Strategy to tether organometallic ruthenium-arene anticancer compounds to recombinant human serum albumin

Article abstract of DOI:10.1021/ic701474m

In order to utilize macromolecules for drug targeting and delivery, a strategy to tether organometallic ruthenium-arene drugs to carrier protein molecules was developed. The approach involves the design of a drug fragment capable of conjugating to linker molecules on a modified carrier protein via hydrazone bond formation. The proof-of-concept using recombinant human serum albumin is described.

Full text of DOI:10.1021/ic701474m

Inorg. Chem. 2007, 46, 9048−9050
Strategy To Tether Organometallic Ruthenium−Arene Anticancer
Compounds to Recombinant Human Serum Albumin
Wee Han Ang,† Elisa Daldini,† Lucienne Juillerat-Jeanneret,‡ and Paul J. Dyson*,†
Institut des Sciences et Inge´nierie Chimiques, Ecole Polytechnique Fe´de´rale de Lausanne (EPFL),
CH-1015 Lausanne, Switzerland, and UniVersity Institute of Pathology, Centre Hospitalier
UniVersitaire Vaudois (CHUV), CH-1011 Lausanne, Switzerland
Received July 25, 2007
In order to utilize macromolecules for drug targeting and delivery,
a strategy to tether organometallic ruthenium arene drugs to carrier
pressure in the blood and to scavenge free radicals as an
antioxidant. It is an attractive macromolecular carrier, given
its nontoxicity and nonimmunogencity and that it is available
in pure form. Chlorambucil and paclitaxel conjugated to HSA
exhibit cytotoxicity comparable to that of the parent drugs
in vitro but are less toxic in vivo.5 In addition, a doxorubicin
prodrug, which exploits endogenous serum albumin as a drug
carrier, also showed a superior antitumor effect on murine
renal cell carcinoma in vivo.6 Recombinant HSA (rHSA)
purified from yeast (Pichia pastoris) is also commercially
available and has been tested in clinical trials, with no adverse
effects reported.4
In recent years, there has been growing interest in studying
ruthenium-based compounds as potential anticancer drug
candidates, following the successful completion of two
ruthenium(III) compounds in phase I clinical trials (see
Figure 1).7 In addition, organometallic ruthenium(II)-arene
complexes bearing the 1,3,5-triaza-7-phosphatricyclo[3.3.1.1]-
decane ligand (known as RAPTA complexes) exhibit favor-
able pharmacological profiles in vitro and in vivo for
application as antitumor compounds.8,9 It is therefore worth-
while to develop a system to conjugate RAPTA moieties to
a carrier protein molecule such as rHSA for passive drug
targeting.
−
protein molecules was developed. The approach involves the
design of a drug fragment capable of conjugating to linker
molecules on a modified carrier protein via hydrazone bond
formation. The proof-of-concept using recombinant human serum
albumin is described.
Drug targeting, i.e., the specific delivery of a drug to
cancer cells, may be achieved by the use of targeting groups
or by tuning the chemical and physical characteristics of the
drug or drug carrier, such as hydrophobicity and molecular
size.1 One passive targeting method that has been widely
utilized exploits the so-called “enhanced permeability and
retention (EPR)” effect of macromolecules on tumors.2 The
EPR effect is based on the observation that macromolecules
are able to penetrate the leaky vasculature surrounding the
tumor. As a result of the increased permeability, the
macromolecules “selectively” permeate the tumor tissues as
compared to healthy tissues. In addition, their lymphatic
drainage system is impaired, which results in an accumulation
of the macromolecules at the tumor site. Different types of
macromolecules have been used as carrier molecules, includ-
ing liposomes, dendrimers, poly(ethylene glycol) polymers,
nanoparticles, and protein biomolecules.3
(5) (a) Dosio, F.; Brusa, P.; Crosasso, P.; Arpicco, S.; Cattel, L. J.
Controlled Release 1997, 47, 293-304. (b) Kratz, F.; Beyer, U.; Roth,
T.; Schutte, M. T.; Unold, A.; Fiebig, H. H.; Unger, C. Arch. Pharm.
(Weinheim) 1998, 331, 47-53.
In particular, human serum albumin (HSA) is known to
accumulate in tumors, being taken up by tumor cells at
increased levels compared to normal cells, and has been
exploited as the carrier conjugate of various organic anti-
cancer drugs such as chlorambucil, doxorubicin, and pacli-
taxel.4 The main role of HSA is to maintain the osmotic
(6) (a) Kratz, F.; Muller-Driver, R.; Hofmann, I.; Drevs, J.; Unger, C. J.
Med. Chem. 2000, 43, 1253-1256. (b) Kratz, F.; Warnecke, A.;
Scheuermann, K.; Stockmar, C.; Schwab, J.; Lazar, P.; Druckes, P.;
Esser, N.; Drevs, J.; Rognan, D.; Bissantz, C.; Hinderling, C.; Folkers,
G.; Fichtner, I.; Unger, C. J. Med. Chem. 2002, 45, 5523-5533.
(7) (a) Hartinger, C. G.; Zorbas-Seifried, S.; Jakupec, M. A.; Kynast, B.;
Zorbas, H.; Keppler, B. K. J. Inorg. Biochem. 2006, 100, 891-904.
(b) Jakupec, M. A.; Arion, V. B.; Kapitza, S.; Reisner, E.; Eichinger,
A.; Pongratz, M.; Marian, B.; Graf v. Keyserlingk, N.; Keppler, B.
K. Int. J. Clin. Pharmacol. Ther. 2005, 43, 595-596. (c) Alessio, E.;
Mestroni, G.; Bergamo, A.; Sava, G. Curr. Top. Med. Chem. 2004, 4,
1525-1535.
(8) (a) Ang, W. H.; Daldini, E.; Scolaro, C.; Scopelliti, R.; Juillerat-
Jeannerat, L.; Dyson, P. J. Inorg. Chem. 2006, 45, 9006-9013. (b)
Ang, W. H.; Dyson, P. J. Eur. J. Inorg. Chem. 2006, 4003-4018. (c)
Dyson, P. J.; Sava, G. Dalton Trans. 2006, 1929-1933. (d) Gossens,
C.; Dorcier, A.; Dyson, P. J.; Rothlisberger, U. Organometallics 2007,
26, 3969-3975.
* To whom correspondence should be addressed. E-mail: paul.dyson@
epfl.ch.
† Ecole Polytechnique Fe´de´rale de Lausanne (EPFL).
‡ Centre Hospitalier Universitaire Vaudois (CHUV).
(1) Yokoyama, M. J. Artif. Organs 2005, 8, 77-84.
(2) Modi, S.; Jain, J. P.; Domb, A. J.; Kumar, N. Curr. Pharmaceut. Des.
2006, 12, 4785-4796.
(3) Haag, R.; Kratz, F. Angew. Chem., Int. Ed. 2006, 45, 1198-1215.
(4) Chuang, V. T. G.; Kragh-Hansen, U.; Otagiri, M. Pharm. Res. 2002,
19, 569-577.
9048 Inorganic Chemistry, Vol. 46, No. 22, 2007
10.1021/ic701474m CCC: $37.00
© 2007 American Chemical Society
Published on Web 10/05/2007

COMMUNICATION
Figure 1. Ruthenium-based complexes investigated for anticancer activity.
Figure 3. MALDI-TOF mass spectra of rHSA and rHSA-RAPTA
conjugates.
Figure 2. Ball-and-stick representation of 3. Atoms are spheres of arbitrary
diameter. The DMF solvent molecule is omitted for clarity. Key bond
distances (Å) and angles (deg): Ru-Cav, 2.228; Ru-P, 2.308(4); Ru-
Clav, 2.438(8); Cl-Ru-Cl, 86.61(14); P-Ru-Clav, 84.8(3).
Scheme 2
Scheme 1
Ideally, the coordination sphere of the RAPTA moiety
should remain as unperturbed as possible and, therefore,
modification at the arene ring is preferred. In addition,
because the reactive ruthenium center is susceptible to both
nucleophilic and redox reagents, a mild conjugation method
that connects the RAPTA moiety to the protein by means of
a linker is required. An optimal conjugation method would
be to cross-link RAPTA to biomolecules via acid-labile
hydrazone bonds using aldehyde and hydrazine functional
groups based on well-established methods. Indeed, the
lability of the hydrazone bond under acidic conditions has
been exploited for targeted release of drugs after cellular
uptake.5 We therefore decided to build a RAPTA fragment
containing an aldehyde bond, which can conjugate to rHSA
functionalized with hydrazine groups, by means of the
hydrazone bond (see Scheme 1).
The ruthenium fragment was synthesized in two main parts
via a reported procedure.9 First, the arene ligand was prepared
from 1-methylamine-1,4-cyclohexadiene (1) in three steps
(see Scheme 2 and the Supporting Information). Second, the
functionalized arene ligand was reacted with hydrated
ruthenium(III) chloride in ethanol under reflux for 16 h to
yield the arene-capped ruthenium(II) dimer 2. The subsequent
reaction with pta was carried out in degassed N,N-dimeth-
ylformamide (DMF) and monitored using 31P{1H} NMR
spectroscopy to yield the target complex RAPTA-FORM (3)
in good yield. The product was characterized spectroscopi-
cally by negative-mode electrospray ionization mass spec-
trometry (nESI-MS) and 1H and 31P{1H} NMR spectroscopy.
In particular, its solid-state structure, determined using X-ray
crystallography,10 revealed that the bond parameters around
the ruthenium(II) are remarkably similar to those of RAPTA-
C, implying that the coordination sphere of the ruthenium-
(II) center is largely preserved (see Figure 2). The structure
(9) (a) Scolaro, C.; Bergamo, A.; Brescacin, L.; Delfino, R.; Cocchietto,
M.; Laurenczy, G.; Geldbach, T. J.; Sava, G.; Dyson, P. J. J. Med.
Chem. 2005, 48, 4161-4171. (b) Scolaro, C.; Geldbach, T. J.; Rochat,
S.; Dorcier, A.; Gossens, C.; Bergamo, A.; Cocchietto, M.; Tavernelli,
I.; Sava, G.; Rothlisberger, U.; Dyson, P. J. Organometallics 2006,
25, 756-765.
(10) Crystal data for 3: C22H27Cl2N4O3PRu‚0.5DMF, Mw ) 634.96, crystal
system ) monoclinic, a ) 20.93(2) Å, b ) 17.304(8) Å, c ) 6.992-
(3) Å, R ) γ ) 90°, â ) 90.20(6)°, V ) 2532(3) Å3, T ) 100(2) K,
space group ) P21/c, Z ) 4, λ(Mo K) ) 0.710 73 Å, 25 582 reflections
collected, 4012 independent reflections, Rint ) 0.1341, R1 [I > 2(I)]
) 0.0886, wR2 (all data) ) 0.2299, GOF ) 1.122.
Inorganic Chemistry, Vol. 46, No. 22, 2007 9049

COMMUNICATION
Table 1. Inhibition of Cancer Cell Growth (IC50) for Test Compounds
and rHSA Conjugates after 72 h
derivative did not significantly affect the cell growth within
the concentration range tested, a positive response was
observed in the A2780 cell line exposed to rHSA conjugated
with RAPTA. The IC50 value is 20-fold lower than that of
3, even if one considers that four RAPTA moieties are
present (Table 1). It is worth noting that RAPTA-C, the
prototype compound with a p-cymene ring, is also nontoxic
toward A2780 cells with IC50 > 300 µM, further indicating
the remarkable effect provided by the conjugation of the
ruthenium(II)-arene unit to rHSA.
In comparison, paclitaxel drug moieties conjugated to HSA
via ester bonds (6 or 30 molecules per HSA) were less active
than the drug in free form.5 This was attributed to the labile
ester bonds, which may be hydrolyzed before cellular uptake.
Such a hypothesis is supported by the observation that while
the HSA-chlorambucil conjugate with ester bond linkers
was not active against cancer cells, a similar derivative with
acetaldehyde carboxylic hydrazone bond linkers was active.
As mentioned above, such hydrazone bonds have been shown
to cleave under acidic conditions, thus providing a means
for the targeted release of the drug in the acidic environment
of lysosomes within the tumor cell. This phenomenon could
explain the improved efficacy in the rHSA-RAPTA con-
jugate reported herein. Because HSA in known to be taken
up by cells via endocytosis, the facilitated uptake of the drug
conjugate coupled with the controlled release of the RAPTA
drug moiety could correspond to the key reason for the
improvement in drug efficacy. Clearly, these potential
mechanisms would need to be investigated further in order
to develop other organometallic drug-protein conjugates
with optimized activities against cancer cells.
A2780 ovarian carcinoma
rHSA
>75 µMa
>75 µMa
11 µM
rHSA-hydrazine
rHSA-RAPTA
RAPTA-FORM 3
RAPTA-C
288 µM
>300 µM
a Maximum concentration possible.
of 1 was also confirmed by X-ray crystallography,11 and the
bond parameters of the functional part of the molecule are
very similar to those of the coordinated system (see the
Supporting Information).
The functionalization of the rHSA protein was carried
using established protocols (see the Supporting Information
for full details). Briefly, the protein was modified with the
succinyl HCl terephthalic hydrazine linker, which reacts with
amine groups on the lysine residues of the protein. Because
excess modification of the hydrophobic linkers can result in
the precipitation of the protein, the optimal reaction condi-
tions were determined empirically to be within 5-10-fold
stoichiometric excess of the linker molecule. Upon modifica-
tion, the protein is purified and conjugated with 3 in
phosphate-buffered saline (pH 7.4). The conjugated protein
is further purified and analyzed using UV-vis absorption
spectroscopy and matrix-assisted laser desorption ionization
time-of-flight (MALDI-TOF) mass spectrometry. The pres-
ence of the conjugated hydrazone bond is indicated by an
increase in absorbance at 317 nm, which is absent in rHSA,
hydrazine-modified rHSA, and 3. The MALDI-TOF mass
spectra obtained indicated an increase of approximately 1900
Da, equivalent to the presence of three to four RAPTA
moieties. The mass spectra also showed that the parent rHSA
molecule was completely consumed during the conjugation
(see Figure 3). However, the broadening of the mass peak
suggests that conjugation was not completely homogeneous
and that the final protein solution probably contained a
mixture of rHSA species with different numbers of RAPTA
conjugates.
In conclusion, a strategy to tether an organometallic
ruthenium(II)-arene anticancer compound to rHSA was
developed with a view to develop a drug that could
selectively accumulate in tumor cells. Preliminary results
indicate that the strategy is viable and that rHSA could be
exploited as a carrier biomolecule for drug delivery of
RAPTA complexes in vivo, with the prospect of adjusting
the loading capacity for tailored effects.
Using the A2780 ovarian carcinoma cell line, rHSA, rHSA
modified with the hydrazine linker, and rHSA conjugated
with RAPTA were tested for their ability to inhibit cancer
cell growth in vitro. The protein concentration was deter-
mined using the Bradford assay, and the cells were tested to
a maximum protein concentration of 5 mg/mL, equivalent
to 75 µM. Whereas rHSA and the hydrazine-modified
Acknowledgment. The authors thank Rosario Scopelliti
(EPFL) and Euro Scolari (EPFL) for assistance in the X-ray
crystallographic determination and Marc Moinette (EPFL)
for carrying out the MALDI measurements. Financial support
from the EPFL and the Swiss Cancer League is also
gratefully acknowledged.
Supporting Information Available: Crystallographic data of
1 and 3 in CIF format, synthesis of 3, and protocol for the inhibition
of cell growth experiments. This material is available free of charge
via the Internet at http://pubs.acs.org.
(11) Crystal data for 1: C16H17NO3, Mw ) 271.31, crystal system )
monoclinic, a ) 5.2366(5) Å, b ) 12.6144(19) Å, c ) 21.364(2) Å,
R ) γ ) 90°, â ) 96.086(8)°, V ) 1403.3(3) Å3, T ) 100(2) K,
space group ) P21/c, Z ) 4, λ(Mo K) ) 0.710 73 Å, 25 814 reflections
collected, 3211 independent reflections, Rint ) 0.0591, R1 [I > 2(I)]
) 0.0511, wR2 (all data) ) 0.1018, GOF ) 1.128.
IC701474M
9050 Inorganic Chemistry, Vol. 46, No. 22, 2007