A galactosidase-responsive "Trojan horse" for the selective targeting of folate receptor-positive tumor cells

Article abstract of DOI:10.1002/cmdc.201100114

Licence to Kill: A β-galactosidase-responsive molecular "Trojan horse" was designed to selectively kill folate receptor-positive tumor cells. The recognition of the folate membrane receptor is followed by the internalization and the subsequent enzymatic a

Full text of DOI:10.1002/cmdc.201100114

MED
DOI: 10.1002/cmdc.201100114
A Galactosidase-Responsive “Trojan Horse” for the Selective Targeting of
Folate Receptor-Positive Tumor Cells
Mickaꢀl Thomas,[a] Jonathan Clarhaut,[b, c] Pierre-Olivier Strale,[b] Isabelle Tranoy-Opalinski,[a] Joꢀlle Roche,[b] and
Sꢁbastien Papot*[a]
The selective targeting of cytotoxic compounds is one of the
major challenges in cancer chemotherapy. Most anticancer
drugs lack any intrinsic selectivity, causing severe side effects
as the result of their toxicity toward nonmalignant cells. To cir-
cumvent this drawback, a wide spectrum of drug carriers has
been investigated with the aim to increase drug deposition in
tumors, while avoiding accumulation of cytotoxic agents in
healthy tissues.[1] Such compounds have been designed to
meet two key requirements: 1) the efficient recognition of ma-
lignant specific characteristics, such as tumor-associated mem-
brane receptors or antigens; 2) the controlled release of anti-
neoplastic agents exclusively at the tumor site. Within this
framework, several chemotherapeutics have been conjugated
to low- molecular-weight ligands that display a high affinity for
the corresponding receptor.[2] In this approach, following re-
ceptor–ligand interaction, the conjugate is internalized via re-
ceptor-mediated endocytosis.[3] Once inside the target cancer
cell, the linker between the ligand and the drug is cleaved to
regenerate the active cytotoxic agent. The vast majority of tar-
geting ligand–drug conjugates designed so far contain hydra-
zone- or disulfide-based linkers. However, many of these chem-
ically cleavable linkers are known to be labile in the blood-
stream resulting in the nonselective release of some drug prior
to reaching the target area.[4] The design of enzyme-responsive
drug conjugates that can be activated exclusively by lysosomal
glycosidases may provide a valuable alternative mechanism for
the liberation of antitumor agents in a stringently controlled
fashion. In this context, Seattle Genetics (Bothell, USA) devel-
oped a promising macromolecular antibody–drug conjugate
system allowing the release of various cytotoxics through
human b-glucuronidase activation.[5]
“Trojan horse” 1, including a folate ligand,[6] a galactoside trig-
ger[7] and the potent doxorubicin articulated around a central
self-immolative linker[8] (Scheme 1). The folate ligand enables
the selective recognition of folate receptor-positive tumor cells
and the subsequent internalization of the whole assembly by
endocytosis. The intracellular b-galactosidase-catalyzed cleav-
age of the carbohydrate unit then triggers the release of the
drug through the self-immolative mechanism depicted in
Scheme 1b. Further translocation of doxorubicin into the nu-
cleus finally induces cell death.
The folate receptor (FR)[9] is a cell-surface marker over-
expressed in several cancer types, such as lung, breast, brain,
colon, kidney, ovarian and leukemia.[10] In contrast, FR is pres-
ent in low or undetectable quantities in most normal tissues.
This unique feature of malignant cells has been exploited
using folate–drug conjugates to target potent cytotoxic agents
at the tumor site.[11] The best illustration of such drug carriers
is probably the folate–desacetylvinblastine monohydrazine
conjugate developed by Leamon et al. that is currently pro-
gressing through clinical trials.[12]
The choice of b-galactosidase as the triggering enzyme
relies on its undetectable level in serum. Its activity is mainly
intracellular but located in both healthy and cancerous tissues.
However, with our design, we anticipated that the hydrophilici-
ty imparted by the galactoside trigger may prevent passive cel-
lular uptake and further intracellular activation of assembly 1
in nonmalignant cells.
In order to prepare the galactoside–folate–doxorubicin con-
jugate 1, we designed the central unit 4 bearing three differ-
ent chemical functionalities[5,13] suitable for the successive in-
troduction of each part of the assembly (Scheme 2). It is worth
mentioning here that this central unit allows versatile access to
a wide variety of drug carriers similar to 1. This may permit the
on-demand synthesis of the perfect drug–trigger–tumor-selec-
tive ligand combination to target a given cancer.
Herein, we present a novel drug-delivery system that com-
bines the selectivities of a low-molecular-weight ligand with
tumor affinity and an enzymatic trigger in a single entity. For
this purpose, we designed the galactosidase-responsive
Compound 4 was first synthesized as a racemic mixture by
addition of freshly prepared aluminum propargyl bromide to
the commercially available 4-hydroxy-3-nitro-benzaldehyde
(94%). This was followed by the chemo- and stereoselective
glycosylation of nitrophenol 4, which afforded the b-galacto-
side 5 in 84% yield. Treatment of benzyl alcohol 5 with p-nitro-
phenyl chloroformate and pyridine led to activated carbonate
6 (92%). Doxorubicin was then introduced via nucleophilic
substitution to give the protected galactoside 7 (65%). Depro-
tection of the hydroxy groups furnished the clickable deriva-
tive 8 (80%) ready for the final coupling reaction with an in-
separable mixture of the a- and g-PEGylated folates 9a and 9b
prepared according to literature procedures. Indeed, previous
studies reported that both the a- and g-regioisomers of folate
[a] Dr. M. Thomas, Dr. I. Tranoy-Opalinski, Dr. S. Papot
Laboratoire de Synthꢀse et Rꢁactivitꢁ des Substances Naturelles
UMR-CNRS 6514, Universitꢁ de Poitiers
40 Avenue du Recteur Pineau, 86022 Poitiers (France)
Fax: (+33)549453501
E-mail: sebastien.papot@univ-poitiers.fr
[b] Dr. J. Clarhaut, P.-O. Strale, Prof. J. Roche
Institut de Physiologie et Biologie Cellulaires
UMR-CNRS 6187, Universitꢁ de Poitiers
40 Avenue du Recteur Pineau, 86022 Poitiers (France)
[c] Dr. J. Clarhaut
INSERM CIC 0802, 2 rue de la Milꢁtrie
CHU de Poitiers, 86021 Poitiers (France)
Supporting information for this article is available on the WWW under
http://dx.doi.org/10.1002/cmdc.201100114.
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ChemMedChem 2011, 6, 1006 – 1010

conjugates exhibited a high binding affinity for the FR.[14] Thus,
four isomers (see Supporting Information) in 75% yield using
the well-known copper-catalyzed azide–alkyne cycloaddition
(CuCAAC).[15]
the targeting assembly 1 was finally obtained as a mixture of
The stability of the galacto-
side–folate–doxorubicine conju-
gate 1 was examined both in
phosphate buffer (pH 7) and
bovine serum at 378C. No de-
composition was observed by
HPLC after 24 h under these
conditions. Enzymatic hydrolysis
of compound 1 was then con-
ducted with either Escherichia
coli or bovine liver b-galactosi-
dase and monitored by HPLC/
MS (Figure 1). As expected, in
each case the galactoside 1 was
rapidly cleaved by the enzyme
leading to the release of doxoru-
bicin (m/z=544) along with the
formation of both the a- and g-
regioisomer of
3 (m/z=831).
This result indicates that the gal-
actoside trigger is readily acces-
sible for the enzyme in spite of
the presence of bulky moieties,
such as doxorubicin or folate
ligand attached on the linker
unit.
We next investigated the se-
lective uptake of our folate-tar-
geted assembly 1 in HeLa FR-
positive cells by confocal micros-
copy, possible because of the
fluorescence of doxorubicin
(Figure 2). As a negative control,
the same experiments were car-
ried out with the low FR-express-
ing A549 cell line, which should
not be targeted by compound 1
(see Supporting Information).
First, when incubated for two
hours in each cell culture media,
the free drug was internalized in
both cell types as proved by the
intracellular location of doxoru-
bicin red fluorescence (Fig-
ure 2a). In contrast, under the
same conditions, the folate con-
jugate
1
exclusively entered
HeLa cells, showing its ability to
target FR-overexpressing tumor
cells selectively (Figure 2b).
To demonstrate the involve-
ment of cell-surface FR in this in-
ternalization process, HeLa cells
were exposed to 1 in the pres-
Scheme 1. a) The principle of tumor targeting. Step 1: ligand–receptor interaction; step 2: endocytosis; step 3:
enzyme-triggered drug release; step 4: translocation of doxorubicin into the nucleus. b) b-Galactosidase-catalyzed
drug release mechanism.
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did not penetrate into either
Hela or A549 cells (Figure 2d).
All together, these results clearly
demonstrate that prior recogni-
tion of FR by the folate moiety
of 1 is responsible for the selec-
tive uptake of assembly 1 by
HeLa FR-positive cells.
The antiproliferative activity of
galactoside–folate–doxorubicin
conjugate 1 was then examined
to confirm that the internaliza-
tion of the whole assembly was
followed by cytotoxicity caused
by the drug. As expected, when
incubated in HeLa cell culture
media, the targeting assembly 1
induced a dramatic antiprolifera-
tive effect, which was close to
that recorded with free doxoru-
bicin (Figure 3a; 1: IC50 =
220 nm; Dox: IC50 =52 nm). Con-
versely, A549 cells were not at all
Figure 1. Enzymatic hydrolysis of 1 with E. coli b-galactosidase in phosphate buffer (0.02m, pH 7.0) at 378C using
40 Ummolꢀ1 of substrate: a) t=0 min; b) t=>60 min.
ence of an excess of the free natural ligand, folic acid (1 mm).
As illustrated in Figure 2c, the uptake of 1 was considerably re-
duced in the course of this experiment supporting the hypoth-
esis of FR-mediated endocytosis of the folate conjugate 1. Ad-
ditionally, confocal microscopy experiments conducted with
the galactoside 8 showed that this nonconjugated derivative
sensitive to incubation of compound 1, whereas the free dox-
orubicin was highly toxic (Figure 3b). Indeed, in accordance
with confocal microscopy experiments, assembly 1, which was
not taken up by this cell type, cannot be activated by intra-
cellular b-galactosidase to release doxorubicin cytoxicity. How-
ever, as illustrated in Figure 3b, addition of b-galactosidase in
Figure 2. Confocal microscopy of doxorubicin (Dox) in HeLa and A549 cells treated for 2 h with 10 mm of a) doxorubicin, b) targeting assembly 1, c) targeting
assembly 1+folic acid (1 mm), d) galactoside 8. Nuclei were stained with 4’,6-diamidino-2-phenyl-indole (DAPI). This experiment was performed twice; scale
bar: 100 mm.
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Figure 3. Proliferative activity of a) HeLa and b) A549 cells treated over four
days with the indicated compounds (0–1000 nm). Three experiments were
performed in triplicate; standard deviations are indicated.
Acknowledgements
The authors thank the Centre National de la Recherche Scientifi-
que (CNRS), La Ligue contre le Cancer (Comitꢁ de Charente), and
the Rꢁgion Poitou-Charentes and Le Cancꢁropꢂle Grand Ouest
for financial support of this study, and Prof. J.-P. Gesson (Univer-
sitꢁ de Poitiers, France) for many fruitful discussions.
Scheme 2. Synthesis of the targeting assembly 1. a) 1-Bromo-(2,3,4,6-O-tetra-
O-acetyl)-b-d-galactopyranoside, Ag2CO3, 1,1,4,7,10,10-hexamethyltriethyl-
enetetramine (HMTTA), CH3CN, 08C!RT, 4 h, 84% ; b) p-Nitrophenyl chloro-
formate, pyridine, CH2Cl2, 08C, 3 h, 92%; c) doxorubicin, Et3N, DMF, RT, 12 h,
65%; d) NaOMe, MeOH/CH2Cl2 (85:15), ꢀ158C, 12 h, 80%; e) CuSO4, sodium
ascorbate, DMSO/H2O (9:1), RT, 6 h, 75%.
Keywords: anticancer agents
receptors · glycosides · prodrugs
·
drug delivery
·
folate
A549 cell culture media induced significant cell death (1+b-
galactosidase: IC50 =302 nm; Dox: IC50 =190 nm) showing that
enzymatic activation of 1 is necessary to trigger antiprolifera-
tive activity.
[1] F. Kratz, I. A. Mꢃller, C. Ryppa, A. Warnecke, ChemMedChem 2008, 3, 20–
53.
In conclusion, we developed a highly selective galactosi-
dase-responsive molecular “Trojan horse”, which can target FR-
positive tumor cells without affecting low FR-expressing cells.
Our b-galactosidase-triggered system should prevent prema-
ture activation of the targeting assembly in circulation, there-
fore avoiding nonspecific drug release in healthy tissues. The
generic linker 4 allows the preparation of a large panel of drug
carriers like 1, and other drug–trigger–targeting ligand combi-
nations are currently under investigation. This strategy might
permit the custom design of targeting assemblies for the treat-
ment of particular cancers based on their unique tumor-associ-
ated specificities. This novel drug targeting system could repre-
sent an important step toward the quest for the magic bullet
in cancer chemotherapy. In vivo studies aimed at evaluating
the potential of this approach have been initiated.
[2] S. Jaracz, J. Chen, L. V. Kuznetsova, I. Ojima, Bioorg. Med. Chem. 2005,
13, 5043–5054.
[3] S. D. Conner, S. L. Schmid, Nature 2003, 422, 37–44.
[4] a) L. Ducry, B. Stump, Bioconjugate Chem. 2010, 21, 5–13, and referen-
ces therein; b) M. N. Saleh, S. Sugarman, J. Murray, J. B. Ostroff, D.
Healey, D. Jones, C. R. Daniel, D. LeBherz, H. Brewer, N. Onetto, A. F. Lo-
Buglio, J. Clin. Oncol. 2000, 18, 2282–2292; c) H. Xie, C. Audette, M.
Hoffee, J. M. Lambert, W. A. Blꢄttler, J. Pharmacol. Exp. Ther. 2004, 308,
1073–1082.
[5] a) S. C. Jeffrey, J. B. Andreyka, S. X. Bernhardt, K. M. Kissler, T. Kline, J. S.
Lenox, R. F. Moser, M. T. Nguyen, N. M. Okeley, I. J. Stone, X. Zhang, P. D.
Senter, Bioconjugate Chem. 2006, 17, 831–840; b) S. C. Jeffrey, M. T.
Nguyen, R. F. Moser, D. L. Meyer, J. B. Miyamoto, P. D. Senter, Bioorg.
Med. Chem. Lett. 2007, 17, 2278–2280; c) P. J. Burke, P. D. Senter, D. W.
Meyer, J. B. Miyamoto, M. Anderson, B. E. Toki, G. Manikumar, M. C.
Wani, D. J. Kroll, S. C. Jeffrey, Bioconjugate Chem. 2009, 20, 1242–1250.
[6] P. S. Low, W. A. Henne, D. D. Doorneweerd, Accounts Chem. Res. 2008,
41, 120–129.
ChemMedChem 2011, 6, 1006 – 1010
ꢂ 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemmedchem.org
1009

MED
[7] a) L. F. Tietze, B. Krewer, Chem. Biol. Drug Des. 2009, 74, 205–211; b) L. F.
Tietze, F. Major, I. Schuberth, D. A. Spiegl, B. Krewer, K. Maksimenka, G.
Bringmann, J. Magull, Chem. Eur. J. 2007, 13, 4396–4409; c) L. F. Tietze,
F. Major, I. Schuberth, Angew. Chem. 2006, 118, 6724–6727; Angew.
Chem. Int. Ed. 2006, 45, 6574–6577; d) L. F. Tietze, T. Feuerstein, A.
Fecher, F. Haunert, O. Panknin, U. Borchers, I. Schuberth, F. Alves,
Angew. Chem. 2002, 114, 785–787; Angew. Chem. Int. Ed. 2002, 41, 759–
761; e) A. Kamal, V. Tekumalla, A. Krishnan, M. Pal-Bhadra, U. Bhadra,
ChemMedChem 2008, 3, 794–802; f) E. Bakina, D. Farquhar, Anti-Cancer
Drug Des. 1999, 14, 507–515; g) M. Thomas, F. Rivault, I. Tranoy-Opalin-
ski, J. Roche, J. P. Gesson, S. Papot, Bioorg. Med. Chem. Lett. 2007, 17,
983–986; h) A. Fernandes, A. Viterisi, F. Coutrot, S. Potok, D. A. Leigh, V.
Aucagne, S. Papot, Angew. Chem. 2009, 121, 6565–6569; Angew. Chem.
Int. Ed. 2009, 48, 6443–6447.
[8] a) S. Papot, I. Tranoy, F. Tillequin, J.-C. Florent, J.-P. Gesson, Curr. Med.
Chem. Anti-Cancer Agents 2002, 2, 155–185; b) I. Tranoy-Opalinski, A.
Fernandes, M. Thomas, J.-P. Gesson, S. Papot, Anti-Cancer Agents Med.
Chem. 2008, 8, 618–637.
[9] H. Elnakat, M. Ratnam, Adv. Drug Delivery Rev. 2004, 56, 1067–1084.
[10] P. S. Low, A. C. Antony, Adv. Drug Delivery Rev. 2004, 56, 1055–1058.
[11] a) C. P. Leamon, J. A. Reddy, Adv. Drug Delivery Rev. 2004, 56, 1127–
1141; b) J. A. Reddy, V. M. Allagadda, C. P. Leamon, Curr. Pharm. Biotech-
nol. 2005, 6, 131–150; c) C. P. Leamon, Curr. Opin. Invest. Drugs 2008, 9,
1277–1286.
[12] J. A. Reddy, R. Dorton, E. Westrick, A. Dawson, T. Smith, L. C. Xu, M.
Vetzel, P. Kleindl, I. R. Vlahov, C. P. Leamon, Cancer Res. 2007, 67, 4434–
4442.
[13] a) A. Gopin, N. Pessah, M. Shamis, C. Rader, D. Shabat, Angew. Chem.
2003, 115, 341–346; Angew. Chem. Int. Ed. 2003, 42, 327–332; b) M.-R.
Lee, K.-H. Baek, H. J. Jin, Y.-G. Jung, I. Shin, Angew. Chem. 2004, 116,
1707–1710; Angew. Chem. Int. Ed. 2004, 43, 1675–1678; c) A. Gopin, C.
Rader, D. Shabat, Bioorg. Med. Chem. 2004, 12, 1853–1858; d) C. Antc-
zak, B. Bauvois, C. Monneret, J.-C. Florent, J. Controlled Release 2001, 74,
255–257.
[14] a) A. Bettio, M. Honer, C. Mꢃller, M. Brꢃhlmeier, U. Mꢃller, R. Schibli, V.
Groehn, A. P. Schubiger, S. M. Ametamey, J. Nucl. Med. 2006, 47, 1153–
1160; b) C. Y. Ke, C. J. Mathias, M. A. Green, Nucl. Med. Biol. 2003, 30,
811–817; c) C. Y. Ke, C. J. Mathias, M. A. Green, Adv. Drug Delivery Rev.
2004, 56, 1143–1160; d) C. P. Leamon, R. B. DePrince, R. W. Hendren, J.
Drug Targeting 1999, 7, 157–169.
[15] a) V. V. Rostovtsev, L. G. Green, V. V. Fokin, K. B. Sharpless, Angew. Chem.
2002, 114, 2708–2711; Angew. Chem. Int. Ed. 2002, 41, 2596–2599;
b) C. W. Tornøe, C. Christensen, M. Meldal, J. Org. Chem. 2002, 67, 3057–
3064.
Received: February 23, 2011
Published online on March 25, 2011
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