Overcoming drug resistance by cell-penetrating peptide-mediated delivery of a doxorubicin dimer with high DNA-binding affinity

Accepted Manuscript Overcoming drug resistance by cell-penetrating peptide-mediated delivery of a doxorubicin dimer with high DNA-binding affinity Marco Lelle, Christoph Freidel, Stefka Kaloyanova, Ilja Tabujew, Alexander Schramm, Michael Musheev, Christof Niehrs, Klaus Müllen, Kalina Peneva PII:

S0223-5234(17)30139-3

DOI:

10.1016/j.ejmech.2017.02.056

Reference:

EJMECH 9252

To appear in:

European Journal of Medicinal Chemistry

Received Date: 10 November 2016 Revised Date:

23 February 2017

Accepted Date: 24 February 2017

Please cite this article as: M. Lelle, C. Freidel, S. Kaloyanova, I. Tabujew, A. Schramm, M. Musheev, C. Niehrs, K. Müllen, K. Peneva, Overcoming drug resistance by cell-penetrating peptide-mediated delivery of a doxorubicin dimer with high DNA-binding affinity, European Journal of Medicinal Chemistry (2017), doi: 10.1016/j.ejmech.2017.02.056. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

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Overcoming drug resistance by cell-penetrating peptidemediated delivery of a doxorubicin dimer with high DNAbinding affinity

Marco Lelle,[a, b, c] Christoph Freidel,[c] Stefka Kaloyanova,[c] Ilja Tabujew,[a, c] Alexander Schramm,[d] Michael Musheev, [e] Christof Niehrs,[e, f] Klaus Müllen,[c] and Kalina Peneva*[a, c]

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[a] Prof. Dr. K. Peneva, Dr. M. Lelle Institute of Organic and Macromolecular Chemistry Jena Center of Soft Matter Friedrich Schiller University Jena Lessingstr. 8, 07743 Jena, Germany E-mail: [email protected] [b] Dr. M. Lelle Institute of Physiology II University Hospital Jena Kollegiengasse 9, 07743 Jena, Germany [c] Dr. M. Lelle, Dr. S. Kaloyanova, M. Sc. C. Freidel, Dipl. Chem. I. Tabujew, Prof. Dr. K. Müllen, Prof. Dr. K. Peneva Max Planck Institute for Polymer Research Ackermannweg 10, 55128 Mainz, Germany [d] PD Dr. A. Schramm Clinic for Pediatrics III University Hospital Essen Hufelandstraße 55, 45122 Essen, Germany [e] Dr. M. Musheev, Prof. Dr. C. Niehrs Institute of Molecular Biology Ackermannweg 4, 55128 Mainz, Germany [f] Prof. Dr. C. Niehrs Division of Molecular Embryology DKFZ-ZMBH Alliance, 69120 Heidelberg, Germany

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Supporting information for this article is given via a link at the end of the document.

Abstract: We describe the synthesis and characterization of a novel bioconjugate, consisting of an octaarginine cell-penetrating peptide and a highly DNA-affine doxorubicin dimer. The linkage between the two components is composed of a cleavable disulfide bond, which enables the efficient intracellular delivery of the cytotoxic payload within the reductive environment of the cytosol, mediated through glutathione. To determine the DNA-binding affinity of the dimeric drug molecule, microscale thermophoresis was applied. This is the first utilization of this method to assess the binding interactions of an anthracycline drug with nucleic acids. The cytotoxic effect of the peptide-drug conjugate, studied with drug-sensitive and doxorubicin-resistant cancer cells, demonstrates that the bioconjugate can successfully overcome drug resistance in neuroblastoma cells.

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1. Introduction The anthracycline doxorubicin belongs to a class of antitumor agents with a wide spectrum of activity against human cancers (e.g. leukemia, breast cancer, soft tissue sarcomas, childhood solid tumors, aggressive lymphomas), while only a few cancer types including colon cancer are unresponsive.[1-3] Nevertheless, the therapeutic efficacy of the anticancer drug doxorubicin is limited by diverse side effects such as the onset of drug resistance in tumors, cardiotoxicity accompanied by congestive heart failure and poor tumor selectivity.[2-5] To overcome the lack of selectivity, doxorubicin and other anticancer agents have been modified with targeting moieties, which can address cancer cells through specific binding sites or overexpressed receptors. In this regard peptide hormone,[6] folic acid,[7] antibody,[8] transferrin and albumin drug conjugates[9] play an undisputed role and enable the site-selective delivery of the antitumor agents. [10] [11] However, the vast majority of these conjugates are unable to overcome drug resistance in cancer cells.

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Figure 1. Chemical structure of doxorubicin composed of a DNA-intercalating tetracyclic ring system and the sugar daunosamine.

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Drug resistance is one of the primary reasons for the failure of many anticancer agents such as doxorubicin (Figure 1). The mechanisms related to this drug inefficiency typically involve membrane proteins (e.g. P-glycoprotein) that belong to the ATP-binding cassette transporter superfamily.[3, 12, 13] These membrane-associated transporter proteins efficiently excrete drugs from the cytoplasm in an energy-dependent manner and thus decrease the intracellular level of anticancer agents, which renders them less effective.[14] This phenomenon is also termed multidrug resistance as doxorubicin is not the only substrate of these membrane transporters, but it also affects numerous structurally unrelated substances, such as etoposide, paclitaxel or vinblastine.[15] There is clear evidence for the overexpression of these membrane proteins in many different cancer cells and often their activity is even increased after exposure to antitumor drugs.[13, 15] Nevertheless, expression of the transporter proteins is not an exclusive feature of cancer cells. Healthy cells use these proteins to remove harmful xenobiotics and their metabolites, whereby, tissue detoxification and protection, respectively, are ensured.[3, 14] By contrast, heart muscle cells have a deficiency in detoxifying mechanisms. The expression of membrane transporters like P-glycoprotein is often reduced. Moreover, the capacity to enzymatically detoxify oxygen radicals, which are typically produced by the quinone-hydroquinone redox system of anthracycline drugs, is limited.[2, 16] Thus, severe heart damage is induced by reactive oxygen species during the anticancer therapy with doxorubicin.[17] In order to diminish the cardiotoxicity, more toxic anthracycline derivatives have to be applied. Higher toxicity enables the utilization of lower drug doses with the consequence that the radical formation will be decreased. Anthracycline dimers exhibit an enhanced cytotoxic effect mediated through their increased DNA affinity by bis-intercalation between the base pairs and have the ability to overcome drug resistance.[18, 19] The strengthened binding to DNA has the potential to create a more efficient topoisomerase poison, due to a stronger inhibition of the enzyme, which is the primary mechanism of action responsible for the antitumor effect of doxorubicin.[3, 20] In spite of that, all previously reported anthracycline dimers have the disadvantage that they cannot enable tumor-targeted drug delivery. This is due to the lack of a further functional group, which could be modified with appropriate targeting sequences. 2

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In this work, we introduce a reductively cleavable bioconjugate that is capable of overcoming all the aforementioned limitations. Applying an amino acid-based heterotrifunctional cross-linking reagent, we prepared a doxorubicin dimer carrying a 2-pyridyl disulfide.[21] This mixed disulfide can be easily and irreversibly modified with various thiol-containing biomolecules.[22, 23] The octaarginine cellpenetrating peptide with an N-terminal cysteine, described here, enables a targeted and rapid internalization of the final bioconjugate into the cytosol of cancer cells.[24, 25] Utilizing this carrier, we could successfully bypass the activity of membrane proteins such as P-glycoprotein, increase effectively the intracellular concentration and thus enhance the efficacy of the drug in anthracycline resistant cells.

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2. Results and Discussion

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2.1 Cross-linker preparation Recently, we have presented the synthesis and characterization of a novel lysine-based heterofunctional cross-linking reagent 8, which can lead to the peptide-drug conjugate.[21] This cross-linker (Scheme 2) contains the necessary chemical scaffold for attaching two doxorubicin molecules and site-selectively introducing a cysteine-containing cell-penetrating peptide. The synthesis was accomplished as follows; L-lysine was modified to carry two aminooxy groups and a 2-pyridyl-disulfide functionality, respectively. In order to introduce the sulfhydryl-reactive functional group, cysteamine hydrochloride 2 was, first, transformed into 2-(2-pyridyldithio)ethylamine hydrochloride 3 with an excess of 2,2’dipyridyl disulfide according to a literature procedure (Scheme 2).[23] In another step, (bocaminooxy)acetic acid 4 was reacted to its N-hydroxysuccinimide ester 5 as described before.[26] This activated ester could be used to introduce the doxorubicin-reactive aminooxy groups onto the peptide scaffold. N SH

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Scheme 1. Synthesis of the reactive precursors for the modification of the amino acid lysine. Reagents and conditions: (a) 2,2’-dipyridyl disulfide (6 equiv), MeOH, argon, overnight, rt, quantitative yield; (b) NHS (1.05 equiv), DIPC (1.15 equiv), DCM, argon, 4 h, rt, 92 %

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After the synthesis of the reactive side chains, the modification of lysine was carried out. Amino acids such as cysteine, glutamic acid or lysine are well suited for the preparation of heterofunctional crosslinkers, if appropriate protective groups are employed, since they exhibit already orthogonal functional groups.[27, 28] The accessible carboxylic acid of the N,N’-Di-boc-L-lysine dicyclohexylammonium salt 6 was coupled to the amine of 3 to attach the thiol-reactive group to the amino acid scaffold (Scheme 2). For the subsequent functionalization of the amino groups of the lysine derivative, the protective groups had to be removed. The deprotection was completed rapidly and in quantitative yield under strongly acidic conditions. Afterwards, the deprotected amino acid was reacted without further purification with the active ester 5, to obtain the desired cross-linking reagent 8. The two Boc protective groups of 8 were cleaved prior to bioconjugation, to avoid side reactions, since aminooxy groups are particularly strong nucleophiles and therefore highly reactive.

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Scheme 2. Synthesis of the amino acid-based heterofunctional cross-linking reagent. Reagents and conditions: (a) N,N’-di-boc-L-lysine dicyclohexylammonium salt, 3 (1.1 equiv), TSTU (1.1 equiv), DIPEA (4 equiv), DMF, 4 h, rt, 78 %; (b) DCM/TFA (1:1), 1 h, rt, quantitative yield; (c) DIPEA (10 equiv), 5 (2 equiv), DMF, argon, 3 h, rt, 81 %

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2.2 Bioconjugation between doxorubicin and cell-penetrating peptide Initially, the doxorubicin dimer carrying a 2-pyridyl disulfide was synthesized. After the deprotection of the heterofunctional cross-linker, the condensation with the aliphatic ketone of two doxorubicin molecules was performed (Scheme 3). The reaction was accomplished under mild acid-catalyzed conditions, which have been already previously established, with a slight excess of the drug (2.2 equiv).[29, 30] Unreacted doxorubicin was removed by gel permeation chromatography and the crude product was purified by reversed phase HPLC. Subsequently, the coupling between the dimeric drug molecule and the polyarginine cell-penetrating peptide was carried out in a neutral buffer solution (Scheme 3). The reaction was rapidly finished in an argon atmosphere and the desired bioconjugate was obtained after HPLC purification in good yield (72 %). The peptide-drug conjugate (10) reported here has the potential to deliver the cytotoxic dimeric anthracycline derivative after cellular internalization and subsequent cleavage of the disulfide.

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Scheme 3. Synthesis of the functional doxorubicin dimer and subsequent bioconjugation with the cellpenetrating peptide octaarginine. Reagents and conditions: (a) DCM/TFA (1:1), 1 h, rt, quantitative yield; (b) doxorubicin hydrochloride (2.2 equiv), DMF/0.4 M sodium acetate buffer (1:1) - pH 4.8, 24 h, rt, 71 %; (c) Ac-CRRRRRRRR-NH2 (1.1 equiv), DMF/25 mM TEAA buffer (1:3) - pH 7, argon, 3 h, rt, 72 % 2.3 Intracellular drug delivery mediated by glutathione To assess the release of the cytotoxic substance from the bioconjugate, a 1 mM solution of 10 was incubated in the presence of a tenfold excess of glutathione. This naturally occurring tripeptide is a ubiquitous reducing agent in mammalian cells.[31] The intracellular level of glutathione is physiologically in the millimolar range (up to 10 mM), whereas micromolar concentrations are found in blood plasma.[32] Moreover, the intracellular concentration in tumor cells is typically elevated, which can enable efficient drug delivery inside the cancer cells.[33] 5

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The reduction of the peptide-drug conjugate was monitored by HPLC at 480 nm, the characteristic absorption of doxorubicin, for 24 h (Figure 2) and the identification of the cleavage products was accomplished via MALDI-TOF mass spectrometry. Even a short incubation time of 1 min caused a complete cleavage of the disulfide bond present in the doxorubicin-octaarginine bioconjugate, which would occur in the chromatogram at around 20 min. The cleavage of the disulfide bond in the presence of an excess of the reducing agent glutathione was highly efficient and led to a dimeric doxorubicin molecule with a free thiol as main product (Figure 2, a). Moreover, a small amount of another dimer appeared, a mixed disulfide between the anthracycline derivative a and glutathione. The concentration of this species (Figure 2, b) increased over time as visible in the chromatogram at 3 h. After 24 h of incubation, the formation this substance came to a halt, but a new compound was identified, corresponding to the „dimer of the dimer“ (Figure 2, c). This dimerization probably took place through intermolecular disulfide formation between two molecules of a. Although molecule c was the predominant anthracycline derivative after 24 h, it is unlikely that this substance will appear under physiological conditions, since a constant glutathione level is maintained in mammalian cells by enzymatic processes.[31] Thus, compound c will be permanently reduced to the doxorubicin dimer with the free sulfhydryl group. It is expected that the same will happen to the mixed disulfide with glutathione, and the desired cytotoxic agent will be released within minutes. During our study, free doxorubicin was not detected. Incubation at lower pH values, such as 5.0, which are present in endosomes and around cancer cells, could not degrade the dimeric structure as well, due to the hydrolytic stability of the oxime bond.[34, 35] Therefore, this is an attractive system for the targeted delivery of a doxorubicin dimer. 2.4 Determination of the DNA-binding affinity

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Having successfully demonstrated that elevated glutathione levels in tumor cells can cleave the disulfide of the peptide-drug conjugate and release a dimeric doxorubicin molecule, the affinity of the anthracycline derivative to DNA was determined. To investigate this binding event, microscale thermophoresis was applied. MST is a novel and powerful tool to assess and quantify binding affinities and has been used to study a wide range of biomolecular interactions.[36, 37] The technique is based on thermophoresis, which is the directed movement of molecules along a temperature gradient.[37-39] Thermophoresis depends on surface characteristics of the molecule, such as size, hydrophobicity and charge.[40-42] Thus, MST is well suited to obtain deeper insights about the binding of doxorubicin to DNA, since the interaction between the DNA-intercalating anthracycline dimer and the double-strand will change the aforementioned parameters. Contrary to other methods that have been utilized for the investigation of binding affinities, MST has several advantages, as it can be employed to quickly measure at a high sensitivity and low costs with a small amount of sample (volume less than 500 nL).[37, 40, 41] In order to examine the thermophoretic movement in small capillaries upon irradiation with an infrared laser, a fluorophore-labeled DNA molecule was applied and the emitted light after excitation was detected. Due to the temperature-dependent diffusion, the fluorescence in the heated area can either drop or increase, a phenomenon based on the Soret effect.[38] For the present binding study a Cy5labeled model DNA consisting of 36 random base pairs was chosen and titrated with the doxorubicin dimer 9. Although the pyridyl disulfide carrying anthracycline derivative is not the main cleavage product, this

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Figure 2. Glutathione-mediated drug release from 10. (A) Confirmation of the disulfide cleavage by HPLC, monitored at 480 nm. (B) Chemical structures of the degradation products of 10 after the incubation with a tenfold excess of glutathione (10 mM) as determined by mass spectrometry.

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substance was selected because the molecule has the highest structural similarity to the dimeric doxorubicin molecule with the free thiol functionality. However, 9 is more convenient to handle, as it has no free sulfhydryl group, which could create side products upon oxidation. The titration reveals that an increase in the dimer concentration leads to an elevated fluorescence signal until saturation is observed. Normalization of these data with the fluorescence signal from the capillaries before heating generates values of the fluorescence that can be plotted against the applied dimer concentration to yield the desired sigmoidal binding curve after adequate fitting (supporting information). The fit function derived from the law of mass action was utilized to obtain the dissociation constant Kd, which was directly used to determine the DNA-binding affinity as Ka. The assigned affinity of 9 to the Cy5-labeled model DNA was 7.9 x 106 M-1. The Ka of doxorubicin alone to the same DNA was determined to be 8.8 x 105 M-1, which means that the binding of the dimeric doxorubicin molecule to the model DNA is around ten times stronger ( Table 1). This method is well suited for a 1:1 binding stoichiometry and the DNA indeed is short enough to bind a maximum of one anthracycline dimer. Additionally, no binding between 9 or doxorubicin and a double-stranded model DNA consisting of 12 random base pairs could be detected with MST. However, a DNA with 36 base pairs is long enough to produce more complex stoichiometries with native doxorubicin or related anthracycline molecules. To take the stoichiometry into account, another method, which was deduced from the Hill equation, was applied. The fit function from this equation does not yield dissociation constants to calculate the DNA-binding affinity, but furnishes the concentration at which 50 % of the titrant is bound to the fluorescent DNA molecule. The EC50 of the anthracycline dimer was determined to be 150 ± 4.3 nM, which is in good agreement with the obtained dissociation constant ( Table 1). 7

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This value was also compared to the data for doxorubicin. For the native anthracycline the EC50 is 1160 ± 8.7 nM, which provides evidence that also in this case the binding strength of 9 is roughly tenfold higher as compared to doxorubicin. A meaningful comparison of the present DNA-binding data of doxorubicin and 9 with literature values is not straightforward due to the heterogeneity of the previously conducted experiments that involved different methods and systems. It is well known that Kd is linked to a given set of buffers and conditions, thus, even if similar DNA and anthracycline molecules have been applied before, the determined affinity constants varies by a factor of 100.[43, 44] However, MST is a robust technique, which gives the opportunity to quickly rank ligands in terms of their affinity to DNA. The obtained binding data clearly indicate that the anthracycline dimer has a much greater affinity to DNA than doxorubicin, a crucial prerequisite for the expected enhanced cytotoxicity.

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Table 1. Dissociation and affinity constants as well as EC50 values for the interaction of 9 or doxorubicin with the Cy5-labeled double-stranded model DNA consisting of 36 random base pairs as determined by MST.

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2.5 Cell viability studies The antiproliferative effect of the peptide-drug conjugate 10 on different cell lines was studied to investigate whether the enhanced binding of the doxorubicin dimer to DNA can lead to a more cytotoxic substance. Additionally, 9 was tested, to assess if the cell-penetrating peptide was an efficient delivery agent, and the determined values were compared to those of the native anthracycline doxorubicin and the polyarginine cell-penetrating peptide (Ac-CRRRRRRRR-NH2) alone. The in vitro cytotoxic effects of the employed substances were studied with cell lines from tissues where doxorubicin is usually applied in vivo and examined with a luminescence-based cell viability assay. One of these cell lines is the MCF-7 breast cancer cell line. As expected, the antiproliferative effect of doxorubicin on these adenocarcinoma cells was very strong. The obtained IC50 values for 9 (6.73 ± 2.44 µM) and 10 (3.66 ± 0.77 µM) were higher than for doxorubicin (0.90 ± 0.12 µM). However, the bioconjugate 10 was more cytotoxic as compared to 9. This supports the conclusion that the cellpenetrating peptide facilitates the cell entry of the anthracycline dimer. Doxorubicin is widely applied as a first-line therapy for the treatment of childhood solid tumors including neuroblastoma. In this work, we have used the wild-type human neuroblastoma cell line Kelly-WT and its adriamycin-resistant subline Kelly-ADR, which is generated by continuously exposing Kelly-WT cells to increasing doses of doxorubicin which is in accord with the IC50 values obtained from the present cytotoxicity study. Doxorubicin had a high activity in Kelly-WT cells, whereas the IC50 value for Kelly-ADR cells was nearly 40-times higher (6.57 ± 0.61 µM). A similar pattern was also observed for the unfunctionalized dimer 9, although the difference between the two sublines was not so pronounced (Table 2). The cytotoxic effect of the peptide-drug conjugate on KellyWT cells was comparable to that of 9, but not as strong as the one of the native drug. Contrary to the other utilized substances, the antiproliferative action of the bioconjugate in wild-type and drug-resistant cells was nearly the same. In comparison with doxorubicin the cytotoxicity of 10 against Kelly-ADR cells was increased by a factor of 3 according to the obtained IC50 values. Even the unmodified dimer had roughly the same cytotoxic effect on Kelly-ADR cells as doxorubicin. 8

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The most reasonable explanation for this phenomenon is the resistance of the investigated cell line towards doxorubicin, which renders the drug less effective, because the anthracycline is efficiently excreted from Kelly-ADR cells by membrane-associated transporter proteins.[15] By contrast, the peptide-drug conjugate can probably avoid the drug efflux pumps by its different uptake mechanisms, which are based on endocytosis as well as direct translocation through the plasma membrane.[45] Moreover, polyarginine cell-penetrating peptides are well known to rapidly distribute into the nucleus, a property that is beneficial for DNA-active drugs like doxorubicin.[46] Thus, the probability that the substance meets the transporter proteins is significantly reduced. Doxorubicin typically enters cells via passive diffusion and is thus an easy target for the drug efflux pumps on the inner side of the plasma membrane.[47] The lowered cytotoxicity of 9 is presumably attributed to poor cellular uptake, which emphasizes once more the potential of the cell-penetrating peptide for the delivery of the cytotoxic freight. Additionally, the antiproliferative effect of the polyarginine peptide itself was studied, to investigate whether the cytotoxicity can be assigned to the dimer or if there is an influence from the peptide. The conducted luminescence-based cell viability assay has revealed only a moderate toxicity of the cellpenetrating peptide in all utilized cell lines. Even at concentrations of 100 µM no IC50 values could be determined. This indicates that the presented bioconjugate 10 is a powerful drug delivery system, which can overcome drug resistance in neuroblastoma cells. Table 2. In vitro cytotoxic effects of 9, 10, doxorubicin and the polyarginine cell-penetrating peptide on MCF-7, Kelly-WT and Kelly-ADR cells, as determined by the CellTiter-Glo luminescent cell viability assay, 72 h after the incubation with the substances (data expressed as IC50 values in µM, n ≥ 3, mean ± standard deviation). MCF-7 IC50 (µM) 6.73 ± 2.44 3.66 ± 0.77 0.90 ± 0.12

Kelly-WT IC50 (µM) 1.79 ± 0.44 2.47 ± 0.91 0.18 ± 0.08

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2.6 Investigation of the intracellular distribution To get deeper insights into the cellular distribution and the mode of action of the peptide-drug conjugate, doxorubicin-sensitive Kelly-WT neuroblastoma cells and its drug-resistant counterpart Kelly-ADR were incubated with a 10 µM solution of 10 and investigated by fluorescence confocal laser scanning microscopy at various points of time (Figure 3). The obtained results were compared to doxorubicin, which was applied under the same conditions, to better understand the cytotoxicity data achieved by the cell viability assay. After 24 h, the doxorubicin-associated fluorescence (red) was already visible for both substances in Kelly-WT and Kelly-ADR cells. The bioconjugate 10 was efficiently taken up by drug-sensitive KellyWT cells, whereas it was mainly located at membrane regions of Kelly-ADR cells, which means that the uptake in wild-type cells is faster. Cell-penetrating peptides like the utilized polyarginine often induce uptake by concentrating at the negatively charged cell membrane either due to the interaction with heparan sulfate proteoglycans or to the disruption of the membrane (inverted micelle model, models involving the formation of membrane pores and the carpet model).[46, 48] In contrast, the native drug was found inside the two different cell types, although no doxorubicin could be detected in the nuclei of the drug-resistant Kelly-ADR cells. This is a good indication for the reduced cytotoxicity of the native anthracycline that is evolving its antiproliferative effect in the cell nucleus. Overall, the 9

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uptake of the peptide-drug conjugate by Kelly-ADR cells was lower than that of doxorubicin, which is probably attributed to the uptake mechanism of the native drug. Doxorubicin can typically cross cell membranes quickly by passive diffusion, to accumulate in cancer cells.[47] Contrary to that, cationic cell penetrating peptides are not only taken up by direct translocation, but also by more time demanding processes such as endocytosis.[45, 49, 50] However, in Kelly-WT cells 10 and doxorubicin showed nearly the same uptake. Prolonged incubation of the anthracycline-sensitive cells for altogether 72 h with the drugs displayed that doxorubicin enters the cancer cells better than 10. Moreover, doxorubicin is more enriched in the nucleus, while the peptide-drug conjugate is also observed in the cytosol. A possible explanation for this finding could be the endosomal entrapment of the anthracycline derivative or the uncleaved peptide-drug conjugate. This observation supports the data from the cell viability studies, where the native drug had a stronger antiproliferative effect. The investigation of Kelly-ADR cells by fluorescence microscopy after 72 h showed a different result, which is nevertheless also supporting the obtained values from the cytotoxicity assay. In this case, doxorubicin could be seen neither in the cytosol nor in the nucleus. The amount of drug inside the cancer cells was even reduced compared to the study after 24 h. This effect is most probably related to the drug efflux by membrane transporters, which is significantly diminishing the intracellular doxorubicin concentration. Compared with the native anthracycline, the bioconjugate 10 exhibited much greater uptake. Additionally, the anthracycline-associated fluorescence was not only located at the cell membrane, but also in the nuclei. Thereby, 10 can lead to a stronger antiproliferative effect than doxorubicin and is capable of overcoming drug resistance in neuroblastoma cells. This follows from the uptake mechanism of the peptide-drug conjugate, whereby drug efflux pumps can be efficiently bypassed.

Figure 3. Examination of the cellular distribution of doxorubicin and the peptide-drug conjugate 10 in Kelly-WT as well as Kelly-ADR cells by fluorescence confocal laser scanning microscopy. Images were taken 24 and 72 h after incubation with 10 µM of the drug at 37°C. The corresponding brightfield images are depicted below. Anthracycline-associated fluorescence is visualized by red color. Scale bars represent 15 µm. 10

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After the membrane-associated transporter proteins have been avoided, the bioconjugate must be cleaved and the cytotoxic agent must enter the nucleus. The successful cleavage by glutathione was already demonstrated and the presence in the nucleus was investigated using fluorescence confocal laser scanning microscopy as well. For that reason, Kelly-WT and Kelly-ADR cells were incubated with 10 as before and the nuclei were simultaneously stained with DRAQ5, an infrared fluorescent stain specific to DNA (blue, Figure 4). The corresponding colocalization is displayed by violet color and highlights that the cleaved anthracycline derivative can enter the cell nucleus of both doxorubicin-sensitive and drug-resistant neuroblastoma cells. This is convincing proof that the highly DNA-affine anthracycline dimer can enter its target localization after glutathione-mediated cleavage and thereby can successfully overcome drug resistance.

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Figure 4. Investigation of the nuclear localization of the anthracycline drug in Kelly-WT and KellyADR cells by fluorescence confocal laser scanning microscopy, 72 h after the incubation with 10 µM of 10. Red color illustrates doxorubicin-related fluorescence, whereas the nuclear stain DRAQ5 is depicted in blue. The right panels display the overlay of the drug-associated fluorescence and the fluorescence of the counterstain. Corresponding colocalization is illustrated by violet color. Scale bars represent 15 µm.

In this work, we have presented the preparation of a dimeric molecule that consists of two doxorubicins and a cell-penetrating moiety connected via a cleavable bond. We have demonstrated a simple and efficient way to prepare this rather complex therapeutically active molecule that was obtained in good yield and high purity. This anthracycline dimer has an increased DNA affinity, as determined by microscale thermophoresis. Unlike all other examples described in the literature, the resulting molecule possesses an additional functional group that enables the attachment of targeting moiety or carriers like cell-penetrating peptides. We also report the first utilization of MST for determination of the binding between an anthracycline drug and DNA. It appears that this method is well suited for investigating the affinity and binding interactions between therapeutic molecules and nucleic acids due to its high sensitivity and low costs. This highly DNA-affine doxorubicin dimer can be efficiently delivered inside neuroblastoma cells, as confirmed by cell viability assays and confocal laser scanning microscopy. A higher toxicity in adriamycin-resistant cells than doxorubicin is detected. The higher toxicity of the conjugate can enable the utilization of lower drug doses. This can result in decreased formation of toxic radicals that are 11

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particularly damaging to the heart muscle cells, as they normally have a deficiency of detoxifying mechanisms. This can in turn lead to a diminished cardiotoxicity, one of the severe side effects observed following anthracycline treatment in the clinic. The utilization of bioconjugate chemistry presented here is not only limited to the synthesis of dimeric molecules but can also allow the preparation of antibody-drug conjugates with increased molar loading of the drug. The trifunctional cross-linker applied here is a small, but central part of the conjugate that incorporates the right balance between plasma stability and efficient drug release at the tumor cells. This feature is paving the way to an improved design of chemotherapeutic drugs that can overcome multidrug resistance in patients. 4. Experimental Section 4.1 GENERAL INFORMATION

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All chemicals, including solvents and reagents, were bought from commercial sources (Acros Organics, Alfa Aesar, AppliChem, Deutero, Fisher Scientific, Fluka, Merck, Sigma Aldrich) and used without further purification. Doxorubicin hydrochloride was purchased from Ontario Chemicals, Inc. (Guelph, Ontario, Canada) and custom peptide synthesis of Ac-CRRRRRRRR-NH2 was carried out from Genosphere Biotechnologies (Paris, France). The fluorophore-labeled double-stranded model DNA consisting of 36 random base pairs (5’-Cy5TAAGCTGACTCAGATATCACGTGGCAGAACGGTCGA-3’) was purchased from Thermo Fisher Scientific GmbH (Ulm, Germany). CellTiter-Glo luminescent cell viability assay was purchased from Promega (Mannheim, Germany). Bio-Gel P-2 for gel permeation chromatography was obtained from Bio-Rad Laboratories, Inc. and silica gel column chromatography purification was performed on Macherey-Nagel silica gel (0.063-0.200 mm). Thin layer chromatography was conducted on ALUGRAM SIL G/UV254 sheets with suitable solvents. Proton and carbon NMR spectra were recorded on Bruker AMX 300, Bruker WS 700 and Bruker WB 850 devices. Chemical shifts are expressed in parts per million (ppm) relative to the residual solvent signals of DMSO-d6 and D2O.[51] Coupling constants (J) are given in hertz (Hz). A Bruker Reflex II-TOF spectrometer equipped with a 337 nm nitrogen laser was utilized for MALDI-TOF mass spectrometry measurements and 2,5dihydroxybenzoic acid as well as α-cyano-4-hydroxycinnamic acid (peptide sample) were applied as matrix. HPLC was accomplished on a Jasco LC-2000Plus System, with a diode array detector (MD2015) and suitable solvent delivery pumps (PU-2086). Analytical HPLC was performed on a ReproSil 100 C18 column (250 x 4.6 mm) with 5 µm particle size as stationary phase at a flow rate of 1 mL/min. Purification by preparative HPLC was conducted on a ReproSil 100 C18 column (250 x 20 mm) at a flow rate of 15 mL/min and 5 µm particle size as stationary phase. 25 mM triethylammonium acetate buffer (pH 7 - A) and acetonitrile (B) were used as eluents in combination with a linear gradient from 0 to 65 % B after 45 minutes. The purity of the synthesized compounds was determined by HPLC and was greater than 95 %. 4.2 SYNTHESIS AND CHARACTERIZATION 2-(2-Pyridyldithio)ethylamine hydrochloride (3).[23] 2,2’-Dipyridyl disulfide (25 g, 113.47 mmol) was stepwise dissolved in 150 mL methanol and degassed in an ultrasonic bath for 30 min. Cysteamine hydrochloride (2.15 g, 18.91 mmol) was gradually added to this solution within half an hour. Subsequently, the flask was sealed with a rubber septum and the reaction mixture was stirred overnight in an argon atmosphere. The yellow solution was precipitated twice in cold diethyl ether. Thereby, the product was obtained as a colorless crystalline solid (4.21 g, 18.91 mmol, quantitative yield). 1H NMR (300 MHz, DMSO-d6): δ = 8.55-8.47 (1H, m, NCH), 8.32 (3H, br s, NH3), 7.89-7.72 (2H, m, 12

ACCEPTED MANUSCRIPT SCCHCH), 7.42-7.35 (1H, m, NCHCH), 3.17-3.02 (4H, m, CH2-CH2); 13C NMR (75 MHz, DMSO-d6): δ = 158.1, 149.8, 137.9, 121.6, 120.0, 37.7, 34.8; m/z (MALDI-TOF): 187.00 [M+H]+.

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(boc-Aminooxy)acetic acid N-hydroxysuccinimide ester (5).[26] Initially, a slurry of (bocaminooxy)acetic acid (4.42 g, 23.12 mmol) and N-hydroxysuccinimide (2.79 g, 24.28 mmol) in 55 mL dry DCM was prepared. N,N’-Diisopropylcarbodiimide (3.06 g, 3.76 mL, 24.88 mmol) was added under argon and the clear solution was stirred for 2 h. Subsequently, additional N,N’Diisopropylcarbodiimide (244 mg, 300 µL, 1.99 mmol) was added and the reaction mixture was stirred for further 2 h. Afterwards, the precipitated urea was removed by filtration and washed with a small amount of DCM. The obtained solution was diluted with DCM to a final volume of 300 mL and washed four times with water. The organic layer was dried with magnesium sulfate and the solvent was removed under reduced pressure. Thus, the product was obtained as colorless solid (6.13 g, 21.27 mmol, 92 %). 1H NMR (300 MHz, DMSO-d6): δ = 10.36 (1H, s, NH), 4.82 (2H, s, O-CH2), 2.84 (4H, s, CH2CH2), 1.42 (9H, s, 3xCH3); 13C NMR (75 MHz, DMSO-d6): δ = 170.0, 165.1, 156.6, 80.6, 69.9, 27.9, 25.5; m/z (MALDI-TOF): 357.04 [M+3Na]+.

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(S)-Di-tert-butyl(6-oxo-6-((2-(2-pyridyldithio)ethyl)amino)hexane-1,5-diyl)dicarbamate (7).[21] N,N’Di-boc-L-lysine dicyclohexylammonium salt (1.52 g, 2.89 mmol), 3 (707 mg, 3.18 mmol) and TSTU (955.9 mg, 3.18 mmol) were dissolved in 35 mL anhydrous DMF. DIPEA (1.49 g, 1.96 mL, 11.55 mmol) was added to the initially prepared solution and the reaction mixture was stirred for 4 h at room temperature in an argon atmosphere. Afterwards, the suspension was diluted with 250 mL ethyl acetate and the organic layer was washed three times with 0.2 M hydrochloric acid. The clear solution was dried with magnesium sulfate and the solvent was removed in vacuo. Subsequently, the residue was purified by silica gel column chromatography using ethyl acetate/hexane (3:1) as eluent, to obtain the product as colorless oil (1.16 g, 2.25 mmol, 78 %). 1H NMR (300 MHz, DMSO-d6): δ = 8.52-8.43 (1H, m, NCH), 8.05 (1H, t, J = 5.4 Hz, CHCONH), 7.88-7.70 (2H, m, SCCHCH), 7.31-7.18 (1H, m, NCHCH), 6.82 (1H, d, J = 7.8 Hz, CHNH), 6.75 (1H, t, J = 5.4 Hz, OCONH), 3.89-3.73 (1H, m, CHNH), 3.47-3.19 (2H, m, CH2CH2S), 2.98-2.77 (4H, m, CH2CH2S, OCONHCH2), 1.58-1.17 (6H, m, CHCH2CH2CH2), 1.36 (18H, s, 6xCH3); 13C NMR (75 MHz, DMSO-d6): δ = 172.3, 159.0, 155.5, 155.3, 149.6, 137.8, 121.2, 119.3, 77.9, 77.3, 54.4, 39.6, 37.7, 37.5, 31.6, 29.2, 28.3, 28.2, 22.8; m/z (MALDITOF): 536.93 [M+Na]+.

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(S)-Di-tert-butyl((6-oxo-6-((2-(2-pyridyldithio)ethyl)amino)hexane-1,5-diyl)diamino)-2oxoethoxydicarbamate (8).[21] 7 (1.10 g, 2.14 mmol) was dissolved in 15 mL dry DCM and the equal amount of TFA was added. The initially prepared solution was vigorously stirred for 1 h at room temperature. Afterwards, the solvent and reagent were removed and the obtained residue was dried for further 24 h in high vacuum. The oily substance was dissolved in 25 mL DMF and DIPEA (2.76 g, 3.63 mL, 21.37 mmol) and 5 (1.23 g, 4.27 mmol) were added, consecutively. Subsequently, the solution was stirred for 3 h at room temperature in an argon atmosphere. The reaction mixture was diluted with 300 mL ethyl acetate and washed two times with 0.2 M hydrochloric acid as well as brine. Afterwards, the organic layer was dried with magnesium sulfate and the solvent was removed in vacuo. The obtained residue was purified by silica gel column chromatography using ethyl acetate/methanol (15:1) as eluent, whereby, the product was isolated as colorless solid (1.14 g, 1.73 mmol, 81 %). 1H NMR (300 MHz, DMSO-d6): δ = 10.32 (1H, s, CONHO), 10.29 (1H, s, CONHO), 8.50-8.41 (1H, m, NCH), 8.24 (1H, t, J = 5.4 Hz, CHCONH), 8.10 (1H, d, J = 8.1 Hz, CHNH), 7.98 (1H, t, J = 5.4 Hz, OCH2CONH), 7.897.70 (2H, m, SCCHCH), 7.29-7.18 (1H, m, NCHCH), 4.38-4.08 (3H, m, CHNH, NHOCH2), 4.12 (2H, s, NHOCH2), 3.49-3.19 (2H, m, CH2CH2S), 3.08 (2H, q, J = 6.6 Hz, NHCH2CH2CH2), 2.89 (2H, t, J = 6.6 Hz, CH2CH2S), 1.71-1.48 (2H, m, CHCH2), 1.47-1.34 (2H, m, NHCH2CH2CH2), 1.33-1.21 (2H, m, NHCH2CH2CH2), 1.40 (18H, s, 6xCH3); 13C NMR (75 MHz, DMSO-d6): δ = 171.3, 167.9, 167.7, 159.1, 13

ACCEPTED MANUSCRIPT 157.0, 156.9, 149.6, 137.8, 121.2, 119.3, 80.6, 80.6, 74.8, 74.6, 52.0, 38.0, 37.8, 37.3, 31.8, 28.7, 27.9, 22.7; m/z (MALDI-TOF): 661.11 [M+H]+.

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Functional doxorubicin dimer (9).[21] 8 (217.5 mg, 330 µmol) was dissolved in 10 mL dry DCM and the equal amount of TFA was added. The solution was stirred for 1 h at room temperature and afterwards, the solvent and reagent were removed in vacuo. Doxorubicin hydrochloride (421.1 mg, 726 µmol) was dissolved in 80 mL DMF/sodium acetate buffer (1:1) - pH 4.8 and was added to the deprotected cross-linking reagent. Subsequently, the reaction mixture was stirred for 24 h and the crude product was purified by GPC on Bio-Gel P-2 using 25 mM TEAA buffer (pH 7) as eluent. The isolated fractions were additionally purified by HPLC, to obtain the product as red solid after precipitation in diethyl ether (354.2 mg, 234.3 µmol, 71 %). HPLC: tR = 29.33 min; 1H NMR (700 MHz, DMSO-d6): δ = 8.42-8.38 (1H, m, NCH), 8.17 (1H, t, J = 4.9 Hz, CHCONH), 7.94 (1H, d, J = 7.7 Hz, CHNH), 7.827.73 (5H, m, SCCHCH, 2xMeOCCHCHCH), 7.72-7.66 (2H, m, OCH2CONH, SCCH), 7.51 (1H, d, J = 7.0 Hz, MeOCCH), 7.47 (1H, d, J = 7.0 Hz, MeOCCH), 7.22-7.17 (1H, m, NCHCH), 5.24-5.17 (2H, m, 2xCHOCHO), 5.16-5.04 (2H, br s, 2xOH), 4.78-4.73 (1H, m, CHOCHO), 4.72-4.66 (1H, m, CHOCHO), 4.46-4.28 (8H, m, 2xNOCH2, 2xN=CCH2), 4.19-4.05 (3H, m, CHNH, 2xMeCH), 3.91 (3H, s, MeO), 3.87 (3H, s, MeO), 3.62-3.56 (2H, m, 2xMeCHCH), 3.39-3.20 (6H, m, 2xOHCHCHNH2, CH2CH2S, OHCCCH2), 3.05-2.89 (4H, m, OHCCCH2, NHCH2CH2CH2), 2.84 (2H, t, J = 7.0 Hz, CH2CH2S), 2.43-2.32 (2H, m, CH2CHOCHO), 2.24-2.15 (2H, m, CH2CHOCHO), 1.89-1.82 (2H, m, NH2CHCH2), 1.68-1.58 (3H, m, NH2CHCH2, 0.5xCHCH2), 1.48-1.41 (1H, m, 0.5xCHCH2), 1.36-1.28 (2H, m, NHCH2CH2CH2), 1.20-1.12 (8H, m, 2xMeCH, NHCH2CH2CH2); m/z (MALDI-TOF): 1533.07 [M+Na]+.

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Doxorubicin-octaarginine bioconjugate (10). Doxorubicin derivative 9 (24 mg, 15.9 µmol) was dissolved in 2 mL DMF and 25 mM TEAA buffer (pH 7) each. To this solution the peptide (AcCRRRRRRRR-NH2, 40.6 mg, 17.5 µmol) dissolved in 4 mL 25 mM TEAA buffer (pH 7) was added. The reaction mixture was stirred for 3 h under argon at room temperature and was subsequently purified by HPLC. Afterwards, the solvent of the isolated fractions was removed under reduced pressure and the residue was dissolved in a small amount of methanol. The solution was precipitated in diethyl ether, to obtain the product as red powder (39.0 mg, 11.4 µmol, 72 %). HPLC: tR = 21.20 min; 1H NMR (850 MHz, D2O): δ = 7.89-7.36 (6H, m, 2xMeOCCHCHCH), 5.47-5.29 (2H, m, 2xCHOCHO), 4.75-4.45 (10H, m, 2xNOCH2, 2xN=CCH2, CHOCHO, CHCH2-Cys), 4.38-4.25 (10H, m, CHOCHO, 8xCHCH2CH2CH2-Arg, CHCH2-Lys), 3.99 (3H, s, MeO), 3.97 (3H, s, MeO), 3.82-3.75 (2H, m, 2xMeCHCH), 3.64-3.41 (4H, m, 2xOHCHCHNH3, NHCH2CH2CH2-Lys), 3.35-3.28 (2H, m, OHCCCH2), 3.27-3.14 (16H, m, 8xCHCH2CH2CH2-Arg), 3.13-3.07 (2H, m, OHCCCH2), 3.00-2.93 (2H, m, 0.5xCH2CH2S, 0.5xCHCH2-Cys), 2.89-2.79 (2H, m, 0.5xCH2CH2S, 0.5xCHCH2-Cys), 2.542.40 (2H, m, CH2CH2S), 2.04 (3H, s, MeCONH), 1.94 (30H, s, 10xMeCO2), 1.99-1.92 (4H, m, NH3CHCH2, CHCH2-Lys), 1.90-1.74 (18H, m, NH3CHCH2, 8xCHCH2CH2CH2-Arg), 1.72-1.56 (18H, m, NHCH2CH2CH2-Lys, 8xCHCH2CH2CH2-Arg), 1.51-1.43 (2H, m, NHCH2CH2CH2-Lys), 1.35 (3H, d, J = 7.1 Hz, MeCH), 1.34 (3H, d, J = 7.1 Hz, MeCH); m/z (MALDI-TOF): 2813.12 [M+H]+. Glutathione-mediated drug release: A 1 mM solution of the peptide-drug conjugate 10 in DPBS (pH 7.4) was incubated with 10 mM glutathione in an Eppendorf Thermomixer compact at 37°C and 300 rpm. The reduction of the disulfide was monitored by HPLC for a time period of 24 h. The cleavage products were analyzed at 480 nm under the above-mentioned HPLC conditions. Microscale thermophoresis: MST measurements were performed on a NanoTemper Monolith NT.115 device. Excitation of the Cy5-labeled double-stranded DNA consisting of 36 base pairs was carried out with 50 % LED power and the emission was detected in the range of 670-690 nm. Thermophoresis was induced starting from room temperature with an infrared laser intensity of 10 %. The titration of a 50 14

ACCEPTED MANUSCRIPT nM solution of DNA was accomplished with 9 and doxorubicin in a buffer solution consisting of 50 mM Tris (pH 6.5), 15 mM sodium chloride, 10 mM magnesium chloride as well as 0.05 % Tween 20. Analysis and subsequent fitting of the detected fluorescence signals were performed with the NanoTemper software NT Analysis 1.4.27.

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Cell cultures: MCF-7 cells (ATCC HTB-22, human breast adenocarcinoma cell line) were grown in Dulbecco´s modified Eagle medium supplemented with 10 % FCS, 2 mM L-glutamine, 100 units/mL penicillin and 100 µg/mL streptomycin antibiotics. The human neuroblastoma cell line Kelly-WT was obtained from ATCC and authenticated using STR genotyping. Kelly-WT cells were cultured in RPMI1640 medium containing 10 % FCS, 2 mM L-glutamine, 100 units/mL penicillin and 100 µg/mL streptomycin antibiotics. The anthracycline-resistant Kelly-ADR cells were maintained in a similar manner and were generated by continuous exposure of Kelly-WT cells to increasing doses of doxorubicin. Repeated selection rounds resulted in Kelly-ADR cells with tenfold increased resistance to doxorubicin and a concomitant increase in the IC50 value from 0.1 µg/mL to 1 µg/mL. These cells were chosen for further experiments. All of the above-mentioned cells were grown at 37°C and 5 % CO2 in a humidified incubator and were subcultured every 2-3 days using 0.25 % trypsin.

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Cell viability assay: The antiproliferative effect of 9, 10, doxorubicin and the polyarginine cellpenetrating peptide on MCF-7, Kelly-WT as well as Kelly-ADR cells in vitro were investigated with the luciferase-based CellTiter-Glo cell proliferation assay according to the manufacturer´s instructions. Briefly, cells were seeded into 96-well plates at a density of 1 x 103 cells per well in 100 µL of medium and incubated for 24 h to enable attachment. Afterwards, the medium was removed and 9, 10 as well as doxorubicin were added at various concentrations (0.05-20 µM). The cells were incubated for 72 h with the drugs and subsequently cell viability was determined by quantitation of ATP with the CellTiter-Glo luminescent cell viability assay and a Tecan plate reader. Viability of cells treated with doxorubicin or derivatives was compared to untreated controls and correct for effects of the medium. The applied concentrations of 9, 10 and doxorubicin were transformed into a logarithmic scale prior to analysis and the obtained data from the survival curves were expressed as IC50 values in µM. Each viability experiment was conducted in independent triplicates.

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Fluorescence microscopy: The intracellular localization of doxorubicin and the peptide-drug conjugate 10 was investigated in Kelly-WT and doxorubicin-resistant Kelly-ADR live cells at 37°C and 5 % CO2 via fluorescence confocal laser scanning microscopy. Images were obtained with a 63x/1.2 waterimmersion objective on a TCS SP5 microscope (Leica) and an incubation chamber for live cell imaging (37°C, 5 % CO2). Doxorubicin and the anthracycline derivative 10 were applied at a concentration of 10 µM to 1 x 104 cells per well (µ-Slide 8 Well, ibiTreat, Ibidi) for 24 and 72 h. The drugs were excited with an argon laser at λex = 488 nm (power set to 20 %) and the emission range was set to λem = 550660 nm. The fluorescence signal was detected by hybrid detectors (HyD) with fixed gain values set to 100. Counterstaining of the nuclei was achieved by incubation with 5 µM DRAQ5 (Thermo Fisher Scientific) 5 min prior to microscopy. Keywords: Doxorubicin, multidrug resistance, DNA-binding, cell-penetrating peptide, microscale thermophoresis References: [1] R.B. Weiss, The Anthracyclines - Will We Ever Find a Better Doxorubicin, Semin Oncol, 19 (1992) 670-686. [2] G. Minotti, P. Menna, E. Salvatorelli, G. Cairo, L. Gianni, Anthracyclines: Molecular advances and pharmacologic developments in antitumor activity and cardiotoxicity, Pharmacol Rev, 56 (2004) 185-229. [3] J. Nadas, D.X. Sun, Anthracyclines as effective anticancer drugs, Expert Opin Drug Dis, 1 (2006) 549-568. [4] G. Minotti, G. Cairo, E. Monti, Role of iron in anthracycline cardiotoxicity: new tunes for an old song?, Faseb Journal, 13 (1999) 199-212. [5] P. Schlage, G. Mezo, E. Orban, S. Bosze, M. Manea, Anthracycline-GnRH derivative bioconjugates with different linkages: Synthesis, in vitro drug release and cytostatic effect, J Control Release, 156 (2011) 170-178.

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Cole, Multidrug resistance proteins: role of P-glycoprotein, MRP1, MRP2, and BCRP (ABCG2) in tissue defense, Toxicol Appl Pharmacol, 204 (2005) 216-237. [14] S.V. Ambudkar, S. Dey, C.A. Hrycyna, M. Ramachandra, I. Pastan, M.M. Gottesman, Biochemical, cellular, and pharmacological aspects of the multidrug transporter, Annu Rev Pharmacol, 39 (1999) 361-398. [15] M.M. Gottesman, T. Fojo, S.E. Bates, Multidrug resistance in cancer: Role of ATP-dependent transporters, Nat Rev Cancer, 2 (2002) 4858. [16] K. Meissner, B. Sperker, C. Karsten, H.M. zu Schwabedissen, U. Seeland, M. Böhm, S. Bien, P. Dazert, C. Kunert-Keil, S. Vogelgesang, R. Warzok, W. Siegmund, I. Cascorbi, M. Wendt, H.K. Kroemer, Expression and Localization of P-glycoprotein in Human Heart: Effects of Cardiomyopathy, J Histochem Cytochem, 50 (2002) 1351-1356. [17] J.H. Doroshow, Effect of Anthracycline Antibiotics on Oxygen Radical Formation in Rat-Heart, Cancer Res, 43 (1983) 460-472. [18] J. Portugal, D.J. Cashman, J.O. 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Hubbell, Synthesis of Pyridyl Disulfide-Functionalized Nanoparticles for Conjugating Thiol-Containing Small Molecules, Peptides, and Proteins, Bioconjugate Chem, 21 (2010) 653-662. [24] I. Nakase, Y. Konishi, M. Ueda, H. Saji, S. Futaki, Accumulation of arginine-rich cell-penetrating peptides in tumors and the potential for anticancer drug delivery in vivo, J Control Release, 159 (2012) 181-188. [25] M. Zorko, U. Langel, Cell-penetrating peptides: mechanism and kinetics of cargo delivery, Adv Drug Deliver Rev, 57 (2005) 529-545. [26] S. Deroo, E. Defrancq, C. Moucheron, A. Kirsch-De Mesmaeker, P. Dumy, Synthesis of an oxyamino-containing phenanthroline derivative for the efficient preparation of phenanthroline oligonucleotide oxime conjugates, Tetrahedron Lett, 44 (2003) 8379-8382. [27] Y. Singh, N. Spinelli, E. Defrancq, P. Dumy, A novel heterobifunctional linker for facile access to bioconjugates, Org Biomol Chem, 4 (2006) 1413-1419. [28] G. Clave, H. Boutal, A. Hoang, F. Perraut, H. Volland, P.-Y. Renard, A. Romieu, A novel heterotrifunctional peptide-based cross-linking reagent for facile access to bioconjugates. Applications to peptide fluorescent labelling and immobilisation, Org Biomol Chem, 6 (2008) 30653078. [29] I. Szabo, M. Manea, E. Orban, C. Antal, S. Bosze, R. Szabo, M. Tejeda, D. Gaal, B. Kapuvari, M. Przybylski, F. Hudecz, G. Mezo, Development of an Oxime Bond Containing Daunorubicin-Gonadotropin-Releasing Hormone-III Conjugate as a Potential Anticancer Drug, Bioconjugate Chem, 20 (2009) 656-665. [30] P. Ingallinella, A. Di Marco, M. Taliani, D. Fattori, A. Pessi, A new method for chemoselective conjugation of unprotected peptides to dauno- and doxorubicin, Bioorg Med Chem Lett, 11 (2001) 1343-1346. [31] S.M. Deneke, B.L. Fanburg, Regulation of Cellular Glutathione, Am J Physiol, 257 (1989) L163-L173. [32] A. Meister, M.E. Anderson, Glutathione, Annu Rev Biochem, 52 (1983) 711-760. [33] G.K. Balendiran, R. Dabur, D. Fraser, The role of glutathione in cancer, Cell Biochem Funct, 22 (2004) 343-352. [34] A. Sorkin, M. von Zastrow, Signal transduction and endocytosis: close encounters of many kinds, Nat Rev Mol Cell Biol, 3 (2002) 600614. [35] I.F. Tannock, D. Rotin, Acid Ph in Tumors and Its Potential for Therapeutic Exploitation, Cancer Res, 49 (1989) 4373-4384. [36] S.A.I. Seidel, P.M. Dijkman, W.A. Lea, G. van den Bogaart, M. Jerabek-Willemsen, A. Lazic, J.S. Joseph, P. Srinivasan, P. Baaske, A. Simeonov, I. Katritch, F.A. Melo, J.E. Ladbury, G. Schreiber, A. Watts, D. Braun, S. Duhr, Microscale thermophoresis quantifies biomolecular interactions under previously challenging conditions, Methods, 59 (2013) 301-315. [37] M. Jerabek-Willemsen, C.J. Wienken, D. Braun, P. Baaske, S. Duhr, Molecular Interaction Studies Using Microscale Thermophoresis, Assay Drug Dev Techn, 9 (2011) 342-353. [38] S. Duhr, D. Braun, Why molecules move along a temperature gradient, Proc Natl Acad Sci USA, 103 (2006) 19678-19682. [39] S.A.I. Seidel, C.J. Wienken, S. Geissler, M. Jerabek-Willemsen, S. Duhr, A. Reiter, D. Trauner, D. Braun, P. Baaske, Label-Free Microscale Thermophoresis Discriminates Sites and Affinity of Protein-Ligand Binding, Angew Chem Int Ed, 51 (2012) 10656-10659. [40] P. Baaske, C.J. Wienken, P. Reineck, S. Duhr, D. Braun, Optical Thermophoresis for Quantifying the Buffer Dependence of Aptamer Binding, Angew Chem Int Ed, 49 (2010) 2238-2241. [41] C.J. Wienken, P. Baaske, U. Rothbauer, D. Braun, S. Duhr, Protein-binding assays in biological liquids using microscale thermophoresis, Nat Commun, 1 (2010) 1. [42] C.J. Wienken, P. Baaske, S. Duhr, D. Braun, Thermophoretic melting curves quantify the conformation and stability of RNA and DNA, Nucleic Acids Res, 39 (2011) 1-10. [43] Y.J. Schneider, R. Baurain, A. Zenebergh, A. Trouet, DNA-Binding Parameters of Daunorubicin and Doxorubicin in the Conditions Used for Studying the Interaction of Anthracycline-DNA Complexes with Cells Invitro, Cancer Chemoth Pharm, 2 (1979) 7-10. [44] F.F. Leng, W. Priebe, J.B. Chaires, Ultratight DNA binding of a new bisintercalating anthracycline antibiotic, Biochemistry, 37 (1998) 1743-1753. [45] A.T. Jones, E.J. Sayers, Cell entry of cell penetrating peptides: tales of tails wagging dogs, J Control Release, 161 (2012) 582-591. [46] F. Duchardt, M. Fotin-Mleczek, H. Schwarz, R. Fischer, R. Brock, A comprehensive model for the cellular uptake of cationic cellpenetrating peptides, Traffic, 8 (2007) 848-866. [47] J.M. Siegfried, T.G. Burke, T.R. Tritton, Cellular-Transport of Anthracyclines by Passive Diffusion - Implications for Drug-Resistance, Biochem Pharmacol, 34 (1985) 593-598.

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[48] A. Ziegler, J. Seelig, Binding and clustering of glycosaminoglycans: a common property of mono- and multivalent cell-penetrating compounds, Biophysical Journal, 94 (2008) 2142-2149. [49] I. Tabujew, M. Lelle, K. Peneva, Cell-penetrating peptides for nanomedicine – how to choose the right peptide, Bionanomaterials, 16 (2015) 59-72. [50] W.P.R. Verdurmen, M. Thanos, I.R. Ruttekolk, E. Gulbins, R. Brock, Cationic cell-penetrating peptides induce ceramide formation via acid sphingomyelinase: Implications for uptake, J Control Release, 147 (2010) 171-179. [51] H.E. Gottlieb, V. Kotlyar, A. Nudelman, NMR chemical shifts of common laboratory solvents as trace impurities, J Org Chem, 62 (1997) 7512-7515.

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A bioconjugate composed of a CPP and a highly DNA-affine doxorubicin dimer is reported Binding between doxorubicin and DNA is studied for the first time with microscale thermophoresis The conjugate can overcome drug resistance in neuroblastoma cells Trifunctional cross-linker provides plasma stability and efficient drug release