Shiga toxin-mediated retrograde delivery of a topoisomerase I inhibitor prodrug

Article abstract of DOI:10.1002/anie.200701270

(Figure Presented) A retrograde strategy: An innovative cancer-cell delivery concept exploits the naturally evolved characteristics of the Shiga toxin B-subunit (STxB) for the intracellular activation of a newly synthesized prodrug at the level of the bio

Full text of DOI:10.1002/anie.200701270

Angewandte
Chemie
DOI: 10.1002/anie.200701270
Chemical Biology
Shiga Toxin-Mediated Retrograde Delivery of a Topoisomerase I
Inhibitor Prodrug**
Abdessamad El Alaoui, FrØdØric Schmidt,* Mohamed Amessou, Marianne Sarr,
Didier Decaudin, Jean-Claude Florent, and Ludger Johannes
Selectivity remains one of the principal challenges in cancer
chemotherapy. With more-selective agents, it should be
possible to minimize side effects and to use more efficient
active-compound concentrations.[1] One way to achieve this
goal is targeted delivery to cancer cells.[2,3] Such targeting
strategies have already been described, and the use of
monoclonal antibodies is by far the best-explored example,[4,5]
with compounds such as Mylotarg having reached the
market.[6]
Here, we present a new delivery strategy based on the
nontoxic B-subunit of Shiga toxin, termed STxB. Shiga toxin
is produced by intestinal pathogenic bacteria.[7] STxB asso-
ciates with the catalytic A-subunit that modifies ribosomal
RNA in the cytosol of target cells, thus leading to protein
Figure 1. Principle of retrograde delivery.
biosynthesis inhibition. To reach the cytosol from the
extracellular space where the toxin is produced by the
bacteria, STxB binds to the cellular toxin receptor, the
glycolipid Gb3. By following a recently discovered pathway,
termed the retrograde route (Figure 1), Shiga toxin is then
transported via endosomes and the Golgi apparatus to the
endoplasmic reticulum, from where the A-subunit is trans-
located across the membrane to the cytosol.[8,9]
We designed a prodrug based on SN-38 (1; Scheme 1), the
active principle of CPT11 (Campto), which is used for the
treatment of colorectal carcinoma.[12] SN-38 belongs to the
class of camptothecin derivatives that are cytotoxic by
inhibition of topoisomerase I, and is one of the most efficient
The Shiga toxin receptor Gb3 is strongly expressed by
certain human cancers,[9] including colorectal carcinoma.[10] In
a mouse model of spontaneous intestinal adenocarcinomato-
sis, we have shown that STxB can reach Gb3-expressing
tumors in vivo.[11] As a delivery tool, STxB exhibits character-
istics that the protein has acquired as an intestinal pathogen in
co-evolution with its hosts: stability at extreme pH and in the
presence of proteases, capacity to cross tissue barriers and to
distribute in the organism, and resistance against extra- and
intracellular inactivation.[9]
[*] Dr. A. El Alaoui, Dr. F. Schmidt, Dr. J.-C. Florent
UMR 176 CNRS
Institut Curie, Centre de Recherche
26 rue d’Ulm 75248 Paris Cedex 05 (France)
Fax: (+33)1-4234-6664
E-mail: frederic.schmidt@curie.fr
Dr. M. Amessou, M. Sarr, Dr. D. Decaudin, Dr. L. Johannes
UMR 144 CNRS
Institut Curie, Centre de Recherche
26 rue d’Ulm 75248 Paris Cedex 05 (France)
[**] We acknowledge the help from Wolfgang Faigle, Jean-Marc Clavel,
and Damarys Loew (mass spectrometry laboratory of the Institut
Curie). Our work was supported by funding from the Institut Curie
(PIC Vectorisation) and Canceropôle Ile-de-France.
Supporting information for this article is available on the WWW
under http://www.angewandte.org or from the author.
Scheme 1. Cleavage reactions of compounds 2 and 3. The double-
headed arrows indicate the bonds that are cleaved first.
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compounds in this family.[13] For coupling to the prodrug, an
STxB variant with a thiol functionality, termed STxB-SH, was
used that was specifically designed for site-directed chemical
cross-linking.[14,15]
The phenolic position of SN-38 was chosen to build self-
immolative spacers that include disulfide bonds. After
cleavage of these bonds and release of a free thiol function,
the free phenol must be released without any other external
reactant. To this end, we envisioned two different spacers with
variable stabilities in the biological system. One is based on an
aromatic ring and the other on an aliphatic chain.
pentamer, by using mass spectrometric analysis (see the
Supporting Information) and fluorimetric dosage.
As a first step towards the biological evaluation of
compounds 2 and 3, the stability of the biotin versions was
tested in different media. Compound 2b turned out to be
readily activated even in the absence of cells, thus precluding
its use in vivo. In contrast, compound 3b was completely
stable over extended periods of up to 48 h at 378C in all
media, including pure fetal calf serum. Prodrug 3a was also
stable in pure fetal calf serum, as shown by fluorimetric
measurements (see the Supporting Information).
These two compounds (2 and 3) and their respective
cleavage reactions are depicted in Scheme 1. Two variants
were synthesized for each spacer arm: one (a) with SN-38 (1),
and the other (b) with biotin derivative 4. The latter model
allowed circumvention of the fact that release of SN-38 from
compounds 2a and 3a could not be monitored in vivo because
of a lack of sensitivity. The biotin group was derivatized with a
phenol spacer to obtain a similar susceptibility to cleavage as
that with the phenol function of SN-38. The compounds were
obtained according to Scheme 2and the Supporting Infor-
mation.
Compound 3 was chosen for an in-depth characterization
on HT-29 colorectal carcinoma cells. ELISA analysis with 3b
demonstrated that cleavage became detectable in the 6–24-h
time interval, and was essentially complete at 48 h (Figure 2).
For the synthesis of compounds 3a and 3b, commercial
amino alcohol 5 was first monoprotected as a tert-butoxycar-
bonyl (Boc) derivative. The hydroxy group was converted to
bromide with CBr4 and then substituted by a thioacetate. The
thiol function of 8 was activated as a pyridine disulfide 9.
Liberation of the free amine was performed in acidic medium
and the formed chlorhydrate 10 was kept as a salt because the
free amine was unstable. Compound 10 was then reacted with
phosgene and triethylamine to give the stable carbamoyl
chloride 11. The phenol (SN-38 or biotin derivative) was first
coupled in the presence of a stoichiometric amount of 4-
dimethylaminopyridine (DMAP) to this bifunctional inter-
mediate 11. Finally, STxB-SH was reacted with carbamate 12
under basic conditions (pH 9).
Figure 2. ELISA analysis of activation of 3b on HT-29 cells. Compound
3b (1 mm) was incubated with HT-29 cells on ice. After washing, the
cells were shifted to 378C for the indicated times, lysed, and the
lysates were analyzed by ELISA for the indicated markers. Means of
two determinations are shown. t=incubation time.
The same results were obtained on HeLa cells. To demon-
strate that cleavage occurred intracellularly, we used immu-
nofluorescence analysis (Figure 3). Consistent with the
ELISA data, no cleavage could be detected after short
times of internalization (45 min), in which STxB (red) and
biotin (green) co-localized with
The substitution levels of the coupling products were
determined as five SN-38 or biotin molecules per STxB
the Golgi marker Rab6 (blue).
After 48 h, STxB (red) could
still be detected in the Golgi
region (blue). However, the
biotin signal was largely gone,
which strongly suggests that the
reduction of the disulfide bond
occurred within membranes of
the biosynthetic/secretory path-
way.
Having established that
biotin model compound 3b is
activated in HT-29 cells, we
sought to determine the cyto-
toxic effect of the correspond-
ing prodrug 3a. As shown in
Figure 4, an IC50 value of 300 nm
(in SN-38 equivalents) was
observed on Gb3-expressing
Scheme 2. Synthesis of compounds 3a and 3b.
HT-29 cells. To establish specif-
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Figure 3. Activation of 3b after retrograde trafficking to the biosynthetic/secretory pathway. Compound 3b (1 mm) was incubated withHT-29 cells
on ice. After washing, the cells were shifted to 378C for the indicated times, fixed, and stained for the indicated markers. Top: 45 min of uptake;
bottom: 48 hof uptake. Biotin: green; STxB: red; Rab6 (Golgi): blue.
expression (Figure 4), thus further establishing the selectivity
of compound 3a for Gb3-expressing tumor cells.
In summary, we have identified a novel tumor-delivery
approach based on retrograde prodrug targeting to mem-
branes of the biosynthetic/secretory pathway, by using STxB.
The disulfide linkage of prodrug 3a is slowly released, most
likely in the endoplasmic reticulum whose function in cellular
redox homeostasis is well-recognized.[17] This slow release
should sustain the continued presence of the active principle
in dividing tumor cells, with prodrug being otherwise rapidly
cleared from the circulation. Retrograde delivery will also
place the site of drug release close to the nucleus, where the
molecular target of hydrophobic SN-38 resides. In the light of
high Gb3 expression levels in tumors (more than 107 binding
Figure 4. Cytotoxic activity of compounds on HT-29 cells. The indi-
cated compounds were incubated for 6 hat 37 8C withHT-29 (withor
sites per cancer cell[18]) and the preferential retrograde
transport in tumor cells, when compared to nontumoral
Gb3-expressing cells,[18,19] we expect that retrograde delivery
will make a major contribution to improve the selectivity of
cancer chemotherapy.
without PPMP) or CHO cells. After washing, incubation was continued
for 7 days at 378C, followed by live-cell counting by using 3-(4,5-
dimethylthiazole-2-yl)-2,5-diphenyltetrazolium bromide (MTT).
icity, two Gb3 negative-control situations were tested: HT-29
cells that were treated with the glycosylceramide synthase
inhibitor 1-phenyl-2-palmitoylamino-3-morpholino-1-propa-
nol (PPMP),[16] or spontaneously Gb3-negative Chinese
hamster ovary (CHO) cells. In both cases, the cells were not
sensitive at all to incubation with prodrug 3a, which was used
in the same concentration range for all experiments with 3a
(Figure 4). Furthermore, we showed that nonderivatized
STxB-SH had no measurable cytotoxicity on Gb3-expressing
HT-29 cells under our experimental conditions (Figure 4).
Importantly, neither nonvectorized SN-38 (IC50: 30 nm)
nor its prodrug CPT-11 used in clinics (IC50: 70 mm) had a
cytotoxic effect on HT-29 cells that were dependent on Gb3
Experimental Section
The synthesis and characterization of all new compounds and the
conditions of the biological experiments are described in the
Supporting Information.
Received: March 22, 2007
Revised: June 6, 2007
Published online: July 23, 2007
Keywords: antitumor agents · drug delivery · prodrugs ·
.
retrograde transport · Shiga toxin
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Communications
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