A binuclear gold(I) complex with mixed bridging diphosphine and bis(N-heterocyclic carbene) ligands shows favorable thiol reactivity and inhibits tumor growth and angiogenesis in vivo

Article abstract of DOI:10.1002/anie.201400142

In the design of anticancer gold(I) complexes with high in vivo efficacy, tuning the thiol reactivity to achieve stability towards blood thiols yet maintaining the thiol reactivity to target cellular thioredoxin reductase (TrxR) is of pivotal importance. Herein we describe a dinuclear gold(I) complex (1-PF₆) utilizing a bridging bis(N-heterocyclic carbene) ligand to attain thiol stability and a diphosphine ligand to keep appropriate thiol reactivity. Complex 1-PF₆ displays a favorable stability that allows it to inhibit TrxR activity without being attacked by blood thiols. In vivo studies reveal that 1-PF₆ significantly inhibits tumor growth in mice bearing HeLa xenograft and mice bearing highly aggressive mouse B16-F10 melanoma. It inhibits angiogenesis in tumor models and inhibits sphere formation of cancer stem cells in vitro. Toxicology studies indicate that 1-PF ₆ does not show systemic anaphylaxis on guinea pigs and localized irritation on rabbits. It′s in the blood: A binuclear gold(I) complex 1-PF₆ is stable towards blood thiols and is a tight-binding inhibitor of thioredoxin reductase (TrxR). In vivo antitumor studies show 81-% inhibition of tumor growth in mice with HeLa xenografts and 62-% inhibition of highly aggressive mouse B16-F10 melanoma.

Full text of DOI:10.1002/anie.201400142

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Angewandte
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DOI: 10.1002/anie.201400142
Gold Medicine
A Binuclear Gold(I) Complex with Mixed Bridging Diphosphine and
Bis(N-Heterocyclic Carbene) Ligands Shows Favorable Thiol
Reactivity and Inhibits Tumor Growth and Angiogenesis In Vivo**
Taotao Zou, Ching Tung Lum, Chun-Nam Lok, Wai-Pong To, Kam-Hung Low, and
Chi-Ming Che*
Abstract: In the design of anticancer gold(I) complexes with
high in vivo efficacy, tuning the thiol reactivity to achieve
stability towards blood thiols yet maintaining the thiol
reactivity to target cellular thioredoxin reductase (TrxR) is of
pivotal importance. Herein we describe a dinuclear gold(I)
complex (1-PF6) utilizing a bridging bis(N-heterocyclic car-
bene) ligand to attain thiol stability and a diphosphine ligand to
keep appropriate thiol reactivity. Complex 1-PF6 displays
a favorable stability that allows it to inhibit TrxR activity
without being attacked by blood thiols. In vivo studies reveal
that 1-PF6 significantly inhibits tumor growth in mice bearing
HeLa xenograft and mice bearing highly aggressive mouse
B16-F10 melanoma. It inhibits angiogenesis in tumor models
and inhibits sphere formation of cancer stem cells in vitro.
Toxicology studies indicate that 1-PF6 does not show systemic
anaphylaxis on guinea pigs and localized irritation on rabbits.
serve to deliver bioactive metal ions to the cellular target(-
s).[2a,e–g] In fact, there are reports showing that metal
complexes containing NHC ligands display potent anticancer
properties and are potential drug candidates for drug resistant
cancers and have mechanisms of action different from that of
cisplatin.[2a,c–h]
Gold(I) complexes are anticancer active.[4] Mirabelli,
Sadler, and co-workers first reported the apparently thiol-
unreactive [AuI(dppe)2]+ (dppe = 1,2-bis(diphenylphospha-
nyl)ethane) to display efficacy towards several solid tumor
models,[5] but toxicity was observed in in vivo studies with
large animals.[6] Later studies revealed that the SH/SeH-
containing thioredoxin reductase (TrxR) is a potential cellular
target of the Au+ ion, and the he Au+ ion is able to block the
C-terminal -Cys-Sec- site of TrxR through covalent binding
with selenol and/or thiol.[4a,c–p] TrxR inhibition requires
a facile ligand-exchange reaction with thiols, but the high
thiol affinity of AuI may lead to covalent binding interactions
with blood thiols including albumin and blood glutathione
(GSH), resulting in limited bioavailability to tumor tis-
sues.[4k,n,7] Despite a large number of gold(I) complexes
(mostly the mononuclear ones) reported to display TrxR
inhibition as well as anti-proliferative properties, examples of
AuI complexes which display in vivo solid tumor inhibition
activity are rare.[8] Thus tuning the thiol reactivity to minimize
off-target binding in blood while maintaining enough reac-
tivity to inhibit cellular TrxR could be a guiding principle in
the design of new anticancer gold(I) complexes.
B
y virtue of being strong s donors to allow formation of
À
stable M C bond(s), stable N-heterocyclic carbenes (NHCs)
have a profound impact in diverse areas of chemistry.[1] Less
developed but with burgeoning interests in recent years is the
application of metal NHC complexes in anticancer treat-
ments.[1a–c,2] The side effects and/or lack of in vivo efficacy of
many reported anticancer metal complexes including the
classical platinum anticancer drugs can be related to their
instability under physiological conditions.[3] NHC ligand(s)
stabilize metal ions against precipitation into metal aggre-
gates under physiological conditions and at the same time can
AuI–AuI interactions can be used to stabilize AuI ions for
medicinal applications. The dinuclear gold(I) complex,
[(Ph3PAu)2(m-DTE)] (H2DTE = 1,4-dimercaptobutane-2,3-
diol), could increase the life span of mice bearing Ehrlich-
Ascites tumors despite its severe toxicity.[9] Several dinuclear
gold(I) complexes containing bridging bidentate NHC
ligands, [Au2(bis(NHC))2]2+, are not reactive towards thiols
and could be used as biological probes.[10] It was reported that
replacing one NHC of the mononuclear [AuI(NHC)2]+
complexes with PPh3 would lead to stronger TrxR inhib-
ition.[11] Herein is described a novel binuclear gold(I) complex
(1-PF6, Figure 1) containing mixed diphosphine and bis-
(NHC) bridging ligands. This complex shows favorable
stability in the presence of thiols at blood concentrations,
potently inhibits TrxR through a tight-binding mode, and
effectively inhibits tumor growth in two independent animal
models. This is the first gold(I) complex reported to inhibit
cancer stem cell activity and to inhibit in vivo angiogenesis in
a tumor model.
[*] T. Zou, Dr. C. T. Lum, Dr. C.-N. Lok, Dr. W.-P. To, Dr. K.-H. Low,
Prof. Dr. C.-M. Che
State Key Laboratory of Synthetic Chemistry
Institute of Functional Materials and Department of Chemistry
The University of Hong Kong
Pokfulam Road, Hong Kong (China)
E-mail: cmche@hku.hk
Prof. Dr. C.-M. Che
HKU Shenzhen Institute of Research and Innovation
Shenzhen 518053 (China)
[**] This work was supported by the Innovation and Technology Fund
(ITF-Tier 2, ITS/134/09FP) administered by the Innovation and
Technology Commission, HKSAR, the National Key Basic Research
Program of China (2013CB834802), and the University Grants
Committee (Area of Excellence Scheme AoE/P-03/08, HKSAR,
China). We thank Dr. Eva Yi-Man Fung and Dr. Kwan-Ming Ng for
help in MS analysis.
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/anie.201400142.
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auranofin (m/z 679.1) with 20% remaining after
45 min. An intermediate with m/z value consistent
with glutathione-gold-phosphine adduct GS-Au-PEt3
(m/z 622.1) appeared initially followed by gradual
decrease of its ion intensity (Figure S4). On the
contrary, no significant change of the ion intensity of
12+ (m/z 531.2) was found during the first 45 min of
incubation with GSH, indicating that 1-PF6 was not
reactive towards GSH at this concentration. When
a higher concentration of GSH (5 ꢁ 10À5 m) was used,
the ion intensity of auranofin decreased with a faster
rate while that of 12+ was retained at approximate the
initial level (Figure S5). As GSH (1–10 mm) and serum
albumin (35–50 mgmLÀ1) are the major thiol-contain-
ing compounds present in blood,[15] the binuclear
gold(I) complex 1-PF6 appears to be moderately
stable towards blood thiols.
Figure 1. Chemical structures of 1–5 and auranofin. Note that though termed
1-PF6, the complex is a dication with two PF6 counterions.
The interaction of 1-PF6 with GSH was further
studied by ESI-QTOF-MS analysis upon further
increasing the concentrations of 1-PF6 and GSH. When
1.5 ꢁ 10À4 m of 1-PF6 was incubated with excess GSH (5 ꢁ
10À4 m) for 1 h, small but distinct peaks at m/z 764.3 (z =+
1), 684.8 (z =+ 2), and 605.3 (z =+ 2) appeared, which are
attributed to [Au(NHC-Im)(GS)]+, [Au2(dcpm)(NHC-
Im)(GS)]2+ (Im = imidazolium), and [Au2(dcpm)2]2+, respec-
tively (Figure S6).
We examined the effect of serum albumin on the
cytotoxicity of the gold(I) complexes. After pre-incubation
of these gold(I) complexes in minimal essential medium
(MEM) with/without BSA (40 mgmLÀ1) for 1 h, and then
adding the resulting media into HeLa cells for further 24 h
incubation, the cell viability was determined by MTT assays.
As shown in Table 1, BSA can significantly suppress the
The binuclear gold(I) diphosphine/bis(NHC) complex (1)
was synthesized by the reaction of [Au2bis(NHC)Cl2] and
dcpm [dcpm = bis(dicyclohexylphosphine)methane] to give
the dicationic species termed 1-Cl, ion exchange with NH4PF6
or LiOTf gave 1-PF6 and 1-OTf (see the Supporting Informa-
tion). Complexes 2–5 (Figure 1) and auranofin were pre-
pared/used for comparative study. The crystal structure of 1-
PF6 (Figure S1 and Table S1 in the Supporting Information)[12]
shows that the Au···Au distance is 3.083 ꢀ, being slightly
longer than that of 5 (2.926–3.013 ꢀ)[13] but 0.46 ꢀ shorter
than that of [Au2(NHC-C1-NHC)2]2+ (3.5425 ꢀ).[14]
The stability of 1–5 and auranofin towards bovine serum
albumin (BSA) was examined by inductively coupled plasma
mass spectrometry (ICP-MS) analysis of the content of
unbound (non-covalent) gold in the supernatant obtained
from acetone precipitation of the albumin. For 4, more than
90% of gold was left unbound after incubation of the complex
with BSA for 5 h. For 2, 3, 5, or auranofin, less than 30% of
unbound gold was observed in each case. In the case of 1-PF6,
more than 70% free-gold content was observed for the same
Table 1: Cytotoxic IC50 (mm) of the gold(I) complexes incubated in BSA-
free and BSA-containing media for 24 h as determined by MTT assays.
1-PF6
2
3
4
5
Auranofin
BSA-free 13.5
3.1
16.5
0.187 0.198
1.5
7.34
46.5
>300
<0.09
0.8
17.9
0.042
1.1
49.3
0.023
incubation condition (Figure S2).
BSA
46.1
0.293
Electrospray ionization quadrupole time-of-flight mass
spectrometry (ESI-QTOF-MS) was used to investigate the
reaction of 1-PF6 and GSH. Mixtures of 3 ꢁ 10À6 m of 1-PF6 or
auranofin (both had a linear response of MS intensity versus
concentrations, Figure S3) with 5-fold excess of GSH (1.5 ꢁ
10À5 m) at pH 8.0 were freshly prepared and the ion intensity
of the gold complex was recorded (Figure 2). After mixing
with GSH, there was a rapid decrease in the ion intensity of
ratio[a]
[a] Ratios were calculated by the IC50 of BSA-free divided by the IC50 of the
BSA-containing condition. All the IC50 values were determined from at
least three independent experiments.
cytotoxicity of the gold(I) complexes. The cytotoxicity of 1-
PF6 was less affected compared to the other more thiol-
reactive 2, 3, 5 and auranofin. The relatively thiol-unreactive
complex 4 is less cytotoxic in both BSA-containing and BSA-
free media. Although 4 is inert towards thiols, serum albumin
has a big impact on its cytotoxicity; the presence of BSA was
observed to render 4 to be almost non-toxic (IC50 > 300 mm).
Complex 1-PF6 is also cytotoxic towards breast adenocar-
cinoma (MCF-7), nasopharyngeal carcinoma (SUNE1), lung
adenocarcinoma (H1975), and mouse melanoma (B16-F10)
with IC50 values ranging from 1.3 Æ 0.2 to 3.2 Æ 1.0 mm; these
IC50 values are up to 13.5-fold greater than those of cisplatin
(Table S2). Complexes 1-Cl and 1-OTf showed cytotoxicity
similar to that of 1-PF6 in in vitro cytotoxicity studies.
&
Figure 2. The residual gold complex after mixing 1-PF6 ( ) or aurano-
~
fin ( ) and GSH as determined by ESI-QTOF-MS. The amount of
residual gold(I) complex was determined as the percentage of initial
ion intensity recorded before GSH addition.
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The inhibitory effect of 1-PF6 on purified TrxR was
studied. Treatment of recombinant rat TrxR1 (2 nm) with
200 nm of 1-PF6 in the presence of NADPH resulted in a time-
dependent reduction of TrxR activity by over 90% in 2 h
(Figure S7). In addition, complex 1-PF6 inhibited TrxR more
potently than the structurally related glutathione reductase
(GR), with the IC50 values being 38 nm and 10 mm respec-
tively, using DTNB as the substrate (Figure S8,9). The TrxR
inhibition mechanism was studied by progressive curve
analysis[8d,16] of the time course of TrxR inhibition. The
progress curves are non-linear (Figure 3a), revealing a two-
phase equilibrium that is typical of slow-onset tight-binding
mode of inhibition. Plotting the observed first-order rate
constants, kapp, against the concentration of 1-PF6 gave
a hyperbolic function curve (Figure 3b), indicating a two-
step binding mechanism, where the initial collision complex
(EI) is rapidly formed followed by a subsequent isomerization
that enhances the tightness of enzyme–inhibitor complex
(EI*, Figure 3b inset). Fitting the time course data gave the
values of k3 = 0.0260 sÀ1, k4 = 0.0020 sÀ1, Ki = 413 nm, Ki* =
28.5 nm. Meanwhile, in the presence of 2 mm GSH, the
inhibitory activities on NADPH-TrxR-Trx-insulin system by
1-PF6 were not attenuated but rather moderately enhanced
with IC50 decreased from 5.56 mm to 3.29 mm, but the
inhibition by auranofin under the same conditions decreased
(Figure S10).
Figure 4. a) Proposed binding interaction of 1-PF6 with TrxR. Inset:
binding mode based on MS results. b) 1H NMR spectra (400 MHz) of
GCUG (top), 1-PF6 (upper middle), equal molar of 1-PF6 with GCUG
(lower middle) and [bis(Im)]2+ ligand (bottom) in D2O/[D6]DMSO=
1:1 (v/v, phosphate buffer, pH*=7.4) after 24 h incubation.
GCUG motif.[17a] In the present case, 1-PF6 is bound to
GCUG motif in 1:1 molar ratio through simultaneous
coordination of the binuclear AuI unit to respective S and
Se of the contiguous Cys and Sec residues, accompanied
by the release of bis(NHC) but not dcpm ligand.
Cancer stem cells (CSCs) having the ability of self-
renewal and differentiation have been proposed to cause
relapse and metastasis by giving rise to new tumors.[18] The
ability to form spheres in non-adherent cultures is
a characteristic of CSCs. Herein we employed sphere-
formation assay to examine the effect of 1-PF6 on the
population of HeLa cells. As shown in Figure 5, free-
floating spheres were observed in the solvent-control
group, and treatment of HeLa cells with 5 or 10 mm of 1-
Figure 3. a) Time dependence to the onset of TrxR inhibition by 1-PF6.
b) Plot of kapp (determined by fitting the data in Figure 3a) against
concentrations of 1-PF6 suggests a two-step binding mechanism. Inset:
two-step inhibition mechanism.
Several peptides have been used in the study of
the binding mode of gold(I) with the active site of
TrxR.[4j,n,17] In this work, the C-terminal GCUG
motif (Gly-Cys-Sec-Gly) was used as the model
(Figure 4). Incubation of 0.5 mm of 1-PF6 with
GCUG at the same concentration in 5 mm
NH4HCO3 with 50% MeOH (v/v) for 1 h, 4 h,
and 24 h resulted in all cases formation of a new
species with m/z 1187.2500 (Figure S11a). Both the
m/z value and isotope pattern of this new species
match the formulation of tetrapeptide with
Figure 5. Inhibition of sphere formation of HeLa cells by 1-PF6. Left: representative
pictures from three independent experiments. Right: relative area (s) occupied by
spheres per microscopic field. Data are shown as mean from three independent
experiments. **** p<0.0001 compared to solvent control.
a bound [Au2dcpm]2+ having calculated m/z 1187.2488 (Fig-
ure S11b, error: 1.0 ppm). Meanwhile, the 1H NMR signals at
6.0–8.0 ppm are indicative of the release of bis(NHC) ligand
PF6 resulted in statistically significant inhibition (p < 0.0001)
of the self-renewal (sphere-forming) ability. Similar results
were found in U-87 MG human glioblastoma cells (Fig-
ure S12). These data revealed that 1-PF6 could inhibit the self-
renewal ability of CSCs.
1
(Figure 4b) and the H signals of b-CH2 of Cys and Sec are
also significantly shifted, further indicating coordination of S
and Se atoms to Au atom/ion. Notably, two [AuPEt3]+ units of
auranofin were reported to bind to both SeH and SH of the
As complexes 1–5 are insoluble in water, biocompatible
PET (60% polyethylene glycol 400; 30% ethanol; 10%
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Tween-80) was used as excipient for in vivo study. The
complexes were dissolved in PET which was diluted with
PBS (final PET: 1.5–4%, v/v). Complexes 1-Cl and 1-OTf
could be dissolved in the buffer to give a clear solution.
Complex 1-PF6 gave a finely dispersed suspension that
remained stable for several hours. Light scattering experi-
ment indicated the formation of nanoparticles (diameter size
of 175 Æ 81 nm). TEM analysis revealed that the particles
formed from 1-PF6 were well dispersed with diameters of
144 Æ 50 nm (Figure S13). Complexes 2, 3, and 5 were
observed to form similar suspensions.
The in vivo antitumor effect of the gold(I) complexes was
examined. After treatment of nude mice bearing HeLa
xenografts with 5 mgkgÀ1 of 1-PF6 through intraperitoneal
(i.p.) injection once every three days, significant tumor
volume inhibition by 79% (p < 0.05) and 81% (p < 0.05)
was found compared to those treated with solvent control
after 6 and 9 days of treatment, respectively (Figure 6). In
addition, no mouse death or body weight loss was observed
achieve in vivo antitumor efficacy, as the highly thiol-reactive
complexes 2, 3, and 5, all of which could also form nano-
particles, did not exhibit inhibition of HeLa xenografts
(Figure S17).
In another treatment, a rather low dosage, 0.6 mgkgÀ1, of
1-PF6 was used to treat mice bearing a HeLa xenograft (i.p.
injection); statistically significant tumor growth inhibition
was obtained after 6 days of treatment, with final inhibition of
59.3% after 14 days of treatment (Figure S18). Collectively,
the therapeutic window of 1-PF6 could be quite large since
0.6 mgkgÀ1 to 15 mgkgÀ1 all resulted in significant antitumor
effects.
The in vivo anti-angiogenic effect of gold(I) complexes
has been reported by Ott and co-workers using zebrafish
model.[4n,20] In our study, the anti-angiogenic effect of 1-PF6
was examined by immune-histochemical detection of the
CD31 (cluster of differentiation 31) of the blood microvessels
in the tumor tissue of mice bearing HeLa xenografts
(5 mgkgÀ1). The average number of microvessels per micro-
scopic field of 1-PF6-treated tumor was 2.35 (Figure 7),
while that treated with solvent control was 4.34 (p < 0.05),
indicating that 1-PF6 could significantly inhibit angiogen-
esis of HeLa xenografts (Figure S19).
To examine the side effects, we have conducted
toxicology studies of 1-PF6 in a State Food and Drug
Administration, P. R. China (SFDA)-approved laboratory
(Tianjin Institute of Pharmaceutical Research). All system
anaphylaxis reaction was tested in guinea pigs. The animals
were treated with 0.9% saline injection (negative control),
10% PET solvent, 1% ovalbumin in saline (positive
control) and 0.12 mgkgÀ1 of 1-PF6 in PET solvent, respec-
tively, through i.p. injection every other day for a total of
Figure 6. Antitumor effect of 1-PF6 on mice bearing HeLa xenografts.
a) Changes of tumor volume (V) after treatment with 1-PF6,* p<0.05.
b) Representative mouse photos after 9 days of treatment.
after the treatment with 1-PF6 at this dosage (Figure S14). We
further tested the inhibition effects towards the highly
aggressive, poorly immunogenic mouse melanoma (B16-
F10) model in C57BL/6N mice. As shown in Figure S15a,
while the tumor in solvent control group grew at least three
times faster than HeLa xenograft, significant tumor inhibition
(62%, p < 0.05) could be achieved after administration (i.p.)
with 15 mgkgÀ1 of 1-PF6 once every 2–3 days for 8 days. The
treatment also did not cause mouse death or body weight loss
(Figure S15b).
Figure 7. a) Immunohistochemical detection of CD31 in the tumor
tissues. Arrows indicate CD31 microvessels. b) Average number (N) of
microvessels per microscopic field. * p<0.05, compared to solvent
control.
Complexes 1-Cl and 1-OTf could also inhibit tumor
growth of mice bearing HeLa xenografts at a higher treatment
frequency. After administration of 1-Cl or 1-OTf at dosage of
5 mgkgÀ1 every day, statistically significant difference could
be obtained compared to solvent control, with final tumor
growth inhibition of 47.7% (p < 0.01) and 35.1% (p < 0.05),
respectively (Figure S16). Again, no mouse death or mouse
body weight loss was found. These findings revealed that both
the gold complex cation and counteranion can significantly
affect the in vivo antitumor effect. Presumably the anion
affects the solubility and hence pharmacokinetics of complex
1 salt in the blood serum. A recent report showed that the
formation of nanoparticles through the incorporation of
appropriate anions could increase the anticancer proper-
ties.[19] Formation of nanoparticles could be beneficial in the
present case. This factor, however, is not sufficient alone to
5 times; after 14 days, forelimbs of guinea pigs were injected
intravenously (i.v.) with 2-fold dosage. All guinea pigs in the
positive control group exhibited severe allergic reaction
(eventually died), but 1-PF6 treatment group did not show
allergic reaction. The blood-vessel irritation test was also
performed. Complex 1-PF6 (0.12 mg) was directly injected
into the auricular veins of the rabbits, and the histopathology
was examined after 4 days and 14 days of treatment. No
apparent ear blood vessel dilatation, bleeding, swelling, or
other morphological changes were found at the administra-
tion sites upon treatment with 1-PF6. (See details in Support-
ing Information). These results revealed that 1-PF6 did not
show systemic anaphylaxis and localized irritation.
In summary, the binuclear gold(I) complex 1-PF6 shows
favorable thiol reactivity and meanwhile is a tight-binding
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inhibitor of TrxR. This complex significantly inhibited tumor
growth of HeLa xenografts and highly aggressive mouse B16-
F10 melanoma with no observable side effects under in vivo
conditions. This finding sheds lights on the clinical prospects
of developing gold(I)-NHC complexes into useful anticancer
drugs. Future works will focus on investigating the binding
interactions of this dinuclear gold(I) complex with other thiol-
containing enzymes/proteins and eliciting the anticancer
mechanism(s) of action through proteomics and transcrip-
tomics studies.
Received: January 7, 2014
Published online: April 11, 2014
Keywords: antitumor agents · gold(I) · N-heterocyclic carbenes ·
.
thiol reactivity · thioredoxin reductase (TrxR)
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