Insights into the mechanism of action and cellular targets of ruthenium complexes from NMR spectroscopy

Article abstract of DOI:10.2533/chimia.2012.775

NMR spectroscopy has proved extremely beneficial in the investigation of inorganic drugs from the time that cisplatin was first introduced into the clinic more than 30 years ago. Both 195Pt and 15N NMR were used in early studies and made a major contribution in the understanding of the molecular mechanism of action from model studies involving reactions with amino acids and nucleotides. Over the past decade, ruthenium drugs have proved to be a valuable alternative to platinum drugs, and NMR has also provided unique insights into their molecular mechanism of action including investigations of simple aquation reactions, protein binding and the kinetics and sequence selectivity of DNA binding interactions. In this article, emphasis is given to define the cellular targets and elucidate some of the mechanistic profiles of recent ruthenium-based organometallic compounds offering efficacy toward cancer cells, by various NMR techniques. Schweizerische Chemische Gesellschaft.

Full text of DOI:10.2533/chimia.2012.775

NMR SpectRoScopy iN SwitzeRlaNd
CHIMIA 2012, 66, No. 10 775
doi:10.2533/chimia.2012.775
Chimia 66 (2012) 775–780 © Schweizerische Chemische Gesellschaft
Insights into the Mechanism of Action and
Cellular Targets of Ruthenium Complexes
from NMR Spectroscopy
Federico Giannini, Lydia E. H. Paul, and Julien Furrer*
Abstract: NMR spectroscopy has proved extremely beneficial in the investigation of inorganic drugs from the
time that cisplatin was first introduced into the clinic more than 30 years ago. Both 195Pt and 15N NMR were used
in early studies and made a major contribution in the understanding of the molecular mechanism of action from
model studies involving reactions with amino acids and nucleotides. Over the past decade, ruthenium drugs
have proved to be a valuable alternative to platinum drugs, and NMR has also provided unique insights into
their molecular mechanism of action including investigations of simple aquation reactions, protein binding and
the kinetics and sequence selectivity of DNA binding interactions. In this article, emphasis is given to define
the cellular targets and elucidate some of the mechanistic profiles of recent ruthenium-based organometallic
compounds offering efficacy toward cancer cells, by various NMR techniques.
Keywords: Cellular targets · Cytotoxicity · Glutathione oxidation · NMR spectroscopy · Ruthenium complexes
compounds, including the study of the in- trials.[14–16] These two compounds possess
teractions of various platinum complexes similar structural features but show very
Introduction
with nucleotides, nucleic acids, carbohy- different activity towards cancerous tissue.
drates, proteins and cells, in order to elu- NAMI-A is most effective against lung
cidate their molecular mechanisms and metastases while KP1019 is used to treat
identify key biomolecular targets for ratio- colon carcinomas. Other ruthenium com-
nal drug design.[3–6] From this tremendous plexes are also very promising, for instance
Nowadays, anticancer platinum com-
plexes (Fig. 1) are still the unique metallo-
drugs used in cancer chemotherapy, main-
ly because of the massive research effort
that followed the opportune discovery of
the anticancer properties of cisplatin in the
late 1960s.[1] One of the most impressive
examples of the cisplatin impact is the case
of testicular cancer. Prior to the introduc-
tion of cisplatin in the clinic about 90% of
patients with testicular cancer died of the
disease, and following its application this
percentage was reversed with 90% of cases
cured.[2] In spite of numerous drawbacks,
such as severe toxicity (nephrotoxicity),
drug resistance, or poor oral bioavailabil-
ity, the application of cisplatin has revo-
lutionized anticancer clinical practices to
such an extent that it is still used nowadays,
albeit mostly in combination with other
amount of research, two compounds, car- the RAPTA family,[17]
ONCO4417,[18,19]
boplatin and oxaliplatin, are currently used and DW 1 & 2[20,21] (Fig. 2).
worldwide, and have demonstrated milder
Interestingly, NAMI-A, KP1019 and
side effects compared to the parent drug.[7] the RAPTA complexes only induce weak
There are other platinum drugs, such as
in vitro cytotoxicity, show only modest and
Nedaplatin, Lobaplatin, and Heptaplatin, reversible DNA adduct formation, but have
used in cancer chemotherapy, but they promising clinical applications leading to
are currently restricted to certain areas or apoptosis activation rather than primary
countries.[7]
tumor inhibition.[4,22] Nevertheless, after
Ruthenium complexes have been more than 15 years the exact target(s) of
known for a long time to possess anti- KP1019 and of NAMI-A remain uniden-
cancer properties,[8,9] but it was shown tified, which points out the difficulties to
only recently that these compounds have study this aspect. The lack of informa-
several advantages compared to the plati- tion on the target(s) undoubtedly hampers
num drugs.[10–12] Ruthenium complexes the possibility to explore new analogues
are generally more selective towards can- of KP1019 or of NAMI-A with superior
organic drugs. The limitations of cisplatin
cer cells and therefore show fewer side pharmacological profiles. The more recent
have led to a massive investigation into the
cytotoxicity and genotoxicity of platinum
effects than cisplatin.[13] Nowadays two RAPTA compounds have a markedly dif-
inorganic ruthenium-based drugs have ferent general chemistry to NAMI-A.[23]
already completed phase I clinical tri- It was recently found that RAPTA com-
als, namely NAMI-A[14] and KP1019[15] pounds have a strong preference for pro-
(Fig. 2). NAMI-A has already started and tein binding over DNA, as shown from the
KP1019 will soon start phase II clinical ca. 200 kDa crystal structure of the nucleo-
Fig. 1. The current
three platinum-based
anticancer drugs
which have gained
marketing approval
for human use world-
wide.
*Correspondence: Dr. J. Furrer
Departement für Chemie und Biochemie der
Universität Bern
Freiestrasse 3
CH-3012 Bern
Tel.: +41 31 631 43 83
Fax: +41 31 631 50 87
E-mail: julien.furrer@dcb.unibe.ch

776 CHIMIA 2012, 66, No. 10
NMR SpectRoScopy iN SwitzeRlaNd
Fig. 2. Chemical
Highly Cytotoxic Trithiophenolato
Diruthenium Complexes
structures of some
prototype anticancer
arene ruthenium com-
plexes RAPTA-C (A),
ONCO4417 (X = Cl–)
In a very interesting perspective review,
Gianferra et al. have suggested a categori-
zation of metal anticancer compounds into
five classes based on their mode of action:
i) functional compounds, like cisplatin,
NAMI-A or KP1019, in which the metal
has a functional role, i.e. it binds to the bio-
logical target; ii) structural compounds, in
–
-
or RM175 (X = PF6 )
X
(B), and DW1, the R
enantiomer of the DW
1/2 racemic mixture,
mimicking the protein
kinase inhibitor stau-
rosporine. (C) The two
compounds NAMI-A
and KP1019 that
(A)
(B)
(C)
which the metal only has a structural role,
i.e. it determines the global shape of the
compound. The binding to the biological
target occurs only through non-covalent
interactions; iii) carrier compounds, in
have entered clinical
trials are also shown.
which the metal acts as a carrier for active
ligands that are expected to be delivered
in vivo; iv) catalytic compounds, in which
the metal compound acts as a catalyst; and
v) photoactive compounds, in which the
metal compound is photoactive and be-
haves as a photo-sensitizer (porphyrins,
Scheme 1. General
synthesis of
PDT therapy).[48]
Recently, we have synthesized and
compounds
6
[(η -p-MeC6H4Pri)2Ru2
characterized
ruthenium
complexes
6
(SC6H4-p-X)3]Cl
with the general formula [(η -
pCH3C6H4Pri)2Ru2(SC6H4-p-X)3]+ (Scheme
1).[49,50] The stability and reactivity of
these complexes towards biomolecules
were assessed by recording various NMR
spectra.[51] Surprisingly, only Cys and GSH
(native glutathione) induced changes in the
1H and 13C NMR spectra compared to the
Fig. 3. 1H NMR spec-
tra of glutathione
(GSH) (A),
free components. Fig. 3 shows the 1H NMR
spectra obtained upon titration of GSH into
a solution of the complex 1 with X = CH3
6
[(η -p-MeC6H4Pri)2Ru2
(SC6H4-p-CH3)3]Cl (B),
in D2O with 50 mM NaCl. As can be seen
and of the mixture
in the 1H NMR spectrum of free GSH, the
6
[(η -p-MeC6H4Pri)2Ru2
addition of 1 equivalent of GSH resulted in
(SC6H4-p-CH3)3]Cl:
GSH (ratio 1:1), (C)
recorded at 37°C in
D2O/ DMSO-d6 (95:5)
after 2 hours incuba-
tion. The resonances
of cysteine (GSH) are
indicated by * and the
resonances of cystine
(GSSG) by +.
the appearance of new Cys resonances at δ
4.78 (Hα), 3.33 (Hβ’), and 3.04 ppm (Hβ),
which increased over time relative to the
signals of free GSH and were assigned as
arising from oxidized glutathione (GSSG).
Likewise, addition of 1 equivalent of Cys
resulted in the appearance of new Cys
resonances at δ 4.17 (Hα), 3.46 (Hβ'), and
3.27 ppm (Hβ). These new 1H signals also
increased over time, relative to the signals
of free Cys, and, in agreement with the
literature, were assigned as arising from
cystine, the oxidized form of cysteine.[52]
ecules using NMR spectroscopy have
some core particle in which RAPTA-C
been published in the last few years.[29–32]
However, the binding properties of ruthe-
nium complexes with biomolecules using
NMR studies seem to be a relatively un-
explored domain, as revealed by the few
publications on this subject.
binds only to the protein histone core.[24]
Furthermore, the mode of action, up-
take and the biological processes in these
systemsremainpoorlyoronlypartlyunder-
stood.[25] NMR spectroscopy is a candidate
of choice to answer these questions, since
one- and two-dimensional NMR spectros-
copy of metal complexes have proven to
be extremely powerful in elucidating inter-
actions between complexes with potential
anticancer activity and biomolecules.[26–28]
Indeed, very interesting reports and
promising results revealing interactions
between metal complexes and biomol-
Interestingly, these titration experi-
ments with Cys and GSH revealed that
only the 1H and 13C chemical shifts of the
α-CH and β-CH2 groups of Cys were af-
fected, whereas the chemical shifts of the
complex and the other amino acids of GSH
(γ-Glu and Gly) remained unperturbed
In recent years, we as well as others
have developed a series of ruthenium-
based organometallic compounds offering
(Fig. 3). These results strongly suggest
that Cys and GSH do not form stable ad-
efficacy toward cancer cells.[33–47] The aim
ducts with the complexes, which was fur-
of our research is to define cellular targets
of the compounds and elucidate some of
their mechanistic profiles by various NMR
techniques.
ther evidenced by DOSY spectra (Fig. 4).
From these NMR experiments, it became
apparent that these complexes could act
as a very efficient catalyst for oxidation

NMR SpectRoScopy iN SwitzeRlaNd
CHIMIA 2012, 66, No. 10 777
of Cys to cystine and of GSH to GSSG
(Scheme 2).
In the light of our studies on these di-
nuclear arene ruthenium trithiolato com-
plexes, it appears that they belong to class
(iv), catalytic compounds.Indeed, we have
shown by NMR that they remain intact for
long periods, and are inert upon the addi-
tion of glucose, all natural amino acids and
(A)
(B)
nucleotides. This category encompasses a
recently discovered mode of action found
for areneruthenium iodoazopyridine com-
plexes, which are surprisingly cytotoxic
Fig. 4. DOSY spectra of the mixture 1:GSH (ratio 1:1) recorded in D2O at 37°C after 5 min (A), and
after 2 h at 100% conversion (B). The diffusion coefficient of 1 is indicated with a straight line and
despite their inertness to ligand substitu-
tion: In a pioneering study, Sadler and co-
workers demonstrated these complexes
to act as catalysts for oxidation of the tri-
peptide glutathione, supposed to be at the
origin of their anticancer activity.[53] Our
results however suggest that the oxidation
of GSH to form GSSG is not the unique
mechanism of action of these compounds,
since we could not correlate the rate of
oxidation with the cytotoxicity.[54] Indeed,
there is no clear correlation between the
the diffusion coefficients of GSH (A) and GSSG (B) are indicated with dashed lines, respectively.
Scheme 2. Oxidation
of cysteine (Cys) and
glutathione (GSH)
to give the disulfide
cystine and GSSG,
1
+H
2
DO, 37°C
2
1
respectively.
+H
2
D O, 37°C
2
IC50 and the TOF50
values. However, the
cytotoxicity can be to some extent corre-
lated to physicochemical properties of the
compounds determined by the electronic
influence of the substituents X (Hammett
constants σp) and the lipophilicity of the
thiols p-XC6H4SH (calculated logP pa-
rameters). In Fig. 5, a three-dimensional
graphical representation of the correlation
between IC50 values against A2780 cancer
cells, the Hammett constants σp, and the li-
pophilicity parameters LogP for the eleven
complexes is displayed. Interestingly, the
optimization problem admits a unique so-
lution described by a second order poly-
Fig. 5. Three-
dimensional repre-
sentation of the cor-
relation between IC50
values against the
A2780 cell line, the
Hammett constants
σp and the lipophilic-
ity parameters LogP
for the eleven ruthe-
nium complexes.
Regressions show a
non-linear determin-
ation coefficient
R2 = 0.92.
σp
LogP
nomial function. The regression shows a
good non-linear determination coefficient
R2 = 0.92. In this representation, the opti-
Fig. 6. Hexanuclear
mal region encompassing the most favor-
metallaprism [1]
[CF3SO3]6 and [2]
[CF3SO3]6
able values for both the Hammett constants
σ (–0.2 < σp < 0) and LogP values (LogP
>p3.0) leading to the lowest IC50 values is
apparent.
Therefore, we expect that these com-
pounds also belong to class (ii), namely
the class of structural compounds, i.e. the
global shape of the complexes shall be
partly responsible for the binding to the
biological targets through non-covalent in-
teractions. Current studies for determining
whether these complexes bind to DNA and assemblies of various sizes and function- stable, with the physical properties of the
proteins are under investigation, as a first alities gained a lot of attention.[56,57] The
instance using NMR spectroscopy.
prism being retained following encapsu-
literature shows many different approaches lation. Interestingly, the activity of [2]6+
towards combining transition metal ions (IC50 = 23 μM) against A2780 cancer cell
with different polydentate ligands to pro- lines of the metallaprism significantly in-
duce large metalla assemblies. Our group creases with the encapsulation of Pt(acac)2
and colleagues in Neuchâtel have designed or Pd(acac)2 (IC50 = 12 and 1 μM, respec-
hexacationic areneruthenium metalla- tively) suggesting transport and leaching
prisms (Fig. 6) able to encapsulate revers- of the guest once inside the cell.[58]
Interaction of Ruthenium
Hexacationic Prisms with
Biological Ligands
Since the discovery of the Enhanced ibly inside the cavity planar metal com-
Permeability and Retention effect in plexes such as Pt(acac)2 or Pd(acac)2.[58] titumor therapy (cisplatin, carboplatin, ox-
1986,[55] the design of supramolecular The encapsulated metal complexes remain
aliplatin) are administered intravenously
The metallodrugs currently used in an-

778 CHIMIA 2012, 66, No. 10
NMR SpectRoScopy iN SwitzeRlaNd
Fig. 7. Suggested
and can therefore encounter various reac-
(p-cymene)Ru-(AA)
tive biomolecules in the bloodstream. The
chelate complexes
obtained upon the
addition of Arg (A),
Lys (B), and His (C)
to [1]6+.
concentration of such biomolecules (ascor-
bic acid, glucose, lactic acid) is such that
they should be considered as primary po-
tential binding partners for metallodrugs.
Some proteins like albumin or transferrin
play an important in deactivating them
even before reaching these targets.[59–61]
In recent studies, we have investi-
gated the behavior of two hexacationic
areneruthenium metallaprisms [Ru6(p-
cymene)6(dhnq)3(tpt)2]6+ ([1]6+)and[Ru (p-
cymene)6(dhbq)3(tpt)2]6+ ([2]6+) aga6inst
Fig. 8. 1H NMR spec-
tra of Arg (A), [1]6+ (B),
and of the mixture
[1]6+:Arg (ratio 1:10),
different biological ligands.[62,63] These in-
recorded after 24 h
vestigations have been undertaken to clari-
fy the much better cytotoxicity of the prism
[1]6+ (IC50 = 2 μM against A2780 cancer
cells) compared to the prism [2]6+, (IC50 =
23 μM against A2780 cancer cells) and to
verify their relative reactivity and stability
upon the addition of biological ligands.
Our NMR investigations showed that
these prisms behave rather differently
than other ruthenium based anticancer
complexes. For instance, they showed no
interactions with amino acids that are
known to form stable complexes with Ru
such as Met[64,65] but strongly reacted with
the basic amino acids His, Lys, and Arg.
Reactions of ruthenium anticancer com-
plexes with Lys and Arg have been hardly
reported. More precisely, our NMR inves-
tigations have revealed that coordination to
the imidazole nitrogen atom in His or to
the basic NH/NH2 groups in Arg and Lys
displaces the dhbq and tpt ligands from
the (p-cymene)Ru units, and subsequent
coordination to the amino and carboxylato
groups forms stable N,N,O metallacycles
(Fig. 7). Interestingly, as determined by
NMR spectroscopy, we have found that the
binding to amino acids proceeds rather rap-
idly with [2]6+,[62] (Fig. 8) or quite slowly
with [1]6+[63] (Fig. 9). In addition, for His,
the NMR spectra showed that only about
17% of His reacts with [1]6+, which is con-
firmed by the presence of the resonances
of both the tpt and dhnq moieties in the
NMR spectra, which are otherwise absent
(C). All spectra were
recorded in D2O at
37 °C.
[2]6+ + Arg
[2]6+ + His
[2]6+ + Lys
24 h
12 h
5 min
Fig. 9. 1H NMR spectra of the mixture [2]6+:Arg, [2]6+:His, and [2]6+:Lys (ratio 1:10) recorded as a
function of the time. Only after 12 h, the occurrence of additional resonances near the original Hα
resonances can be clearly observed.
Fig. 10. Excerpt of
the 2D 1H-13C HSQC
NMR spectrum of the
mixture [1]6+:ascor-
Ascorbic acid (AA)
bic acid (ratio 1:6)
recorded after 24 h
(red). For comparison,
excerpts of the 1H-13C
HSQC NMR spectra
of [1]6+ (black) and
when the reaction is complete.[62] Taking
free ascorbic acid
(blue) were added.
into account the much longer reaction time
and the incomplete reaction of His, [1]6+
appears much less reactive than the small-
er metallaprism [2]6+ against amino acids
with basic side chain residues. The higher
Dehydroascorbic acid (DAA)
cytotoxicity of [1]6+ compared to [2]6+ may
appear surprising at first glance, since [1]6+
is apparently more chemically inert com-
pared to [2]6+. However, a recent study by
Hartinger et al. suggests an inverse correla-
tion between metallodrug–protein interac-
tion and cytotoxicity against tumor cells.[66]
Similarly, ourrecenttrithiolatoareneruthe-
nium complexes are highly cytotoxic de-
spite their inertness towards biomolecules
such as amino acids and nucleotides.[51,54]
which is supposed to be at least partly at
the origin of the observed cytotoxicity
of ruthenium complexes, has been rarely
reported so far.[51,53] Interestingly, as de-
termined by NMR spectroscopy, we have
Moreover, we have found that these
metallaprisms are also able to catalyze the
oxidation of ascorbic acid, Cys and glu-
tathione (GSH) to form dehydroascorbic
acid, cystine and GSSG, respectively[62,63]
found that the oxidation of GSH proceeds
(Fig. 10). This mode of action involving
rather slowly with [2]6+,[62] or fairly rap-
redox processes and oxidation of GSH,

NMR SpectRoScopy iN SwitzeRlaNd
CHIMIA 2012, 66, No. 10 779
Fig. 11. 2D 1H DOSY
NMR spectrum of the
solution containing
[1]6+ and GSH record-
ed in D2O at 37°C
fusion coefficients often allow to readily
identify whether a drug forms adducts or
not with its presumed target(s).
For macromolecules (e.g. DNA and
proteins) NMR offers the possibility of de-
termining the three-dimensional structure
of the macromolecule in solution and hence
of characterizing drug-induced changes in
macromolecule structure (which can in
turn trigger biological effects). It is clear,
however, that NMR does not always pro-
vide all the details of particular reactions,
particularly because of limited sensibility
and size limit.
after 24 h (black), as
well as superimposed
2D 1H DOSY NMR
spectra of [1]6+ (blue)
and free GSH (green)
for comparison. The
resonances of Cys
in GSSG, which are
indicated by arrows,
clearly indicate that
an adduct is formed
upon the addition of
GSSG to a water so-
lution of [1]6+.
Acknowledgments
We thank the Departement für Chemie und
Biochemie of the University Berne and the
Swiss National Foundation (Grant N° 200021-
131867) for financial support.
idly with [1]6+.[63] In addition, the reaction release of encapsulated guest molecules.
of GSH with metallaprism [1]6+ reveals a Oxidation of Cys and GSH to give the cor-
rather unexpected result, since the DOSY responding disulphides may explain the
spectrum of the mixture recorded after 24 higher in vitro anticancer activity of [1]6+
h (Fig. 11) indicates that the metallaprism compared to [2]6+.
[1]6+ loses both the tpt and the dhnq ligand
Received: July 17, 2012
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