molecules
Review
Targeting Toxins toward Tumors
Henrik Franzyk
and Søren Brøgger Christensen *
Department of Drug Design and Pharmacology, University of Copenhagen, Universitetsparken 2,
DK-2100 Copenhagen Ø, Denmark; henrik.franzyk@sund.ku.dk
* Correspondence: soren.christensen@sund.ku.dk
Citation: Franzyk, H.; Christensen,
S.B. Targeting Toxins toward Tumors.
Abstract: Many cancer diseases, e.g., prostate cancer and lung cancer, develop very slowly. Common
chemotherapeutics like vincristine, vinblastine and taxol target cancer cells in their proliferating states.
In slowly developing cancer diseases only a minor part of the malignant cells will be in a proliferative
state, and consequently these drugs will exert a concomitant damage on rapidly proliferating benign
tissue as well. A number of toxins possess an ability to kill cells in all states independently of whether
they are benign or malignant. Such toxins can only be used as chemotherapeutics if they can be
targeted selectively against the tumors. Examples of such toxins are mertansine, calicheamicins and
thapsigargins, which all kill cells at low micromolar or nanomolar concentrations. Advanced prodrug
concepts enabling targeting of these toxins to cancer tissue comprise antibody-directed enzyme
prodrug therapy (ADEPT), gene-directed enzyme prodrug therapy (GDEPT), lectin-directed enzymeactivated prodrug therapy (LEAPT), and antibody-drug conjugated therapy (ADC), which will be
discussed in the present review. The review also includes recent examples of protease-targeting
chimera (PROTAC) for knockdown of receptors essential for development of tumors. In addition,
targeting of toxins relying on tumor-overexpressed enzymes with unique substrate specificity will
be mentioned.
Keywords: chemotherapy; prodrug; drug targeting; overexpressed enzymes; ADC; ADEPT; GDEPT;
LEAPT; PROTAC
Molecules 2021, 26, 1292. https://
doi.org/10.3390/molecules26051292
Academic Editors:
1. Introduction
Marialuigia Fantacuzzi and
According to the International Union of Pure and Applied Chemistry (IUPAC), prodrugs are defined as chemically modified drugs that undergo biological and/or chemical
transformation(s) before eliciting pharmacological responses [1]. Drugs may be converted
into prodrugs in order to: (i) increase their bioavailability, (ii) target the drugs toward
tissues such as tumors, (iii) decrease toxicity, (iv) increase chemical stability, (v) increase
solubility, or (vi) mask unpleasant taste [2,3]. Prodrugs are formed by covalent attachment
of the drug to a carrier, also often termed as the promoiety, which is subjected to cleavage
within the body to release the active drugs. Promoieties and their degradation products
should be nontoxic and nonimmunogenic [2]. Pharmacologically inactive compounds,
which in the organism are modified into active drugs, are known as bio-precursor prodrugs.
Examples of such prodrugs are proguanil that in the liver is converted into the antimalarial
drug cycloguanil [4], salicin that is converted into salicylic acid [5], and acetanilide that is
converted into acetaminophen [3]. Both salicylic acid and acetaminophen are antipyretics
and analgesics. Another class of prodrugs is the co-drugs that consist of two drugs covalently attached to each other—either directly or via a linker in a way so that they act as
promoieties for each other [2,6]. Examples of co-drugs comprise sulfasalazine, which in the
body is degraded to 5-aminosalicylic acid and sulfapyridine [6,7], and benorylate, which is
an ester of acetylsalicylic acid with paracetamol [6].
A review focused on the prodrug approach revealed the state of the art in 2017 [8].
In the present review new developments in the fields of boronic acids as prodrugs, of
anthracyclines, and of antibody–drug conjugates, targeting of paclitaxel and refined use of
Alessandra Ammazzalorso
Received: 9 January 2021
Accepted: 22 February 2021
Published: 27 February 2021
Publisher’s Note: MDPI stays neutral
with regard to jurisdictional claims in
published maps and institutional affiliations.
Copyright: © 2021 by the authors.
Licensee MDPI, Basel, Switzerland.
This article is an open access article
distributed under the terms and
conditions of the Creative Commons
Attribution (CC BY) license (https://
creativecommons.org/licenses/by/
4.0/).
Molecules 2021, 26, 1292. https://doi.org/10.3390/molecules26051292
https://www.mdpi.com/journal/molecules
Molecules 2021, 26, 1292
2 of 23
prostate-specific membrane antigen (PSMA) for delivery of payloads are described. The
problem of targeting oncogenes for neoplastic tissue in gene-directed enzyme prodrug
therapy is discussed (which often has been omitted in previous reviews). Finally, a new
group of prodrugs, which link two pharmacophores, i.e., PROtease Targeting Chimeras
(PROTACs; [9,10]) and lectin-directed enxym activated prodrugs, are discussed. One
of these pharmacophores has a high affinity for an E3 ubiquitin ligase (an enzyme that,
with assistance of an E2 ubiquitin-conjugating enzyme, transfers ubiquitin to its protein
substrate), while the other has an affinity for the targeted protein e.g., a receptor or ion
channel. After binding of the protein and the ubiquitin ligase, the biomolecule will be
modified with ubiquitin and subsequently cleaved by a 26S proteasome, which degrades
ubiquitinated proteins [9,10].
Thus, the present review comprises the following: (i) prodrugs cleaved in the acidic
microenvironment of cancer cells (Section 2.1.1), (ii) prodrugs cleaved by reactive oxygen species (ROS) in cancer cells (Section 2.1.2), (iii) prodrugs cleaved by glutathione
(Section 2.1.3), (iv) prodrugs cleaved by enzymes overexpressed in cancer cells ( Section 2.1.4),
(v) prodrugs cleaved by glucuronidase (Section 2.1.5), (vi) prodrugs cleaved by prostatespecific antigen (PSA) or PSMA (Section 2.1.6), (vii) antibody–drug conjugates (Section 2.2),
(viii) antibody-directed enzyme prodrug therapy (Section 2.3), (ix) gene-directed enzyme prodrug therapy (Section 2.4), (x) lectin-directed enzyme-activated prodrug therapy
(Section 2.5), and (xi) protease-targeting chimeras (Section 3).
2. Prodrugs
2.1. Targeting by Selective Cleavage of Prodrugs in the Microenvironment of Cancer Cells
The metabolism in cancer cells involves a high rate of anaerobic glycolysis resulting in
an overproduction of lactic acid and carbonic acid. Since the acid protons of these acids are
exported into the extracellular medium, intracellular pH of cancer cells typically is higher
(pH 7.4 versus 7.2 in normal cells). [8,11,12]. As a consequence of the proton transport the
microenvironment surrounding cancer cells has a pH of 6.8 in contrast to the is estimated
pH of blood 7.4 [13]. A high rate of glycolysis without oxygen supply causes hypoxia
inside cancer cells [8,11,14,15]. The ensuing rise in the intracellular level of reactive oxygen
species (ROS), such as hydrogen peroxide, formed during hypoxia conditions may be
employed as a mode of targeting drugs toward cancer cells. Cancer cells also have an
β
[17], and some specific proteolytic
increased level of glutathione [16], β-glucuronidase
enzymes [17–19].
2.1.1. Prodrugs Cleaved in Acidic Media
Salts of dithiocarbamates (e.g., 1) are stable at physiological pH, but they will after
protonation in the acidic microenvironment surrounding cells undergo cleavage to release
the free amine (2) and carbondisulfide (Scheme 1). In a similar way the emitine monoamide
(3) with 2-methylmaleic acid was used to simultaneously increase the solubility of emitine
in water and enable its release from the prodrug in slightly acidic media [20].
Scheme 1. Dithiocarbamates and methylmaleic amides cleaved at low pH [20].
Molecules 2021, 26, 1292
3 of 23
A prodrug of doxorubicin (DOXO-EMCH, 4) is linked via a hydrazone promoiety to
a maleimide N-substituted with a 6-aminohexanoic spacer. This prodrug is designed to
enable reaction with Cys-34 of serum albumin (to give 5), present as the most abundant
protein in blood. The resulting albumin-linked drug (5) cannot penetrate into cells until the
hydrazone is hydrolyzed in the acidic environment around cancer cells to provide the free
doxorubicin (6; Scheme 2). The clinical phase III trial, however, did not enable registration
of the compound as a drug [14,21,22]. Despite these preliminary results, attempts to
overcome the cardiotoxicity of DOX-EMCH continue.
Scheme 2. Linkage of doxorubicin to serum albumin to prevent its penetration into benign cells; only in the acidic
microenvironment of cancer cells is doxorubicin released [14,21,22].
Polyethylene glycol-coated liposomes encapsulating doxoxrubicin (Caelyx) show less
cardiotoxicity than doxorubicin itself. The drug is used for treatment of breast cancer and
ovarian cancer [23].
Paclitaxel is widely used for treatment of various cancers; however, poor solubility
limits its use. The formulation vehicle Cremophor EL (CrEL; macrogolglycerol ricinoleate;
polyethylene glycol (PEG)-35 castor oil) and ethanol are used to increase its solubility, but
unfortunately CrEL causes side effects like hypersensitivity, neurotoxicity and nephrotoxicity [24]. These side effects have been overcome by a new formulation of lyophilized
paclitaxel with serum albumin (i.e., Abraxane® ) that provides nanoparticles (average size:
130 nm). This drug formulation has been approved by the U. S. Food and Drug Administration (FDA) for treatment of pancreatic cancer and non-small cell lung cancer (NSCLC) [25].
Moreover, paclitaxel (7) has been attached to nanoparticles via acetal linkages (8). Acetals
are cleaved in the acidic environment of cancer cells (Scheme 3) [26]. This method is also
used for conjugation of paclitaxel to nanoparticles prepared from polyethylene glycols
(PEGs) [13].
Polymer
O
O
O
2.1.2. Prodrugs Cleaved by ROS
In normal cells, ATP is primarily produced by oxidative phosphorylation, whereas in
O
cancer cells O
ATP primarily is produced by anaerobic glycolysis (the
WarburgOeffect) [27,28].
O
O
O
O
O
O
O generation of ROS such as hydrogen
O O
TheHOanaerobic
pathway Polymer
stimulates
peroxide [28].
HO
O
OH
+
O
The presence of hydrogen
peroxide
under
certain
physiological conditions can be used to
H3O
O
+
+
O
Ofacilitate cleavage of arylboronic acids, or esters
H
thereof, to give phenols and OH
boric acid [15].
OH
OH
O
NHestimated
O
O
NH O The concentration
of hydrogen peroxide
in benign cells
is
to Oapprox.
1 µM, but in
O
O
cancer cells
it may reach even 10 µM [15]. Some boronic acids are oxidized by cytochrome
O
P450 [15].O Boronic acids may also be oxidized by peroxynitrite [15]. It may
be questioned
8 whether arylboronic functionalities act as true promoieties,
7 since the oxidation provides
Molecules 2021, 26, 1292
4 of 23
boric acid and the corresponding phenol. As an example, camptothecin-10-boronic (9)
acid is oxidatively cleaved by hydrogen peroxide to give 10-hydroxycamptothecin (10;
Scheme 4) [29]. The resulting 10-hydroxycamptothecin (10) proved to be a more potent
topoisomerase inhibitor, and to be more cytotoxic in a number of cell systems than the
original drug. Furthermore, this hydroxylated derivative exhibited tumor growth inhibition
in xenograft models [29].
Polymer
O
O
O
O
O
O
O O
HO
O
O
H3O+
O
O
NH
O
O
O
Polymer
OH
O
O
+
OH
O
H
OH
+
O
O
NH
O
O
O
OH
O
O
8
O
O
O O
HO
7
Scheme 3. Conjugation of paclitaxel (7) to polymers that increase solubility [26].
Scheme 4. Oxidation of camptothecin-10-boronic acid (9) to give 10-hydroxycamptothecin (10) [15].
Arylboronic acid prodrugs of doxorubicin (e.g., 11) have also been reported. Oxidative
cleavage of the boronic acid moiety releases a phenol that spontaneously cleaves itself from
the self-immolative spacer 4-hydroxybenzyl carbamate (12; Scheme 5) [15]. Doxorubicin
(6) very efficiently kills cancer cells, however, a severe cardiotoxicity limits its use as a
chemotherapeutic drug [30]. Targeting of the drug may reduce this side effect. The prodrug
was found to induce regression of pancreatic tumors in mice, and further analysis revealed
that the prodrug was cleaved to doxorubicin inside tumors (Scheme 5) [31].
Similarly, an aryl boronic acid prodrug (13) of paclitaxel (7), also containing a selfimmolative linker, has been reported (Scheme 6) [15]. The size of the PEG moiety was
adjusted so that the prodrug self-assembles into micelles with a size of ca. 50 nm. Native
paclitaxel (7) was only released in the acidic microenvironment of the cells containing a
high level of ROS. Consequently, reduced toxicity of the prodrug as compared to treatment
with paclitaxel was observed in mice while retaining similar tumor regression [32]. At
present no boronic acid prodrugs have been approved, despite intensive research being
performed in the field [15].
2.1.3. Prodrugs Cleaved by Glutathione
Glutathione (H-γGlu-Cys-Gly-OH) is present in almost all mammalian tissues, but
usually it is overexpressed in cancer cells [33]. The active functionality of glutathione is the
thiol, which enables the molecule to participate in redox reactions, and may thus protect
the cell from a high level of ROS [33]. The molecule is able to cleave disulfides including
linkages within prodrugs. Hence, this feature has been utilized in the construction of
prodrugs attached to a promoiety via a disulfide linkage, and e.g., camptothecin (14)
has been linked to a near-infrared (NIR) dicyanomethylenebenzopyran fluorophore (15;
Scheme 7). An in vivo experiment using a mouse BCap-37 tumor xenograft model showed a
significantly improved regression of tumors on mice treated with the prodrug as compared
Molecules 2021, 26, 1292
5 of 23
to that found for those treated with camptothecin or another prodrug in which the linkage
consisted of a stable carbon–carbon bond instead of the disulfide bond [16].
Scheme 5. Prodrug of doxorubicin (6) cleaved by ROS [15].
Scheme 6. Polyethylene glycol (PEG)-containing prodrug (13) of paclitaxel (7) that is cleaved by reactive oxygen species
(ROS) [13].
2.1.4. Prodrugs Cleaved by Expressed Enzymes
A number of enzymes are overexpressed in cancer cells. These enzymes include
oxidoreductases, hydrolases, and matrix metalloproteinases (MMPs) [8]. A prodrug based
on the overexpression of MMPs was designed for doxorubicin (Scheme 8) [34]. The peptide
promoiety conjugated to the amine in doxorubicin prevents entry into cells, and consequently the compound is harmless to benign cells. By contrast, in the microenvironment of
cancer cells the peptide is cleaved at the Gly-hPhe position. Proteases subsequently remove
γ
Molecules 2021, 26, 1292
6 of 23
the remaining amino acids. In a HT1080 xenograft mouse preclinical model, the prodrug
was more efficient in reducing tumor growth than doxorubicin itself, and less undesired
toxicity was observed [34].
Scheme 7. Prodrug (i.e., 15) of camptothecin (14) that is cleaved by glutathione [16].
Scheme 8. A doxorubicin (6) prodrug (i.e., 16) cleaved by matrix metalloproteinase (MMP) and other proteases
(Cit = citrulline; hPhe = homophenylalanine) [34].
Molecules 2021, 26, 1292
7 of 23
Cathepsin B is involved in cancer invasion and metastasis, and it is overexpressed
in cancer tissue [35]. A prodrug (17) consisting of doxorubicin conjugated via the selfimmolative linker 4-aminobenzyl alcohol to a dipeptide fragment (Ac-Phe-Lys-OH), which
is a substrate for cathepsin B, has been designed (Scheme 9). In a mouse model this
prodrug inhibited development of peritoneal carcinomatosis as well as its progression
more efficiently and with fewer side effects than doxorubicin itself [35].
Scheme 9. Prodrug of doxorubicin (i.e., 17) that is cleaved by cathepsin B [35].
2.1.5. Prodrugs Cleaved by ββ-glucuronidase
β
Glucuronides are formed as a phase II metabolism of drugs. β-Glucuronidases
are
expressed excessively in a number of tumors such as breast, lung and gastrointestinal tract
carcinomas as well as in melanomas, where they are particularly abundant in necrotic
areas [17,36,37]. Prodrugs based on this selective enzyme distribution include glucuronides
of doxorubicin (6) and 4′ -epi-doxorubicin
(19) [17]. A self-immolative linker was intro′
duced, since the β-glucuronide
(i.e.,
18)
conjugated
directly to the sugar part of doxorubicin
β
was not a substrate for β-glucuronidase
(Scheme
10)
[17].
β
Scheme 10. Glucuronides (18 and 20) of doxorubicin (6) and 4′′-epi-doxorubicin (19) [17].
In an attempt to circumvent the poor solubility of paclitaxel (7) in water a glucuronide
(i.e., 21) was made. The resulting glucuronide (21) proved indeed to be soluble in water,
β
′
Molecules 2021, 26, 1292
8 of 23
and it was rapidly converted into paclitaxel (7) in the presence of high concentrations of
β-glucuronidase
(Scheme 11) [38].
β
Scheme 11. Water-soluble glucuronide-based prodrug (21) of paclitaxel (7) [38].
Likewise, 7-Aminocamptothecin was conjugated to glucuronic acid via another selfimmolative linker (Scheme 12).
Scheme 12. Glucuronide of 7-aminocamptothecin (23) cleaved by β-glucuronidase [39].
β
2.1.6. Prodrugs Cleaved by PSA or PSMA
Prostate cancer is a slowly developing cancer disease. In high-income countries it is the
cancer disease that causes second-most deaths among men. In its initial stages the disease
can be treated with androgen ablation therapy. However, if progression of disease occurs
Molecules 2021, 26, 1292
9 of 23
during treatment with anti-androgens, resulting in development of distant metastasis, the
prostate cancer is defined as metastatic Castration-Resistant Prostate Cancer (mCRPC)
which is not sensitive to hormone ablation [40]. All tumors of mCRPCs secrete the enzyme
prostate-specific antigen (PSA) into their microenvironment. PSA is a chymotrypsin-like
protease with a unique substrate specificity [18]. PSA also diffuses into the bloodstream, but
PSA in the blood is inactivated by complexing with blood proteins like serum albumin [40].
Conjugation of cytotoxins with different selectively labile peptides has been used for
targeting of mCRPCs. Thus, O-desacetylvinblastine (24) has been targeted to mCRPCs by
conjugation to a PSA-specific peptide substrate to give a prodrug (i.e., 25) (Scheme 13) [19].
Scheme 13. Prodrug (i.e., 25) of desacetylvinblastine (24) cleaved by prostate-specific antigen (PSA) [19].
PSA cleaves the peptide between the two Ser residues adjacent to the C-terminal Pro
residue, whereupon a spontaneous intramolecular attack of the amino group of the terminal Ser on the Pro carboxylate affords desacetylvinblastine (24) and a diketopiperazine.
This intramolecular diketopiperazine formation appeared to depend on the presence of
the Pro residue, as it did not occur when Leu was incorporated instead. Desacetylvinblastine (24) proved equally efficient in inducing mitotic arrest as the original vinblastine
(Scheme 13) [19].
A number of peptides have been conjugated to doxorubicin (6) [41]. Among these
different promoities the peptide H2 N-Glu-Hyp-Ala-Ser-Chg-Ser-Leu-OH was found to
afford a prodrug that was efficiently cleaved by PSA, and it showed a dramatically increased
activity in reducing LNCaP xenografts in mice as compared to that of native doxorubicin
(6). The released active drug consists of a mixture of doxorubicin and H-Leu-doxorubicin
(Scheme 14) [41].
A drawback of using vinblastine or doxorubicin (6) as drugs for treatment of prostate
cancer is that both compounds cause mitotic arrest, and consequently they primarily target
proliferating cells. A more pronounced cell death is expected when toxins capable of killing
cells in all stages are used. Thapsigargin (27) is a cytotoxic compound that kills cells in
all states by blocking the sarco/endoplasmic reticulum Ca2+ ATPase (SERCA), thereby
Molecules 2021, 26, 1292
10 of 23
inducing the unfolded protein response leading to apoptosis [42]. The general toxicity
of the compound requires targeting via a prodrug in order to avoid general systemic
toxicity [43]. No obvious anchoring point for conjugation to a peptide exists in native
thapsigargin, but replacement of the butanoyl moiety with a 12-aminododecanoic acid
spacer (to give 28) allows for introduction of an amine functionality that may serve as
attachment point for a peptide promoiety (Scheme 15).
OH O
O
HO
HO
H-Glu-Hyp-Ala-Ser-Chg-Ser-Leu
H
N
HO
O
O
OH O
O
26
PSA
OH O
O
HO
HO
HO
+
OH O
O
H 2N
HO
OH O
O
HO
O
6
O
+
H-Glu-Hyp-Ala-Ser-Chg-Ser-Leu-OH
H-Leu
H
N
HO
OH O
O
O
O
+
H-Glu-Hyp-Ala-Ser-Chg-Ser-OH
Scheme 14. Doxorubicin prodrug (26) cleaved by PSA [41].
Scheme 15. Prodrug of ω
ω-aminododecanolydesbutanoylthapsigargin (28) cleaved by PSA [43].
Conjugation of a thapsigargin analogue to a polar peptide was expected to inhibit
diffusion into cells, thereby preventing the prodrug from reaching the intracellular SERCA
pump. The hexapeptide H-His-Ser-Ser-Lys-Leu-Gln-OH is very efficiently released from
Molecules 2021, 26, 1292
11 of 23
the prodrug by PSA, but only to a limited extent by any other proteases in the human
body [18]. The C-terminal Leu residue was introduced in order to make the prodrug
a substrate for PSA. In vivo experiments in mice confirmed that the prodrug was only
cleaved in the blood to a limited extent, but very efficiently cleaved by PSA to release the
active Leu derivative within tumors. Thus, this prodrug prevented growth of tumors in
mice [44].
KLK2 is another enzyme (previously named human kallikrein 2, hK2) that may be
used for targeting drugs toward prostate cancers [45,46]. Similarly to PSA, KLK2 is also
secreted from the prostate and prostate cancer cells. The level of KLK2 in blood can be used
as a biomarker for prostate cancer, and KLK2 is inactivated upon entering the bloodstream
by binding to blood proteins [47].
An alternative enzyme, characteristic for the prostate glandule, is prostate-specific
membrane antigen (PSMA). The catalytic site of this enzyme extends outwards into the
extracellular environment. The enzyme is not only expressed in the prostate glandule, but
also in human prostatic carcinoma and in neovascular tissue of a number of tumors [48,49].
In a healthy individual the enzyme is exclusively expressed in the prostate, ensuring
that cleavage of the peptide conjugated to its payload solely occurs in the prostata glandula [49–53]. As the only enzyme outside the central nervous system PSMA cleaves the
amide linkage in the γ-Glu tetramer [54]. Taking advantage of this feature, a prodrug
of the 12-aminododeanoate of desbutanoylthapsigargin (28), (i.e., mipsagargin, 31) was
made (Scheme 16). The C-terminal βAsp residue was introduced in order to make the
prodrug a substrate for PSMA [49]. This prodrug, which has been prepared by solid-phase
synthesis [55], was cleaved rapidly in tumors to release the βAsp derivative, which slowly
was cleaved to provide the free mipsagargin. A solid-phase synthesis of these guaianolide
prodrugs were developed [55].
In the clinical phase II trial this prodrug conferred a prolonged stabilization of the
disease in patients with hepatocellular carcinoma [56]. Hepatocellular cancer also expresses
PSMA in neovascular tissue, which thus should be sensitive to mipsagargin [57]. However,
the results obtained in clinical phase II trial did not meet the expectations, and hence the
drug was not marketed [58]. A major problem might be that the prodrug, despite its five
negative charges on its peptide side chains, has been found to be able to penetrate cell
membranes in benign cells, thereby causing unspecific toxicity [59].
2.2. Antibody–Drug Conjugates (ADCs)
An antibody–drug conjugate (ADC) is a prodrug consisting of a monoclonal antibody
conjugated to a cytotoxin (i.e., the payload) via a linker. The prodrug is designed based
on the hypothesis that appropriate antibodies that preferentially bind to cancer-specific
antigens (located on the surface of cancer cells) can be obtained. Indeed, the two first ADCs,
Adcetris® (brentuximab vedotin) for Hodgkin lymphoma and Kadcyla® (trastuzumab
emtansine) for breast cancer, were approved by the FDA in 2011 and in 2013, respectively.
Since then six additional ADCs have been approved by the FDA (i.e., Besponsa® (inotuzumab ozogamicin), Mylotarg® (gemtuzumab ozogamicin), Polivy® , Enhertu® , Padcev® ,
and Trodelvy® ) [61]. More than 60 ADC prodrugs are under clinical development [62–64].
The linker should be stable in circulation to avoid release of free cytotoxin causing
systemic toxicity, as is often seen with conventional chemotherapeutics (Figure 1).
Monoclonal antibodies of rodent origin may cause severe immunogenic reactions
in humans. The use of chimeric humanized antibodies has to some extent solved this
problem [65].
The ideal cytotoxin should be very toxic since only a limited number of antigens are
present on the surface of malignant cells, and consequently only a limited number of ADCs
can be internalized to exert the cytotoxic effect [66]. A number of toxins have been used as
payloads, e.g., the peptide monomethyl auristatin E, the polyketide macrolides ansamitocin
(as a mixture of aliphatic alkyl esters) and maytansins (= maitansins) as well as doxorubicin
Molecules 2021, 26, 1292
12 of 23
(6), duocarmycin (32), and enediynes like calicheamicin γ1 I (33) have been converted into
ADCs (Figure 2) [61,63].
Scheme 16. The PSMA-sensitive prodrug mipsagargin (31) [56,60].
Figure 1. Cartoon illustrating the structure of an antibody–drug conjugate (ADC)γdrug.
Figure 2. Examples of payloads in ADCs.
Molecules 2021, 26, 1292
13 of 23
The calicheamycins is a group of extremely potent cytotoxins. Originally the compounds were tested as antibiotics, and their minimum inhibitory cancentration (MIC)
values were found to be from 0.5 µg/mL toward Eschericia coli to less than 0.2 ng/mL
against Bacillus subtilis [67]. Antitumor activity was tested against P388 leukemia and B16
melanoma in mice by intraperitoneal injection. The optimum dose was found to be 5 µg/kg
as compared to 1.6 mg/kg for cisplatin against P388 cells, and 1.25 µg/kg as compared
to 300 mg/kg forγcisplatin against B16 cells [67]. All mice died, indicating a high general
toxicity. Calicheamicin γ1 I is an interesting payload because of its extreme cytotoxicity [66].
This molecule represents an extraordinary example of natural bioengineering that has
occurred during evolution. The sugar part including the iodinated aromatic residue confers
affinity for DNA. After complexing with DNA, a nucleophilic attack, e.g., by glutathione on
the central sulfur in the trisulfide, leads to the cleavage of this linkage, thereby releasing the
free thiol, which undergoes an intramolecular thiol Michael addition to the α,β-unsaturated
β
ketone. By changing the trigonal bridgehead β-carbon to a tetragonal
carbon, sufficient
tension is induced in the 10-membered ring to initiate a Bergman cyclization. The resulting
intermediate diradical finally cleaves the DNA [66] (Scheme 17).
Scheme 17. Diradical formation from calicheamicin γ1γI after nucleophilic attack on the α,βαβ
unsaturated ketone [66].
In particular two functional groups are used for conjugation of drugs via a linker
to the antibody, namely the thiol group of cysteine and/or the amino groups of lysine
residues [61]. Traut’s reagent or carbodiimides with or without hydroxysuccinimide have
been used for coupling of a carboxylic acid to lysine side chains [63]. As an example,
Scheme 18 depicts how calicheamicin γ1 I (33) has been coupled to an antibody in Mylotarg
(gemtuzumab ozogamicin) [63].
After internalization of the antibody–antigen complex, enzymes within the cell facilitate hydrolysis of the hydrazone moiety [63]. The disulfide will be cleaved by glutathione,
enabling Bergman cyclization. Other linkers have been designed to be cleaved by intracellular proteases like cathepsin B (e.g., Adcetris). After internalization of the ADC Kadcyla,
in which mertansine (34) is linked to an antibody, the linker including the lysine residue
remains attached to the payload after decomposition of the antibody (Scheme 19). This
extended linker moiety appears not to compromise the effect of the drug. Other examples
of ADCs have been reported by Nicolaou and Rigol [63].
αβ
Molecules 2021, 26, 1292
γ
14 of 23
Scheme 18. Coupling of a calicheamicin to antibodies via lysine residues (the O-Sugar moiety is defined in Scheme 17) [63].
Scheme 19. Internalization of an antibody–mertansine conjugate occurs while the payload remains
attached to the antibody via a lysine residue [63].
Molecules 2021, 26, 1292
15 of 23
2.3. Antibody-Directed Enzyme Prodrug Therapy
Similarly to ADCs, the concept of antibody-directed prodrug therapy (ADEPT) is
based on the ability of antibodies to selectively target antigens expressed abundantly on the
surface of cancer cells [68,69]. The principle involves a preferential binding of a non-human
enzyme to the surface of cancer cells via an antibody–antigen complex. The choice of a nonhuman enzyme makes it possible to choose a linker which solely is cleaved by this enzyme
and not by any endogenous enzymes. On the other hand, a potential drawback of using a
non-human enzyme may be a strong allergic reaction due to unforeseen immunogenicity.
In contrast to the ADC approach, non-internalizing antigens can be targeted. The enzyme is
linked to the antibody by using a bisfunctional linker, where one functionality can be linked
to the lysine side chains present on the enzyme, while the other functionality can be linked
to thiols of cysteines on the antibody (Figure 3) [68–70]. After administration to the patient
the antibody binds to the surface of the cancer cell. When the excess free antibody–enzyme
conjugate is completely cleared from the body the prodrug is administered.
Figure 3. Antibody–enzyme conjugate bound to antigens on the surface of malignant cells.
As mentioned above, a prerequisite for the use of enzymes not present in the human
body is that they are non-immunogenic [70]. Enzymes belonging to the families of alkaline
phosphatases (cleaving phosphate from prodrugs), peptidases, sulfatases (for cleavage of
sulfate monoesters), carboxylesterases and carboxypeptidases (for cleavage of e.g., glutamic
amides), have been investigated in this respect [70].
Some antibodies themselves possess catalytic properties, e.g., the antibody 38C2
catalyzes retroaldol and retro-Mannich reactions; for example, a prodrug of doxoxrubicin
is cleaved by 38C2 (Scheme 20). [70,71].
Scheme 20. Doxorubicin prodrug (35) cleaved by 38C2 [70,71].
Molecules 2021, 26, 1292
16 of 23
Even though some promising clinical results have been obtained, no drugs based on
ADEPT are in clinical use at present [68,69].
2.4. Gene-Directed Enzyme Prodrug Therapy (GEPDT)
In gene-directed enzyme prodrug therapy (GEPDT), a gene encoding for a unique
enzyme is introduced into the tumor cells by using a vector. The technique was already
introduced in 1986, and the gene introduced into cells is called a suicide gene [72]. Upon
expression of the enzyme on the cancer cell surface, the enzyme enables cleavage of the
linkage between the payload and the promoiety, after which the payload may diffuse into
the cancer cell (Figure 4) [69,70]. A major drawback in GDEPT is the prerequisite of achieving selective transfer of a gene into malign cells. Retroviruses, adenoviruses and herpes
viruses have been studied as potential vectors [73]. Retroviral vectors have some selectivity,
since they are only incorporated into the genome of actively dividing cells [73]. Attachment
of tissue-specific promoters may allow for transgenic expression only in neoplastic cells.
The use of receptor-specific vectors has also been proposed [73]. Mesenchymal stem cells,
exhibiting strong tropism toward tumors and metastases expressing receptors on their
surface, can efficiently be transduced with vectors [72]. Virus-like particles have also been
used to internalize the gene into cells [74]. No drugs based on the principle of GDEPT have
been approved so far.
Figure 4. Cartoon illustrating the principle of gene-directed enzyme prodrug therapy (GEPDT) [69,70].
2.5. Lectin-Directed Enzyme-Activated Prodrug Therapy (LEAPT)
In lectin-directed enzyme-activated prodrug therapy (LEAPT), a drug or an enzyme is
targeted toward cancer cells by using sugar–protein recognition, whereas antigen–antibody
recognition is used for targeting in ADEPT and GDEPT. Lectins are proteins involved in
biological carbohydrate recognition comprising cellular processes such as growth, differentiation, proliferation or apoptosis [75]. In order to improve the selectivity of doxorubicin, a
galacturonamide derivative (i.e., 36) was prepared (Figure 5) [76]. Here, the expression of
asialoglycoprotein receptors (ASGPRs) 1 and 2 on the surface of hepatocytes with a high
affinity for D-galactose and L-rhamnose was exploited. After binding to the receptor, the
appropriate carbohydrate-containing ligand is internalized. The ASGPRs are expressed on
the surface of HT-29, MCF-7 and A549 cells to a much higher extent than in normal liver
cells [76]. The Gal-Dox derivative proved to exhibit higher selectivity toward cancer cell
lines than doxorubicin (6) itself. In S180 tumor-bearing mice, the Gal-Dox-treated group
had a higher accumulation of the drug in the malignant tissue than the doxorubicin-treated
group as well as an improved survival rate [76]. However, in this case the doxorubicin
Molecules 2021, 26, 1292
17 of 23
derivative may in fact not be a true prodrug, since probably the Gal derivative may also
interact with the topoisomerase target.
Figure 5. Galacturonamide of doxorubicin (36) for lectin-directed enzyme-activated prodrug therapy
(LEAPT) [76].
Another approach utilizes the overexpression of glucose transporters (GLUT) in cancer
tissue. By preparing glucose or glucuronic acid derivatives of paclitaxel, two advantages
are obtained: (i) the compounds become more soluble in water, and (ii) increased uptake
through the GLUT into the cancer tissue (Figure 6) [77].
Figure 6. Glucose and glucuronic acid derivatives (i.e., 37 and 38) of paclitaxel (7) displaying
improved selectivity via LEAPT [77].
The glucose and glucuronic acid derivatives (37 and 38) were found to exert a low
cytotoxicity on benign cells, but an activity similar to that of paclitaxel (7) itself on cell lines
expressing GLUT. It is assumed that the prodrug is cleaved by intracellular β-glucosidases.
β
A mechanism involving cleavage of the methyl glucoside followed by self-immolative
cleavage to give paclitaxel (7) has been proposed [77]. However, the glucose derivative
reported was an α-glycoside.
α
A two-phase LEAPT mechanism overcoming the requirement for intracellular cleavage of the prodrug has been suggested. In the first phase a glycosylated enzyme interacts
with a carbohydrate-recognizing lectin on the surface of the cells in the targeted tissue.
After similar interactions, a glycosylated prodrug, which is a substrate for the glycosylated
enzyme, becomes internalized as well. Inside the cells, the internalized enzyme cleaves the
glycosylated prodrug to liberate the active drug (Scheme 21).
A procedure for pergalactosylation of a naringinase produced by Penicillium decumbens
has been developed to give a pergalactosylated enzyme, which showed high affinity for
ASGPRs on the surface of hepatocytes. Binding to ASPGR triggers internalization of the
bound ligand. The naringinase possesses α-rhamnosidase and β-glucosidase activities.
Thus, a rhamnose derivative of doxorubicin (6) was prepared, and the stability of this
derivative was tested [78]. At present, no drugs based on the LEAPT principle have
been approved.
Molecules 2021, 26, 1292
18 of 23
Scheme 21. Two-phase LEAPT. First a glycosylated enzyme binds to the cell surface and becomes internalized. Secondly, a glycosylated prodrug binds to the surface, and is then internalized whereafter
it is cleaved by the internalized enzyme [78].
3. Protease-Targeting Chimeras (PROTAC)
In living cells, misfolded, damaged or mutated proteins are removed from the cells by
natural processes, in which the protein first becomes covalently bound to one of a number
of ubiquitins, which are highly conserved 76-residue peptides [79]. This conjugation
α which transfer ubiquitin
β
process involves ubiquitin-activating enzymes E1,
to E2 from
where ubiquitin is transferred to a von Hippel–Lindau(VHL)-cullin-RING ligase complex
including E3 that conjugates ubiquitin to the target protein mainly via lysine residues [9,10].
Subsequently, the ubiquitin-modified protein is degraded by a 26S proteasome to give a
number of small peptides and a number of lysine-modified ubiquitins [9,10,80]. Other
proteases finally cleave the oligopeptides into free amino acids [81].
Advantages of this system are that drugs may be developed to selectively remove
intracellular proteins. A chimera consisting of a residue with high affinity for the VHL
complex was via a linker attached to a moiety with high affinity for the estrogen-related
receptor α (ERRα). After complexing with VHL, knockdown of the ERRα level was
observed. The first experiment was performed in MCF7 cells after incubation with the
chimeras to knock down ERRα [9]. Moreover, a serine-threonine kinase (RIPK2) was
knocked down after incubation of MCF7 cells with a chimera consisting of a moiety with
high affinity for the VHL complex (Figure 7) [9].
The effect of small-molecule drugs as ligands for biomolecules in treatment of cancer
diseases can be limited by mutations in the gene encoding the biomolecule, thereby making
the modified target insensitive to the agent. Such mutations are observed for the epidermal
growth factor receptors and androgen receptors [82]. PROTAC has been used to enable
knockdown of steroid receptors and for non-small lung cancer by knockdown of epidermal
growth factor. In addition, the anaphylactic lymphoma kinase can similarly be removed as
a possible α
treatment
is
α of different types of human cancers [82]. The PROTAC technique
α
still at an early stage, and at present no such drugs are currently in clinical use.
α
Molecules 2021, 26, 1292
19 of 23
Figure 7. Cartoon illustrating the principle of protease-targeting chimera (PROTAC) [9,10,80]. A
compound with affinity for the Hippel–Lincau-cullin-Ring (VHL) complex and the target protein
α is used to attach the protein to the E3 ligase in the VHL
α (ERRα))
(e.g., estrogen-related receptor α
complex. After complexation the E3 conjugates ubiquitin to the protein making it a target for
proteasomal degradation.
4. Conclusions
In recent decades, several diverse methods have been developed for the targeting of
toxins to cancer tissue to avoid their general systemic toxicity. In the present review, the
initial sections concern new attempts developed for prodrugs to be cleaved predominantly
in the microenvironment of cancer cells and tumors. Thus, the lower pH characterizing
cancer tissue has been explored for selective cleavage of prodrugs based on amides of a
substituted maleic acid [20], a hydrazone promoiety [14,21,22], and labile acetal linkage to
polymers [26]. A prodrug of doxorubicin (Aldoxorubicin) designed to prevent cardiotoxicity expected to be cleaved by the acidic microenvironment of cancer cells failed in clinical
trial III [22]. Moreover, proof-of-concept studies of prodrugs relying on selective cleavage
due to the increased ROS production in cancer cells comprise examples of arylboronic acid
derivatives [13,15,29]. An example of glutathione-promoted cleavage of a disulfide-based
prodrug has also appeared [16].
Next, enzymes, overexpressed by cancer cells or neovascular tissue in tumors, capable
of selective cleavage of prodrugs carrying a peptide substrate moiety, offer several examples: e.g., MMP [34], cathepsin B [35], β-glucuronidase [17,38,39], and PSA [19,41,43] and
PSMA [56,60]. One prodrug, mipsagargin, actually went into clinical phase III trials but,
despite the polarity of the γGlu-γGlu-γGlu-γGlu-βAsp peptide moiety, the compound
appeared to be able to penetrate cell membranes of benign cells also, and thus cause general
toxicity [58].
In addition, the progress within the field of ADCs (with an anticancer drug as payload)
comprise >60 entities in clinical development [62–64]. In total eight ADCs have been
approved by the FDA as new and improved cancer therapies, albeit not in the period 2014
to 2019 [83–88]. Calicheamicin and maytansine have been used as the payload in many of
these drugs [63].
Another approach also involving antibodies
is ADEPT [70,71]; however, even though
β
promising clinical results have been reported, no drugs based on ADEPT are currently
approved for clinical use [68,69]. γ
γ
γ
γ
β
In addition, an advanced approach requiring selective introduction of a gene, coding
for an enzyme capable of cleaving a prodrug, into cancer cells (i.e., GDEPT) [69,70] is considered a promising approach, but so far no drugs based on this concept have been approved.
Similarly, targeting to cancer cells via sugar–protein recognition processes involving lectins
Molecules 2021, 26, 1292
20 of 23
present on the surface of cancer cells (i.e., LEAPT) have been explored [76–78]. Nevertheless, no drugs based on these principles have been approved as yet.
Finally, recent examples of protease-targeting chimeras (PROTACs) involve ubiquitination enzyme complexes that undergo proteolytic degradation to release the drug [9,10,80],
however, this technique is at an early stage, and no examples of its clinical use have appeared.
Even though the described techniques have been utilized to improve the solubility of
paclitaxel and selectivity of doxorubicin, the associated prodrugs have not as yet shown
sufficiently improved properties to convince medical agencies that they can be approved
as drugs. In conclusion, the new approaches reviewed here may indeed lead to future new
anticancer drugs that are urgently needed for treatment of cancer diseases for which no
cure exists. Nevertheless, most of these recently developed targeting principles remain to
result in approved drugs, which emphasizes the need for further research to unleash the
full potential of these concepts currently considered for experimental therapies.
Author Contributions: Both authors have contributed to the manuscript. Both authors have read
and agreed to the published version of the manuscript.
Funding: This research received no external funding.
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
Data Availability Statement: No supporting information for this work.
Conflicts of Interest: The authors declare no conflict of interest.
References
1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
11.
12.
13.
14.
15.
16.
Mishra, A.P.; Chandra, S.; Tiwari, R.; Srivastava, A.; Tiwari, G. Therapeutic potential of prodrugs towards targeted drug delivery.
Open Med. Chem. J. 2018, 12, 111–123. [CrossRef]
Elsharif, N.A. Review: Prodrug concept in drug design. Res. Rev. J. Pharm. Sci. 2018, 9, 22–28.
Zawilska, J.B.; Wojcieszak, J.; Olejniczak, A.B. Prodrugs: A challenge for the drug development. Pharm. Rep. 2013, 65, 1–14.
[CrossRef]
McKeage, K.; Scott, L.J. Atovaquone/proguanil: A review of its use for the prophylaxis of Plasmodium falciparum malaria. Drugs
2003, 63, 597–623. [CrossRef]
Oketch-Rabah, H.A.; Marles, R.J.; Jordan, S.A.; Low Dog, T. United States pharmacopeia safety review of willow bark. Planta Med.
2019, 85, 1192–1202. [CrossRef]
Das, N.; Dhanawat, M.; Dash, B.; Nagarwal, R.C.; Shrivastava, S.K. Codrug: An efficient approach for drug optimization. Eur. J.
Pharm. Sci. 2010, 41, 571–588. [CrossRef] [PubMed]
Greenstein, R.J.; Su, L.; Shahidi, A.; Brown, S.T. On the action of 5-amino-salicylic acid and sulfapyridine on M. avium including
subspecies paratuberculosis. PLoS ONE 2007, 2, e516.
Zhang, X.; Li, X.; You, Q.; Zhang, X. Prodrug strategy for cancer cell-specific targeting: A recent overview. Eur. J. Med. Chem. 2017,
139, 542–563. [CrossRef] [PubMed]
Bondeson, D.P.; Mares, A.; Smith, I.E.D.; Ko, E.; Campos, S.; Miah, A.H.; Mulholland, K.E.; Routly, N.; Buckley, D.L.;
Gustafson, J.L.; et al. Catalytic in vivo protein knockdown by small-molecule PROTACs. Nat. Chem. Biol. 2015, 11, 611–
617. [CrossRef] [PubMed]
Paiva, S.-L.; Crews, C.M. Targeted protein degradation: Elements of PROTAC design. Curr. Opin. Chem. Biol. 2019, 50, 111–119.
[CrossRef]
Weber, C.E.; Kuo, P.C. The tumor microenvironment. Surg. Oncol. 2012, 21, 172–177. [CrossRef] [PubMed]
Corbet, C.; Feron, O. Tumour acidosis: From the passenger to the driver’s seat. Nat. Rev. Cancer 2017, 17, 577–593. [CrossRef]
[PubMed]
Mu, J.; Zhong, H.; Zou, H.; Liu, T.; Yu, N.; Zhang, X.; Xu, Z.; Chen, Z.; Guo, S. Acid-sensitive PEGylated paclitaxel prodrug
nanoparticles for cancer therapy: Effect of PEG length on antitumor efficacy. J. Control. Release 2020, 326, 265–275. [CrossRef]
[PubMed]
Souza, C.; Pellosi, D.S.; Tedesco, A.C. Prodrugs for targeted cancer therapy. Expert Rev. Anticancer 2019, 19, 483–502. [CrossRef]
[PubMed]
Maslah, H.; Skarbek, C.; Pethe, S.; Labruere, R. Anticancer boron-containing prodrugs responsive to oxidative stress from the
tumor microenvironment. Eur. J. Med. Chem. 2020, 207, 112670. [CrossRef] [PubMed]
Wu, X.; Sun, X.; Guo, Z.; Tang, J.; Shen, Y.; James, T.D.; Tian, H.; Zhu, W. In Vivo and In Situ tracking cancer chemotherapy by
highly photostable NIR fluorescent theranostic prodrug. J. Am. Chem. Soc. 2014, 136, 3579–3588. [CrossRef]
Molecules 2021, 26, 1292
17.
18.
19.
20.
21.
22.
23.
24.
25.
26.
27.
28.
29.
30.
31.
32.
33.
34.
35.
36.
37.
38.
39.
40.
41.
21 of 23
De Graaf, M.; Boven, E.; Scheeren, H.W.; Haisma, H.J.; Pinedo, H.M. Beta-glucuronidase-mediated drug release. Curr. Pharm. Des.
2002, 8, 1391–1403. [CrossRef]
Denmeade, S.R.; Lou, W.; Lovgren, J.; Malm, J.; Lilja, H.; Isaacs, J.T. Specific and efficient peptide substrates for assaying the
proteolytic activity of prostate-specific antigen. Cancer Res. 1997, 57, 4924–4930. [PubMed]
Brady, S.F.; Pawluczyk, J.M.; Lumma, P.K.; Feng, D.M.; Wai, J.M.; Jones, R.; Feo-Jones, D.; Wong, B.K.; Miller-Stein, C.; Lin, J.H.;
et al. Design and synthesis of a pro-drug of vinblastine targeted at treatment of prostate cancer with enhanced efficacy and
reduced systemic toxicity. J. Med. Chem. 2002, 45, 4706–4715. [CrossRef]
Akinboye, E.S.; Rosen, M.D.; Denmeade, S.R.; Kwabi-Addo, B.; Bakare, O. Design, synthesis, and evaluation of pH-dependent
hydrolyzable emetine analogues as treatment for prostate cancer. J. Med. Chem. 2012, 55, 7450–7459. [CrossRef]
Kratz, F. Doxo-emch (INNO-206): The first albumin-binding prodrug of doxorubicin to enter clinical trials. Expert Opin. Investig.
Drugs 2007, 16, 855–866. [CrossRef] [PubMed]
Cranmer, L.D. Spotlight on aldoxorubicin (INNO-206) and its potential in the treatment of soft tissue sarcomas: Evidence to date.
OncoTargets Ther. 2019, 12, 2047–2062. [CrossRef] [PubMed]
O’Brien, M.E.R.; Wigler, N.; Inbar, M.; Rosso, R.; Grischke, E.; Santoro, A.; Catane, R.; Kieback, D.G.; Tomczak, P.; Ackland, S.P.;
et al. Reduced cardiotoxicity and comparable efficacy in a phase III trial of pegylated liposomal doxorubicin HCl (CAELYX/Doxil)
versus conventional doxorubicin for first-line treatment of metastatic breast cancer. Ann. Oncol. 2004, 15, 440–449. [CrossRef]
Gelderblom, H.; Verwij, J.; Nooter, K.; Sparreboom, A. Cremophor EL: The drawbacks and advantages of vehicle selection for
drug formulation. Eur. J. Cancer 2001, 37, 1590–1598. [CrossRef]
Miele, E.; Spinelli, G.P.; Miele, E.; Tomao, F.; Tomao, S. Albumin-bound formulation of paclitaxel (Abraxane®ABI-007) in the
treatment of breast cancer. Int. J. Nanomed. 2009, 4, 99–105.
Gu, Y.; Zhong, Y.; Meng, F.; Cheng, R.; Deng, C.; Zhong, Z. Acetal-linked paclitaxel prodrug micellar nanoparticles as a versatile
and potent platform for cancer therapy. Biomacromolecules 2013, 14, 2772–2780. [CrossRef]
Björkling, F.; Moreira, J.; Stenvang, J. Anticancer Agents. In Textbook of Drug Design and Discovery, 5th ed.; Strømgaard, K.,
Krogsgaard-Larsen, P., Madsen, U., Eds.; CRC Press: Boca Raton, FL, USA, 2017; pp. 369–386.
Pascale, R.M.; Calvisi, D.F.; Simile, M.M.; Feo, C.F.; Feo, F. The warburg effect 97 years after its discovery. Cancers 2020, 12, 2819.
[CrossRef]
Wang, L.; Xie, S.; Ma, L.; Chen, Y.; Lu, W. 10-Boronic acid substituted camptothecin as prodrug of SN-38. Eur. J. Med. Chem. 2016,
116, 84–89. [CrossRef]
Octavia, Y.; Tocchetti, C.G.; Gabrielson, K.L.; Janssens, S.; Crijns, H.J.; Moens, A.L. Doxorubicin-induced cardiomyopathy: From
molecular mechanisms to therapeutic strategies. J. Mol. Cell. Cardiol. 2012, 52, 1213–1225. [CrossRef]
Skarbek, C.; Serra, S.; Maslah, H.; Rascol, E.; Labruere, R. Arylboronate prodrugs of doxorubicin as promising chemotherapy for
pancreatic cancer. Bioorg. Chem. 2019, 91, 103158. [CrossRef] [PubMed]
Dong, C.; Zhou, Q.; Xiang, J.; Liu, F.; Zhou, Z.; Shen, Y. Self-assembly of oxidation-responsive polyethylene glycol-paclitaxel
prodrug for cancer chemotherapy. J. Control. Release 2020, 321, 529–539. [CrossRef] [PubMed]
Gamcsik, M.P.; Kasibhatla, M.S.; Teeter, S.D.; Colvin, O.M. Glutathione levels in human tumors. Biomarkers 2012, 17, 671–691.
[CrossRef]
Albright, C.F.; Graciani, N.; Han, W.; Yue, E.; Stein, R.; Lai, Z.; Diamond, M.; Dowling, R.; Grimminger, L.; Zhang, S.-Y.; et al.
Matrix metalloproteinase-activated doxorubicin prodrugs inhibit HT1080 xenograft growth better than doxorubicin with less
toxicity. Mol. Cancer 2005, 4, 751–760. [CrossRef]
Shao, L.-H.; Liu, S.-P.; Hou, J.-X.; Zhang, Y.-H.; Peng, C.-W.; Zhong, Y.-J.; Liu, X.; Liu, X.-L.; Hong, Y.-P.; Firestone, R.A.; et al.
Cathepsin B cleavable novel prodrug Ac-Phe-Lys-PABC-ADM enhances efficacy at reduced toxicity in treating gastric cancer
peritoneal carcinomatosis An experimental study. Cancer 2012, 118, 2986–2996. [CrossRef] [PubMed]
Fujita, M.; Taniguchi, N.; Makita, A.; Oikawa, K. Cancer-associated alteration of β-glucuronidase in human lung cancer: Elevated
activity and increased phosphorylation. GANN Jpn. J. Cancer Res. 1984, 75, 508–517.
Sperker, B.; Werner, U.; Murdter, T.E.; Tekkaya, C.; Fritz, P.; Wacke, R.; Adam, U.; Gerken, M.; Drewelow, B.; Kroemer, H.K.; et al.
Expression and function of β-glucuronidase in pancreatic cancer: Potential role in drug targeting. Naunyn Schmiedeberg’s Arch.
Pharm. 2000, 362, 110–115. [CrossRef]
De Bont, D.B.A.; Leenders, R.G.G.; Haisma, H.J.; van der Meulen-Muileman, I.; Scheeren, H.W. Synthesis and biological activity
of β-glucuronyl carbamate-based prodrugs of paclitaxel as potential candidates for ADEPT. Bioorg. Med. Chem. 1997, 5, 405–414.
[CrossRef]
Leu, Y.-L.; Roffler, S.R.; Chern, J.-W. Design and synthesis of water-soluble glucuronide derivatives of camptothecin for cancer
prodrug monotherapy and antibody-directed enzyme prodrug therapy (ADEPT). J. Med. Chem. 1999, 42, 3623–3628. [CrossRef]
[PubMed]
Akinboye, E.S.; Brennen, W.N.; Denmeade, S.R.; Isaacs, J.T. Albumin-linked prostate-specific antigen-activated thapsigargin- and
niclosamide-based molecular grenades targeting the microenvironment in metastatic castration-resistant prostate cancer. Asian J.
Urol. 2019, 6, 99–108. [CrossRef]
Garsky, V.M.; Lumma, P.K.; Feng, D.-M.; Wai, J.; Ramjit, H.G.; Sardana, M.K.; Oliff, A.; Jones, R.E.; DeFeo-Jones, D.;
Freidinger, R.M.; et al. The synthesis of a prodrug of doxorubicin designed to provide reduced systemic toxicity and greater
target efficacy. J. Med. Chem. 2001, 44, 4216–4224. [CrossRef]
Molecules 2021, 26, 1292
42.
43.
44.
45.
46.
47.
48.
49.
50.
51.
52.
53.
54.
55.
56.
57.
58.
59.
60.
61.
62.
63.
22 of 23
Lindner, P.; Christensen, S.B.; Nissen, P.; Møller, J.V.; Engedal, N. Cell death induced by the ER stressor thapsigargin involves
death receptor 5, anon-autophagic function of MAP1LC3B, anddistinct contributions from unfoldedprotein response components.
Cell. Commun. Signal. 2020, 18, 12. [CrossRef]
Denmeade, S.R.; Isaacs, J.T. The SERCA pump as a therapeutic target: Making a “Smart bomb” for prostate cancer. Cancer Biol.
2005, 4, 14–22. [CrossRef]
Denmeade, S.R.; Jakobsen, C.M.; Janssen, S.; Khan, S.R.; Garrett, E.S.; Lilja, H.; Christensen, S.B.; Isaacs, J.T. Prostate-specific
antigen-activated thapsigargin prodrug as targeted therapy for prostate cancer. J. Natl. Cancer Inst. 2003, 95, 990–1000. [CrossRef]
Darson, M.F.; Pacelli, A.; Roche, P.; Rittenhouse, H.G.; Wolfert, R.L.; Young, C.Y.; Klee, G.G.; Tindall, D.J.; Bostwick, D.G. Human
glandular kallikrein 2 (hK2) expression in prostatic intraepithelial neoplasia and adenocarcinoma: A novel prostate cancer marker.
Urology 1997, 49, 857–862. [CrossRef]
Darson, M.F.; Pacelli, A.; Roche, P.; Rittenhouse, H.G.; Wolfert, R.L.; Saeid, M.S.; Young, C.Y.; Klee, G.G.; Tindall, D.J.;
Bostwick, D.G. Human glandular kallikrein 2 expression in prostate adenocarcinoma and lymph node metastases. Urology 1999,
53, 939–944. [CrossRef]
Janssen, S.; Rosen, D.M.; Ricklis, R.M.; Dionne, C.A.; Lilja, H.; Christensen, S.B.; Isaacs, J.T.; Denmeade, S.R. Pharmacokinetics,
biodistribution, and antitumor efficacy of a human glandular kallikrein 2 (hK2)-activated thapsigargin prodrug. Prostate 2006, 66,
358–368. [CrossRef] [PubMed]
Pinto, J.; Suffoletto, B.; Berzin, T.; Qiao, C.; Lin, S.; Tong, W.; Heston, W. Identification of a membrane-bound pteroyl poly
gamma-glutamyl carboxypeptidase (folate hydrolase) that is highly expressed in human prostatic carcinoma cells. FASEB J. 1996,
10, A496.
Denmeade, S.R.; Mhaka, A.M.; Rosen, D.M.; Brennen, W.N.; Dalrymple, S.; Dach, I.; Olesen, C.; Gurel, B.; DeMarzo, A.M.;
Wilding, G.; et al. Engineering a prostate-specific membrane antigen-activated tumor endothelial cell prodrug for cancer therapy.
Sci. Transl. Med. 2012, 4. [CrossRef] [PubMed]
Wang, H.-L.; Wang, S.-S.; Song, W.-H.; Pan, Y.; Yu, H.-P.; Si, T.-G.; Liu, Y.; Cui, X.-N.; Guo, Z. Expression of prostate-specific
membrane antigen in lung cancer cells and tumor neovasculature endothelial cells and its clinical significance. PLoS ONE 2015,
10. [CrossRef]
Nomura, N.; Pastorino, S.; Jiang, P.; Lambert, G.; Crawford, J.R.; Gymnopoulos, M.; Piccioni, D.; Juarez, T.; Pingle, S.C.; Makale, M.;
et al. Prostate specific membrane antigen (PSMA) expression in primary gliomas and breast cancer brain metastases. Cancer Cell
Int. 2014, 14, 1–9. [CrossRef]
Kasoha, M.; Unger, C.; Solomayer, E.-F.; Bohle, R.M.; Zaharia, C.; Khreich, F.; Wagenpfeil, S.; Juhasz-Boess, I. Prostate-specific
membrane antigen (PSMA) expression in breast cancer and its metastases. Clin. Exp. Metastasis 2017, 34, 479–490. [CrossRef]
[PubMed]
Nimmagadda, S.; Pullambhatla, M.; Chen, Y.; Parsana, P.; Lisok, A.; Chatterjee, S.; Mease, R.; Rowe, S.P.; Lupold, S.; Pienta, K.J.;
et al. Low-level endogenous PSMA expression in nonprostatic tumor xenografts is sufficient for In Vivo tumor targeting and
imaging. J. Nucl. Med. 2018, 59, 486–493. [CrossRef]
Pinto, J.T.; Suffoletto, B.P.; Berzin, T.M.; Qiao, C.H.; Lin, S.; Tong, W.P.; May, F.; Mukherjee, B.; Heston, W.D.W. Prostate-specific
membrane antigen: A novel folate hydrolase in human prostatic carcinoma cells. Clin. Cancer Res. 1996, 2, 1445–1451.
Zimmermann, T.; Christensen, S.B.; Franzyk, H. Preparation of enzyme-activated thapsigargin prodrugs by solid-phase synthesis.
Molecules 2018, 23, 1463. [CrossRef] [PubMed]
Mahalingam, D.; Tubb, B.; Nemunaitis, J.J.; Cen, P.; Rowe, J.H.; Sarantopoulos, J.; Kurman, M.R.; Allgood, V.; Campos, L.T.
Clinical activity and correlative DCE-MRI imaging of G-202, a thapsigargin-based prostate-specific membrane antigen-activated
prodrug, in progressive hepatocellular cancer. J. Clin. Oncol. 2015, 33, 301. [CrossRef]
Denmeade, S.R.; Isaacs, J.T. Engineering enzymatically activated “molecular grenades” for cancer. Oncotarget 2012, 3, 666–667.
[CrossRef]
Mahalingam, D.; Mahalingam, D.; Arora, S.P.; Sarantopoulos, J.; Peguero, J.; Campos, L.; Cen, P.; Rowe, J.; Allgood, V.; Tubb, B.;
et al. A phase ii, multicenter, single-arm study of mipsagargin (G-202) as a second-line therapy following sorafenib for adult
patients with progressive advanced hepatocellular carcinoma. Cancers 2019, 11, 833. [CrossRef]
Tarvainen, I.; Zimmermann, T.; Heinonen, P.; Jantti, M.H.; Yli-Kauhaluoma, J.; Talman, V.; Franzyk, H.; Tuominen, R.K.;
Christensen, S.B. Missing selectivity of targeted 4β-phorbol prodrugs expected to be potential chemotherapeutics. ACS Med.
Chem. Lett. 2020, 11, 671–677. [CrossRef]
Jakobsen, C.M.; Denmeade, S.R.; Isaacs, J.T.; Gady, A.; Olsen, C.E.; Christensen, S.B. Design, synthesis, and pharmacological
evaluation of thapsigargin analogues for targeting apoptosis to prostatic cancer cells. J. Med. Chem. 2001, 44, 4696–4703. [CrossRef]
[PubMed]
Dan, N.; Setua, S.; Kashyap, V.K.; Khan, S.; Jaggi, M.; Yallapu, M.M.; Chauhan, S.C. Antibody-drug conjugates for cancer therapy:
Chemistry to clinical implications. Pharmaceuticals 2018, 11, 1–22.
Poudel, Y.B.; Chowdari, N.S.; Cheng, H.; Iwuagwu, C.I.; King, H.D.; Kotapati, S.; Passmore, D.; Rampulla, R.; Mathur, A.; Vite, G.;
et al. Chemical modification of linkers provide stable linker-payloads for the generation of antibody-drug conjugates. ACS Med.
Chem. Lett. 2020, 11, 2190–2194. [CrossRef] [PubMed]
Nicolaou, K.C.; Rigol, S. The role of organic synthesis in the emergence and development of antibody-drug conjugates as targeted
cancer therapies. Angew. Chem. Int. Ed. 2019, 58, 11206–11241. [CrossRef]
Molecules 2021, 26, 1292
64.
65.
66.
67.
68.
69.
70.
71.
72.
73.
74.
75.
76.
77.
78.
79.
80.
81.
82.
83.
84.
85.
86.
87.
88.
23 of 23
Tumey, L.N. Thinking Small and Dreaming Big: Medicinal Chemistry Strategies for Designing Optimal Antibody-Drug Conjugates
(ADC’s). In 2017-Medicinal Chemistry Reviews; American Chemical Society: Washington, DC, USA, 2017; Volume 52, pp. 363–381.
Morrison, S.L.; Johnson, M.J.; Herzenberg, L.D.; Oi, V.T. Chimeric human antibody molecules: Mouse antigen-binding domains
with human constant region domains. Proc. Natl. Acad. Sci. USA 1984, 81, 6851–6855. [CrossRef] [PubMed]
Nicolaou, K.C.; Smith, A.L.; Yue, E.W. Chemistry and biology of natural and designed enediynes. Proc. Natl. Acad. Sci. USA 1993,
90, 5881–5888. [CrossRef]
Maiese, W.M.; Lechevalier, M.P.; Lechevalier, H.A.; Korshalla, J.; Kuck, N.; Fantini, A.; Wildey, M.J.; Thomas, J.; Greenstein, M.
Calicheamicins, a novel family of antitumor antibiotics: Taxonomy, fermentation and biological properties. J. Antibiot. 1989, 42,
558–563. [CrossRef]
Sharma, S.K.; Bagshawe, K.D. Antibody directed enzyme prodrug therapy (ADEPT): Trials and tribulations. Advan. Drug Deliv.
Rev. 2017, 118, 2–7. [CrossRef]
Aloysius, H.; Hu, L. Targeted prodrug approaches for hormone refractory prostate cancer. Med. Res. Rev. 2015, 35, 554–585.
[CrossRef]
Jung, M. Antibody directed enzyme prodrug therapy (ADEPT) and related approaches for anticancer therapy. Mini-Rev. Med.
Chem. 2001, 1, 399–407. [CrossRef] [PubMed]
Tranoy-Opalinski, I.; Fernandes, A.; Thomas, M.; Gesson, J.P.; Papot, S. Design of self-immolative linkers for tumour-activated
prodrug therapy. Anticancer Agents Med. Chem. 2008, 8, 618–637. [CrossRef] [PubMed]
Moradian Tehrani, R.; Verdi, J.; Noureddini, M.; Salehi, R.; Salarinia, R.; Mosalaei, M.; Simonian, M.; Alani, B.; Ghiasi, M.R.;
Jaafari, M.R.; et al. Mesenchymal stem cells: A new platform for targeting suicide genes in cancer. J. Cell. Physiol. 2018, 233,
3831–3845. [CrossRef] [PubMed]
Singhal, S.; Kaiser, L.R. Cancer chemotherapy using suicide genes. Surg. Oncol. Clin. N. Am. 1998, 7, 505–536. [CrossRef]
Sanchez-Sanchez, L.; Tapia-Moreno, A.; Juarez-Moreno, K.; Patterson, D.P.; Cadena-Nava, R.D.; Douglas, T.; Vazquez-Duhalt, R.
Design of a VLP-nanovehicle for CYP450 enzymatic activity delivery. J. Nanobiotechnol. 2015, 13, 1–10. [CrossRef] [PubMed]
Sharon, N.; Lis, H. History of lectins: From hemagglutinins to biological recognition molecules. Glycobiology 2004, 14, 53R–62R.
[CrossRef]
Ma, Y.; Chen, H.; Su, S.; Wang, T.; Zhang, C.; Fida, G.; Cui, S.; Zhao, J.; Gu, Y. Galactose as broad ligand for multiple tumor
imaging and therapy. J. Cancer 2015, 6, 658–670. [CrossRef] [PubMed]
Lin, Y.-S.; Tungpradit, R.; Sinchaikul, S.; An, F.-M.; Liu, D.-Z.; Phutrakul, S.; Chen, S.-T. Targeting the delivery of glycan-based
paclitaxel prodrugs to cancer cells via glucose transporters. J. Med. Chem. 2008, 51, 7428–7441. [CrossRef]
Garnier, P.; Wang, X.-T.; Robinson, M.A.; van Kasteren, S.; Perkins, A.C.; Frier, M.; Fairbanks, A.J.; Davis, B.G. Lectin-directed
enzyme activated prodrug therapy (LEAPT): Synthesis and evaluation of rhamnose-capped prodrugs. J. Drug Target. 2010, 18,
794–802. [CrossRef]
Mukhopadhyay, D.; Riezman, H. Proteasome-independent functions of ubiquitin in endocytosis and signaling. Science 2007, 315,
201–205. [CrossRef]
Teicher, B.A.; Tomaszewski, J.E. Proteasome inhibitors. Biochem. Pharm. 2015, 96, 1–9. [CrossRef]
Ciechanover, A. The unravelling of the ubiquitin system. Nat. Rev. Mol. Cell Biol. 2015, 16, 322–324. [CrossRef] [PubMed]
Bashraheel, S.S.; Domling, A.; Goda, S.K. Update on targeted cancer therapies, single or in combination, and their fine tuning for
precision medicine. Biomed. Pharm. 2020, 125, 110009. [CrossRef]
Bronson, J.J.; Peese, K.M.; Black, A.; Dhar, M.; Pashine, A.; Ellsworth, B.A.; Merritt, J.R. To Market, To Market. In 2015 Medicinal
Chemistry Reviews; Desai, M.J.C., Ed.; American Chemcal Society: Washington, DC, USA, 2015; Volume 50, pp. 461–576.
Bronson, J.J.; Peese, K.M.; Dhar, M.; Pashine, A.; Duclos, F.J.; Ellsworth, B.A.; Garcia, R.; Merritt, J.R. To Market, To Market. In
2016 Medicinal Chemistry Reviews; Desai, M.C., Ed.; Amercian Chemical Society: Washington, DC, USA, 2016; Volume 51, pp.
439–540.
Bolger, C.A.; Dhar, T.G.M.; Pashine, A.; Dragovich, P.S.; Mallet, W.; Robert, M.J.; Peese, K.M. To Market, To Market. In 2017
Medicinal Chemistry Review; Bronson, J.J., Ed.; American Chemical Society: Washington, DC, USA, 2017; Volume 52, pp. 537–601.
Bolger, C.A.; Carpenter, J.E.; Dhar, T.G.M.; Pashine, A.; Dragovich, P.S.; Cook, J.H.; Gillis, E.P.; Peese, K.M.; Merritt, J.R. To Market,
To Market. In 2018 Medicinal Chemistry Reviews; Bonson, J., Ed.; American Chemical Society: Washington, DC, USA, 2018; Volume
53, pp. 587–696.
Bolger, C.A.; Kahn, S.A.; Lipovšek, D.; Mallet, W.; Wieler, J. To Market, To Market. In 2019 Medicinal Chemistry Reviews; Bonson, J.J.,
Ed.; American Chemical Society: Washington, DC, USA, 2019; Volume 54, pp. 601–639.
Araujo, E.; Braun, M.-G.; Dragovich, P.S.; Converso, A.; Nantermet, P.G.; Roecker, A.J.; Dhar, T.G.M.; Haile, P.; Hurtley, A.;
Merritt, J.R. To Market, To Market-2018: Small Molecules. In 2019 Medicinal Chemistry Reviews-2019; Bonson, J.J., Ed.; American
Chemical Society: Washington, DC, USA, 2019; Volume 54, pp. 469–596.