biotiny-
lated ApA analogs, we recently identified actin-related proteins
2/3 (Arp2 and Arp3) as presumed targets of 1.10 Arp2 and
Arp3 constitute the Arp2/3 complex, which binds to the sides
of an existing actin filament and initiates growth of a new actin
filament to form branched actin filament networks.11,12 Due to
the high structural similarity between G-actin and actin-related
proteins, especially around the hydrophobic clefts between
subdomains 1 and 3 (ApA-binding site of actin), we expected
that 1 might bind to Arp2 or Arp3 to give 1:1 complexes and
inhibit the ability of the Arp2/3 complex. These properties
might enhance the potent disassembly of actin filaments caused
by 1. However, the detailed molecular mechanism and binding
modes of 1 with actin and actin-related proteins are still unclear
due to their polymerization properties and instability as a
complex in vitro. We prepared aplyronine photoaffinity
derivatives that can covalently bind to targets and investigated
the binding modes of 1 to these proteins as well as its
mechanisms of action.
plyronine A (ApA, 1), a 24-membered macrolide isolated
A
from the sea hare Aplysia kurodai, exhibits remarkable
antitumor activities in vivo against P388 murine leukemia cells
(T/C 545%, 0.08 mg/kg) and several cancers.1 The primary
target of 1 was found to be actin, a cytoskeleton protein that is
essential for various biological functions, such as muscle
contraction, cell motility, and cell division.2 In fact, ApA
potently depolymerizes fibrous actin (F-actin) and inhibits the
polymerization of actin by forming a 1:1 complex with the
monomeric globular molecule (G-actin, Kd = 100 nM).3 Studies
on structure−activity relationships,4 an X-ray analysis of the
actin−ApA complex,5 and photoaffinity labeling experiments6
have established that the tail part (C24/C34) of 1 is important
for its actin-binding property. Meanwhile, aplyronine C (ApC,
2), an extremely minor congener of 1 in A. kurodai that lacks
the C7 trimethylserine moiety, shows significant actin-
depolymerizing activity and is highly accumulated in the
cytoplasm, similar to 1 but is much less cytotoxic than 1 (∼1/
1000).1a,d,7 Interestingly, this C7 ester moiety of 1 protrudes
toward the bulk solvent region in the actin−ApA complex.5
Thus, the significant antitumor activities of 1 may not be due
solely to its F-actin-severing properties and instead may involve
its interactions with protein(s) other than actin.
Based on the finding that the C34 N-formyl enamide moiety
of 1 can be replaced with oximes without a significant loss of
activity,7a we introduce here an aryltrifluoromethyldiazirine13
(photoreacting) group via L-lysine (L-Lys) linkers (Figure 1).
To detect photolabeled proteins, we use fluorescent labeling
with azide/acetylene 1,3-dipolar cyclization (Huisgen reaction)
̈
after the photoreactions as well as affinity purification and the
immunoblotting detection of biotin groups using resin-bound
or enzyme-conjugated avidins. Thus, we designed an ApA
photoaffinity acetylene analog (ApA/PA, 4) and an ApA
photoaffinity PEG-linked biotin analog (ApA/PB, 5). To
compare, we also synthesized a tolyl analog 3, which lacks
Received: October 24, 2012
© XXXX American Chemical Society
A
dx.doi.org/10.1021/ja310495p | J. Am. Chem. Soc. XXXX, XXX, XXX−XXX

Journal of the American Chemical Society
Communication
they were immediately used for the next coupling reactions
with aldehydes.
With diazirine reagents 14 and 19 in hand, we synthesized
photoaffinity derivatives of aplyronines (Scheme 2). Acidic
Scheme 2. Preparation of Aplyronine Photoaffinity
Derivatives
Figure 1. Structures of PA and PB derivatives of aplyronines.
Photoreacting (aryl diazirine) and detecting (acetylene or biotin)
groups are highlighted in pink and green, respectively.
the diazirine moiety of 4, and an ApC photoaffinity biotin
analog (ApC/PB, 6).
hydrolysis of 1 afforded the C34 aldehyde 20,10 which was
condensed with alkoxyamine 13 in EtOH to give the tolyl
analog 3 (36%). With the addition of acetate buffer (5 mM),
however, the coupling reactions of aldehyde 20 with alkoxy-
amines 14 or 19 were accelerated to provide 4 (71%) and 5
(69%), respectively. Due to the scarcity of 2 from natural
sources (e.g., 3 × 10−7% yield based on wet wt),1a we examined
the conversion from ApA (1). After several attempts, we found
that methanolysis of 1 with 0.1% triethylamine preferentially
cleaved the trimethylserine ester to give 2 in 44% yield (with
42% of recovered 1) (Figure S1). Subsequent acidic hydrolysis
of 2 followed by condensation with alkoxyamine 19 gave 6
(47%).
Biological activities of aplyronine analogs were compared
with those of natural compounds (Table 1). The ApA analogs
3−5 all showed potent cytotoxicities against HeLa S3 cells
(IC50 0.16−1.2 nM), whereas the ApC analog 6 was ∼260
times less cytotoxic than 5. By using pyrene-labeled (pyrenyl)
actin, we established that ApA tolyl analog 3 significantly
depolymerized F-actin in vitro (EC50 2.0 μM against 3 μM
actin), as with 1 (EC50 1.3 μM) (Figure S2). Also, 5 potently
Scheme 1 summarizes the synthesis of the two diazirine-
containing alkoxyamines 14 and 19. Condensation of the
carboxylic acid 714 derived from L-Lys with propargylamine
using EDC and HOBt gave amide 8 (71%). Acidic cleavage of
the Boc group in 8 with TFA followed by coupling with the
succinyl ester 915 (R = Me) in the presence of NMM and
DMAP yielded tolyl amide 11 in a moderate yield (49%).
Similarly, aryldiazirine amide 12 was prepared from 8 and
succinyl ester 1016 (R = C(NN)CF3) (48%). Fmoc
deprotection in 11 and 12 with piperidine in DMF afforded
alkoxyamines 13 and 14 in quantitative yield, respectively, while
phthalimide (Phth)-protected carboxylic acid 1517 was
condensed with the primary amine 1618 bearing a PEG-linked
biotin by using EDC and HOAt to give amide 17 (74%).
Subsequent acidic cleavage of the Boc group in 17 with TFA
followed by coupling with compound 10 in the presence of
DIPEA yielded aryldiazirine amide 18 (82%). Finally,
deprotection of the Phth group in 18 with hydrazine
monohydrate quantitatively yielded alkoxyamine 19. Since all
three of the neutral alkoxyamines 13, 14, and 19 were unstable,
Scheme 1. Synthesis of the Diazirine-Containing Alkoxyamines 14 and 19
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Journal of the American Chemical Society
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Table 1. Biological Activities of ApA and Its Derivatives
cytotoxicity (HeLa S3) IC50
(nM)
actin-depolymerizing activity EC50
a
(μM)
1
2
3
4
5
6
0.010
17
1.3
1.4
2.0
0.16
0.76
1.2
b
−
bc
,
−
Figure 2. Photolabeling experiments for actin with 4. After actin (10
μM) was incubated with or without 4 (20 μM) in DMSO/G-buffer
(1:9), samples were irradiated with UV365, TAMRA azide, sodium
ascorbate, CuSO4, and TBTA were added, and the mixture was
incubated for the times shown at the bottom. Labeled actin was
detected by TAMRA fluorescence and CBB (Coomassie brilliant blue)
stain. The control (lane 9) consisted of actin without UV irradiation or
fluorescence labeling.
b
310
−
a
Activity was monitored by measuring the fluorescence intensity of
pyrenyl actin. Values indicate the concentrations required to
depolymerize F-actin (3 μM) to 50% of its control amplitude. Not
examined. Confirmed by ultracentrifugation.
b
c
inhibited F-actin sedimentation, which was confirmed by
ultracentrifugation (Figure S3).7a
derivatives. Upon photoreaction with 5 followed by affinity
purification using NeutrAvidin agarose, three strong bands
corresponding to β-actin, Arp2, and Arp3 (43, 40, and 47 kDa,
respectively) were detected with silver stain as well as with anti-
Arp2 and Arp3 (Figure 3a,b, lane 1). Binding of these three
To develop effective actin labeling and detection methods,
we next examined the model Huisgen reactions using ApA/
tolyl analog 3 (Scheme 3). While 1,3-dipolar cycloaddition
̈
Scheme 3. Fluorescent Labeling Experiments for Alkyne 3 in
an Aqueous Phase
Figure 3. Purification of ApA-binding proteins in tumor cells. Target
proteins in HeLa S3 cell lysate were photolabeled with the 5 or 6
preloaded on the NeutrAvidin agarose in the absence or presence of
excess 1. (a) Resulting biotin-bound proteins were eluted by boiling in
SDS buffer and detected with silver stain. (b,c) Immunoblotting
analysis. Proteins in (a) were detected with (b) anti-Arp2/Arp3 (1:1
mix) or (c) streptavidin-HRP conjugate. (d,e) Stepwise elution.
Binding proteins on the resin were eluted by treatment with 1 (10 μM,
lanes 1−4) followed by boiling in SDS buffer (lanes 5−8) and
detected with (d) silver stain or (e) anti-Arp2/Arp3. Arrowheads in
(a,d) indicate Arp2 and Arp3.
between the alkyne in 3 and tertamethylrhodamine (TAMRA)/
azide using sodium ascorbate and copper sulfate gave an ApA/
TAMRA conjugate 21, the reaction was very slow in G-buffer-
rich [2 mM Tris·HCl (pH 8.0), 0.2 mM CaCl2, 0.2 mM ATP,
0.5 mM 2-mercaptoethanol] solution (30%). However, the
addition of tris[(1-benzyl-1H-1,2,3-triazol-4-yl)methyl]amine
(TBTA)19 to stabilize the Cu(I) active species considerably
improved the yield of tetrazole 21 (78%, Figure S5).
Photolabeling experiments of actin using 4 were then carried
out (Figure 2). After covalent bond formation by irradiation
with UV (365 nm) at 0 °C for 15 min, the probe-bound actin
was further labeled with TAMRA by the Huisgen reaction.
proteins with compound 5 was competitively inhibited by
excess 1 (lane 2), and the results were similar to those with the
biotinylated ApA analogs.10 These results established that there
are specific interactions between the ApA analogs and actin or
actin-related proteins, rather than their simple absorptions on
the agarose resin.
To confirm the properties of binding between ApA and its
target proteins, photoreacted products were detected by
streptavidin-HRP conjugate. As expected, a strong band
corresponding to actin was observed upon treatment with 5
(Figure 3c, lane 1), and the band disappeared with the addition
of excess 1 (lane 2). In contrast, neither Arp2 nor Arp3 was
detected as a biotinylated protein. Thus, actin surely formed a
covalent bond with 5, while actin-related proteins did not.
̈
Notably, the most highly fluorescent actin bands were observed
when the Huisgen reactions were applied for 30 min (lane 3).
̈
Meanwhile, prolonged azide treatment over 1 h resulted in the
significant attenuation of actin fluorescence, due to the
degradation of actin protein itself (lanes 5−8). While the
amount of TAMRA−bound actin was slightly increased without
4 in association with the Huisgen reactions (lanes 2, 4, and 6),
̈
we concluded that ApA analog 4, bearing an aryldiazirine group
at the C34 terminus, formed a covalent bond with actin, in
which the protein/probe complex was efficiently fluorescence-
labeled for 30 min.
Furthermore, we sought to identify the target proteins of
aplyronines in tumor cell lysate using biotinylated photoaffinity
C
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Journal of the American Chemical Society
Communication
(h) Yamada, K.; Ojika, M.; Kigoshi, H.; Suenaga, K. Proc. Jpn. Acad.,
Ser. B 2010, 86, 176−189.
When the photolabeled products were competitively eluted
with 1, both Arp2 and Arp3 were liberated from the resin with a
quantity of actin (Figure 3d,e, lane 1), while the actin covalently
bound to ApA derivatives was eluted only after the boiling
process (lane 5). These results indicated that Arp2 and Arp3
might indirectly bind to ApA as the ternary adducts of the
actin/ApA complex or through the oligomeric actin that
interacts with ApA derivatives. Furthermore, through affinity
purification with 6, both Arp2 and Arp3 were obtained with
actin from cell lysate, as with 5, in which only the actin was
photolabeled (Figure 3a−e, lane 3). Since both 5 and 6 with
different cytotoxicity bound to Arp2 and Arp3 in same extent,
we concluded that actin-related proteins were not critical target
proteins of 1 that are essential for its potent antitumor activity.
In summary, through the use of photoaffinity derivatives of
aplyronines, we have established their interactions with actin
and actin-related proteins. We concluded that actin-related
proteins might interact with ApA via the actin/ApA complex.
Further investigations on the target proteins of ApA in living
cell systems as well as its mechanisms of action are currently
underway. Since the alkoxyamine reagents 14 and 19 can be
easily coupled with various substances that contain aldehyde or
hemiacetal groups and the conjugated oximes were consid-
erably stable under physiological conditions (pH 6−8),20,21
they may serve as quite useful photolabeling tools for analyzing
the binding sites and properties of various biomacromolecules
and active substances.
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ASSOCIATED CONTENT
* Supporting Information
Experimental details and characterization data. This material is
available free of charge via the Internet at http://pubs.acs.org.
■
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AUTHOR INFORMATION
Corresponding Author
mkita@chem.tsukuba.ac.jp; kigoshi@chem.tsukuba.ac.jp
■
Notes
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The authors declare no competing financial interest.
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ACKNOWLEDGMENTS
■
We thank Profs. Motonari Uesugi (Kyoto University, Japan)
and Takeo Usui (University of Tsukuba, Japan) for their
valuable suggestions regarding the identification of target
proteins and their interactions. This work was supported by
Grants-in-Aid for Scientific Research from JSPS and MEXT
(21681028 for M.K.; 23102014 and 23310148 for H.K.).
Support was also provided by the Naito Foundation, the
Uehara Memorial Foundation, and the Takeda Science
Foundation.
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