Synthesis of marmycin A and investigation into its cellular activity

Article abstract of DOI:10.1038/nchem.2302

Anthracyclines such as doxorubicin are used extensively in the treatment of cancers. Anthraquinone-related angucyclines also exhibit antiproliferative properties and have been proposed to operate via similar mechanisms, including direct genome targeting.

Full text of DOI:10.1038/nchem.2302

ARTICLES
PUBLISHED ONLINE: 20 JULY 2015 | DOI: 10.1038/NCHEM.2302
Synthesis of marmycin A and investigation
into its cellular activity
Tatiana Cañeque1, Filipe Gomes1†, Trang Thi Mai1†, Giovanni Maestri1,2, Max Malacria1,3
and Raphaël Rodriguez1,4,5
*
Anthracyclines such as doxorubicin are used extensively in the treatment of cancers. Anthraquinone-related
angucyclines also exhibit antiproliferative properties and have been proposed to operate via similar mechanisms,
including direct genome targeting. Here, we report the chemical synthesis of marmycin A and the study of its cellular
activity. The aromatic core was constructed by means of a one-pot multistep reaction comprising a regioselective
Diels–Alder cycloaddition, and the complex sugar backbone was introduced through a copper-catalysed Ullmann cross-
coupling, followed by a challenging Friedel–Crafts cyclization. Remarkably, fluorescence microscopy revealed that
marmycin A does not target the nucleus but instead accumulates in lysosomes, thereby promoting cell death
independently of genome targeting. Furthermore, a synthetic dimer of marmycin A and the lysosome-targeting agent
artesunate exhibited a synergistic activity against the invasive MDA-MB-231 cancer cell line. These findings shed light
on the elusive pathways through which anthraquinone derivatives act in cells, pointing towards unanticipated biological
and therapeutic applications.
he angucyclines comprise a large number of natural products as yet unidentified molecular mechanisms (Fig. 1a)11. Similarly,
formally arising from polyketide biosynthesis1. This family has the exact mechanism by which the marmycins operate has remained
T
attracted considerable attention due to the diverse nature of its elusive. As part of our continuing efforts towards the design and
structural variants and the widespread biological activities associated synthesis of biologically active small molecules capable of selective
with them. In particular, the angucyclines display antiproliferative, genome targeting12–15, we became interested in dissecting the mam-
antibiotic and antiviral properties, but the underlying mechanisms malian cell biology of the marmycins.
are not always fully understood1. Angucyclines are characterized
by an anthraquinone core, reminiscent of that of known anticancer Results
drugs, embedded within an extended angular aromatic backbone. Chemical synthesis of the marmycins. The limited supply of
Typically, the aromatic core is decorated by C- and O-glycosides naturally occurring marmycin A (1) prompted us to establish a
as well as unsaturated and oxidized functionalities. Marmycin A synthetic route and set up a sustainable source. Significant work
(1) and B (2) are novel angucyclines, recently isolated from a in this area16–19 led to the construction of 7′-nor-marmycin A. For
marine Streptomyces-related actinomycete, which exhibit relevant instance, remarkable Lewis acid-catalysed one-pot assemblies of
cytotoxic properties (Fig. 1a)2. These natural products have caught suitable anilines and glycals have enabled the construction of the
our attention partly as a result of their unique molecular architec- core structure of marmycin A devoid of the C7′ methyl group17,18
.
ture, which contains both C- and N-glycosidic bonds organized as The presence of alkyl substituents at the junction of bicyclic
a complex hexacyclic framework. Based on structural similarities systems is a ubiquitous feature of natural products, as illustrated
with other angucyclines, pluramycins and anthracyclines, the mar- by taxanes20 and steroids21, and represents a significant synthetic
mycins have been proposed to kill cancer cells through DNA target- obstacle in some cases. Regardless, these pioneering studies have
ing. For instance, the angucycline-derived jadomycin B (3) provided relevant clues and set important milestones towards
promotes the cleavage of double-stranded DNA in vitro upon achieving these complex structures. Previous expertise in natural
exposure to copper and light (Fig. 1a)3. The pluramycin derivative product chemistry22–24 led us to consider a biomimetic route,
hedamycin (4) intercalates within double-stranded DNA in a whereby the marmycins could arise from the stepwise assembly of
sequence-specific manner and covalently reacts with guanine resi- the aromatic core and
a sugar derived from 3-epi,4-epi-
dues in vitro, indicating that the flat core is prone to π–π stacking vancosamine2. From a retrosynthetic standpoint (Fig. 1b), we
and some degree of hydrophobic interactions (Fig. 1a)4,5. reasoned that a seco-marmycin intermediate could be obtained by
Doxorubicin (DXR, 5) triggers the production of deleterious reac- reacting the aromatic triflate 7 with the free amine 8. Such a
tive oxygen species (ROS)6, mediates the eviction of histones7,8 substrate was predicted to exhibit a sufficiently high electron
and interferes with topoisomerase II to promote genomic instability density at C9 to allow for a late-stage cyclization upon activation
and cell death (Fig. 1a)9,10. The notion of direct genome targeting is, of the anomeric position, despite the presence of the electron-
however, confounded by landomycin E (6), a potent anticancer withdrawing quinone fragment. However, the quaternary centre
compound known to induce mitochondrial dysfunction through carrying the amine of 8 was expected to impose a high level of
1Centre de Recherche de Gif, Institut de Chimie des Substances Naturelles du CNRS, 1 Avenue de la Terrasse, Gif sur-Yvette 91198, France. 2Department of
Chemistry, Università degli Studi di Parma, Parco Area delle Scienze 17/a, Parma 43124, Italy. 3Institut Parisien de Chimie Moléculaire, Sorbonne
Universités, UPMC Univ Paris 06, UMR CNRS 8232, Paris CEDEX 05 75252, France. 4Institut Curie Research Center, Organic Synthesis and Cell Biology
Group, 26 rue d’Ulm, Paris Cedex 05 75248, France. 5CNRS UMR 3666, Paris 75005, France. †These authors contributed equally to this work.
e-mail: raphael.rodriguez@curie.fr
*
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NATURE CHEMISTRY DOI: 10.1038/NCHEM.2302
ARTICLES
Me
a
O
O
OH
OH
O
b
O
Me
R
O
O
OH
11
OH
H
1´
H
9
O
Me
O
Me
OMe
O
NH
O
3´
NH
4´
HO
O
Me
Me
7´
Marmycin A (1) R = H
Marmycin B (2) R = Cl
HO
1
Me
NH2
Doxorubicin (DXR, 5)
HO
7´
Electrophilic
cyclization
Cross-coupling
HO
Me
O
HO
Me
OH
O
O
Me
H
O
Me
O
OMe
N
O
O
O
OH
O
MOMO
O
O
OH
Me
HO
Me
Me
Me NH2
OTf
O
7
Me
O
O
8
Jadomycin B (3)
Landomycin E (6)
Me
Me
O
OH
O
O
O
OH
O
O
Me
HO
O
OMe
Me
OH
Br
O
O
Me
Me
Me2N
HO
OH
Cycloaddition
O
Me
OH
Me
OH
O
OAc
O
O
O
11
9
10
Methyl-α-L-rhamnopyranoside
OH
Hedamycin (4)
Me
Me
HO
NMe2
Figure 1 | Anthraquinone-containing natural products. a, Molecular structures of marmycin A (1) and B (2), jadomycin B (3), hedamycin (4), the clinically
approved anthracycline doxorubicin (DXR, 5) and landomycin E (6). b, Retrosynthetic analysis towards marmycin A precursors featuring a stepwise fusion
of the aromatic and chiral sugar moieties and a regioselective cycloaddition.
steric hindrance that could preclude an effective metal-catalysed Methyl-α-L-rhamnopyranoside 11 was used as a chiral starting
cross-coupling of the aminosugar with the anthraquinone 7. material, and was first reacted with benzaldehyde then chloromethyl
Additionally, the absence of
a
known natural analogue methyl ether (MOMCl), thereby enabling us to undress the chiral
functionalized with glycoside at C11 hinted towards
a
a
sugar via a Klemer–Rodemeyer elimination30 upon exposure of
biosynthesis involving first a C–N coupling, which in turn would the diastereomeric mixture of benzylidene 15 to sec-buthyllithium
impose the formation of the C–C bond at C9 and yield the in THF. The primary amine was installed in three steps through
β-epimer as the sole reaction product. Next, we hypothesized that prior conversion of the ensuing ketone into hydroxylimine 16,
the aromatic building block could be built from known dienophile which was then methylated in the presence of methylcerium
925 and diene 1026 through a Diels–Alder cycloaddition. This dichloride with high diastereoselectivity. The intermediate com-
disconnection was, however, distinct from a biomimetic step, pound was finally subjected to hydrogenolysis to provide the
which formally involves polyketide chemistry rather than pericyclic target diastereoisomer 8.
processes. Finally, we anticipated that the aminosugar 8 could be
With the advanced building blocks 7 and 8 in hand, we explored
various metal-catalysed cross-coupling conditions including classi-
readily obtained from the chiral pool 11 based on previous work27,28
.
With this strategy in mind, we first prepared the requisite diene25 cal palladium-mediated Buchwald–Hartwig chemistry31. Despite
and dienophile26 from commercially available starting materials several attempts by varying ligands, solvents, bases, reaction temp-
(Fig. 2a). Dienophile 9 was synthesized in two steps from naphtha- eratures and the use of microwave and flow chemistry, we were
lene-1,5-diol 12, whereas diene 10 was prepared in five steps from unable to produce sufficient quantities of the desired aniline, result-
1,3-cyclohexanedione 13 using modified procedures27. The bromi- ing instead in the quantitative hydrolysis of triflate 7 to regenerate
nated dienophile 9 and unprotected diene 10 were then reacted in phenol 14 along with unreacted amine 8. In contrast, tert-butyl
hot toluene and stirred in methanolic potassium carbonate to amine and other less hindered primary aliphatic amines were
afford a mixture of reaction intermediates and the desired cyclo- coupled in excellent yields using palladium catalysts. Further inves-
adduct 14 in good overall yields. Interestingly, we found that stirring tigations led to the identification of copper as an effective metal for
the crude cycloaddition mixture in methanol for several hours this reaction32. We found that a catalytic amount of copper iodide in
before the addition of potassium carbonate was necessary for this hot toluene led to the production of the protected seco-marmycin 17
multistep process to occur in one pot, suggesting that traces of in acceptable yields, providing sufficient material to carry on with
hydrobromic acid may be released after cycloaddition, acting as a the next step. Notably, copper-catalysed Ullmann aminations are
catalyst for dehydration and aromatization in methanol. usually conducted in the presence of aromatic halides rather than
Furthermore, using 10 as a protected tert-butyldimethylsilyl ether triflates. Nevertheless, these conditions were suitable to couple the
increased the yields of cycloaddition, but precluded dehydration/ electron-deficient substrate 7 with the sterically demanding amine
aromatization, illustrating the virtue of protecting group-free chem- 8, where palladium chemistry had proven ineffective.
istry in some cases29. The corresponding triflate 7 was synthesized
using a standard procedure and directly engaged in the next step. Brønsted and Lewis acids including p-toluenesulfonic acid (PTSA),
Marmycin intermediate 17 was then exposed to a series of
2
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NATURE CHEMISTRY DOI: 10.1038/NCHEM.2302
ARTICLES
a
OH
O
Br
Ref. 25
2 steps
OH
12
(Commercial)
OAc
O
9
Me
Me
O
(i) Toluene, 100 °C
MeOH, 4 h, rt
then K2CO3
(ix) 7, CuI, K2CO3, toluene
160 °C, sealed tube
33% (uncorrected)
(Known)
O
O
33% one-pot
(uncorrected)
MeO
NH
Me
O
OR
(ii)
Me
OH
O
Ref. 26
14 R = H
7 R = Tf
O
OMOM
Me
5 steps
OH
13
(Commercial)
17
10
(Known)
(Unnatural)
Me
O
OMe
Me
O
OMe
Me
O
OMe
Me
O
OMe
OH
(iii)
(iv)
(v)
(vii)
MOMO
(vi)
(viii)
MOMO
MOMO
O
HO
N
O
Me NH2
O
OH
11
15
16
8
(Commercial)
Me
Me
c
b
O
O
Me
(xi) 18, NaH
DMF, 0 °C
75%
(xii) 17, aq. HBF4
MeCN, 82 °C
13%
R
O
H
O
9
Me
O
Me
NH
O
MeO
NH
Me
OR
O
Top view
NH
Me
O
Me
O
O
HO
OMe
Me
19
17 R = MOM
18 R = H
(+)-1 Marmycin A (R = H)
(+)-2 Marmycin B (R = Cl)
(x)
(xiii)
Oxamethoxymarmycin
(unnatural)
Side view
Figure 2 | Chemical synthesis of the marmycins. a, Synthesis of precursors of 1. (i) 9, 10, toluene, 100 °C, 16 h, then concentrated to dryness; MeOH, rt,
4 h, then K2CO3, rt, 1 h, 33%. (ii) Tf2O, Et3N, CH2Cl2, –78 °C, 6 h, 78%. (iii) Benzaldehyde dimethylacetal, PTSA, dimethylformamide (DMF), 95 °C
(reduced pressure), 12 h, 98%. (iv) (iPr)2EtN, MOMCl, CH2Cl2, rt, 72 h, 91%. (v) sec-Butyllithium (1.6 M, hexane), THF, –40 °C, 90 min, 61%. (vi) O-benzyl
hydroxylamine HCl salt, NaOAc, EtOH, rt, 2 h, 90%. (vii) Anhydrous CeCl3, methyllithium (3 M, diethoxymethane), THF, –78 °C, 1 h; then 16, –78 °C to rt,
1 h, 72%. (viii) H2, Pd/C (10 mol%), MeOH, rt, 12 h, 88%. (ix) 7, 8, CuI (20 mol%), K2CO3, toluene, 160 °C, sealed tube, 72 h, 33%. b, Modular
cyclization of aromatic and sugar moieties. (x) BF3·Et2O, CH2Cl2, –78 °C to rt, 24 h, 42%. (xi) NaH, DMF, 0 °C, 2 h, 75%. (xii) 50% wt/wt aq. HBF4,
MeCN, 82 °C, 8 h, 13%. (xiii) NCS, C2H4Cl2, 84 °C, 24 h, 61%. c, ORTEP drawing of the X-ray crystal structure of 1 showing top and side views.
rt, room temperature.
camphorsulfonic acid, pyridinium p-toluenesulfonate, indium molecular weight of 1. Hence, when 17 was subjected to aq.
bromide, indium triflate and scandium triflate, varying organic sol- HBF4, we observed the formation of (+)-1 in moderate yields
vents and reaction temperatures. These conditions led either to (Fig 2b,c). This outcome was in agreement with a mechanism in
unreacted starting material, products of hydrolysis at C1′ and C4′, which stringent acidic conditions activate the anomeric position,
or the almost quantitative production of the free aromatic amine concurrently decreasing the electron density at C9 as a result of
resulting from C3′–N bond cleavage, together with other uncharac- the protonation/chelation of the aromatic amine and/or the anthra-
terized by-products. Thus, we postulated that varying the nature of quinone moiety. Conversely, milder acidic conditions (such as
the protecting group of the alcohol at C4′ might provide control aq. HBF4) target a window of reactivity in favour of the desired
over conformational states in favour of a kinetically driven cycliza- intramolecular Friedel–Crafts reaction. Moreover, the use of water
tion. To this end, the protected intermediate 17 was first treated with instead of dry organic solvents probably influenced the distribution
boron trifluoride diethyl etherate to selectively release the free of conformational states and thus the fate of this reaction. These
alcohol 18 (Fig. 2b). To our surprise, all attempts to introduce results emphasize the dual electrophilic/nucleophilic nature of C9
another protecting group onto alcohol 18 under basic conditions and suggest that structures such as 19 may also be natural metab-
resulted in formation of the O-cyclized product 19 (Fig. 2b), high- olites. As we were unable to produce marmycin B (2) from the syn-
lighting the dominant electrophilic nature of C9. This observation thetic sample of 1 using a nucleophilic reagent (for example,
encouraged us to take advantage of this reactivity through the gen- aq. HCl), we found that 2 could instead be obtained from 1 in the
eration of the nucleophile at the anomeric position. Strikingly, presence of N-chlorosuccinimide (NCS). This supports the notion
analysis of the reaction mixture of 17 exposed to a salt of triphenyl- that the aromatic amine increases the electron density at both C9
phosphonium hydrogen tetrafluoroborate (HPPh3·BF4) by mass and C11, hence conferring the desired reactivity giving access to
spectrometry revealed the presence of a product displaying the both natural small molecules. Although moderate reaction yields
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NATURE CHEMISTRY DOI: 10.1038/NCHEM.2302
ARTICLES
c
a
DAPI
Marmycin A
DMSO DXR Marmycin A
b
DAPI
DXR
–
–
–
Z-DEVD-FMK
p21
150
+
p-p53
(Ser15)
100
50
0
0.1 μM
10.3 μM
p53
γH2AX
(Ser139)
H2AX
0.0001
0.01
1
100
β-actin
Concentration (μM)
Figure 3 | Marmycin A kills human cancer cells independently of genome targeting. a, Percentage of viable U2OS cells after 72 h treatment with DXR
(dashed line) or 1 (solid line). n = 3; error bars, s.d. b, Confocal microscopy images showing accumulation of DAPI (pan-nuclear blue), DXR (pan-nuclear
red) and 1 (red puncta) in U2OS cells. Yellow boxes indicate the area of magnification of the main images. Zoomed images are ×5. Scale bar, 10 μm.
c, Western blot analysis showing levels of γH2AX, p-p53 and p21 in U2OS cells treated with dimethylsulfoxide (DMSO), DXR or 1 (50 μM, 24 h) including
a co-treatment with Z-DEVD-FMK.
could be broadly considered intractable, we were able to produce and induced phosphorylation of tumour suppressor p53 and the
appropriate quantities of rare natural products to explore the associ- production of p21.
ated cell biology and did not seek to improve the reaction
conditions further.
The fact that 1 is inherently fluorescent allowed us to perform
live cell imaging experiments without the use of reagents that
would otherwise alter the plasma membrane of cells. By doing so,
Cellular activity of marmycin A. Previous work has shown that 1 we could visualize the accumulation of 1 throughout the apolar
exhibits cytotoxic properties against several cancer cell lines membrane, reflecting the lipophilic nature of the small molecule
associated with a G1/S cell cycle arrest in ovarian A2780 cancer (Fig. 4a). Furthermore, this staining was more pronounced at
cells, consistent with the idea that 1 operates via a well-defined some hotspots defining small vesicles of 1 sporadically disseminated
mechanism2. In our hands, 1 induced cell death, but no cell cycle throughout the plasma membrane, which suggested that 1 was, at
arrest was detected by fluorescence-activated cell sorting least in part, internalized along with other components of the mem-
(Supplementary Figs 1 and 2). Side-by-side comparison of 1 and brane by means of endocytosis (Fig. 4a). We therefore attempted to
the clinically approved DNA intercalating agent DXR revealed identify the nature of the organelles in which 1 accumulated, with
that higher doses of 1 were required to kill human osteosarcoma particular focus on endocytic compartments, in search of putative
U2OS cells, with a half-maximum inhibitory concentration (IC50) cell death mechanisms. Simultaneous treatment of living cells
value of ∼10 μM (against ∼0.1 μM for DXR) at 72 h treatment with 1 (red staining) and the lysosomal marker DND-22 (blue stain-
(Fig. 3a). This result, and the fact that 1 has a rather curved ing) led to a significant co-localization of both substrates (merged
molecular structure (Fig. 2c), suggested that 1 may operate pink staining), characteristic of a physical proximity of these com-
independently of DNA intercalation. Interestingly, visual inspection pounds in the lysosomes (Fig. 4a). Conversely, pretreatment with
of growing cells indicated a diffuse staining of the red natural 1 prevented the accumulation of DND-22 in the vesicles, further
product inside the cell. This prompted us to exploit this indicating that marmycin A targets the lysosomal compartment.
property to identify the subcellular localization of 1 without the To confirm this we evaluated the localization of 1 with a biological
need to chemically label the natural product in cells, as marker of this organelle. Cells were virally infected using the
previously done by us to detect otherwise invisible small BacMam system to transiently express a green fluorescent protein-
molecules14,33. Analysis of 1 showed an excitation at ∼525 nm fused lysosomal-associated membrane protein 1 (GFP-Lamp1).
and fluorescence emission at ∼650 nm (Supplementary Fig. 3). Lamp1 is involved in the structural integrity of the lysosomal com-
Comparative analysis of cellular staining of the minor groove partment and the regulation of lysosome/autophagosome fusion35,36
.
DNA binder 4,6-diamidino-2-phenylindole (DAPI), the DNA Although the transduction efficiency was moderate, some cells con-
intercalating agent DXR and natural product 1 using fluorescence tained green hollow vesicles filled with 1 (red staining), further
microscopy revealed that DAPI and DXR gave a similar nuclear demonstrating that 1 accumulated within the lumen of lysosomes
staining, while 1 did not penetrate into the nucleus but (Fig. 4b and Supplementary Fig. 4). In contrast, marmycin A did
accumulated instead as small vesicles in the cytosol (Fig. 3b). not accumulate in the Golgi apparatus, demonstrating that the
These data strongly challenge the notion of genome targeting by small molecule is not a generic lipophilic membrane interacting
angucycline 1. To confirm this, we studied markers of the DNA agent (Supplementary Fig. 5).
damage response. Little increase of phosphorylated histone
To evaluate the functional consequences of the accumulation of 1
variant H2AX (γH2AX), a surrogate mark of DNA double- in lysosomes, we monitored (by western blotting) the turnover of
strand breaks, was observed upon treatment with 1, as monitored the ubiquitin-binding and oxidative stress response protein p62 as
by western blotting (Fig. 3c). In line with this, no phosphorylation well as the autophagy marker protein LC3-II (Fig. 4c)37. We
of p53 or significant induction of p21 could be detected, consistent found that treatments with 1 resulted in a modest increase in p62
with the idea that 1 does not directly promote DNA damage or a and LC3-II levels, indicating that 1 had little influence on the auto-
checkpoint-dependent cell cycle arrest. It is noteworthy that the phagic flux. In comparison, the lysosome targeting agent chloroquine
low amount of γH2AX induced by 1 was prevented by co- (CQ) induced significantly higher levels of both p62 and LC3-II,
treatment with the caspase 3 inhibitor Z-DEVD-FMK, indicating indicating a strong inhibition of the flux. Interestingly, co-treatment
some level of caspase activation and nuclease-mediated DNA of cells with 1 and the ROS scavenger N-acetyl cysteine (NAC) pre-
cleavage34, characteristic of an apoptotic response. In comparison, vented p62 induction by 1, but did not rescue cell viability. This
the DNA intercalating drug DXR increased the level of γH2AX result indicated that 1 induced some level of ROS as a peripheral
4
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NATURE CHEMISTRY DOI: 10.1038/NCHEM.2302
ARTICLES
a
c
DMSO CQ
Marmycin A
Zoom
DND-22
Marmycin A
Merge
–
–
–
NAC
+
p62
LC3-I
LC3-II
β-actin
b
DAPI
GFP-Lamp1
Marmycin A
Merge
d
DMSO
Marmycin A
E-64 CA-074 TPCK
n.i.
Bid
β-actin
Me
e
f
O
150
H
O
Me
NH
Me
O
100
50
0
OH
O
O
O
0.09 μM
0.9 μM
10 μM
O
O
O
O
O
O
H
H
O
O
Me
Me
O
Me
Me
H
H
O
O
O
H
H
0
0.0001 0.001 0.01
0.1
1
10
Me
Me
Concentration (μM)
Artesumycin (21)
Artesunate (20)
(unnatural)
Figure 4 | Marmycin A accumulates in the lysosomes and triggers programmed cell death. a, Live imaging of U2OS cells co-treated with 1 and DND-22.
The yellow box indicates the area of magnification of the main image. The zoomed image is ×3. White arrowheads indicate sites of co-localization. The white
box represents a further ×3 magnification. b, Confocal microscopy images of U2OS cells expressing GFP-Lamp1 treated with 1. Yellow boxes indicate the area
of magnification of the main images. Zoomed images are ×5. White arrowheads indicate sites of co-localization. The white box shows a further ×3
magnification. Scale bars, 10 μm. c, Western blot analysis of p62 and LC3 in U2OS cells treated with DMSO, CQ or 1 (50 µM, 24 h) including a co-treatment
with NAC. d, Western blot analysis of Bid in U2OS cells treated with DMSO or 1 (10 µM, 96 h) including a co-treatment with CB or CT inhibitors. n.i., no
inhibitor. e, Molecular structures of 20 and 21. f, Percentage of viable MDA-MB-231 cells after 72 h treatment with DXR (dashed black line), 1 (solid black
line), 20 (cyan), 21 (red) and an equimolar mixture of 1 and 20 (blue). n = 3; error bars, s.d.
event and not the primary cause of cell death. Additionally, while polar aminosugar fused to a lipophilic aromatic core and accumu-
CQ is known to inhibit the autophagic flux by increasing the pH lates within the lysosomal compartment, we reasoned that 1 may
in lysosomes37, the inability of 1 to inhibit autophagy suggested also act as a detergent and trigger a similar response. Western
that this natural product had no effect on the lysosomal pH at the blot analysis revealed a clear reduction of Bid protein upon treat-
concentration used.
ment with 1 for 96 h (Fig. 4d). It is noteworthy that cleavage of
Lipophilic small molecules such as sphingosine have been shown Bid was prevented when cells were co-treated with the cathepsin
to induce lysosomal membrane permeabilization (LMP) in some B (CB) inhibitors E-64 and CA-074, or the chymotrypsin (CT)
circumstances38–40, resulting in the translocation of lysosomal pro- inhibitor TPCK, in strong agreement with LMP and the release
teases within the cytosol. This, in turn, can promote the cleavage of these lysosomal proteases within the cytosol. Consistent with
and activation of specific cytosolic proteins including the pro-apop- this, 1 induced a destabilization of the lysosomal membrane, as
totic BH3 interacting-domain death agonist protein (Bid), leading to indicated by the release of bulky FITC-dextran from lysosomes
mitochondrial outer membrane permeabilization41,42, cytochrome c (Supplementary Fig. 6)39. Additionally, we also observed reduced
release and caspases-dependent cell death. Because 1 contains a levels of caspase 3, and cell viability was partially rescued when
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NATURE CHEMISTRY DOI: 10.1038/NCHEM.2302
ARTICLES
Artesunate
complex. Small molecules capable of targeting this organelle can
induce pleiotropic responses, making it difficult to detect and quan-
tify each single event. This includes a partial or complete LMP, the
release of proteases with redundant functions, and the activation of
caspase-dependent and -independent pathways, among other pro-
cesses. The current study shows that 1 induces the cleavage of Bid
and caspase-dependent cell death. More work is required to delin-
eate the fine details of this response, such as the occurrence of pro-
teases/caspase-independent cell death pathways40, which could take
place on the basis that cell killing by 1 was not completely prevented
by caspase inhibitors. It is also conceivable that the sudden release of
the acidic content and metal ions from lysosomes upon treatment
with 1 contributed to cell death in a manner that did not directly
implicate the translocation of lysosomal proteases, and so explicit
cell death pathways remain open to question. Regardless, our
results provide substantial evidence of an alternative mechanism
of action of a natural angucycline that does not involve genomic
DNA. Targeting the lysosome to elicit cell death is emerging as an
Marmycin A
Artesumycin
Landomycin E
Lysosome
Chloroquine
(clinically approved)
?
?
Caspase-dependent
cell death
Protease/caspase-
independent cell death
Hedamycin
DNA
damage
Doxorubicin
(clinically approved)
Figure 5 | Antiproliferative mechanisms of anthraquinone-containing
drugs. Black arrows indicate previously established mechanisms. Red arrows
highlight the cellular activity of marmycin A. Black arrows with question
marks indicate putative mechanisms of action of other anthraquinone-
containing small molecules.
attractive therapeutic strategy for the treatment of cancers39,45,46
.
cells were co-treated with the caspase 3 inhibitors Z-DEVD-FMK or
Z-VAD-FMK (Supplementary Figs 7 and 8).
For example, the clinically approved drug CQ has been shown to
prevent the relapse observed in certain cancers refractory to a
DXR regimen47–50. Accordingly, marmycin A—and perhaps other
angucyclines—represent valuable probes with which to study the
biology of the lysosome, leaning towards unanticipated therapeutic
applications for this class of natural products.
The antimalarial and antiproliferative drug artesunate (20,
Fig. 4e) has been shown to exert its activity by targeting the lysoso-
mal compartment through Fenton-type chemistry43. We therefore
reasoned that a synthetic dimer of 1 and 20 would combine the
properties of each substrate embedded within a single scaffold.
Remarkably, we found that the synthetic dimer 21 (which we
named artesumycin) exhibited enhanced antiproliferative activity
compared to 1 and 20 when used either independently or as a co-
treatment (Fig. 4e). For example, we found that 21 displayed an
IC50 value of ∼0.9 μM against the metastatic MDA-MB-231 breast
cancer cell line at 72 h incubation (Fig. 4f). In contrast, compounds
1 and 20 were poorly effective against this cell line at up to 10 μM
when used independently, and 10 μM of each drug was required
to reduce the number of viable cells by ∼50%. These data indicate
that 21 does not undergo hydrolysis in cells to release 1 and 20,
Methods
Cell culture and proliferation assays. U2OS, A2780 and MDA-MB-231 cells
were purchased from ATCC and were maintained in McCoy’s 5A or RPMI
1640, respectively, supplemented with 10% fetal bovine serum (FBS) and
1× antibiotic–antimycotic (Gibco) and incubated at 37 °C with 5% CO2.
Cell viability assays were carried out by plating 2,000 cells per well in 96-well
plates. Cells were treated with the relevant drug for 24 h or 72 h, then incubated
with CellTiter-Blue (20 μl/well) for 1 h before recording fluorescence
(560(20)Ex/590(10)Em) using a PerkinElmer Wallac 1420 Victor2 Microplate
Reader. For cell cycle analysis, cells were fixed in ice-cold 70% ethanol, stained
with propidium iodide (10 mg ml–1 PI, 250 mg ml–1 RNase A, 0.5% BSA, 0.02%
sodium azide in 1× PBS) and analysed by FACS on a CyFlow Ploidy Analyser
strongly supporting a mechanistic synergy mediated by 21 against from Partec.
lysosomal integrity rather than independent additive effects of 1
Drugs and inhibitors. Marmycin A was prepared in the laboratory according to
and 20. It is noteworthy that 21 accumulated in the lysosomes, as
confirmed by its co-localization with GFP-Lamp1, in line with the
organelle-specific targeting capacity of 1 (Supplementary Fig. 9).
Together, these data provide solid evidence for a novel model,
whereby 1 induces cell death through lysosomal dysfunction,
excluding direct genome targeting.
Fig. 2. Cells were treated with 10 μM marmycin A for 24 h unless stated otherwise.
Doxorubicin (Sigma) was used at 1 μM for 6 h (cell imaging) or 1 μM for 24 h
(western blotting); chloroquine (Sigma) was used at 100 μM for 24 h; N-acetyl
cysteine (Sigma) was used at 2 mM for 24 h (2 h pre-treatment). Z-DEVD-FMK
(BD Biosciences) was used at 100 μM for 24 h (30 min pre-treatment). Z-VAD-FMK
(BD Biosciences) was used at 100 μM for 24 h (30 min pretreatment). E-64
(Sigma) was used at 15 μM (added 48 h after 1, cells harvested at 96 h). CA-074 Me
(Enzo Life Sciences) was used at 1 μM (added 48 h after 1, cells harvested at 96 h).
N-tosyl-L-phenylalanine chloromethyl ketone (TPCK, Sigma) was used at
5 μM (added 48 h after 1, cells harvested at 96 h). Artesunate was purchased
from Sigma.
Discussion
We describe an expeditious synthetic route to 1. Our strategy fea-
tures a straightforward one-pot access to the angular aromatic
core and its assembly with a vancosamine-derived aminosugar
using a copper-catalysed cross-coupling followed by a challenging
acid-mediated cyclization. This synthetic sequence lends strong
support to a plausible biosynthetic route involving similar inter-
mediates, thereby establishing the absolute configuration of the
marmycins by means of chemical synthesis from known precursors.
Importantly, although natural sources of 1 are scarce, this synthesis
allowed the production of sufficient quantities of material to identify
some of the cellular events through which the natural product exerts
its activity and the preparation of the more potent derivative 21.
Unbiased strategies based on quantitative proteomics44 and high-
throughput sequencing14,15 have previously enabled the identifi-
cation of biological targets of small molecules in cells. Here, we
have used high-resolution microscopy14,33 to identify, instead, a cel-
lular organelle as the biologically relevant target of 1. In particular,
we have discovered that 1 operates as a lysosomotropic small mol-
ecule (Fig. 5), suggesting that other structurally related compounds
Immunofluorescence analysis. U2OS cells were cultured and treated with 1 or DXR
at ∼40% confluence. LysoTracker Blue DND-22 (L7525, Life Technologies) and 1
were added and used without fixation in live cell imaging experiments. Fluorescence
was recorded 10 min after the addition of both reagents. GFP-Lamp1 was transiently
expressed in U2OS cells following the manufacturer’s instructions. Briefly, 5 μl
of CellLight Lysosomes-GFP BacMam 2.0 (C10596, Life Technologies) was mixed
well with 200 μM U2OS culture medium and added to each well of a 24-well plate
loaded poly-L-lysine-treated coverslips. After 16 h incubation, cells were washed with
PBS and treated with 1 (10 μM, 6 h). Cells were then washed with PBS, fixed for
12 min with 2% formaldehyde/PBS and permeabilized for 10 min with 0.1% Triton
X-100/PBS, then washed with PBS. The Golgi apparatus was detected using an
anti-RCAS1 antibody (1/100, #12290, Cell Signaling) and anti-rabbit Alexa Fluor
secondary antibody (1/500, A-11008, Life Technologies). Coverslips were
mounted with VectaShield mounting medium with or without DAPI (Vector
Laboratories). Images were taken with Leica SP8 microscope or Nikon Te(-E)
inverted confocal microscopes equipped with a spinning disk Yokogawa
CXU-X1 A1 (DND-22 experiment only). Data were analysed with ImageJ
software (NIH).
Western blotting. Cells were treated as indicated in Figs 3 and 4, then washed twice
with PBS and lysed with 2× Laemmli buffer. Cell extracts were heated for 5 min at
could trigger similar responses. The biology of the lysosome is 95 °C, sheared through a 26-gauge needle and quantified with a Nanodrop
6
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© 2015 Macmillan Publishers Limited. All rights reserved

NATURE CHEMISTRY DOI: 10.1038/NCHEM.2302
ARTICLES
2000 (Thermo Scientific). Protein lysates (∼100 μg) were loaded on a 4–20%
Mini-PROTEAN TGX Stain-Free Gel (BioRad), resolved by SDS-PAGE
electrophoresis and transferred onto a nitrocellulose membrane (Amersham).
Proteins were probed with the following antibodies: anti-β-actin (ab8226, Abcam),
anti-H2AX (PA1-14198, Thermo Scientific), anti-γH2AX (#2577, Cell Signaling),
anti-p21 (#2947, Cell Signaling), anti-p53 (#2524, Cell Signaling), anti-p-p53
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secondary antibodies were anti-rabbit HRP (A120-108P, Bethyl), anti-mouse HRP
(A90-116P) and anti-donkey HRP (A140-107P, Bethyl). Antigens were detected
by electrochemiluminescence (Amersham). Imaging was performed using a
ChemiDoc XRS+ System.
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Received 31 December 2014; accepted 10 June 2015;
published online 20 July 2015
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Acknowledgements
The authors thank the CNRS for funding, and the Imagif Cell Biology Unit, J.-F. Gallard
and F. Blanchard for assistance with cell imaging, NMR spectroscopy and X-ray
crystallography, respectively. R.R. thanks J.A. Yeoman, S. Müller, J.E. Moses, S.L. Buchwald,
L. Johannes, M. Mehrpour and members of R.R.’s laboratory for discussions and
proofreading of this manuscript. Research in R.R.’s laboratory is supported by the
European Research Council (grant no. 647973), the Fondation pour la Recherche Médicale
(grant no. AJE20141031486), the Emergence Ville de Paris Program and the Ligue
Nationale Contre le Cancer.
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NATURE CHEMISTRY DOI: 10.1038/NCHEM.2302
ARTICLES
Author contributions
Additional information
R.R., with contributions from T.C., G.M. and M.M., conceived and designed the
experiments. T.C., with assistance from F.G., synthesized the marmycins and artesumycin.
T.T.M. with contribution from T.C. performed proliferation assays, fluorescence-activated
cell sorting, western blotting and cellular imaging. R.R., G.M. and M.M. supervised the
research. All the authors analysed the data. R.R. wrote the manuscript. All the authors
commented on the manuscript. T.T.M. and F.G. contributed equally to this work.
Supplementary information and chemical compound information are available in the
online version of the paper. Reprints and permissions information is available online at
www.nature.com/reprints. Correspondence and requests for materials should be addressed to R.R.
Competing financial interests
The authors declare no competing financial interests.
8
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