Structure-activity-distribution relationship study of anti-cancer antimycin-type depsipeptides

Article abstract of DOI:10.1039/c9cc03051d

Small-molecule natural products have been an essential source of pharmaceuticals to treat human diseases, but very little is known about their behavior inside dynamic, live human cells. Here, we demonstrate the first structure-activity-distribution relationship (SADR) study of complex natural products, the anti-cancer antimycin-type depsipeptides, using the emerging bioorthogonal Stimulated Raman Scattering (SRS) Microscopy. Our results show that the intracellular enrichment and distribution of these compounds are driven by their potency and specific protein targets, as well as the lipophilic nature of compounds.

Full text of DOI:10.1039/c9cc03051d

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Structure–activity–distribution relationship study
of anti-cancer antimycin-type depsipeptides†
Cite this:DOI: 10.1039/c9cc03051d
a
b
a
a
Jeremy Seidel, ‡ Yupeng Miao,‡ William Porterfield, Wenlong Cai,
a
a
b
a
b
Received 19th April 2019,
Accepted 11th July 2019
Xuejun Zhu, Seong-Jong Kim, Fanghao Hu,
and Wenjun Zhang
Santi Bhattarai-Kline, Wei Min*
a
*
DOI: 10.1039/c9cc03051d
rsc.li/chemcomm
8
–14
Small-molecule natural products have been an essential source of offered by the latest SRS technology.
pharmaceuticals to treat human diseases, but very little is known about reporter can be as simple as an alkyne,
The vibrational Raman
6
,15
delivering chemical
their behavior inside dynamic, live human cells. Here, we demonstrate specificity and biocompatibility for natural product visualiza-
the first structure–activity–distribution relationship (SADR) study of tion and quantification in complex living systems with minimal
complex natural products, the anti-cancer antimycin-type depsipep- activity perturbation of compounds. Compared to fluorescent
tides, using the emerging bioorthogonal Stimulated Raman Scattering imaging, SRS imaging offers additional advantages of minimal
(SRS) Microscopy. Our results show that the intracellular enrichment phototoxicity and photobleaching, allowing prolonged dynamic
and distribution of these compounds are driven by their potency and imaging of tagged natural products within live cells. SRS
specific protein targets, as well as the lipophilic nature of compounds. microscopy has recently been used to image various alkyne-
tagged small-molecule derivatives that have high local intracel-
1
6–21
Nature’s small molecules have played an enormous role in the lular concentrations.
Despite the great potential, few natural
history of medicinal and pharmaceutical chemistry. For example, products have been imaged using SRS microscopy to probe their
it has been estimated that over 70% of anti-cancer small intracellular behavior.
molecule treatments are natural products, their derivatives or
mimics. Although the pharmaceutical value of natural products depsipeptides, a class of complex natural products that have
has been widely recognized, it is still difficult to transform attracted recent attention due to their anti-cancer potential.
Here, we’ve applied SRS imaging to study antimycin-type
1
2
2
medicinally active natural products into drugs. One of the major This family of natural products share a common structural
challenges is to understand the complex interplay between skeleton consisting of a macrocyclic ring with an amide linkage
natural products and the network of cellular machinery beyond to a 3-formamidosalicylate unit, and primarily differ in the size
2
the specific protein targets. This has spurred the development of their macrolactone ring (Fig. 1). The well-recognized members
of advanced imaging techniques to obtain views of natural of this family are the 9-membered antimycins, for which multi-
products in cells, but often in a static and destructive manner ple modes of action have been proposed, including inhibition of
3
,4
using bulky fluorescent probes. An improved imaging techni-
que, which provides dynamic views of natural product uptake
and distribution in live cells, will have a profound impact on
5
natural product-based drug discovery and development. Tagging
natural products with a bio-orthogonal alkyne functionality
coupled with the Stimulated Raman Scattering (SRS) microscopy
6
,7
offers such promise.
Raman imaging has evolved greatly over the past decade,
with improved sensitivity, resolution, and scanning speeds
a
Department of Chemical and Biomolecular Engineering, University of California,
Berkeley, CA 94720, USA. E-mail: wjzhang@berkeley.edu
b
Department of Chemistry, Columbia University, NY 10025, USA.
E-mail: wm2256@columbia.edu
†
‡
Electronic supplementary information (ESI) available. See DOI: 10.1039/c9cc03051d
These authors contributed equally.
Fig. 1 Structures of selected antimycin-type depsipeptides and their
alkyne-tagged derivatives. The ring size is indicated in red numbers.
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2
3
a
mitochondrial electron transport chain, anti-apoptotic proteins Table 1 GR50 values (mM) for selected antimycins and neoantimycins
against two cancer cell lines in vitro
2
4
25
Bcl
2
/Bcl-x
L
,
K-Ras plasma membrane localization, and ATP
2
6
citrate lyase activity. The levels of contributions of these
different mechanisms are unclear. Much less is known about
the 15-membered neoantimycins, despite the fact that they
have also shown promising anti-cancer activities toward various Neoantimycin (3)
cancer cell lines. The inhibitory activity of K-Ras plasma
membrane localization was shown to be shared between anti-
mycins and neoantimycins, but neoantimycins lacked the
Bcl-x inhibitory activity and were demonstrated to inhibit the
expression of GRP-78,
Compound
HeLa
MCF-7
b
Antimycin (1)
PhDY-Ant (2)
20.2 Æ 4.3
2.1 Æ 0.5
28.8 Æ 9.7
24.4 Æ 5.5
40.9 Æ 22.8
41000
31.8 Æ 10.4
38.7 Æ 8.2
c
2
7
PhDY-NeoA (4)
Deformylated NeoA (5)
4100
41000
a
2
5
GR50 is the concentration at which the growth rate is half of that
under untreated conditions. All data were collected in at least tripli-
b
L
cate and GR50 values were averaged between at least three biological
2
8,29
c
a molecular chaperone in the endo- replicates with error given in standard error of the mean (SEM). GR50
could not be determined due to solubility limitations.
plasmic reticulum (ER) that promotes protein folding and
provides resistance to both chemotherapy and hypoglycemic
3
0
stress. To gain additional insights into anti-cancer activities
of antimycin-type depsipeptides, we performed an SADR study
of both antimycin and neoantimycin against live cancer cells.
To increase the sensitivity of SRS imaging toward bioactive
small molecules which are typically used in the low to mid
3
1
micromolar range, we chose to employ a conjugated diyne
with a terminal phenyl ring as a Raman tag. This tag is known
to possess an increased Raman scattering cross section due to
conjugation within the poly-yne chain and the presence of an
1
6
aryl end-capping group also improves the stability of poly-ynes.
This tag has recently been used in separate studies to image a
phenyl-diyne anisomycin derivative in mammalian cells and to
track the distribution of a cholesterol derivative in Caenorhabdi-
1
6,32
tis elegans where a detection limit of B30 mM was attained.
Since bioactive antimycins naturally have high structural varia-
tions at the C-7 alkyl and C-8 acyloxy moieties (Fig. 1) and the
previous introduction of an alkyne side chain at C-8 did not
significantly change cytotoxic activity nor binding of compounds
4
to cancer cells, we reasoned that the Raman tag could be readily
introduced at this position with minimal functional perturba-
tion. To prepare phenyl-diyne antimycin (PhDY-Ant, 2), C-8
deacylated antimycin was first purified from the culture of 2251 cm . (c) Off-resonance channel at 2000 cm . (d) CH
Streptomyces albus DantB in which the last step of C-8 acyloxy
Fig. 2 SRS and fluorescence imaging of PhDY-Ant (2) in HeLa cells.
À1
(
a) CH
3
channel at 2940 cm representing proteins. (b) Diyne label at
À1
À1
2
channel at
À1
2
845 cm , representing lipids. (e) Confocal fluorescence imaging of ER-
Tracker excited at 488 nm. (f) Confocal fluorescence imaging of Mito-
Tracker excited at 635 nm. (g) Overlay image of (d) lipids and (b) diyne
label. (h) Overlay image of (e) ER-Tracker and (b) diyne label. (i) Overlay
image of (f) Mito-Tracker and (b) diyne label.
formation is abolished in antimycin biosynthesis due to the
3
3
deletion of the dedicated C-8 acyltransferase AntB. A phenyl-
diyne carboxylic acid was chemically synthesized and then
coupled to the purified deacylated antimycin via Steglich ester-
ification to yield 2 (Scheme S1 and Fig. S1, ESI†). As expected, the
MTT proliferation assays with both HeLa (human cervical cancer) absolute intracellular concentration of 2 was determined to be
and MCF-7 (human breast cancer) cell lines confirmed that PhDY- B1.74 mM from the dosing concentration of 50 mM, showing a
Ant retained a comparable activity to the natural antimycin 35-fold enrichment of this compound in cells. To confirm that
(Table 1 and Fig. S2, ESI†).
the observed signal was driven by the activity of antimycin, a
PhDY-Ant (2) was then incubated with HeLa cells and SRS control experiment was performed by incubating 50 mM PhDY
images were acquired by tuning the frequency difference tag with cells. No signal of compound was detected inside cells
between the pump and Stokes lasers to be resonant with (Fig. S3, ESI†), suggesting that the observed Raman signal was
À1
intracellular components such as proteins (CH
3
, 2940 cm
)
not an off-target affect caused by the PhDY tag alone. We
À1
and lipids (CH
2
, 2845 cm ), and in the bio-orthogonal region further re-isolated small molecules from the 2-treated HeLa cells
of the Raman spectrum (alkyne, 2251 cm ; off-resonance, and confirmed that 2 was not rapidly metabolized to products
000 cm ). 2 was used at solution concentrations ranging containing the PhDY tag (Fig. S4, ESI†), suggesting that the
À1
À1
2
from 1–100 mM to probe the detection limit. Intracellular signal observed Raman signal was directly due to the presence of PhDY-
could be distinguished at concentrations as low as 10 mM, and Ant. We next probed the antimycin uptake rate and mechanism.
contrast was dramatically improved by increasing the solution Time-resolved imaging of 2 uptake into live HeLa cells showed
concentration of 2 to 50 mM (Fig. 2 and Fig. S3, ESI†). The that compound uptake was nearly immediate, reaching 75% of
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the maximum within six minutes (Fig. S5, ESI†). 2 appeared to has been identified. In addition, limited structure–activity
rapidly distribute throughout the cytoplasm of the cells and persist relationship studies have been performed on the molecular
through prolonged incubation. In addition, a low-temperature scaffold of neoantimycin. It is yet to be determined if a similar
(4 1C) uptake study was performed to investigate possible mechan- tagging strategy, the esterification of the macrolactone C-11
isms of compound uptake. 2 was absorbed at comparable levels at hydroxyl moiety that is naturally present in neoantimycin
both 4 1C and 37 1C (Fig. S6, ESI†), suggesting that PhDY-Ant (Fig. 1), can be adopted to produce a neoantimycin derivative
crossed the cell membrane through passive diffusion.
that is suitable for imaging analysis while retaining its anti-
The non-destructive nature of SRS imaging allows follow-up cancer activity. Neoantimycin was purified from the culture of
studies on compound distribution inside the cell, as well as S. orinoci and subjected to esterification by a phenyl-diyne
correlating compound uptake with any possible phenotypic carboxylic acid to generate phenyl-diyne neoantimycin (PhDY-
changes to cellular composition using dual-color and multi- NeoA, 4) (Scheme S2 and Fig. S11, ESI†). The MTT proliferation
modal approaches. For example, using a multi-modal approach assays with both HeLa and MCF-7 cells indicated that 4 had a
to probe the subcellular localization of 2, HeLa cells were slightly decreased but significant bioactivity, although its GR50
treated with ER-Tracker Green and Mito-Tracker Deep Red, value against HeLa cells could not be determined due to solubi-
cell-permeable fluorescent stains selective for the endoplasmic lity limitation (Table 1 and Fig. S12, ESI†). This response of cell
reticulum and mitochondria, respectively. Inspection of the lines to the C-11 modification is consistent with a recent report in
merged images demonstrated that 2 correlated well with ER- which oxidation of the same hydroxyl to ketone of neoantimycin
Tracker (Fig. 2). This colocalization agrees with one of its led to slightly increased IC50 values against multiple cancer cell
2
7
known direct protein targets, Bcl , an anti-apoptotic protein lines. The N-formyl group has been conserved in the antimycin-
2
3
4
localized primarily in the ER. Notably, a prolonged incubation type depsipeptides and linked to respiration inhibition for
led to a decrease of correlation between ER-Tracker and 2 antimycin. To probe the role of the N-formyl group in anti-
Fig. S7, ESI†), possibly due to compound dissociation from cancer activities of neoantimycin, we produced deformylated
22
(
targets and/or translocation of protein targets. In spite of the neoantimycin (5) and its tagged version (6) through acid degrada-
known activity of antimycin in inhibiting the mitochondrial tion of 3 and 4, respectively (Scheme S3 and Fig. S13, S14, ESI†).
electron transport chain by binding to the quinone reduction The growth of HeLa and MCF-7 cells was not inhibited upon
23
i 1
site Q of the cytochrome bc complex, no significant colocaliza- treatment of up to 1 mM of 5 (Table 1 and Fig. S12, ESI†),
tion of 2 with Mito-Tracker was found (Fig. 2). In addition, no demonstrating the critical role of this moiety for anti-cancer
obvious phenotypic changes were observed in lipids or proteins activity of the 15-membered neoantimycin. This is in contrast to
during the eight-hour incubation, although the merged images for the 18-membered antimycin-type depsipeptides of which defor-
2
and the lipid channel showed a strong correlation, especially in mylation did not significantly decrease the inhibitory activity
3
5,36
lipid droplets (Fig. 2). This correlation is likely caused by the toward various cancer cell lines,
suggesting different modes
lipophilic nature of 2 rather than any specific binding. Further of action for 15- and 18-membered compounds. Nonetheless, the
image analysis using profile plots demonstrated that the localiza- generation of both active and inactive tagged neoantimycins
tion of 2 was best explained by a combination of ER-Tracker and provided an opportunity to investigate possible differential
lipids (Fig. S8, ESI†). This result showed that the intracellular uptake of these compounds in live cells.
behavior of antimycin was not totally dictated by specific protein
PhDY-NeoA (4) and deformylated PhDY-NeoA (6) were then
binding nor non-specific absorption. Instead, there was a complex subjected to SRS imaging analysis with both HeLa and MCF-7
interplay between antimycin, its protein targets, and the lipid-rich cells. Enrichment of both compounds in lipid droplets was
regions of the cell.
observed for both cell lines, which is likely due to the lipophilic
We next analyzed the distribution of PhDY-Ant (2) in MCF-7 nature of compounds rather than any specific binding to
and compared it to HeLa cells to probe if the distribution is cell- targets (Fig. S15 and S16, ESI†). In addition, 4 was detected
line specific. Similar to HeLa cells, 2 colocalized with ER-Tracker, by SRS throughout the cytoplasm of the MCF-7 cells with an
but not Mito-Tracker (Fig. S9, ESI†). These data suggest that estimated intracellular concentration of B0.82 mM from a
localization of antimycin in the ER is conserved across different dosing concentration of 100 mM. 4 was also detected throughout
cancer cell lines. In addition, MCF-7 cells showed a much smaller the cytoplasm of the HeLa cells, although with a decreased
number of lipid droplets than HeLa cells, but instead contained signal intensity, demonstrating a positive correlation of com-
highly lipid-rich regions at the intercellular boundaries that did pound intracellular enrichment to its cytotoxic activity. This is
not attract 2 (Fig. S9, ESI†). This result further suggests that the particularly true for intracellular enrichment of 6, for which
characteristics and location of lipids are also important for significantly weaker SRS signals were detected (except within
enrichment of antimycin. Indeed, a profile analysis showed that lipid droplets) in either cell line (Fig. S15, ESI†). We hypothesize
certain areas of localization of 2 in MCF-7 cells were best that the loss of cytotoxic activity after deformylation is likely
correlated with ER-Tracker while others were best correlated due to decreased binding to the molecular targets of neoanti-
with the lipid channel (Fig. S10, ESI†).
mycin, leading to a decreased intracellular enrichment. Further
Compared to antimycin, the molecular mechanism for the analysis using dual-color and multi-modal approaches showed
ring-expanded neoantimycin to inhibit cancer cell growth is that the intracellular distribution of PhDY-NeoA (4) differed from
much less known, and no direct protein target of neoantimycin that of PhDY-Ant (2). In particular, 4 showed no correlation with
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Mito-Tracker and a very weak correlation with ER-Tracker
in MCF-7 cells (Fig. S16, ESI†). Line plot analysis showed that
4 Y. Yan, J. Chen, L. Zhang, Q. Zheng, Y. Han, H. Zhang, D. Zhang,
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Fig. S17, ESI†), suggesting that neoantimycin does not target
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This research was financially supported by grants to W. Z.
from the American Cancer Society, Alfred P. Sloan Foundation,
and the Chan Zuckerberg Biohub Investigator Program. W. M.
acknowledges support of R01EB020892 and R01GM128214
from NIH, and the Camille and Henry Dreyfus Foundation.
J. A. S. is supported by the National Science Foundation
Graduate Research Fellowship Program. We thank the Berkeley
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