synthesis of marmycin a and investigation into its cellular activity

Synthesis of marmycin A and investigationinto its cellular activityTatiana Caeque1, Filipe Gomes1, Trang Thi Mai1, Giovanni Maestri1,2, Max Malacria1,3

and Raphal Rodriguez1,4,5*

Anthracyclines such as doxorubicin are used extensively in the treatment of cancers. Anthraquinone-relatedangucyclines 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 cellularactivity. The aromatic core was constructed by means of a one-pot multistep reaction comprising a regioselectiveDielsAlder cycloaddition, and the complex sugar backbone was introduced through a copper-catalysed Ullmann cross-coupling, followed by a challenging FriedelCrafts cyclization. Remarkably, uorescence microscopy revealed thatmarmycin A does not target the nucleus but instead accumulates in lysosomes, thereby promoting cell deathindependently of genome targeting. Furthermore, a synthetic dimer of marmycin A and the lysosome-targeting agentartesunate exhibited a synergistic activity against the invasive MDA-MB-231 cancer cell line. These ndings shed lighton the elusive pathways through which anthraquinone derivatives act in cells, pointing towards unanticipated biologicaland therapeutic applications.

The angucyclines comprise a large number of natural productsformally arising from polyketide biosynthesis1. This family hasattracted considerable attention due to the diverse nature of itsstructural variants and the widespread biological activities associatedwith them. In particular, the angucyclines display antiproliferative,antibiotic and antiviral properties, but the underlying mechanismsare not always fully understood1. Angucyclines are characterizedby an anthraquinone core, reminiscent of that of known anticancerdrugs, embedded within an extended angular aromatic backbone.Typically, the aromatic core is decorated by C- and O-glycosidesas well as unsaturated and oxidized functionalities. Marmycin A(1) and B (2) are novel angucyclines, recently isolated from amarine Streptomyces-related actinomycete, which exhibit relevantcytotoxic properties (Fig. 1a)2. These natural products have caughtour attention partly as a result of their unique molecular architec-ture, which contains both C- and N-glycosidic bonds organized asa complex hexacyclic framework. Based on structural similaritieswith other angucyclines, pluramycins and anthracyclines, the mar-mycins have been proposed to kill cancer cells through DNA target-ing. For instance, the angucycline-derived jadomycin B (3)promotes the cleavage of double-stranded DNA in vitro uponexposure to copper and light (Fig. 1a)3. The pluramycin derivativehedamycin (4) intercalates within double-stranded DNA in asequence-specic manner and covalently reacts with guanine resi-dues in vitro, indicating that the at core is prone to stackingand some degree of hydrophobic interactions (Fig. 1a)4,5.Doxorubicin (DXR, 5) triggers the production of deleterious reac-tive oxygen species (ROS)6, mediates the eviction of histones7,8

and interferes with topoisomerase II to promote genomic instabilityand cell death (Fig. 1a)9,10. The notion of direct genome targeting is,however, confounded by landomycin E (6), a potent anticancercompound known to induce mitochondrial dysfunction through

as yet unidentied molecular mechanisms (Fig. 1a)11. Similarly,the exact mechanism by which the marmycins operate has remainedelusive. As part of our continuing efforts towards the design andsynthesis of biologically active small molecules capable of selectivegenome targeting1215, we became interested in dissecting the mam-malian cell biology of the marmycins.

ResultsChemical synthesis of the marmycins. The limited supply ofnaturally occurring marmycin A (1) prompted us to establish asynthetic route and set up a sustainable source. Signicant workin this area1619 led to the construction of 7-nor-marmycin A. Forinstance, remarkable Lewis acid-catalysed one-pot assemblies ofsuitable anilines and glycals have enabled the construction of thecore structure of marmycin A devoid of the C7 methyl group17,18.The presence of alkyl substituents at the junction of bicyclicsystems is a ubiquitous feature of natural products, as illustratedby taxanes20 and steroids21, and represents a signicant syntheticobstacle in some cases. Regardless, these pioneering studies haveprovided relevant clues and set important milestones towardsachieving these complex structures. Previous expertise in naturalproduct chemistry2224 led us to consider a biomimetic route,whereby the marmycins could arise from the stepwise assembly ofthe aromatic core and a sugar derived from 3-epi,4-epi-vancosamine2. From a retrosynthetic standpoint (Fig. 1b), wereasoned that a seco-marmycin intermediate could be obtained byreacting the aromatic triate 7 with the free amine 8. Such asubstrate was predicted to exhibit a sufciently high electrondensity at C9 to allow for a late-stage cyclization upon activationof the anomeric position, despite the presence of the electron-withdrawing quinone fragment. However, the quaternary centrecarrying 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 ofChemistry, Universit degli Studi di Parma, Parco Area delle Scienze 17/a, Parma 43124, Italy. 3Institut Parisien de Chimie Molculaire, SorbonneUniversits, UPMC Univ Paris 06, UMR CNRS 8232, Paris CEDEX 05 75252, France. 4Institut Curie Research Center, Organic Synthesis and Cell BiologyGroup, 26 rue dUlm, Paris Cedex 05 75248, France. 5CNRS UMR 3666, Paris 75005, France. These authors contributed equally to this work.*e-mail: [email protected]

ARTICLESPUBLISHED ONLINE: 20 JULY 2015 | DOI: 10.1038/NCHEM.2302

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steric hindrance that could preclude an effective metal-catalysedcross-coupling of the aminosugar with the anthraquinone 7.Additionally, the absence of a known natural analoguefunctionalized with a glycoside at C11 hinted towards abiosynthesis involving rst a CN coupling, which in turn wouldimpose the formation of the CC bond at C9 and yield the-epimer as the sole reaction product. Next, we hypothesized thatthe aromatic building block could be built from known dienophile925 and diene 1026 through a DielsAlder cycloaddition. Thisdisconnection was, however, distinct from a biomimetic step,which formally involves polyketide chemistry rather than pericyclicprocesses. Finally, we anticipated that the aminosugar 8 could bereadily obtained from the chiral pool 11 based on previous work27,28.

With this strategy in mind, we rst prepared the requisite diene25

and dienophile26 from commercially available starting materials(Fig. 2a). Dienophile 9 was synthesized in two steps from naphtha-lene-1,5-diol 12, whereas diene 10 was prepared in ve steps from1,3-cyclohexanedione 13 using modied procedures27. The bromi-nated dienophile 9 and unprotected diene 10 were then reacted inhot toluene and stirred in methanolic potassium carbonate toafford a mixture of reaction intermediates and the desired cyclo-adduct 14 in good overall yields. Interestingly, we found that stirringthe crude cycloaddition mixture in methanol for several hoursbefore the addition of potassium carbonate was necessary for thismultistep process to occur in one pot, suggesting that traces ofhydrobromic acid may be released after cycloaddition, acting as acatalyst for dehydration and aromatization in methanol.Furthermore, using 10 as a protected tert-butyldimethylsilyl etherincreased the yields of cycloaddition, but precluded dehydration/aromatization, illustrating the virtue of protecting group-free chem-istry in some cases29. The corresponding triate 7 was synthesizedusing a standard procedure and directly engaged in the next step.

Methyl--L-rhamnopyranoside 11 was used as a chiral startingmaterial, and was rst reacted with benzaldehyde then chloromethylmethyl ether (MOMCl), thereby enabling us to undress the chiralsugar via a KlemerRodemeyer elimination30 upon exposure ofthe diastereomeric mixture of benzylidene 15 to sec-buthyllithiumin THF. The primary amine was installed in three steps throughprior conversion of the ensuing ketone into hydroxylimine 16,which was then methylated in the presence of methylceriumdichloride with high diastereoselectivity. The intermediate com-pound was nally subjected to hydrogenolysis to provide thetarget diastereoisomer 8.

With the advanced building blocks 7 and 8 in hand, we exploredvarious metal-catalysed cross-coupling conditions including classi-cal palladium-mediated BuchwaldHartwig chemistry31. Despiteseveral attempts by varying ligands, solvents, bases, reaction temp-eratures and the use of microwave and ow chemistry, we wereunable to produce sufcient quantities of the desired aniline, result-ing instead in the quantitative hydrolysis of triate 7 to regeneratephenol 14 along with unreacted amine 8. In contrast, tert-butylamine and other less hindered primary aliphatic amines werecoupled in excellent yields using palladium catalysts. Further inves-tigations led to the identication of copper as an effective metal forthis reaction32. We found that a catalytic amount of copper iodide inhot toluene led to the production of the protected seco-marmycin 17in acceptable yields, providing sufcient material to carry on withthe next step. Notably, copper-catalysed Ullmann aminations areusually conducted in the presence of aromatic halides rather thantriates. Nevertheless, these conditions were suitable to couple theelectron-decient substrate 7 with the sterically demanding amine8, where palladium chemistry had proven ineffective.

Marmycin intermediate 17 was then exposed to a series ofBrnsted and Lewis acids including p-toluenesulfonic acid (PTSA),

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Figure 1 | Anthraquinone-containing natural products. a, Molecular structures of marmycin A (1) and B (2), jadomycin B (3), hedamycin (4), the clinicallyapproved anthracycline doxorubicin (DXR, 5) and landomycin E (6). b, Retrosynthetic analysis towards marmycin A precursors featuring a stepwise fusionof the aromatic and chiral sugar moieties and a regioselective cycloaddition.

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camphorsulfonic acid, pyridinium p-toluenesulfonate, indiumbromide, indium triate and scandium triate, varying organic sol-vents and reaction temperatures. These conditions led either tounreacted starting material, products of hydrolysis at C1 and C4,or the almost quantitative production of the free aromatic amineresulting from C3N bond cleavage, together with other uncharac-terized by-products. Thus, we postulated that varying the nature ofthe protecting group of the alcohol at C4 might provide controlover conformational states in favour of a kinetically driven cycliza-tion. To this end, the protected intermediate 17was rst treated withboron triuoride diethyl etherate to selectively release the freealcohol 18 (Fig. 2b). To our surprise, all attempts to introduceanother protecting group onto alcohol 18 under basic conditionsresulted in formation of the O-cyclized product 19 (Fig. 2b), high-lighting the dominant electrophilic nature of C9. This observationencouraged us to take advantage of this reactivity through the gen-eration of the nucleophile at the anomeric position. Strikingly,analysis of the reaction mixture of 17 exposed to a salt of triphenyl-phosphonium hydrogen tetrauoroborate (HPPh3BF4) by massspectrometry revealed the presence of a product displaying the

molecular weight of 1. Hence, when 17 was subjected to aq.HBF4, we observed the formation of (+)-1 in moderate yields(Fig 2b,c). This outcome was in agreement with a mechanism inwhich stringent acidic conditions activate the anomeric position,concurrently decreasing the electron density at C9 as a result ofthe protonation/chelation of the aromatic amine and/or the anthra-quinone moiety. Conversely, milder acidic conditions (such asaq. HBF4) target a window of reactivity in favour of the desiredintramolecular FriedelCrafts reaction. Moreover, the use of waterinstead of dry organic solvents probably inuenced the distributionof conformational states and thus the fate of this reaction. Theseresults emphasize the dual electrophilic/nucleophilic nature of C9and suggest that structures such as 19 may also be natural metab-olites. As we were unable to produce marmycin B (2) from the syn-thetic sample of 1 using a nucleophilic reagent (for example,aq. HCl), we found that 2 could instead be obtained from 1 in thepresence of N-chlorosuccinimide (NCS). This supports the notionthat the aromatic amine increases the electron density at both C9and C11, hence conferring the desired reactivity giving access toboth natural small molecules. Although moderate reaction yields

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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-benzylhydroxylamine 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, Modularcyclization of aromatic and sugar moieties. (x) BF3Et2O, 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.

NATURE CHEMISTRY DOI: 10.1038/NCHEM.2302 ARTICLES



could be broadly considered intractable, we were able to produceappropriate quantities of rare natural products to explore the associ-ated cell biology and did not seek to improve the reactionconditions further.

Cellular activity of marmycin A. Previous work has shown that 1exhibits cytotoxic properties against several cancer cell linesassociated with a G1/S cell cycle arrest in ovarian A2780 cancercells, consistent with the idea that 1 operates via a well-denedmechanism2. In our hands, 1 induced cell death, but no cell cyclearrest was detected by uorescence-activated cell sorting(Supplementary Figs 1 and 2). Side-by-side comparison of 1 andthe clinically approved DNA intercalating agent DXR revealedthat higher doses of 1 were required to kill human osteosarcomaU2OS cells, with a half-maximum inhibitory concentration (IC50)value of 10 M (against 0.1 M for DXR) at 72 h treatment(Fig. 3a). This result, and the fact that 1 has a rather curvedmolecular structure (Fig. 2c), suggested that 1 may operateindependently of DNA intercalation. Interestingly, visual inspectionof growing cells indicated a diffuse staining of the red naturalproduct inside the cell. This prompted us to exploit thisproperty to identify the subcellular localization of 1 without theneed to chemically label the natural product in cells, aspreviously done by us to detect otherwise invisible smallmolecules14,33. Analysis of 1 showed an excitation at 525 nmand uorescence emission at 650 nm (Supplementary Fig. 3).Comparative analysis of cellular staining of the minor grooveDNA binder 4,6-diamidino-2-phenylindole (DAPI), the DNAintercalating agent DXR and natural product 1 using uorescencemicroscopy revealed that DAPI and DXR gave a similar nuclearstaining, while 1 did not penetrate into the nucleus butaccumulated instead as small vesicles in the cytosol (Fig. 3b).These data strongly challenge the notion of genome targeting byangucycline 1. To conrm this, we studied markers of the DNAdamage response. Little increase of phosphorylated histonevariant H2AX (H2AX), a surrogate mark of DNA double-strand breaks, was observed upon treatment with 1, as monitoredby western blotting (Fig. 3c). In line with this, no phosphorylationof p53 or signicant induction of p21 could be detected, consistentwith the idea that 1 does not directly promote DNA damage or acheckpoint-dependent cell cycle arrest. It is noteworthy that thelow amount of H2AX induced by 1 was prevented by co-treatment with the caspase 3 inhibitor Z-DEVD-FMK, indicatingsome level of caspase activation and nuclease-mediated DNAcleavage34, characteristic of an apoptotic response. In comparison,the DNA intercalating drug DXR increased the level of H2AX

and induced phosphorylation of tumour suppressor p53 and theproduction of p21.

The fact that 1 is inherently uorescent allowed us to performlive cell imaging experiments without the use of reagents thatwould otherwise alter the plasma membrane of cells. By doing so,we could visualize the accumulation of 1 throughout the apolarmembrane, reecting the lipophilic nature of the small molecule(Fig. 4a). Furthermore, this staining was more pronounced atsome hotspots dening small vesicles of 1 sporadically disseminatedthroughout the plasma membrane, which suggested that 1 was, atleast in part, internalized along with other components of the mem-brane by means of endocytosis (Fig. 4a). We therefore attempted toidentify the nature of the organelles in which 1 accumulated, withparticular focus on endocytic compartments, in search of putativecell death mechanisms. Simultaneous treatment of living cellswith 1 (red staining) and the lysosomal marker DND-22 (blue stain-ing) led to a signicant co-localization of both substrates (mergedpink staining), characteristic of a physical proximity of these com-pounds in the lysosomes (Fig. 4a). Conversely, pretreatment with1 prevented the accumulation of DND-22 in the vesicles, furtherindicating that marmycin A targets the lysosomal compartment.To conrm this we evaluated the localization of 1 with a biologicalmarker of this organelle. Cells were virally infected using theBacMam system to transiently express a green uorescent protein-fused lysosomal-associated membrane protein 1 (GFP-Lamp1).Lamp1 is involved in the structural integrity of the lysosomal com-partment and the regulation of lysosome/autophagosome fusion35,36.Although the transduction efciency was moderate, some cells con-tained green hollow vesicles lled with 1 (red staining), furtherdemonstrating that 1 accumulated within the lumen of lysosomes(Fig. 4b and Supplementary Fig. 4). In contrast, marmycin A didnot accumulate in the Golgi apparatus, demonstrating that thesmall molecule is not a generic lipophilic membrane interactingagent (Supplementary Fig. 5).

To evaluate the functional consequences of the accumulation of 1in lysosomes, we monitored (by western blotting) the turnover ofthe ubiquitin-binding and oxidative stress response protein p62 aswell as the autophagy marker protein LC3-II (Fig. 4c)37. Wefound that treatments with 1 resulted in a modest increase in p62and LC3-II levels, indicating that 1 had little inuence on the auto-phagic ux. In comparison, the lysosome targeting agent chloroquine(CQ) induced signicantly higher levels of both p62 and LC3-II,indicating a strong inhibition of the ux. Interestingly, co-treatmentof cells with 1 and the ROS scavenger N-acetyl cysteine (NAC) pre-vented p62 induction by 1, but did not rescue cell viability. Thisresult indicated that 1 induced some level of ROS as a peripheral

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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-nuclearred) and 1 (red puncta) in U2OS cells. Yellow boxes indicate the area of magnication of the main images. Zoomed images are 5. Scale bar, 10 m.c, Western blot analysis showing levels of H2AX, p-p53 and p21 in U2OS cells treated with dimethylsulfoxide (DMSO), DXR or 1 (50 M, 24 h) includinga co-treatment with Z-DEVD-FMK.




event and not the primary cause of cell death. Additionally, whileCQ is known to inhibit the autophagic ux by increasing the pHin lysosomes37, the inability of 1 to inhibit autophagy suggestedthat this natural product had no effect on the lysosomal pH at theconcentration used.

Lipophilic small molecules such as sphingosine have been shownto induce lysosomal membrane permeabilization (LMP) in somecircumstances3840, resulting in the translocation of lysosomal pro-teases within the cytosol. This, in turn, can promote the cleavageand activation of specic cytosolic proteins including the pro-apop-totic BH3 interacting-domain death agonist protein (Bid), leading tomitochondrial outer membrane permeabilization41,42, cytochrome crelease and caspases-dependent cell death. Because 1 contains a

polar aminosugar fused to a lipophilic aromatic core and accumu-lates within the lysosomal compartment, we reasoned that 1 mayalso act as a detergent and trigger a similar response. Westernblot analysis revealed a clear reduction of Bid protein upon treat-ment with 1 for 96 h (Fig. 4d). It is noteworthy that cleavage ofBid was prevented when cells were co-treated with the cathepsinB (CB) inhibitors E-64 and CA-074, or the chymotrypsin (CT)inhibitor TPCK, in strong agreement with LMP and the releaseof these lysosomal proteases within the cytosol. Consistent withthis, 1 induced a destabilization of the lysosomal membrane, asindicated by the release of bulky FITC-dextran from lysosomes(Supplementary Fig. 6)39. Additionally, we also observed reducedlevels of caspase 3, and cell viability was partially rescued when

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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 magnication of the main image. The zoomed image is 3. White arrowheads indicate sites of co-localization. The whitebox represents a further 3 magnication. b, Confocal microscopy images of U2OS cells expressing GFP-Lamp1 treated with 1. Yellow boxes indicate the areaof magnication of the main images. Zoomed images are 5. White arrowheads indicate sites of co-localization. The white box shows a further 3magnication. Scale bars, 10 m. c, Western blot analysis of p62 and LC3 in U2OS cells treated with DMSO, CQ or 1 (50 M, 24 h) including a co-treatmentwith NAC. d, Western blot analysis of Bid in U2OS cells treated with DMSO or 1 (10 M, 96 h) including a co-treatment with CB or CT inhibitors. n.i., noinhibitor. 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 blackline), 20 (cyan), 21 (red) and an equimolar mixture of 1 and 20 (blue). n = 3; error bars, s.d.




cells were co-treated with the caspase 3 inhibitors Z-DEVD-FMK orZ-VAD-FMK (Supplementary Figs 7 and 8).

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 thereforereasoned that a synthetic dimer of 1 and 20 would combine theproperties of each substrate embedded within a single scaffold.Remarkably, we found that the synthetic dimer 21 (which wenamed artesumycin) exhibited enhanced antiproliferative activitycompared to 1 and 20 when used either independently or as a co-treatment (Fig. 4e). For example, we found that 21 displayed anIC50 value of 0.9 M against the metastatic MDA-MB-231 breastcancer cell line at 72 h incubation (Fig. 4f). In contrast, compounds1 and 20 were poorly effective against this cell line at up to 10 Mwhen used independently, and 10 M of each drug was requiredto reduce the number of viable cells by 50%. These data indicatethat 21 does not undergo hydrolysis in cells to release 1 and 20,strongly supporting a mechanistic synergy mediated by 21 againstlysosomal integrity rather than independent additive effects of 1and 20. It is noteworthy that 21 accumulated in the lysosomes, asconrmed by its co-localization with GFP-Lamp1, in line with theorganelle-specic 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.

DiscussionWe describe an expeditious synthetic route to 1. Our strategy fea-tures a straightforward one-pot access to the angular aromaticcore and its assembly with a vancosamine-derived aminosugarusing a copper-catalysed cross-coupling followed by a challengingacid-mediated cyclization. This synthetic sequence lends strongsupport to a plausible biosynthetic route involving similar inter-mediates, thereby establishing the absolute conguration of themarmycins by means of chemical synthesis from known precursors.Importantly, although natural sources of 1 are scarce, this synthesisallowed the production of sufcient quantities of material to identifysome of the cellular events through which the natural product exertsits 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 identi-cation of biological targets of small molecules in cells. Here, wehave 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 compoundscould trigger similar responses. The biology of the lysosome is

complex. Small molecules capable of targeting this organelle caninduce pleiotropic responses, making it difcult to detect and quan-tify each single event. This includes a partial or complete LMP, therelease of proteases with redundant functions, and the activation ofcaspase-dependent and -independent pathways, among other pro-cesses. The current study shows that 1 induces the cleavage of Bidand caspase-dependent cell death. More work is required to delin-eate the ne details of this response, such as the occurrence of pro-teases/caspase-independent cell death pathways40, which could takeplace on the basis that cell killing by 1 was not completely preventedby caspase inhibitors. It is also conceivable that the sudden release ofthe acidic content and metal ions from lysosomes upon treatmentwith 1 contributed to cell death in a manner that did not directlyimplicate the translocation of lysosomal proteases, and so explicitcell death pathways remain open to question. Regardless, ourresults provide substantial evidence of an alternative mechanismof action of a natural angucycline that does not involve genomicDNA. Targeting the lysosome to elicit cell death is emerging as anattractive therapeutic strategy for the treatment of cancers39,45,46.For example, the clinically approved drug CQ has been shown toprevent the relapse observed in certain cancers refractory to aDXR regimen4750. Accordingly, marmycin Aand perhaps otherangucyclinesrepresent valuable probes with which to study thebiology of the lysosome, leaning towards unanticipated therapeuticapplications for this class of natural products.

MethodsCell culture and proliferation assays. U2OS, A2780 and MDA-MB-231 cellswere purchased from ATCC and were maintained in McCoys 5A or RPMI1640, respectively, supplemented with 10% fetal bovine serum (FBS) and1 antibioticantimycotic (Gibco) and incubated at 37 C with 5% CO2.Cell viability assays were carried out by plating 2,000 cells per well in 96-wellplates. Cells were treated with the relevant drug for 24 h or 72 h, then incubatedwith CellTiter-Blue (20 l/well) for 1 h before recording uorescence(560(20)Ex/590(10)Em) using a PerkinElmer Wallac 1420 Victor2 MicroplateReader. For cell cycle analysis, cells were xed in ice-cold 70% ethanol, stainedwith propidium iodide (10 mg ml1 PI, 250 mg ml1 RNase A, 0.5% BSA, 0.02%sodium azide in 1 PBS) and analysed by FACS on a CyFlow Ploidy Analyserfrom Partec.

Drugs and inhibitors. Marmycin A was prepared in the laboratory according toFig. 2. Cells were treated with 10 M marmycin A for 24 h unless stated otherwise.Doxorubicin (Sigma) was used at 1 M for 6 h (cell imaging) or 1 M for 24 h(western blotting); chloroquine (Sigma) was used at 100 M for 24 h; N-acetylcysteine (Sigma) was used at 2 mM for 24 h (2 h pre-treatment). Z-DEVD-FMK(BD Biosciences) was used at 100 M for 24 h (30 min pre-treatment). Z-VAD-FMK(BD Biosciences) was used at 100 M for 24 h (30 min pretreatment). E-64(Sigma) was used at 15 M (added 48 h after 1, cells harvested at 96 h). CA-074 Me(Enzo Life Sciences) was used at 1 M (added 48 h after 1, cells harvested at 96 h).N-tosyl-L-phenylalanine chloromethyl ketone (TPCK, Sigma) was used at5 M (added 48 h after 1, cells harvested at 96 h). Artesunate was purchasedfrom Sigma.

Immunouorescence analysis.U2OS cells were cultured and treated with 1 or DXRat 40% conuence. LysoTracker Blue DND-22 (L7525, Life Technologies) and 1were added and used without xation in live cell imaging experiments. Fluorescencewas recorded 10 min after the addition of both reagents. GFP-Lamp1 was transientlyexpressed in U2OS cells following the manufacturers instructions. Briey, 5 lof CellLight Lysosomes-GFP BacMam 2.0 (C10596, Life Technologies) was mixedwell with 200 M U2OS culture medium and added to each well of a 24-well plateloaded poly-L-lysine-treated coverslips. After 16 h incubation, cells were washed withPBS and treated with 1 (10 M, 6 h). Cells were then washed with PBS, xed for12 min with 2% formaldehyde/PBS and permeabilized for 10 min with 0.1% TritonX-100/PBS, then washed with PBS. The Golgi apparatus was detected using ananti-RCAS1 antibody (1/100, #12290, Cell Signaling) and anti-rabbit Alexa Fluorsecondary antibody (1/500, A-11008, Life Technologies). Coverslips weremounted with VectaShield mounting medium with or without DAPI (VectorLaboratories). Images were taken with Leica SP8 microscope or Nikon Te(-E)inverted confocal microscopes equipped with a spinning disk YokogawaCXU-X1 A1 (DND-22 experiment only). Data were analysed with ImageJsoftware (NIH).

Western blotting. Cells were treated as indicated in Figs 3 and 4, then washed twicewith PBS and lysed with 2 Laemmli buffer. Cell extracts were heated for 5 min at95 C, sheared through a 26-gauge needle and quantied with a Nanodrop

Marmycin A ArtesumycinLandomycin E

Hedamycin

Doxorubicin(clinically approved)

Artesunate

Chloroquine(clinically approved)

Caspase-dependentcell death

Protease/caspase-independent cell death

?

Lysosome

DNAdamage

?

Figure 5 | Antiproliferative mechanisms of anthraquinone-containingdrugs. Black arrows indicate previously established mechanisms. Red arrowshighlight the cellular activity of marmycin A. Black arrows with questionmarks indicate putative mechanisms of action of other anthraquinone-containing small molecules.




2000 (Thermo Scientic). Protein lysates (100 g) were loaded on a 420%Mini-PROTEAN TGX Stain-Free Gel (BioRad), resolved by SDS-PAGEelectrophoresis and transferred onto a nitrocellulose membrane (Amersham).Proteins were probed with the following antibodies: anti--actin (ab8226, Abcam),anti-H2AX (PA1-14198, Thermo Scientic), anti-H2AX (#2577, Cell Signaling),anti-p21 (#2947, Cell Signaling), anti-p53 (#2524, Cell Signaling), anti-p-p53(#9284, Cell Signaling), anti-p62 (610,833, BD Transduction Laboratories), anti-LC3(#2775, Cell Signaling), anti-Bid (#2002, Cell Signaling) and anti-Caspase 3 (#9665,Cell Signaling) and were diluted 1/1,000 in 5% BSA, 0.1% Tween-20/TBS. Thesecondary antibodies were anti-rabbit HRP (A120-108P, Bethyl), anti-mouse HRP(A90-116P) and anti-donkey HRP (A140-107P, Bethyl). Antigens were detectedby electrochemiluminescence (Amersham). Imaging was performed using aChemiDoc XRS+ System.

Received 31 December 2014; accepted 10 June 2015;published online 20 July 2015

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AcknowledgementsThe authors thank the CNRS for funding, and the Imagif Cell Biology Unit, J.-F. Gallardand F. Blanchard for assistance with cell imaging, NMR spectroscopy and X-raycrystallography, respectively. R.R. thanks J.A. Yeoman, S. Mller, J.E.Moses, S.L. Buchwald,L. Johannes, M. Mehrpour and members of R.R.s laboratory for discussions andproofreading of this manuscript. Research in R.R.s laboratory is supported by theEuropean Research Council (grant no. 647973), the Fondation pour la Recherche Mdicale(grant no. AJE20141031486), the Emergence Ville de Paris Program and the LigueNationale Contre le Cancer.




Author contributionsR.R., with contributions from T.C., G.M. and M.M., conceived and designed theexperiments. T.C., with assistance from F.G., synthesized the marmycins and artesumycin.T.T.M. with contribution from T.C. performed proliferation assays, uorescence-activatedcell sorting, western blotting and cellular imaging. R.R., G.M. and M.M. supervised theresearch. All the authors analysed the data. R.R. wrote the manuscript. All the authorscommented on the manuscript. T.T.M. and F.G. contributed equally to this work.

Additional informationSupplementary information and chemical compound information are available in theonline version of the paper. Reprints and permissions information is available online atwww.nature.com/reprints. Correspondence and requests for materials should be addressed to R.R.

Competing nancial interestsThe authors declare no competing nancial interests.




Synthesis of marmycin A and investigation into its cellular activityResultsChemical synthesis of the marmycinsCellular activity of marmycin A

DiscussionMethodsCell culture and proliferation assaysDrugs and inhibitorsImmunofluorescence analysisWestern blotting

Figure 1 Anthraquinone-containing natural products.Figure 2 Chemical synthesis of the marmycins.Figure 3 Marmycin A kills human cancer cells independently of genome targeting.Figure 4 Marmycin A accumulates in the lysosomes and triggers programmed cell death.Figure 5 Antiproliferative mechanisms of anthraquinone-containing drugs.ReferencesAcknowledgementsAuthor contributionsAdditional informationCompeting financial interests

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synthesis of marmycin a and investigation into its cellular activity

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