On the design principles of peptide–drug conjugates for targeted drug delivery to the malignant tumor site

Abstract

Cancer is the second leading cause of death affecting nearly one in two people, and the appearance of new cases is projected to rise by >70% by 2030. To effectively combat the menace of cancer, a variety of strategies have been exploited. Among them, the development of peptide–drug conjugates (PDCs) is considered as an inextricable part of this armamentarium and is continuously explored as a viable approach to target malignant tumors. The general architecture of PDCs consists of three building blocks: the tumor-homing peptide, the cytotoxic agent and the biodegradable connecting linker. The aim of the current review is to provide a spherical perspective on the basic principles governing PDCs, as also the methodology to construct them. We aim to offer basic and integral knowledge on the rational design towards the construction of PDCs through analyzing each building block, as also to highlight the overall progress of this rapidly growing field. Therefore, we focus on several intriguing examples from the recent literature, including important PDCs that have progressed to phase III clinical trials. Last, we address possible difficulties that may emerge during the synthesis of PDCs, as also report ways to overcome them.

Full text

930
On the design principles of peptide–drug conjugates for
targeted drug delivery to the malignant tumor site
Eirinaios I. Vrettos1, Gábor Mező2,3 and Andreas G. Tzakos*1
Review Open Access
Address:
1University of Ioannina, Department of Chemistry, Section of Organic
Chemistry and Biochemistry, Ioannina, GR-45110, Greece, 2Eötvös
Loránd University, Faculty of Science, Institute of Chemistry,
Pázmány P. stny. 1/A, H-1117 Budapest, Hungary and 3MTA-ELTE
Research Group of Peptide Chemistry, Hungarian Academy of
Sciences, Eötvös Loránd University, Pázmány P. stny. 1/A, H-1117
Budapest, Hungary
Email:
Andreas G. Tzakos* - agtzakos@gmail.com
* Corresponding author
Keywords:
bioconjugates; cancer; drug delivery; PDC; peptide; peptide–drug
conjugate; side-products in PDCs
Beilstein J. Org. Chem. 2018, 14, 930–954.
doi:10.3762/bjoc.14.80
Received: 29 January 2018
Accepted: 04 April 2018
Published: 26 April 2018
This article is part of the Thematic Series "Peptide–drug conjugates".
Guest Editor: N. Sewald
© 2018 Vrettos et al.; licensee Beilstein-Institut.
License and terms: see end of document.
Abstract
Cancer is the second leading cause of death affecting nearly one in two people, and the appearance of new cases is projected to rise
by >70% by 2030. To effectively combat the menace of cancer, a variety of strategies have been exploited. Among them, the devel-
opment of peptide–drug conjugates (PDCs) is considered as an inextricable part of this armamentarium and is continuously
explored as a viable approach to target malignant tumors. The general architecture of PDCs consists of three building blocks: the
tumor-homing peptide, the cytotoxic agent and the biodegradable connecting linker. The aim of the current review is to provide a
spherical perspective on the basic principles governing PDCs, as also the methodology to construct them. We aim to offer basic and
integral knowledge on the rational design towards the construction of PDCs through analyzing each building block, as also to high-
light the overall progress of this rapidly growing field. Therefore, we focus on several intriguing examples from the recent litera-
ture, including important PDCs that have progressed to phase III clinical trials. Last, we address possible difficulties that may
emerge during the synthesis of PDCs, as also report ways to overcome them.
930
Introduction
Current cancer chemotherapy
Cancer is one of the leading causes of death globally behind the
heart and circulatory disorders based on statistics of World
Health Organization (WHO) [1]. Among all different types of
cancer, the most fatal for males are lung and prostate cancer,
while for females are breast cancer, colon & rectum cancer [1].
Notably, more than 12 million cancer cases and 7 million

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931
Figure 1: Conventional chemotherapy versus targeted chemotherapy. Black color = Solid malignant tumor; red = conventional untargeted cytotoxic
agent; blue = targeted cytotoxic agent.
cancer deaths are estimated to have occurred both in males and
females in 2008 worldwide [2]. These numbers have mounted
up to 15 million cases and 8.8 million deaths in 2015. These
statistics clearly indicate that cancer is not retreating but is
creeping up, tending to become the leading cause of mortality.
Thus, it can be concluded that the current therapeutic formula-
tions utilized in oncology are not adequately effective against
the complexity of cancer, mostly due to the associated collat-
eral toxicity present in healthy tissues. It is estimated that about
30% of the clinical trials on ClinicalTrials.gov are related to
cancer, while only 10% of them eventually gain market
approval [3], rendering the drug development, especially in this
therapeutic direction, costly and inefficient. Specifically, 12
cancer drugs were approved by the FDA in 2017 [4], com-
prising 26% of the total amount of approvals with respect to
other therapeutic areas. These figures suggest that it is of great
importance to turn the focus of the global market on targeted
therapies. In 2009, the total earnings in the United States,
derived from targeted cancer drugs, have reached $10.4 billion,
showing an almost 2.2-fold increase since 2005 [5]. However,
despite the significant attention that field has gained the past
decades, it still remains unfulfilled.
Current treatment processes involve a combination of surgical
intervention, radiation and chemotherapy. Drugs used for this
purpose are inevitably cytotoxic in order to eliminate cancer
cells, but they lack selectivity that could be developed through
targeting malignant cells (Figure 1). Due to the uncontrolled
peripheral toxicity, anticancer drugs usually kill healthy tissues,
resulting in severe effects on the patient’s health. One represen-
tative example is gemcitabine, which demonstrates higher toxic-
ity for healthy cells, after long-term administration, with respect
to cancer cells. This happens since cancer cells evolve more
rapidly and develop drug resistance by diminishing expressed
nucleoside receptors responsible for the cell uptake of gemcita-
bine [6].
Additionally, chemotherapy with anticancer agents is often
hampered by their poor aqueous solubility, low oral bioavail-
ability and metabolic instability. These drawbacks are linked to
the unfavorable ADME (absorption distribution metabolism
excretion) that are basically described in the following four
consecutive axes: 1) Absorption is directly connected with the
transportation process of the drug from the site of administra-
tion to the systemic circulation [7]. 2) Distribution refers to the
delivery of the drug to the tissues which usually occurs via the
bloodstream. Conventional chemotherapeutic drugs (gemcitabi-
ne, paclitaxel, doxorubicin, etc.) are not capable to be selec-
tively delivered to the tumor sites and they end up scattered in
the whole body. 3) Metabolism is a standard biological strategy
for detoxification, breaking down of the administrated drugs,
once inserted into the human body. The drugs get decomposed
and converted to their metabolites. These metabolites can be
pharmacologically inactive, e.g., gemcitabine converted to 2',2'-
difluorodeoxyuridine (dFdU) [8] or possess enhanced activity
with respect to the parent drug, e.g., temozolomide converted to
5-(3-methyltriazen-1-yl)imidazole-4-carboxamide (MTIC) [9].
4) Excretion is the final step and is responsible for the removal
of the parent drug and/or its metabolites from the human body.
Renal excretion is the predominant route of elimination, occur-
ring via urine.
Therefore, most conventional cytotoxic agents applied in
chemotherapy lack optimum pharmacokinetic properties

Beilstein J. Org. Chem. 2018, 14, 930–954.
932
(ADME) and thus are not very effective to treat malignancies.
Moreover, despite the intensive research to construct new cyto-
toxic drugs, survival rates in most cancers remain low [10] and
clinical attrition rates in oncology have been devastating [11].
These data render obvious that the currently developed
drugs, as also the continuous attempt to discover new
ones, have not provided the expected therapeutic impact in
oncology.
It is clear that we do have access to an enormous pool of unspe-
cific cytotoxic agents that can efficiently kill cancer cells. What
is currently needed is not to invest so intensively in generating
more cytotoxic agents but to re-use and re-cycle available ones
and tailor them to be transformed into targeted chemotherapeu-
tics. Along these lines, drug delivery vehicles that can be
tailored for different types of cancer and shape personalized
therapeutics are continuously gathering attention. Such drug
delivery systems are of ultimate importance to effectively
surpass these hurdles and eventually improve drug potency.
Charting the malignant tumor
microenvironment
In order to selectively deliver cytotoxic drugs to malignant
tumor sites, scientists can take advantage and map first the
differential microenvironment between cancer and normal cells.
The first one to report a fundamental difference between malig-
nant and normal cells was Otto Heinrich Warburg in the early
1900s, who was awarded the Nobel Prize in 1931. He proposed
that malignant tumor growth relies on aerobic glycolysis, in
contrast to normal cells that generate energy by mitochondrial
oxidative phosphorylation. The fact that cells converted pyru-
vate to lactate, even in the presence of oxygen, rendered his ob-
servation puzzling for scientists, who still struggle to elucidate
the complete mechanism of action of diseased cells. Following
the Warburg effect, 18F-deoxyglucose positron emission tomog-
raphy (FDG–PET) imaging was developed in order to visualize
the phenomenon of increased glucose uptake by cancer cells
[12].
Nowadays, it has been demonstrated that malignant cells differ
markedly in many metabolic aspects compared to normal cells
[13], thus offering the opportunity to target them in various
ways. Most cancer tissues exhibit the following characteristics
that can be exploited for developing targeted cytotoxic agents:
1. Dysregulation of translation initiation factors and regula-
tors [14].
2. Mutations in epigenetic regulatory genes [15].
3. Overexpression of surface receptors like HER2R [16],
folate receptor [17], GnRH receptor [18,19] and amino
acid transporters [20].
4. Overwhelming production of stimulus agents and en-
zymes [21]. For instance, many types of cancer show en-
hanced levels of reactive oxygen species (ROS) which
are reactive molecules and play a crucial role in cell
proliferation [22].
5. The slightly acidic pH of the tumor microenvironment
[23] (Warburg effect).
These are some noteworthy differences that underlie the dis-
crimination between cancerous and normal cells and are often
exploited in order to control the site of the drug release during
targeted cancer chemotherapy.
Review
Strategies for targeted delivery of toxic
warheads to malignant tumor sites
The main challenge of the drug delivery concept is to transport
sufficient amount of the cytotoxic agent to a specific location
with minimum adverse side effects. To conquer this, various ap-
proaches are being exploited at the moment. These include, but
are not limited to: a) utilization of drug delivery vehicles and
formulates like nanoparticles [24] and calixarenes or cyclo-
dextrins [25,26], where the cytotoxic drug is loaded and can be
released at the malignant tumor site; b) installation of labile
chemical groups to the tumor microenvironment (i.e., low pH)
able to mask the cytotoxic drug and form a prodrug with en-
hanced plasma stability and/or cell permeability [27] and in the
same time diminished toxicity for normal cells; c) covalent
attachment of a drug on a tumor-targeting element (small mole-
cule, peptide or antibody) able to selectively target and
permeate cancer cells. The conjugation is being conducted via a
rationally designed linker able to release the drug inside the
cancer microenvironment [19].
The ideal targeting molecular device would consist of the
following modules: a) the cytotoxic agent (drug), b) the trans-
porting - drug delivery vehicle (i.e., lipid, mannan [28-30]),
c) the linker tethering the transporting vehicle to the cytotoxic
warhead, d) the “programmable” navigating/targeting moiety
(i.e., receptor-specific ligand) and e) the “stealth” carrier (i.e.,
PEG) transfusing enhanced bioavailability. These modules are
encoded in Figure 2A with different colors: the transporting
vehicle in green color, the drug in blue color, the linker in red
color, the navigating/targeting agent in black color and the
“stealth” carrier in grey color [31]. The specific color coding
will be followed, for simplicity purposes, in all examples of
targeting devices that will be presented throughout this review.
Among the most intriguing navigating delivery systems that can
combine the transporting vehicle and the navigating/targeting
moiety in a single module are the tumor-homing peptides [32].

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933
Figure 2: A. General structural architecture of the ideal navigated drug delivery system [31]. B. General structure of a peptide–drug conjugate (PDC).
These peptides are exploited to assemble the peptide–drug
conjugates (PDCs) which are considered as prodrugs, due to the
covalent coupling of a peptide to a drug via specific linkers. The
main building blocks of a simple PDC include a cytotoxic agent
(drug), a tumor-homing peptide (navigating/targeting moiety)
and a linker between them (Figure 2B).
This class of prodrugs is continuously gaining attention since
peptides can be easily produced in large quantities and their
purification is simple. Moreover, an array of different tumor-
targeting peptides has been discovered [32] for multifarious
types of cancer. This bountiful palette can permit the construc-
tion of personalized cancer therapeutics upon selecting a tumor-
homing peptide that will be most appropriate for the type of
cancer needed. In addition, peptide sequences can be selected
according to the required physicochemical properties such as
solubility, stability and overall charge or the characteristic
groups necessary for the conjugation with the therapeutic
payload. The overall experimental procedure to synthesize a
PDC is usually rapid and facile. Notably, the overall cost to
produce a PDC, where an already approved drug can be
selected and re-used from a pool of available cytotoxic agents,
is much lower compared to the cost of synthesizing a new cyto-
toxic agent, as it is based on an already applied drug with the
addition of a small peptide. Nevertheless, the last years more
complex bioconjugates have been synthesized to allow the si-
multaneous diagnosis and therapy (theranostics) of diseases.
The therapeutic efficacy of a PDC is predominantly associated
with the potency of the drug and the targeting efficiency of the
assembled conjugate. Thus, PDCs should possess certain fea-
tures to render them appealing candidates for treatment:
1. The peptide contained in the PDC must bind selectively
and with the optimal affinity to a certain receptor,
present on the cell surface of the targeted tissues and not
within their cytosol or nucleus (i.e., steroid receptors
[33]).
2. The selected receptor should be uniquely expressed or
overexpressed on cancer cells (usually 3-fold or higher in
comparison with normal cells). Additionally, it should be
expressed in sufficient levels to pump inside the cell effi-
cacious doses of the drug.
3. The peptide-carrier should be constructed in such way
that the conjugation with a drug or/and a fluorophore is
feasible. Conjugation usually occurs on lysine, cysteine
and glutamic acid [34] via orthogonal coupling or on the
free N-terminus of the peptide during solid phase peptide
synthesis. Though, the conjugation site should be care-
fully selected, since perturbations induced in the peptide
structural microenvironment may result in the abolish-
ment of its binding affinity/selectivity to the targeted re-
ceptor.
4. The linker should be carefully selected to succeed the
optimal performance of the PDC. An injudicious selec-
tion may cause diminished binding affinity of the peptide
to the receptor or/and reduction of the therapeutic
window of the drug. Additionally, it should be enzymati-
cally stable during the blood circulation in order to effi-
ciently reach the malignant tumor site and release the
payload in its microenvironment, reducing the off-target
toxicity.
5. The cytotoxic agent should contain proper functional
group that can be linked to the tumor homing peptide
(i.e., gemcitabine [19]) or if it is not present it should be
rationally installed taking into consideration the final de-
rivative of the cytotoxic agent to retain the original cyto-
toxic activity. The sections below summarize the basic
design principles of peptide–drug conjugates to selec-
tively target the malignant cells.

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934
Selecting the proper tumor-targeting peptide
to generate the PDCs
There is an immense variety of peptides (linear or cyclic) that
have been exploited as carriers/targeting elements to successful-
ly deliver the cytotoxic warhead to cancer cells [32]. These
peptides are cell-specific and bind to certain receptors
promoting their internalization. They are usually inserted into
the cell via endocytosis and then they are transported to intra-
cellular compartments with higher concentration of enzymes
and lower values of pH, where they disassociate from the recep-
tor and afterward from the anticancer agent. The most represen-
tative examples of peptides utilized for PDCs are highlighted
below.
Linear peptides are included among the rich reservoir of
options, finding applications in tumor targeting. They exist in
different lengths, structures and with various physicochemical
properties.
Attempting to ameliorate certain disadvantages of linear
peptides like fast renal clearance or low binding selectivity/
affinity due to the unstable structure of the linear peptides,
cyclic peptides have been introduced. An immense number of
cyclic peptides have been synthesized [35-37] and many of
them have displayed superior affinity and selectivity for the re-
ceptor than their parent linear counterparts [38]. Cyclic peptides
are usually synthesized by reacting the N-terminus with the
C-terminus or by exploiting specific functional groups of
certain amino acids present in the sequence. A representative
example is the sulfhydryl group of cysteine-containing peptides
which may cyclize through the formation of intramolecular
disulfide bonds [39].
The most commonly used linear peptides and cyclic peptides
that can be delivered inside cancer cells via endocytosis and one
that smuggles into glioma tissues via transcytosis (angiopep-2)
are presented below:
Arginine-glycine-aspartic acid (RGD): A widely applied
peptide carrier is the tripeptide arginine-glycine-aspartic acid
(RGD) motif, which was first identified by Ruoslahti and
Pierschbacher in the early 1980s [40] within fibronectin that
mediates cell attachment and was known to target integrin α5β1
[41]. In general, the ‘integrin’ nomenclature was first used in
1987 to describe a family of receptors, appearing as
heterodimers of noncovalently associated α and β subunits, able
to link the extracellular matrix (ECM) with the intracellular
cytoskeleton to mediate cell adhesion, migration and prolifera-
tion [42]. The RGD motif is contained in various proteins like
fibrinogen, fibronectin, prothrombin, tenascin and other glyco-
proteins [43] and is known to be recognized by over 10 inte-
grins, among the over 24 known integrins [44,45], including all
αv integrins, α5β1, α8β1 and αIIbβ3 [46].
The fact that carcinogenesis is highly dependent on migration,
invasion and angiogenesis renders integrins important anti-
cancer targets. Integrin αvβ3 is an important factor in tumor
angiogenesis and metastasis [45], two common characteristics
of cancer that discriminates it from other diseases. Notably,
integrin αvβ3 (also known as the vitronectin receptor) appears
to be the most important among all integrins regarding cell
proliferation, invasion and angiogenesis [47]. This integrin is
overexpressed on activated endothelial cells, new-born vessels
and other tumor cells [48,49], but it is found to be expressed at
undetectable levels in most adult epithelial cells, making it a
suitable target for anti-angiogenic therapy [50]. Due to its high
levels of expression in cancer cells, several peptides containing
the RGD motif have been exploited for the formulation of
PDCs, with the most representative example to be the peptide
CDCRGDCFC [46,51,52].
Gonadotropin-releasing hormone (GnRH): Gonadotropin-
releasing hormone (GnRH), also known as luteinizing hormone-
releasing hormone (LHRH), is a hormone responsible for the
secretion of two gonadotropins: follicle-stimulating hormone
(FSH) and luteinizing hormone (LH) from the anterior pituitary
gland. GnRH is synthesized and released from GnRH neurons
within the hypothalamus and selectively binds to its receptor
(GnRH-R), a seven-transmembrane G-protein-coupled receptor.
The structure of the GnRH hormone (pGlu-His-Trp-Ser-Tyr-
Gly-Leu-Arg-Pro-Gly-NH2) was first discovered in 1971 by
Baba et al. [53]. Besides this form, there is GnRH-II (pGlu-His-
Trp-Ser-His-Gly-Trp-Tyr-Pro-Gly-NH2) discovered in most
vertebrates as well as in humans [54]. This peptide acts through
a similar receptor (type II GnRH-R), which is expressed in dif-
ferent tissues, including tumor cells. Another natural isoform of
GnRH is GnRH-III (pGlu-His-Trp-Ser-His-Asp-Trp-Lys-Pro-
Gly-NH2), which has been isolated from sea lamprey. GnRH-III
binds to GnRH-R overexpressed on the cancer cell surface, re-
sulting in an antiproliferative effect but seems to be less potent
than the rest GnRH analogs regarding stimulating gonadotropin
release at the pituitary level [55].
GnRH peptide analogs constitute an emerging class of tumor
homing peptides for malignant tissues expressing the GnRH-R.
Their development is based on the fact that specific human
cancer cells (mostly ovarian, prostate, lung and breast) uniquely
express or overexpress GnRH-R with respect to normal tissues
[55-57]. Therefore, covalent attachment of a cytotoxic agent to
these peptides provides the possibility to produce potent tumor-
targeting PDCs. Various amino acid alterations have been per-
formed with respect to the native hormone [58], while the most

Beilstein J. Org. Chem. 2018, 14, 930–954.
935
frequently used GnRH analog is D-Lys6-GnRH-I, which is
known to bind selectively to GnRH-R. The substitution of Gly6
of the native hormone with D-Lys6 provided an analog with
higher binding affinity, stabilized β-bend and resistance to
proteolytic cleavage. Moreover, the side chain of lysine
contains a free amine group (εNH2) allowing orthogonal cou-
pling with a cytotoxic warhead [19]. A considerable number of
PDCs based on GnRH [59-63] exist and our group has exploited
this peptide to construct two PDCs [18,19].
Somatostatin (SST): Somatostatin is a neuropeptide produced
by neuroendocrine, inflammatory and immune cells and has an
important role in various physiological functions acting as a
classical endocrine hormone, a paracrine regulator or a neuro-
transmitter [64]. Somatostatin appears in two distinct active
forms: somatostatin-14 (SST-14) and somatostatin-28 (SST-28).
Both SST-14 and SST-28 exhibit biological activity through
high-affinity membrane receptors (somatostatin receptor 1–5;
SSTR1–5), that are widely distributed throughout the human
body in various tissues like the nervous, pituitary, kidney, lung
and immune cells [65,66].
SSTRs are overexpressed in various neuroendocrine malignant
tumors (NETs) including pancreatic, pituitary, prostate, lung
carcinoids, osteosarcoma etc. and other non-NETs including
breast, colorectal, ovarian, cervical etc. [67]. Therefore, these
receptors can be targeted for selective delivery of efficient con-
centrations of cytotoxic warheads to the tumor sites. However,
native somatostatin gets rapidly hydrolyzed due to enzymatic
degradation and therefore, more stable and potent analogs have
been developed. These analogs were synthesized by replacing
L-amino acids with their D-isomers and reducing the length by
keeping only the peptide epitope responsible for the biological
activity. The most widely known analogs of somatostatin are
cyclic peptides named octreotide (d-Phe-c[Cys-Phe-d-Trp-Lys-
Thr-Cys]-Thr-ol), lanreotide (d-2Nal-c[Cys-Tyr-d-Trp-Lys-Val-
Cys]-Thr-NH2) and vapreotide (d-Phe-c[Cys-Tyr-d-Trp-Lys-
Val-Cys]-Trp-NH2), which bind mainly to the subtype 2 recep-
tor (SSTR2) that is known to be the most frequently overex-
pressed SSTR [68]. There are several examples of PDCs
consisting of the aforementioned somatostatin targeting
peptides [67,69,70], as also other somatostatin peptide analogs,
e.g., pentetreotide (DTPA-d-Phe-c[Cys-Phe-d-Trp-Lys-Thr-
Cys]-Thr-ol) [71].
Epidermal growth factor (EGF): Epidermal growth factor re-
ceptor (EGFR) is a transmembrane protein belonging to the
ErbB family of receptor tyrosine kinases which consists of
4 structurally-related members: EGFR/HER1 (ErbB-1), HER2/
neu (ErbB-2), HER3 (ErbB-3) and HER4 (ErbB-4). Cohen and
Rita Levi-Montalcini shared the Nobel Prize in Medicine in
1986 for discovering growth factors. EGFR is upregulated in a
wide pool of cancer tissues and is able to enter cells usually via
clathrin-mediated endocytosis [72]. Many peptides have been
discovered to bind the EGFR with high affinity and selectivity
through screening phage display libraries and have been used
like a viable approach for targeted drug delivery: YHWYGYT-
PQNVI [73], CMYIEALDKYAC [74], LTVSPWY [75],
YWPSVTL [76].
Angiopep-2: A peptide that has recently attracted attention is a
19-mer peptide named angiopep-2 (TFFYGGSRGKRNNFK-
TEEY), due to its ability to cross the blood-brain barrier (BBB).
The BBB is formed by the endothelial cells of the brain,
restricting and controlling the exchange of molecules between
the central nervous system and the rest body. Angiopep-2 is
able to cross the BBB via receptor-mediated transcytosis after
binding to the low-density lipoprotein receptor-related protein-1
(LRP-1), which is overexpressed in brain cells [77]. Moreover,
the two lysines available in its sequence render angiopep-2 an
appealing PDC candidate, with the aim to smuggle therapeutic
payloads to brain malignancies [78,79].
Cyclic peptide variants have been developed for the RGD
peptide motif, reported above. The most commonly used cyclic
peptide is iRGD (CRGDKGPDC), a 9-amino acid cyclic
peptide, with tumor tissue penetration activity [80]. iRGD
initially binds to αVβ3 and αVβ5 integrins that are overex-
pressed in tumor endothelial cells. Afterward, a proteolytical
cleavage takes place to reveal a cryptic RXXK/R motif located
at the C-terminus (CendR motif, C-End Rule), which then binds
to neuropilin-1 (NRP-1), activating an endocytic transport path-
way responsible for the enhanced transport of anti-cancer drugs
into tumors (Figure 3) [80].
In Table 1 are reported the most common peptides (linear and
cyclic) utilized in PDCs.
Selecting the proper cytotoxic agent to
generate the PDCs
According to the National Cancer Institute (cancer.gov), there
are more than 250 FDA-approved anticancer drugs utilized to
treat malignancies at the moment. Among this large pool of
cytotoxic drugs, an array of them has been utilized as toxic
warheads in PDCs and five representative examples are gemci-
tabine, doxorubicin, daunorubicin, paclitaxel and camptothecin
(Figure 4). The main drawback of these original anticancer
agents is their uncontrolled toxicity which results in severe side
effects. Without the addition of a targeting moiety, they bear
low capacity to discriminate cancerous from normal cells.
Moreover, the addition of a peptide as a targeting vehicle can
enhance the pharmacokinetic and therapeutic window of the

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936
Table 1: The most common peptides (linear and cyclic) utilized for the formulation of PDCs used in cancer. Letters with bold color stand for D-amino
acids.
peptide name peptide sequence targeted receptor reference
RGD R-G-D integrin αvβ3 [37,46,51,52]
iRGD CRGDK/RGPD/EC integrin αvβ3/αvβ5 [81]
octreotide SSTR2/5 [69]
D-Lys6-LHRH Glp-H-W-S-Y-K-L-R-P-G LHRH-R [18,19,61]
angiopep-2 T-F-F-Y-G-G-S-R-G-K-R-N-N-F-K-T-E-E-Y LRP-1 [78,79]
GE11 Y-H-W-Y-G-Y-T-P-Q-N-V-I ErbB1 (EGFR) [73]
Figure 3: Binding and penetration mechanism of iRGD. The iRGD
peptide is accumulated on the surface of αv integrin-expressing endo-
thelial and other cells in malignancies. The RGD motif is responsible
for binding to integrins. Afterward, the peptide is cleaved by cell sur-
face-associated protease(s) to eventually expose the cryptic CendR el-
ement, RXXK/R, at the C-terminus (red dotted line). The CendR ele-
ment then interferes with the binding to neuropilin-1, resulting in tissue
and cell penetration. The tumor-penetrating peptide can be used to
decorate a cargo (a simple chemical moiety or a nanoparticle), but only
in the case that the cargo is attached to the N-terminus of the iRGD
peptide as the disulfide bond is cleaved before the peptide is internal-
ized (black line). The figure was adopted from reference [81] (© 2009
Elsevier Ltd.).
parent cytotoxic agent. Since different drugs may employ a dif-
ferent mechanistic approach to kill cells, the appropriate drug is
selected according to features characterizing the targeted
cancerous cells. For instance, daunorubicin and doxorubicin
possess similar mechanisms of action [82], whereas gemcitabi-
ne [83], camptothecin [84] and paclitaxel [85] function through
different mechanisms.
The selected drug must comply with certain design principles in
order to serve as an appealing candidate for PDCs. The selected
drug must be amenable to the linker chemistry. It must bear an
intrinsic functional group for direct conjugation with the
peptide/linker (Figure 4) or a functional group able to be deriva-
tized for further conjugation (i.e., click chemistry [86]). In the
latter case, the site of derivatization has to be carefully selected
so that the biological activity of the drug and the release of the
active drug will not be perturbed. In case that the drug binds
through recognition of a specific receptor, in silico approaches
have to be recruited in order to rationally select the location of
the drug that will be chemically modified [18].
Furthermore, it must be sufficiently cytotoxic versus the
selected malignant tumor cells in order to eliminate them and
consequently promote tumor regression. The selected drug
should ideally possess low-nanomolar IC50 values for the
targeted malignant tumor. A legitimate strategy to overcome a
low drug potency problem is by increasing the drug loading of
the peptide-carrier. For example, in the PDC ANG1005, 3 drug
molecules (paclitaxel) were loaded on a single angiopep-2
peptide which has completed phase II clinical trials [87]. Never-
theless, the concept of higher drug loading is hard to be imple-
mented, in contrast with single drug loading that is usually
preferred, mostly due to poor physicochemical properties.
Below we analyze a set of drugs that have been tailored and in-
corporated in PDCs.
Gemcitabine (Gem): Gemcitabine (dFdC) is a nucleoside
analog of deoxycytidine in which the hydrogen atoms on the
2' carbon are replaced by fluorine. It is sold under the brand
name Gemzar by Eli Lilly and Company and has been FDA ap-
proved for the treatment of various cancers including breast,
ovarian, non-small cell lung and pancreatic cancer. The main
drawbacks for its use are the high and non-selective toxicity to
normal cells, the deactivation through deamination in its inac-
tive metabolite dFdU, the acquired multidrug resistance (MDR)
and its high hydrophilicity deterring its prolonged drug release
from various vehicles [88], which therefore reduces the effec-
tive concentration of gemcitabine. It enters cells through
nucleoside transporters hENTs (human equilibrative nucleoside

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Figure 4: Representative examples of anticancer drugs utilized for the construction of PDCs. The most usual conjugation sites are marked with red
cycles.
transporters) and hCNTs (human concentrative nucleoside
transporters) and mostly through hENT1 (human equilibrative
nucleoside transporter 1) [89,90]. After internalization, gemcita-
bine is sequentially mono-, di- and tri-phosphorylated by phos-
phorylating kinases. Gemcitabine diphosphate (dFdCDP) and
gemcitabine triphosphate (dFdCTP) are the active metabolites
which inhibit processes required for DNA synthesis [91]. The
incorporation of dFdCTP into DNA during polymerization,
which causes DNA polymerases unable to proceed, is the major
mechanism by which gemcitabine causes cell death (masked
termination) [83]. Regarding the possible functional sites in
gemcitabine that can be used for the construction of PDCs are
its primary and secondary alcohols as also the amine (Figure 4).
Paclitaxel (PTX): Paclitaxel (PTX) is a member of the taxane
family and one of the most common anticancer agents used
against a wide variety of tumors. It is sold under the brand name
Taxol by Bristol-Myers Squibb Company and is FDA approved
for the treatment of breast cancer, ovarian cancer, non-small cell
lung cancer and AIDS-related Kaposi's sarcoma. The main
disadvantages in the utilization of paclitaxel are its high hydro-
phobicity, requiring suitable vehicles to effectively deliver it to
tumor tissues, and the development of multidrug resistance due
to the P-glycoprotein-mediated efflux [85,92]. Paclitaxel stabi-
lizes microtubules by binding specifically to the beta-tubulin
subunit, promoting mitotic halt and consequently cell death
[93]. The difference with other known drugs that act on micro-
tubules (vinca alkaloids) is that paclitaxel does not induce the
disassembly of microtubules but boosts the polymerization of
tubulin [94]. Sites available in PTX for the formation of PDCs
are highlighted in Figure 4.
Anthracyclines: Anthracyclines are among the main anti-
cancer drugs that are applied in combinations with other
chemotherapeutic agents. They are utilized against a variety of
cancers including leukemias, lymphomas, breast, ovarian,
bladder and lung. Daunorubicin (Dau) was the first anthracy-
cline discovered that was extracted from Streptomyces
peucetius, a species of actinobacteria, at the beginning of the
1960s. Shortly after, the isolation of doxorubicin (Dox) from a
mutated Streptomyces strain was accomplished. Anthracyclines
are consisted of a tetracyclin aglycon part and a daunosamine
sugar moiety. The difference between Dau and Dox is a
hydroxy group substituted at the C-14 carbon atom on Dox pro-
viding an extra conjugation site for ester linkage (Figure 6). The
mechanism of action of anthracyclines is based on their interca-

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938
lation to DNA inhibiting the macromolecular biosynthesis.
Furthermore, they stabilize the topoisomerase II DNA complex
preventing the transcription. They may also increase quinone
type free radical production, however, this plays a role rather in
their cytotoxic side effects. Daunorubicin is mainly used in the
treatment of leukemia [95] while doxorubicin in the cure of
other types of cancers (breast cancer, bladder cancer, Kaposi's
sarcoma) in combination with other anti-cancer agents.
Camptothecin (CPT): Camptothecin is a cytotoxic alkaloid
collected from extraction of the bark and stem of the Chinese
tree ‘Camptotheca acuminata’. It was first isolated and charac-
terized in 1966 by Wall et al. [96,97]. The main mechanism of
action involves binding to the reversible complex of topoisom-
erase I (topo I) and the 3′-phosphate group of the DNA back-
bone through hydrogen bonding, resulting in accumulation of a
persistent ternary complex (the cleavable complex). This stabi-
lized complex prevents the re-ligation step of DNA, catalyzed
by topo I, resulting in DNA damage and therefore cell death
(apoptosis). CPT is predominantly cytotoxic during the S phase
replication of DNA because of the collision of the replication
fork with the cleavable complex, converting the single-strand
breaks into double-strand breaks and eventually causing cell
death [98]. CPT can be conjugated to targeting elements to en-
hance its efficacy via its primary alcohol marked in Figure 4.
Although CPT showed remarkable results during its phase I
clinical trials against a variety of solid tumors, its low water-
solubility and stability led to the formulation of various new
analogs with the same mechanism of action. The two most
progressed analogs of CPT are topotecan and irinotecan.
Topotecan (hycamtin) has been approved by the FDA for the
treatment of ovarian and cervical cancer, as also small cell lung
carcinoma. Irinotecan (camptosar) has been approved by the
FDA for the treatment of metastatic carcinoma of the colon or
rectum, alone or in combination with fluorouracil (5-FU).
Camptothecin has been utilized as an anticancer agent in
various PDC formulations, such as conjugation with the
targeting peptides D-Lys6-LHRH [99], somatostatin [100] and
c(RGDyK) [101].
Linker design for PDCs: Principles and
representative examples
Another crucial aspect that should be considered during the
design of a PDC is the linker tethering the peptide and the drug.
The linker has to be carefully shaped so as not to perturb the
binding affinity of the peptide to its receptor and the drug effi-
cacy. An inappropriate linker may impede the release of the
drug from the PDC and therefore diminish its overall thera-
peutic potency. Linkers utilized in PDCs exist in different cate-
gories and vary on their length, stability, release mechanism,
functional groups, hydrophilicity/hydrophobicity etc.
This linker can be designed to bear an enzyme-hydrolyzable
unit (EHU) like a carboxylic ester or an amide bond, cleaved by
esterases and amidases, respectively. The most commonly
utilized linkers that bear a carboxylic ester bond, as the enzyme-
hydrolyzable unit, are succinyl (derived from succinic acid) and
glutaryl (derived from glutaric acid). Concerning the utilization
of amide bond in the linker as the unit tethering the drug and the
peptide, it can be tailored to be cleaved based on the targeted
tissue and/or type of cancer where a specific protease is statisti-
cally upregulated (i.e., cathepsin B upregulated in various
malignancies including lung, brain, prostate and breast [102]).
Also, during the design of the PDC specific attention has to be
given on the selection of the bonds that will be used in the
linker. Specifically, in several currently available PDCs, at least
two different bonds are used: one to connect the linker to the
peptide and the other to connect the drug to the linker. Such
cases have to consider, during the design process, the microen-
vironment that the assembled PDC is to be located, since differ-
ent enzymes and/or the tumor microenvironment might trigger
the improper release of the drug from the PDC, i.e., to end up
with the drug-carrying part of the linker.
Another class of linkers is the stimuli-responsive/degradable
linkers, designed to achieve an efficient release of the drug from
the bioconjugate in the tumor microenvironment. Such linkers
are rationally designed to be cleaved when they sense specific
stimuli in the environment of cancerous cells (slightly acidic
pH, enhanced levels of reducing agents and/or enzymes) or
external stimuli (ultrasound, temperature, irradiation). Specifi-
cally, there are certain bonds like imine, oxime, hydrazone,
orthoester, acetal, vinyl ether and polyketal [103] that are
known to undergo hydrolysis at acidic pH, while being
extremely stable during blood circulation. Therefore, acid-labile
bonds could be hydrolyzed in the slightly acidic microenviron-
ment and/or in the acidic cellular compartments of cancer cells
and consequently release the active drug. Additionally, disul-
fide linkers are often adopted in PDCs, since they are cleaved
by reducing agents like cysteine and glutathione, present in high
concentrations in malignant cells.
Linkers bearing enzyme-hydrolyzable units (EHU) responsive
to proteases are degradable peptide linkers that have attracted
significant interest due to the specificity of certain enzymes and
there has been a dramatic escalation over in the past years. The
most representative examples in this field are the MMP-2/9
(matrix metalloproteinases) and cathepsin B peptide substrates.
MMP-2/9 and cathepsin B are proteolytic enzymes present at
elevated levels in cancer cells known to participate in human
tumor invasion and metastasis. Cathepsin B is able to recognize
specific peptide sequences like Val-Cit (valine-citrulline) [104]
and GFLG [105]. On the other hand, GPLGIAGQ [106],

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Figure 5: Illustration of the drug release mechanism from the self-immolative spacer PABC conjugated to a tumor homing peptide via an enzyme-
hydrolyzable unit. Red color = the self-immolative spacer PABC; blue color = drug; green color = enzyme-hydrolyzable unit (EHU); black color the
tumor-homing peptide.
Table 2: Representative examples of biodegradable/responsive linkers utilized for the formulation of PDCs in cancer.
linker drug release mechanism reference
succinyl action of esterases/amidases [19]
glutaryl action of esterases/amidases [67]
PABC 1,6-elimination [109,110]
oxime bond hydrolysis in acidic pH [111]
peptide GFLG action of cathepsin B [105]
peptide PLGLAG action of MMP-2/9 [107]
PLGLAG [107] and GPVGLIGK [108] are some common
peptide substrates for MMP-2 and MMP-9.
Another rapidly emerging category in PDC linkers that has
gained much attention in the last years are the self-immolative
or self-destructive spacers/linkers [109,110]. This type of
linkers/spacers offers the capability to release the active drug
after simultaneous cascade reactions, as shown in Figure 5.
Para-amino benzyl alcohol (PABC; colored in red) is a repre-
sentative example that can be connected in the amino group via
an amide bond to an enzyme-hydrolyzable unit (EHU; colored
in green) and to a tumor-targeting element (i.e. tumor homing
peptide; colored in black). The alcohol group at the opposite
site can be connected via a carbonate ester/carbamate bond to
the cytotoxic agent (colored in blue). The EHU is designed so
as to be a substrate for proteases overexpressed in the targeted
tumor microenvironment (i.e cathepsin B). Once EHU will be
recognized by these enzymes it is cleaved off resulting in the
consequent release of the active drug through rapid cascade
reactions (Figure 5).
The most representative examples of various types of linkers
are summarized in Table 2.
Representative examples of PDCs targeting
cancer cells
Integrating the basic design principles in PDCs pinpointed
above, a list of representative developed examples is analyzed
below, so as to provide a spherical perspective regarding
peptide–drug conjugation chemistry.
Currently, there are two PDCs that have been developed
utilizing peptides as tumor targeting elements that selectively
bind to specific receptors and small molecules as anticancer
agents that have reached phase III clinical trials (Table 3) for
the treatment of various types of cancer. ClinicalTrials.gov have
also announced the initiation of a clinical trial based on
various PDCs consisted of two novel peptides selected
after phage display that target murine A20 leukemic
cells (ClinicalTrials.gov Identifier: NCT02828774). These
clinical trials will focus on chronic lymphocytic leukemia
(CLL).
Except these two PDCs, there are other types of PDCs that do
not consist of peptides as targeting moieties and small mole-
cules as drugs and have reached even up to phase III clinical
trials. These PDCs are summarized in Table 4.
Notably, there is only one PDC in the market designated 111In-
DTPA-d-Phe1-octreotide, which is utilized for diagnostic radi-
ology in somatostatin receptor-positive tumors [118]. It consti-
tutes a complex of 111Indium bound to diethylenetriaminopen-
taacetic acid (DTPA), which is conjugated to the targeting
somatostatin peptide [D-Phe1]-octreotide. Recently, another
similar analog, designated 111In-DTPA-d-Phe-1-Asp0-d-Phe1-
octreotide, has been evaluated and presented enhanced tumor
accumulation in pancreatic tumor cells and simultaneously
lower renal radioactivity [119].
Herein, we will analyze in depth the two PDCs in clinical trials
consisted of peptides and small molecules (Table 3), as also

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Table 3: Peptide–drug conjugates consisting of peptides and small molecules that have been used in clinical trials.
peptide cytotoxic agent linker drug release mechanism name target CCTareference
D-Lys6-LHRH Dox (SM)bglutaryl esterases/amidases AEZS-108 LHRH-R phase III [112]
angiopep-2 PTX (SM) succinyl esterases/amidases ANG1005 LRP-1 phase II [79]
aCCT= current clinical trials; bSM= small molecule.
Table 4: Various other types of peptide–drug conjugates in clinical trials.
peptide cytotoxic agent linker drug release
mechanism
target name CCTareference
CNGRCG hTNFα (Protein) – amidases CD13 receptor NGR015 phase III [113]
polyglutamic acid PTX (SM)b– esterases – CT2103 phase III [114]
LHRH CLIP71c (lytic peptide) – amidases LHRH-R EP-100 phase I [115]
DRDDS (spacer) DAVBLHd (SM)b2-mercapto-
ethanol
glutathione folate receptor EC145 phase III [116]
D-γ-E-γ-E-γ-E-E
(masking moiety)
12ADTe-Asp – PSMA PSMA G-202 phase II [117]
aCCT = current clinical trials; bSM = small molecule; cCLIP71 = KFAKFAKKFAKFAKKFAK; dDAVBLH = desacetyl vinblastine hydrazide;
e12ADT = 8-O-(12-aminododecanoyl)-8-O-debutanoyl thapsigargin.
Figure 6: Structures of the PDCs named AN-152 and AN-207.
various other similar PDC formulations existed in the current
literature that have been evaluated in preclinical models.
First, two widely-known peptide–drug conjugates named
AN-152 (AEZS-108) and AN-207 will be analyzed. These
conjugates contain the luteinizing hormone-releasing hormone
(LHRH) as the peptide-targeting module and doxorubicin
(DOX) or its daunosamine-modified derivative 2-pyrrolino-
DOX as the cytotoxic agent, respectively (Figure 6). Specifi-
cally, Andrew V. Schally and his group first synthesized the
corresponding analogs [120] where they covalently coupled the
two drugs to the epsilon-amino group of the D-Lys side chain of
the peptide D-Lys6-LHRH.
Notably, both conjugates fully preserved the cytotoxic activity
of the parent drugs, DOX or 2-pyrrolino-DOX, respectively, in
vitro and also retained the high binding affinity of their peptide
carrier to receptors for LHRH on rat pituitary [120]. The two
conjugates were subjected to stability tests and they showed
slow drug release in human serum in contrast with nude mice
that carboxylesterase enzymes are about 10 times higher [121].
Consequently, the two analogs were heavily evaluated in in

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941
vivo models in nude mice bearing various types of cancer. Mice
bearing OV-1063 (LHRH receptor positive) or UCI-107
(LHRH receptor negative) human epithelial ovarian cancers
were treated with AN-152 or DOX with systematic intraperi-
toneal administration. The growth of UCI-107 cells was not in-
hibited by AN-152 but systemic administration of AN-152 in
OV-1063 cells proved that AN-152 is less toxic but inhibits
tumor growth better than equimolar doses of DOX [122]. These
results were confirmed in nude mice bearing other ovarian
human cancers (ES-2), where AN-207 caused up to 59.5% inhi-
bition in tumor growth [123]. Also, AN-207 and AN-152 were
tested in female BDF mice bearing estrogen independent MXT
mouse mammary cancers, presenting stronger tumor inhibitory
effects than their respective cytotoxic radicals up to 93%, while
equimolar quantities of their respective radicals were more toxic
[124]. Moreover, PDC AN-207 was significantly more potent,
regarding the growth inhibition of hormone-dependent Dunning
R-3327-H prostate cancers in rats, reaching up to 50% of the
initial tumor volume in comparison with 2-pyrrolino-DOX.
Shortly afterward, they tested the two conjugates in membranes
of human breast cancer cells: MCF-7 hormone-dependent and
MDA-MB-231 hormone- independent [125]. They proved that
the specific analogs retained the high binding affinity of the
D-Lys6-LHRH carrier to the relevant receptors. Both
conjugates displayed IC50 values in the low nanomolar
concentration range for MCF-7 (13.7 ± 1.09 nM for AN-152
and 6.08 ± 0.5 nM for AN-207) and MDA-MB-231
(5.60 ± 1.24 nM for AN-152 and 1.89 ± 0.4 nM for AN-207)
cells. AN-152 was tested regarding the inhibition of tumor
growth of subcutaneously (sc) implanted androgen-dependent
LNCaP and MDA-PCa-2b and androgen-independent C4-2
prostate cancers, xenografted into nude mice. The results
demonstrated the stronger inhibition of AN-152 on the tumor
with respect to the free DOX [126]. Similarly, in vivo experi-
ments were conducted regarding AN-207 in nude mice bearing
xenografts of MDA-PCa-2b prostate cancer cells, showing iden-
tical results like AN-152 [127]. Gründker et. al. evaluated the
antitumor effects of AN-152 in vivo in human LHRH-R-posi-
tive HEC-1B endometrial and NIH:OVCAR-3 ovarian cancers,
and in the LHRH-R-negative SK-OV-3 ovarian cancer cell line
via intravenous injections [128]. The tumor volumes of HEC-
1B and NIH:OVCAR-3 cancers were reduced significantly even
after 1 week of treatment with AN-152 while presenting no
toxic side effects. Treatment with DOX arrested tumor growth
but did not reduce tumor volume. The growth of SK-OV-3
cancers was not affected by AN-152. Based on the presented
results, it can be concluded that these two analogs possess
higher antitumor activity but less toxicity with respect to the
parent drugs DOX and 2-pyrrolino-DOX and can be used
versus a wide variety of ovarian, prostate, endometrial and
breast tumors.
Notably, after the extensive evaluation of analog AN-152 in
preclinical models, starting from 2006 it has been tested in
phase I and phase II studies (AN-152 was renamed to AEZS-
108 for the clinical trials) of LHRH-R positive recurrent
endometrial and ovarian cancers. The phase I/II study in castra-
tion-resistant prostate cancer (CRPC) and chemotherapy refrac-
tory bladder cancer also showed promising results. Due to the
promising results from phase II trials in endometrial cancer, a
multinational phase III clinical study is underway [112].
It is important to note that despite the fact that analog AN-207
presented a better biological profile, evident in all the preclin-
ical models, its further development fell short due to chemical
and plasma instability.
According to ClinicalTrials.gov, during phase I analog AEZS-
108 was tested in 17 women with epithelial cancer of the ovary,
endometrium or breast and for which standard treatment could
not be used or was no longer effective. The results showed
promising tolerance from the patients with fewer side effects
than the commonly applied drugs. Moreover, AEZS-108 was
evaluated in phase I clinical trial on patients with castration-
and taxane-resistant prostate cancer and the results proved that
AEZS-108 possesses a sufficient safety profile and efficacy. It
succeeded in lowering the PSA levels in some patients with
prostate cancer and it became evident that the internalization of
AEZS-108 in prostate cancer circulating tumor cells (CTCs)
may be a viable pharmacodynamic marker [129].
These promising results led to phase II clinical trials to patients
with castration- and taxane-resistant prostate cancer and their
disease showed progression after taxane-based chemotherapy.
AEZS-108 showed significant activity in these patients who
were pretreated with taxanes and maintained an acceptable
safety profile [130].
Last, phase II clinical trials were conducted in collaboration
with the German Gynecological Oncology Group (AGO) and
3 other centers from Bulgaria on 43 women. The patients had
platinum-resistant advanced ovarian cancer, FIGO (Fédération
Internationale de Gynécologie et d'Obstétrique) III or IV or
recurrent endometrial cancer (EC) and LHRH receptor-positive
tumor status. The treatment with AEZS-108 had significant ac-
tivity and low toxicity in these women [131,132].
Based on the fact that the previously described analog AN-207
showed superior in vitro and in vivo results compared to
AN-152 but lacked stability, Andrew V. Schally and his group
turned their focus on its building block 2-pyrrolino-DOX and
tried to construct new PDCs using other peptides. Therefore,
they synthesized a new analog, designated AN-238, consisting

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942
Figure 7: Structure of the PDC named AN-238.
of the octapeptide RC-121 linked through the α-amino group of
its N-terminal D-Phe moiety and a glutaric acid spacer to the
14-OH group of 2-pyrrolino-DOX (Figure 7). The octapeptide
RC-121 was utilized due to its high binding affinity to the
somatostatin receptor (SST-R) [133].
The anti-cancer activity was first evaluated in various rat/human
cancer lines xenografted into nude mice with breast human
tumors (MDA-MB-238, MCF-7, MX-1) and prostate rat/human
tumors (Dunning AT-1, PC-3). All cell lines showed a great
response to the treatment with AN-238 with high inhibition of
the tumor, while 5 of 10 mice with MX-1 tumor were totally
cured [61]. The cytotoxic profile of this analog was similarly
evaluated in additional cancer cell lines xenografted into nude
mice including prostate, renal, mammary, ovarian, gastric,
colorectal and pancreatic [134]. Various types of renal,
colorectal, pancreatic and gastric cancers showed a major
response to the treatment with more than 70% inhibition while
all the other types showed a good response to the treatment with
an average of 60% inhibition. AN-238 was also evaluated in
U87-MG brain cancer cells with good response, inducing
82% growth inhibition of subcutaneous tumors [134]. There-
fore, AN-238 has been proved to be a promising candidate for a
large number of tumors, being able to suppress the growth of
these tumors and their metastases. Last, Engel et. al. showed
that AN-238 inhibits tumor growth in human experimental
endometrial carcinomas which express SST receptors, regard-
less of the expression levels of multidrug resistance protein
MDR-1 [135]. The analog AN-238 is still pending for clinical
trials.
An interesting example of a PDC able to cross the blood-brain
barrier (BBB), is ANG1005 [136], composed of three mole-
cules of paclitaxel linked by a cleavable succinyl ester linkage
to the angiopep-2 peptide (Figure 8).
BBB is formed by the brain capillary endothelium with very
low permeability as it excludes about 100% of the large mole-
cules and about 98% of the small molecules attempting to pass
to the brain [137]. Being mandatory to surpass the BBB in order
to deliver pharmaceuticals to the brain, scientists have strug-
gled to discover either novel small molecules able to cross it
through various mechanisms [138] or novel techniques able to
disrupt its dense structure like ultrasound-mediated drug
delivery [139,140]. The design principles on the synthesis of the
specific conjugate, ANG1005, were the following: the peptide
angiopep-2 is able to cross the BBB via receptor-mediated tran-
scytosis after binding to LRP-1 and consequently it is often
used as drug delivery vehicle, while paclitaxel bears cytotoxici-
ty against glioblastoma. It has been shown that the brain uptake
of ANG1005 was 4.5-fold higher compared to paclitaxel and
the cytotoxicity remained higher in all cancer cell lines tested
(glioblastoma U87 MG, U118, U251; lung carcinoma A549,
NCI-H460, Calu-3; ovarian carcinoma SK-OV-3). It has been
also proved that human tumor xenografts were inhibited more
with ANG1005 than paclitaxel. Finally, mice with intracerebral
implantation of U87 MG glioblastoma cells or NCI-H460 lung
carcinoma cells exhibited increased survival rates after
ANG1005 administration.
Because of these promising results, ANG1005 progressed to
phase I clinical trials in 2007 in 63 patients with recurrent or
progressive malignant glioma. It was found that ANG1005
delivers paclitaxel across the BBB and achieves therapeutic
concentrations in the tumor site. It became evident that this
PDC possessed similar toxicity to paclitaxel as also enhanced
activity in recurrent glioma [141]. Phase II clinical trials were
then initiated on patients with recurrent high-grade glioma and
on breast cancer patients with recurrent brain metastases. The
results have not been published yet but it has already been
stated that very promising results were collected and phase III
clinical trials will start shortly. Based on the overall progress of
ANG1005, other similar molecules have been synthesized and
studied in preclinical models [142].
The group of Prof. G. Mező has achieved a great progression in
the field of PDCs the last years working mostly on GnRH-III
(Glp-His-Trp-Ser-His-Asp-Trp-Lys-Pro-Gly-NH2), which was

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943
Figure 8: Chemical structure and synthetic scheme for the PDC ANG1005. (A) ANG1005 is composed of three molecules of paclitaxel linked by a
cleavable succinyl ester linkage to the angiopep-2 peptide. (B) Schematic representation of ANG1005 synthesis steps. Paclitaxel was first reacted
with succinic anhydride and then activated with N-hydroxysuccinimide to form 2′-succinyl-NHS-paclitaxel in two steps. In the conjugation step (step 3),
amines of the angiopep-2 peptide react with 2′-succinyl-NHS-paclitaxel. The scheme was modified according to Br. J. Pharmacol. 2008, 155, 185–197
[136].
exploited as a tumor homing device for drug targeting 10 years
ago [111]. The aim of this was to apply a peptide hormone with
lower endocrine effect than GnRH-I that might be useful espe-
cially for hormone-independent tumors like colon cancer [143].
In addition, GnRH-III has Lys at position 8 of the sequence pro-
viding a conjugation site without inducing perturbation in the
receptor recognition. In the first conjugates, daunorubicin (Dau)
was attached to the lysine side chain via oxime linkage through
an aminooxyacetyl (Aoa) moiety (Glp-His-Trp-Ser-His-Asp-
Trp-Lys(Dau=Aoa)-Pro-Gly-NH2). The oxime bond, de-
veloped between the aminooxyacetyl function and the carbonyl
group of C-13 on Dau is stable under physiological conditions
and prevents the early drug release in contrast to the ester bond
(Figure 9).
Thus, no free drug release can be detected from such type of
conjugates before reaching their targets. However, oxime bond
is also stable in lysosomes where the conjugates decomposed
after receptor-mediated endocytosis. Among the fragments
arose during lysosomal degradation, H-Lys(Dau=Aoa)-OH was

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944
Figure 9: Structure of oxime linked Dau–GnRH-III conjugate with or without cathepsin B labile spacer and their metabolite released in lysosomal
homogenate [144].
observed as the smallest Dau containing metabolite [144].
Therefore, the DNA binding propensity of this metabolite was
also examined and it was found that although it is efficient it
presented lower binding capacity with respect to the parent
drug.
The in vitro antitumor activity of the above-mentioned conju-
gate was studied on MCF-7 human breast and HT-29 human
colon adenocarcinoma cells [144]. The IC50 values showed two
orders of magnitude lower effect compared to the free Dau.
Thus, a systematic comparative study of various anthracycline-
GnRH conjugates was conducted in order to conduct their com-
plete evaluation as potential targeted cancer chemotherapeutics.
The influence of different: (i) anthracycline drugs, (ii) linkers
among the tumor-homing peptide moiety and the drug, and (iii)
tumor-homing peptides (e.g., GnRH-III and D-Lys6-GnRH-I)
was examined regarding their in vitro cellular uptake, drug
release and cytostatic effect [145]. Doxorubicin (Dox) was
coupled to both GnRH-III and D-Lys6-GnRH-I through a
glutaric acid linker via ester bond. AN-152, the GnRH-I based
PDC (see above), served as a control. No significant differ-
ences in cellular uptake and cytostatic effect were observed be-
tween the two PDCs. Recently, it was also indicated that the
cellular uptake of carboxyfluorescein-labeled GnRH-I, GnRH-II
and GnRH-III conjugates might be influenced not only by the
targeting moiety, but also by the type of cancer cells [146].
However, no significant differences could be observed
regarding the cellular uptake of the three GnRH conjugates by
MCF-7 and HT-29 cells. It is worth mentioning that the highest
water solubility was detected for the GnRH-III conjugate. The
ester bond can be cleaved by esterases not only in cancer cells,
but also in human plasma during blood circulation. The early
drug release in the bloodstream may cause unwanted side
effects. Furthermore, O–N acyl shift was detected both during
the synthesis and the storage of ester-linked doxorubicin conju-
gate, resulting to an inactive compound where the tumor-
homing peptide acylated the amine of the daunosamine sugar
moiety. This was found through the mass spectrometric (MS)
fragmentation profile of the PDC [146].
In a different PDC, Dau was linked to GnRH-III through a
hydrazone bond or by incorporation of a self-immolative spac-
er [145]. The hydrazone linkage was formed similarly to the
oxime bond on C-13 atom of Dau but it allows the effective
drug release under slightly acidic conditions in lysosomes. The
p-aminobenzylalcohol-based self-immolative spacer, combined
with the dipeptide Lys-Phe (cathepsin-B lysosomal enzyme
cleavable spacer), was connected to the amino functional group
of daunosamine moiety. The last construct also provided the
free drug release. Both conjugates illustrated similar cytostatic
effects and cellular uptake as the conjugates with ester bonds.
All these conjugates showed IC50 values in the range of

Beilstein J. Org. Chem. 2018, 14, 930–954.
945
0.2–0.5 μM on MCF-7 cells while 1–3 μM on HT-29 cells. The
free Dau or Dox had higher in vitro cytostatic effect than the
conjugates, especially on HT-29 cells. Nevertheless, the synthe-
sis of these conjugates was not so efficient and their chemical,
biological and long-term shelf-stability of these PDCs were not
so sufficient for drug development.
In another construct, daunorubicin and doxorubicin were at-
tached to the ε-amino group of Lys of GnRH-III through oxime
linkage [111,145]. The conjugation of the drug and the
aminooxyacetylated tumor homing peptide was almost quantita-
tive under slightly acidic conditions. Interestingly, the conju-
gate with Dox illustrated much lower antitumor effect than the
Dau conjugate. The oxime-linked Dau-GnRH-III conjugate
(non-cleavable linker) had one order of magnitude lower anti-
tumor activity than the conjugates with the cleavable linkers.
The cellular uptake of the oxime-linked conjugates was lower,
too, but this effect might come from the different fluorescent
properties of the free Dau and the peptide/metabolite-linked
Dau. Because of the high synthetic yield and stability of oxime-
linked conjugates, it can be suggested that such conjugates
might be good candidates for the development of targeted tumor
therapeutics. Therefore, efforts were made to develop further
conjugates with higher antitumor activity.
To achieve higher antitumor activity, the sequence of the
peptide GnRH-III was modified. Previous studies indicated that
only a few changes are acceptable without significant loss of the
anti-proliferative effect of the hormone peptide. Interestingly,
Ser at position 4 could be replaced by Lys or acetylated Lys
[147]. It is worth mentioning that the Ser in GnRH agonist and
antagonist analogs are rarely modified [148]. The incorporation
of Lys or Lys(Ac) in position 4 of GnRH-III increased the anti-
tumor activity of the conjugate GnRH-III(Dau=Aoa). However,
in the case of [4Lys]-GnRH-III(Dau=Aoa) enzyme stability of
the conjugate was decreased while [4Lys(Ac)]-GnRH-
III(Dau=Aoa) showed higher stability [149]. When the acetyl
group was exchanged to other short-chain fatty acids (SCFAs)
the enzyme stability was enhanced by the length of hydro-
carbon chain of SCFAs [150]. According to the cellular uptake
and cytostatic in vitro studies, the optimal compound was the
butyric acid containing [4Lys(Bu)]-GnRH-III(Dau=Aoa) conju-
gate that almost reached the in vitro biological effects of the
conjugates with a cleavable linker. This conjugate showed sig-
nificant tumor growth inhibition in vivo, not only on subcuta-
neous implanted but also on orthotopically developed HT-29
colon cancer-bearing mice [151]. The PDC in the applied dose
(15 mg Dau content/kg body weight) showed similar or higher
antitumor activity than the free Dau at a maximal tolerated dose
(MTD) without significant toxic side effects on organs. In
contrast to the conjugate, Dau presented toxicity on the liver
causing worse condition and higher mortality during the treat-
ment.
In addition, the incorporation of Lys at position 4 provided a
new conjugation site. Therefore, Dau or methotrexate (MTX)
were attached to the ε-amino group of 4Lys resulting in conju-
gates with two identical ([4Lys(Dau=Aoa), 8Lys(Dau=Aoa)]-
GnRH-III), or different drug molecules ([4Lys(MTX),
8Lys(Dau=Aoa)]-GnRH-III) [152,153]. Some improvement in
the cytostatic effect could be detected compared with the conju-
gates containing only one drug molecule, but they were not
better than the conjugate with butyric acid. This observation led
to retain the Lys(Bu) at position 4 and the two Dau molecules
were attached to the amino groups of an additional Lys
through the enzyme labile GFLG spacer coupled to 8Lys
(Figure 10).
The resulted PDC presented a reduced aqueous solubility, thus
an oligoethylene glycol linker was inserted between the spacer
and the tumor-homing peptide [154]. This PDC showed the best
in vitro cytostatic effects among the oxime-linked Dau-contain-
ing conjugates, but the improvement of the synthetic process to
lead to higher amounts of this PDC is required to proceed for in
vivo studies. Thus, it can be concluded that oxime linked
Dau–homing peptide conjugates could be good candidates for
targeted tumor therapy.
Furthermore, the tumor homing peptide D-Lys6-GnRH-I has
been exploited by our group to selectively deliver the anti-
cancer agent gemcitabine to the tumor site. We, therefore, de-
signed and synthesized four different bioconjugates consisting
of D-Lys6-GnRH-I and the anticancer agent gemcitabine
(named GSG, GSG2, 3G, 3G2) through different conjugation
sites (the primary and secondary alcohol groups of gemcitabine)
and using linkers of different lengths (succinyl and glutaryl) as
shown in Figure 11.
In order to evaluate whether the tethering of the cytotoxic agent
to the D-Lys6-GnRH-I peptide induces any perturbation on the
local microenvironment of the peptide that is responsible for re-
ceptor recognition, we utilized 1H 1H 2D-TOCSY NMR [19].
Upon superimposing the relevant spectra of the different PDCs
on the relevant spectrum of the native hormone we found that
these PDCs didn’t alter the microenvironment of D-Lys6-
GnRH-I allowing to suggest that they will not influence the
binding affinity of the targeting peptide unit of these PDC to the
GnRH-R. This was further validated since the new conjugates
were found to possess higher binding affinity with respect to the
parent peptide, with IC50 ranging even up to 1.9 nM for the
conjugate 3G. The conjugates were evaluated regarding their
antiproliferative effect on prostate cancer cells (DU145 and

Beilstein J. Org. Chem. 2018, 14, 930–954.
946
Figure 10: Synthesis of the most effective GnRH-III–Dau conjugate with two drug molecules [153].
PC-3) and the PDC GSG showed IC50 values similar to gemci-
tabine GSG that possessed the highest antiproliferative effect
was utilized for further pharmacokinetic studies in mice. These
pinpointed that GSG is able to release a high amount of gemci-
tabine (averaging 500 ng/mL) for a period of over ≈250 min,
while administered free gemcitabine was consumed in less than
100 min. At the same time, the levels of the inactive metabolite
of gemcitabine (dFdU) were maintained at very low levels for
the GSG conjugate in contrast to the direct administration of
free gemcitabine. Finally, when injected into mice with
xenografted tumors, GSG inhibited the tumor growth more
effectively than gemcitabine when using equimolar quantities.
Therefore, GSG could pave the way for the construction of
other similar bioconjugates in order to effectively enhance the
concentration of the cytotoxic drug in the tumor cells and
inhibit their uncontrolled growth.
OuWe have also designed and synthesized a PDC containing
the cytotoxic agent sunitinib and the D-Lys6-GnRH peptide-
targeting-unit tethered through a succinyl linker [7]. Sunitinib is
a small orally administrated drug that inhibits the phosphoryla-
tion of several receptor tyrosine kinases (RTKs). It was ap-
proved by the FDA in 2006 for the treatment of renal cell carci-
noma (RCC) and imatinib-resistant gastrointestinal stromal
tumor (GIST). Though, sunitinib has proved to cause severe
side effects like cardiac and coronary microvascular dysfunc-
tion [155]. Therefore, these data rendered sunitinib as an
appealing candidate for targeted therapy using a PDC.
Native sunitinib (Figure 12A) does lack functional groups that
could be exploited for conjugation to the peptide-targeting unit,
thus, a novel analog had to be constructed (SAN1, Figure 12B).
This was constructed based on in silico studies and modifying
properly the drug scaffold so as not to perturb the drug binding
to the targeted receptors. In silico, in vitro and pharmacokinetic
evaluation of the synthesized SAN1 were conducted and com-
pared with native sunitinib. The results indicated that SAN1
exhibited similar properties and thus could serve as an alterna-

Beilstein J. Org. Chem. 2018, 14, 930–954.
947
Figure 11: Structures of the four different PDCs of D-Lys6-GnRH-I and gemcitabine (GSG, GSG2, 3G, 3G2) [19].
Figure 12: Structures of (A) native sunitinib; (B) SAN1 analog of sunitinib and (C) assembled PDC named SAN1GSC [18].
tive to the parent drug for the formulation of the final PDC. Ad-
ditionally, SAN1 was further explored in in vivo models: mice
xenografted with a castration-resistant CaP (CRPC) cell line
were subjected to treatment based on SAN1 and sunitinib. Mice
were dosed daily via intraperitoneal injection and the results
unveiled the potency of SAN1, which showed to inhibit the

Beilstein J. Org. Chem. 2018, 14, 930–954.
948
tumor growth in a similar way like native sunitinib. In the
installed hydroxy group in the core of SAN1, a succinyl linker
was conjugated that was then connected to the free amine group
of Lys8 of D-Lys6-GnRH to form the PDC named SAN1GSC
(Figure 12C).
SAN1GSC was evaluated in vitro and then in vivo, in mice
xenografted with the CRPC model, showing similar bioactivity
as SAN1. The most promising results arose from the measured
concentration of SAN1 in the blood circulation and in the tumor
site. The levels of free SAN1 released from SAN1GSC were 4
times higher inside the malignant cells with respect to SAN1
levels from the unconjugated SAN1. It is worth mentioning that
in the frame of our construct, cardiotoxic and hematotoxic
effects in treated mice were minimal and elevations of blood
pressure that contribute to cardiac dysfunction were absent [18].
Problems and solutions during synthesis of
PDCs
Although the synthesis of PDCs is usually a rapid and facile
procedure, various synthetic problems may arise. The most
common ones appear during peptide synthesis and might refer
to low aqueous solubility and/or difficulty to synthesize. Insolu-
bility issues can be overcome by altering the C-/N-terminus
and/or substituting specific residues. Difficulties in the synthe-
sis can be handled by decreasing the number of hydrophobic
residues and/or shortening the sequence. Similar synthetic prob-
lems have been encountered during peptide synthesis the last
decades and have been fully addressed.
During the conjugation of 5’-O-gemcitabine hemisuccinate to
the D-Lys6-GnRH peptide towards the synthesis of the PDC
GSG (presented in Figure 10) we recently unveiled the forma-
tion of a previously unnoticed side product in addition to the
desired product [156]. Specifically, we found that when guani-
dinium salts are utilized in peptide coupling conditions, a
uronium derivative can be installed on specific amino acid scaf-
folds, beside to the formation of the expected amide bonds. This
side product was persistent even after HPLC purification and
was also apparent in the recorded mass spectrum of GSG as a
second peak, besides the expected product, bearing the mass of
the expected PDC plus 100 amu, leading to the reduction of the
overall yield below 10%. Specifically, the guanidinium/uronium
coupling reagent (HATU) was utilized for the formation of the
amide bond between D-Lys6 of the peptide carrier and the
carboxylic acid of the succinate linker connected to gemcitabi-
ne to synthesize the PDC GSG (Figure 13). We hypothesized
that the side product was originated from the coupling reagent
(HATU) and after conducting template reactions with every
amino acid present in the sequence of D-Lys6-GnRH with
Fmoc-Ser(t-Bu)-OH in the presence of HATU or HBTU and
DIPEA, it became evident that the aminium moiety of HATU/
HBTU could be installed either on the amino (–NH2) group of
Lys or on the phenol (–OH) group of Tyr [156].
Our findings were further verified by reacting other tumor-
homing peptides like D-Lys6-GnRH and Fmoc-HER2-BP1
(LTVSPWY, a heptapeptide known for its activity against
erbB2) with HATU/DIPEA and characterizing the final prod-
ucts by ESIMS and 1H NMR spectroscopy where the same
aminium side product was also recorded. Interestingly, when
the dipeptide Fmoc-Cys-Tyr-NH2 was reacted with HATU, we
found that the side product could be installed both on the phenol
group of Tyr as also on the sulfhydryl group of Cys. Therefore,
we tested these reaction conditions on the peptide C1B5141–151
subdomain peptide (RCVRSVPSLCG) of protein kinase C
(PKC) γ isozymes, which possess 2 cysteines (but no tyrosine
or lysine or free N-terminus amine) and bears anticancer prop-
erties [157]. Again, a side product with two aminium moieties
on the two cysteines was formed and characterized with
ESIMS. This observation was also applied to a simple phenol
where again a side product was recorded pinpointing the broad
impact of our findings beyond traditional peptide chemistry. We
thus revealed the formation of a previously unnoticed side prod-
uct during the synthesis of PDCs, derived from guanidinium/
uronium peptide coupling reagents that occurs on phenols, pri-
mary amines and sulfhydryl groups (Figure 14).
Along these lines, we suggested a mechanism that this side-
product formation is taking place, directly after the formation of
the amide bond that occurs from structure (II) to structure (III),
as shown in Figure 15.
We discovered that the side product, which is difficult to be
separated from the expected PDC and therefore results in
reduced synthetic yield, could be avoided by using 1 equiv of
HATU/HBTU, instead of the classical and established proto-
cols using 1.5 equiv [156]. Taking into account that uronium/
guanidinium coupling reagents are among the most expensive
ones, using the specified conditions (equimolar quantity) may
also reduce the total cost of the synthesis.
Conclusion
Currently used chemotherapeutics are in their majority highly
toxic, causing severe side effects. Thus, with the aim to en-
hance their narrow therapeutic index, a wide variety of strate-
gies have been explored. Selective drug delivery via special
carriers represents a viable approach to deal with tumors with
higher efficacy while using lower doses of the anticancer agent.
Specifically, peptide–drug conjugates (PDCs) operate as potent
drug delivery carriers and thus have attracted considerable
attention over the last decades. The simplicity, versatility and

Beilstein J. Org. Chem. 2018, 14, 930–954.
949
Figure 13: Synthetic scheme for the formation of GSG and the unexpected side product [156].
Figure 14: Illustration of uncharted guanidinium peptide coupling reagent side reactions during PDCs synthesis [156].

Beilstein J. Org. Chem. 2018, 14, 930–954.
950
Figure 15: Putative mechanism for the formation of the uronium side product [156].
the relatively low cost for the construction of PDCs have
rendered them appealing candidates. In the present review,
basic and integral knowledge has been accumulated towards the
PDCs design through examining every module required to
assemble the fully decorated PDC: the peptide, the cytotoxic
agent and the linker. We highlighted the overall progress of this
field through selective analysis of noteworthy examples in the
literature, as also possible synthetic problems that may arise and
their solutions. Based on the fact that several PDCs have been
selected for clinical trials and presented tumor inhibition with
minimum side effects, this field needs to be further refined and
explored. Through this review, we made efforts to provide an
influential impetus for the construction of new peptide–drug
conjugates, which could eventually transform undesired toxic
drugs to highly potent formulations for the effective treatment
of cancer.
Acknowledgements
This work was co-financed by the European Union (European
Social Fund ESF) and Greek national funds through the Opera-
tional Program “Education and Lifelong Learning” of the
National Strategic Reference Framework (NSRF) - Research
Funding Program: ARISTEIA II [grant number: 5199]. The
authors would also like to thank National Research, Develop-
ment and Innovation Office (NKFIH K119552), Hungary.
ORCID® iDs
Eirinaios I. Vrettos - https://orcid.org/0000-0002-4801-1900
Gábor Mező - https://orcid.org/0000-0002-7618-7954
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doi:10.3762/bjoc.14.80