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

Article abstract of DOI:10.1016/j.ejmech.2017.02.056

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.

Full text of DOI:10.1016/j.ejmech.2017.02.056

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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
10.1016/j.ejmech.2017.02.056
EJMECH 9252
DOI:
Reference:
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.
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Overcoming drug resistance by cell-penetrating peptide-
mediated delivery of a doxorubicin dimer with high DNA-
binding 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]
[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: kalina.peneva@uni-jena.de
[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
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.
1

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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.
O
O
OH
OH
OH
H
O
O
OH
O
O
1
OH
NH2
Figure 1. Chemical structure of doxorubicin composed of a DNA-intercalating tetracyclic ring system
and the sugar daunosamine.
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 cell-
penetrating 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.
2. Results and Discussion
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, (boc-
aminooxy)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
a
S
H3N
Cl
Cl H3N
S
2
3
O
O
O
O
O
b
O
O
N
O
N
H
OH
O
N
O
H
5
4
O
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 %
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 cross-
linkers, 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.
3