Probing the Anticancer Mechanism of (-)-Ainsliatrimer A through Diverted Total Synthesis and Bioorthogonal Ligation

Article abstract of DOI:10.1002/anie.201407225

Herein, we report an efficient approach for exploring the novel anticancer mechanism of (-)-ainsliatrimer A, a structurally complex and unique trimeric sesquiterpenoid, through a combined strategy of diverted total synthesis (DTS) and bioorthogonal ligation (TQ ligation), which allowed us to visualize the subcellular localization of this natural product in live cells. Further biochemical studies facilitated by pretarget imaging revealed that PPARγ, a nucleus receptor, was a functional cellular target of ainsliatrimer A. We also confirmed that the anticancer activity of ainsliatrimer A was caused by the activation of PPARγ.

Full text of DOI:10.1002/anie.201407225

Angewandte
Chemie
DOI: 10.1002/anie.201407225
Target Identification
Probing the Anticancer Mechanism of (À)-Ainsliatrimer A through
Diverted Total Synthesis and Bioorthogonal Ligation**
Chao Li, Ting Dong, Qiang Li, and Xiaoguang Lei*
Abstract: Herein, we report an efficient approach for explor-
ing the novel anticancer mechanism of (À)-ainsliatrimer A,
a structurally complex and unique trimeric sesquiterpenoid,
through a combined strategy of diverted total synthesis (DTS)
and bioorthogonal ligation (TQ ligation), which allowed us to
visualize the subcellular localization of this natural product in
live cells. Further biochemical studies facilitated by pretarget
imaging revealed that PPARg, a nucleus receptor, was a func-
tional cellular target of ainsliatrimer A. We also confirmed that
the anticancer activity of ainsliatrimer A was caused by the
activation of PPARg.
Despite these challenges, several excellent studies have
been able to facilitate the target identification of complex
natural products.[4] A number of remarkable functionalization
strategies aimed at modifying natural products directly have
been developed to tackle synthetic challenges.[2a,5] However,
diverted total synthesis (DTS) remains an effective approach
for the syntheses of natural product analogues or natural
product-based chemical probes,[6] because DTS can expand
the flexibly of the chemical space around the natural products
from advanced intermediates. It can thus provide many
valuable analogues for structure–activity relationship (SAR)
studies and affinity-tag labeling. The emerging bioorthogonal
ligation approach affords a new avenue for cellular studies of
natural products,[7] as the incorporation of small chemical
reporters into complex scaffolds minimally affects their
original bioactivity. Subsequent target localization and iden-
tification studies can thus be performed by completing
bioorthogonal reactions with a cognate tag. Recently, our
research group developed a novel bioorthogonal ligation
method that was enabled by a click hetero-Diels–Alder
cycloaddition between a thio vinyl ether and an ortho-
quinolinone quinone methide (TQ ligation).[8] We envisioned
that TQ ligation might provide us with a useful means to study
the subcellular localization of complex natural products in
live cells, which would facilitate subsequent target identifica-
tion.
H
istorically, natural products and their derivatives have
been an invaluable source for drug discovery.[1] However,
identifying functional targets and clarifying the mechanisms
of action of bioactive natural products have proven to be
particularly challenging.[2] First, structural modifications of
complex natural products for the synthesis of useful chemical
probes are difficult to achieve due to the intrinsic architec-
tural complexity of many natural products. Furthermore,
target identification, which is the foundation of a chemical
biology research program, remains challenging for several
reasons including low target affinities, low abundance of
targets in vivo, and limitations of bioanalytical methods.[3]
Enabled by total synthesis,[9] we recently not only
confirmed that (À)-ainsliatrimer A (1; Figure 1), an architec-
turally complex and unique trimeric sesquiterpenoid, dis-
played potent cytotoxicity against several cancer cell lines
(see Table S1 in the Supporting Information), but also
demonstrated that the activity resulted from the induction
of apoptosis.[10] However, the exact mode of anticancer action
remained elusive. Herein, we introduce a combined strategy
of diverted total synthesis (DTS) and bioorthogonal ligation
(TQ ligation) to facilitate identification of the target of this
complex natural product.
To perform the pretarget imaging and target identification
of 1 using the TQ ligation, it was first necessary to prepare
1 modified with a thio vinyl ether group. Initially, we
attempted to modify the natural product directly to obtain
a pendant functional group that could react with a thio vinyl
ether fragment. However, the similar properties of four a,b-
unsaturated ketones can lead to selectivity issues, and one or
more of them might act as a Michael acceptor in reactions
with biomolecules. We therefore proposed that other feasible
approaches might be possible including alkylation and
hydroxylation at C2’’, reduction of the C3’’-carbonyl group,
allylic oxidation of the C9’’-OH group, and cross-metathesis
and hydroboration of the exo-methylene group at C14’’
[*] Dr. C. Li,[+] Prof. Dr. X. Lei
Beijing National Laboratory for Molecular Sciences
Key Laboratory of Bioorganic Chemistry and Molecular Engineering
of Ministry of Education, Department of Chemical Biology, College
of Chemistry and Molecular Engineering, Synthetic and Functional
Biomolecules Center
and Peking-Tsinghua Center for Life Sciences, Peking University
Beijing 100871 (China)
E-mail: xglei@pku.edu.cn
Homepage: http://www.chem.pku.edu.cn/leigroup/
T. Dong,[+] Q. Li, Prof. Dr. X. Lei
Graduate School of Peking Union Medical College and Chinese
Academy of Medical Sciences
Beijing 100730 (China)
and
National Institute of Biological Sciences (NIBS)
Changping District, Beijing 102206 (China)
[+] These authors contributed equally to this work.
[**] We thank Dr. She Chen (NIBS) for protein MS analysis, Mingyan
Zhao (NIBS) for NMR spectroscopy and HPLC-MS analysis, and Dr.
Jiang Zhou (Peking University) for HRMS analysis. Financial
support from the National High Technology Projects 973
(2012CB837400) and NNSFC (21222209, 91313303) is gratefully
acknowledged.
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/anie.201407225.
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or recovery of the starting material.[10,13]
Ultimately, we were pleased to discover that
treatment of 3 with Ag2O resulted in the
successful elimination to afford 3’-hydroxy-
gochnatiolide B (4) in high yield.[14] Treatment
of 4 with the freshly prepared diene 5 under
anaerobic conditions afforded 6 in moderate
yield as a single isomer. The anaerobic con-
ditions prevented autoxidation at C10 in 6,
and pyridine was added to inhibit the poly-
merization of 5, both of which resulted in
a higher yield of 6 than previously reported.[9b]
Moreover, alcohol 6 could be converted into
(À)-ainsliatrimer A (1) by Dess–Martin oxi-
dation,[10] thereby demonstrating that the
newly generated configurations at C4’ and
C10 fully coincided with those in 1. Fortu-
nately, the reduction of the C3’’-carbonyl
group in 1 did not compromise the antitumor
activity (see Figure S1 in the Supporting
Figure 1. Target identification of (À)-ainsliatrimer A (1) enabled by diverted total syn-
thesis (DTS) and bioorthogonal ligation (TQ ligation).
(Figure 1). However, none of these methods provided sat-
isfactory results because of the lability of 1 and the poor
reactivity and selectivity of the proposed procedures. Inspired
by the remarkable diverted total synthesis strategy, we
envisioned that (À)-gochnatiolide B (2), an advanced inter-
mediate in the synthesis of 1,[9b] could be modified. In turn, the
modified 2 could be used in the syntheses of analogues of
ainsliatrimer A.
Information),[10] which allowed for the further attachment of
thio vinyl ether fragments.
We then set out to synthesize the chemical probe of 1. To
enhance the hydrophilicity of the chemical probe and
facilitate the reaction between the labeled ortho-quinolinone
quinone methide (oQQM) and the thio vinyl ether fragment,
a tri(ethyleneglycol) linker was designed to be inserted
between the thio vinyl ether and 6 to form the chemical
probe 7. However, direct esterification between the thio vinyl
ether modified linker acid and 6 was unsuccessful under
Therefore, we aimed to modify 2 by using approaches
similar to those described above. Although unsuccessful
results were obtained, we noticed
that the 3’-carbonyl group in 2 could
be reduced under treatment with
NaBH4/CeCl3·7H2O concomitantly
with the 1,4-reduction of the exo-
methylene group at C4. We next
decided to protect this methylene
moiety. Thiophenol is commonly
applied for the protection of a,b-
unsaturated ketones;[11] however, in
this case, its regioselectivity was
poor due to concomitant addition
to the enone moieties in the lactone
rings. To our delight, after fine-
tuning the nucleophile, the selective
Michael
addition
proceeded
smoothly using 4-nitrothiophenol,
and the thioether was produced in
high yield as a single diastereoiso-
mer (Scheme 1). Luche reduction of
the thioether provided 3 in moder-
ate yield without the detection of
other diastereoisomers.[12] However,
the elimination of thiophenol from 3
proved nontrivial, and extensive
experimentation with different
bases and oxidative/b-elimination
and alkylative thioether elimination
procedures led to either low yields
Scheme 1. Synthesis of the chemical probe TV-ainsliatrimerA (7). Reagents and conditions: a) p-
nitrothiophenol, CHCl3, RT, 97%; b) NaBH4, CeCl3·7H2O, MeOH/CH2Cl2 (1:1), À788C, 46% (68%
brsm); c) Ag2O, MeOH, RT, 87%; d) 5, toluene/pyridine (100:1), glove box, 358C, 62–76%; e) 1. 9,
2,4,6-trichlorobenzoyl chloride, iPr2EtN, toluene, 308C then 2. DMAP, benzene, 358C, 56%; f) 3%
TFA/CH2Cl2, RT; g) 10, Et3N, DMF, 358C, 82% over 2 steps. DMAP=4-dimethylaminopyridine,
TFA=trifluoroacetic acid, DMF=N,N-dimethylformamide, Moz=[(p-methoxybenzyl)oxy]carbonyl,
brsm=based on recovered starting material.
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various conditions because of the lability of alcohol 6 and the
low solubility of the acid fragment. Therefore, we focused our
attention on a stepwise procedure, and the Moz-protected
linker 9 was attached to 6 in moderated yield.[15] After
deprotection, the thio vinyl ether fragment was assembled to
furnish the desired TV-modified ainsliatrimer A (7) in a facile
manner.[16] Furthermore, we observed that 7 was stable in
DMSO solution for two weeks under air at room temperature,
thus indicating that the potential occurrence of inter- or
intramolecular Diels–Alder reactions between the thio vinyl
ether and the dienes in the ainsliatrimer A could be ruled out.
With probe 7 in hand, we further demonstrate that 7
possesses equipotent activity at approximately double the
concentration of 1, and retained its apoptosis-inducing ability
(see Figure S1 in the Supporting Information).[10] Further cell-
cycle analysis experiments revealed that both 1 and 7 could
cause cell-cycle arrest in the G2/M phase (see Figure S2 in the
Supporting Information).[10] Collectively, these studies dem-
onstrated that 7 is a suitable chemical probe for the anticancer
natural product 1 for subsequent bioimaging studies in live
cells.
Encouraged by this result, we concentrated our efforts on
live-cell imaging studies. HeLa cells were first treated with
TV-ainsliatrimer A (7) under conditions that were expected
to produce high intracellular levels without killing the cells.
After extensive exploration, we chose to treat the HeLa cells
with 1.2 mm 7 for 2 h at 378C. After washing the cells with
DMEM media three times, they were exposed to the media
containing the 10 mm fluorescein-modified oOQM precursor
(11) for 18 h. After thorough washing of the cells to remove
the excess labeling mixture and subsequent co-staining with
the nuclear dye DAPI, the cells were imaged with a confocal
fluorescence microscope. Clearly stained, intracellular struc-
tures of the nucleus were distinguishable in the group treated
with 7, and the 7-associated fluorescence fully merged with
DAPI (Figure 2; see also Figure S3 in the Supporting
Information). In contrast, in the DMSO-treated group,
whereby the cells were incubated with 11 in the absence of
7, the fluorescence from 11 alone generated extremely low
levels of cellular nuclear labeling, and the majority of the
signals were enriched in the cytoplasm. This indicated that 7
was specific for the cellular targets of interest. As an
additional control, we performed tagging with 11 under
identical conditions, but in cells treated with 1; in this case,
nuclear labeling was not observed, again indicating that
successful labeling was enabled by TQ ligation. These results
definitively show that 7 accumulates primarily in the nuclei of
HeLa cells, further suggesting that the potential target(s) of
1 are localized in the nucleus.
Figure 2. Pretarget imaging studies using 7 in live cells. Live HeLa
cells were treated with fluorescein-modified tag 11 after treatment with
DMSO, 1, and 7. All cells were treated with the nuclear dye DAPI
before being imaged on a confocal microscope. DAPI=4’,6-diamidino-
2-phenylindole, FITC=fluorescein isothiocyanate, DIC=differential
interference contrast.
ure 3B), thereby indicating that the unsaturated enone
moieties were essential for the biological activity of 1 (see
Figure S4 in the Supporting Information).[10]
With the functional chemical probes 13 and 14 in hand, we
performed pull-down experiments by incubating HeLa cell
nuclear extracts with 13 and 14. The resulting lysates were
subsequently incubated with streptavidin-labeled beads.
After washing the beads, the proteins precipitated were
resolved by SDS-PAGE and stained with silver (Figure 3C).
The purity of the nuclear protein extraction was also
examined by detecting for contamination from a-tubulin
and the nuclear marker histone H1, which were easily
detected (see Figure S5 in the Supporting Information).[10]
Finally, we chose the gel bands preferentially pulled down by
probe 13 rather than 14, and cut down both of them for MS
analysis. Several proteins were identified by peptide-mass
fingerprinting analysis. The proteins included in the list were
represented by more than 6 unique peptides (see Table S2 in
the Supporting Information).[10] After excluding the proteins
present in the 14 group from those of the 13 group, we finally
focused on two potential target proteins: peroxisome prolif-
erator activated receptors g (PPARg) and histone deacety-
lase 2 (HDAC2; see Tables S2 and S3 in the Supporting
Information).[10]
We next target identification of the functional target(s) of
1. Since we assumed that some of the extracted nuclear
proteins might be unstable at 378C for 12 h, we decided to
complete the TQ ligation in vitro beforehand. Accordingly, 7
was treated with the biotin-tagged oQQM precursor 12 to
afford biotin-labeled probe 13 (Figure 3A).[8] In parallel, we
also synthesized biotin-labeled 14 as the negative control
from saturated 6 by using the same synthetic strategy.[10]
Examination of the bioactivity showed that 13 retained its
original activity, whereas 14 was biologically inactive (Fig-
We next evaluated the functional roles of these two
candidate targets in the anticancer activity of ainsliatrimer A
(1). Initially, we performed target-knockdown experiments
using siRNA in HeLa cells, and measured the cell survival
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To determine whether ainsliatri-
mer A directly binds to PPARg, we
generated recombinant Flag-tagged
PPARg protein. We first immobilized
PPARg on Flag affinity beads, then
incubated the immobilized PPARg
with an increasing concentration of
probe 13. Subsequently, the precip-
itates were blotted for biotin or Flag
with a short exposure. As shown in
Figure 4C, a strong interaction could
be observed between 13 and the
Flag-tagged PPARg. Moreover, this
binding could be out-competed by an
excess of unlabeled 1 (Figure 4D).
We further confirmed that 1 cannot
bind to other PPAR subtypes, includ-
ing PPARa or PPARd/b (see Fig-
ure S8 in the Supporting Informa-
tion).[10] Collectively, these results
demonstrate that 1 can efficiently
interact with PPARg.
Finally, we observed that the
death of HeLa cells induced by
1
could be significantly reduced
when cells were pretreated with
GW9662, previously reported
a
PPARg-selective antagonist. This
suggested that 1 might act as an
agonist of PPARg (see Figure S9 in
the Supporting Information).[10] Con-
sistent with this result, we further
demonstrated that 1 induced the
transcriptional activity of PPARg
(see Figure S10 in the Supporting
Figure 3. A) Chemical structures of 12 as well as probes 13 and 14. B) HeLa cells were preincubated
with the indicated concentrations of 1, 13, or 14 for 48 h and the cell viability was determined by
MTT methods. C) The nuclear extracts of HeLa cells were incubated with 13 or 14 at 48C overnight,
followed by pull-down with streptavidin-agarose beads. The precipitates were resolved by SDS-
PAGE, and the gel was stained with silver. The indicated silver-stained protein bands were analyzed
by mass spectrometry. MTT=3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide.
rate. We found that the knockdown of PPARg, a nuclear
receptor protein that functions as a transcription factor,
resulted in a significant decrease in the cytotoxicity caused by
1 (Figure 4A). This finding indicated that PPARg might be
a functional target of 1. Knockdown at the protein level
following siRNA treatment was confirmed by immunoblot-
ting analysis (Figure 4B). In contrast, knockdown of HDAC2
did not rescue the cell death caused by 1, thus demonstrating
that HDAC2 was not a functional target (see Figure S6 in the
Supporting Information).[10] Subsequently, we selected the
melanoma cell line SK-MEL-28 for further testing because of
the relatively low PPARg expression level in HeLa cell.[17]
SK-MEL-28 is known to have a high PPARg expression
level[18] and has been proven to be very sensitive to treatment
with ainsliatrimer A (see Table S1 in the Supporting Infor-
mation).[10] Pull-down experiments using chemical probes 13
and 14 with the nuclear extracts of SK-MEL-28 revealed only
one observable band that was specifically precipitated by 13,
but not by 14. We confirmed that this band was PPARg by MS
analysis (see Figure S7 in the Supporting Information).[10]
Figure 4. A) HeLa cells were transfected with the indicated siRNA.
48 h post-transfection, the cells were treated under the indicated
conditions for an additional 48 h, and then the cell viability was
determined by an MTT assay. B) The cell lysates were collected and
harvested for Western blot analysis by using the indicated antibodies.
C,D) The recombinant Flag-PPARg proteins were incubated with 13,
14, or in the absence of 1 for 1.5 h at 378C. The mixtures were blotted
for biotin or Flag. All experiments were repeated at least three times,
with similar results obtained.
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the expression of COX-2, a downstream signaling target of
PPARg,[19] in the 184B5 cell line (see Figure S11 in the
Supporting Information).[10,20] Taken together, these results
establish that 1 acts as an agonist to PPARg.
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In summary, we used an efficient workflow to complete
the target identification of the bioactive and structurally
complex natural product (À)-ainsliatrimer A (1). This
approach was facilitated by a combined strategy of diverted
total synthesis and the pretarget imaging enabled by TQ
ligation, which allowed us to visualize the subcellular local-
ization of the natural product before pull-down experiments.
By applying this strategy, we identified PPARg as a functional
target of 1. Through subsequent biochemical studies, we also
confirmed that the anticancer activity of ainsliatrimer A is
caused by the activation of PPARg. We also observed the
occurrence of intriguing reactions during the synthesis,
including the selective addition of a,b-unsaturated ketones
by fine-tuning the nucleophile and Ag2O-dependent thiophe-
nol elimination, which should have potential synthetic utility
in the transient protection of enones. Notably, PPARg is
regarded as a promising anticancer target in view of its crucial
regulatory role in cell metabolism. To date, several anticancer
mechanisms involving the activation of PPARg have been
reported, such as the induction of apoptosis and regulation of
metabolic pathways.[21] Further studies towards the elucida-
tion of how exactly the activation of PPARg by ainsliatri-
mer A regulates cell proliferation through transcription level
or protein–protein interactions are in progress and will be
reported in due course.
[10] For details, see the Supporting Information.
[11] For selected examples of the transient protection of enones with
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Received: July 15, 2014
Published online: && &&, &&&&
Keywords: antitumor agents · bioorganic chemistry ·
.
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Communications
Target Identification
C. Li, T. Dong, Q. Li,
X. Lei*
&&&& — &&&&
Probing the Anticancer Mechanism of
(À)-Ainsliatrimer A through Diverted
Total Synthesis and Bioorthogonal
Ligation
The target of the structurally complex
natural product (À)-ainsliatrimer A has
been identified by a systematic and
efficient approach enabled by diverted
total synthesis and bioorthogonal liga-
tion. This approach enabled visualization
of the subcellular localization of the
natural product in live cells and identified
activation of PPARg as leading to the
anticancer activity of ainsliatrimer A.
6
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Angew. Chem. Int. Ed. 2014, 53, 1 – 6
These are not the final page numbers!