Gold(i) complex of N,N′-disubstituted cyclic thiourea with in vitro and in vivo anticancer properties-potent tight-binding inhibition of thioredoxin reductase

Article abstract of DOI:10.1039/c0cc01058h

Coinage metal complexes of an N,N′-disubstituted cyclic thiourea exert significant cytotoxicities to cancer cells and, in particular, the gold(i) thiourea complex exhibits a potent tight-binding inhibition of the anticancer drug target thioredoxin reductase with an inhibitory constant at nanomolar level. The Royal Society of Chemistry.

Full text of DOI:10.1039/c0cc01058h

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Chemical Communications
www.rsc.org/chemcomm
Volume 46 | Number 41 | 7 November 2010 | Pages 7641–7844
ISSN 1359-7345
COMMUNICATION
Chi-Ming Che et al.
Gold(I) complex of N,N’-disubstituted cyclic thiourea with in vitro and in vivo anti-cancer properties - potent
tight-binding inhibition of thioredoxin reductase

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Gold(I) complex of N,N0-disubstituted cyclic thiourea with in vitro and
in vivo anticancer properties—potent tight-binding inhibition of
thioredoxin reductasew
Kun Yan,a Chun-Nam Lok,a Katarzyna Bierlab and Chi-Ming Che*a
Received 21st April 2010, Accepted 14th June 2010
DOI: 10.1039/c0cc01058h
Coinage metal complexes of an N,N0-disubstituted cyclic
thiourea exert significant cytotoxicities to cancer cells and, in
particular, the gold(I) thiourea complex exhibits a potent tight-
binding inhibition of the anticancer drug target thioredoxin
reductase with an inhibitory constant at nanomolar level.
complexes,8 other types of TU ligands are sparsely seen in
bioactive metal complexes.9 Several Au(III) dithiocarbamate
complexes8a and to the best of our knowledge only one
metal–TU complex, [AuI(PEt3)(SQC(NH2)2)]+,9e have been
documented to inhibit TrxR.
Herein we report the biological activities of homoleptic
metal–TU complexes [AuI(TU)2]Cl (1), [AgI(TU)2]OTf (2)
and [CuI(TU)2]PF6 (3), of which 1 and 2 are potent inhibitors
of purified and cellular TrxR. Interestingly, the TrxR inhibitor
1, like its NHC analogue,6 did not inhibit cellular GR. Also, a
significant in vivo anticancer activity of 1 has been observed.
Coinage metal (Au, Ag, Cu) ions, such as d10 M+, exhibit
distinct biological activities that could be harnessed to develop
effective therapeutic agents including antiarthritic, anti-
microbial and anticancer drugs.1 Under physiological
conditions, however, the hydrated d10 M+ ions except Ag(I)
are unstable toward precipitation, reduction or aerobic
oxidation. Such instability can be circumvented by using
appropriate auxiliary ligands. In the literature, phosphine
ligands have been employed to develop bioactive complexes
of d10 M+ ions.2 Recent works have also witnessed other
ligand systems, particularly, N-heterocyclic carbenes (NHC)
for this endeavor.3
A compelling finding in the anticancer activity of d10 M+
complexes is the remarkable potency of Au(I)–phosphine
complexes, such as auranofin (a [AuI(PEt3)(SR)] species), in
Treatment of the previously reported N,N0-disubstituted
imidazolidine-2-thione10 with [Au(THT)Cl] (THT = tetra-
hydrothiophene), AgOTf and [Cu(CH3CN)4]PF6 afforded 1,
2 and 3, respectively. The structures of 1 and 2 have been
determined by X-ray crystallography (ESIw, Table S1 and Fig.
S1 and S2), both featuring a linear two-coordinate M+ ion
with monodentate S-donor TU ligands. The M–S distances
(2.287(3) A for 1, average 2.4071(11) A for 2) and S–M–S
angles (180.0(1)1 for 1, 172.77(4)1 for 2) are similar to those in
the [MI(TU)2]+ complexes with TU = imidazolidine-2-thione
inhibiting thioredoxin reductase (TrxR),4
a
NADPH
dependent selenoenzyme that plays a pivotal role in cancer
progression and represents an increasingly attractive target for
anticancer drugs.5 Recently, Berners-Price and co-workers6
reported that
a
homoleptic Au(I)–NHC complex,
[AuI(NHC)2]+, can selectively inhibit TrxR in cells without
inhibiting the closely related non-selenoenzyme glutathione
reductase (GR).
We are interested in exploring the possible use of homoleptic
d10 M+ thiourea (TU) complexes, [MI(TU)2]+, as potent
inhibitors of TrxR, in view of the well documented coordination
chemistry of TU ligands,7 including imidazolidine-2-thiones,7a
N-acyl thioureas7b and thiosemicarbazones.7c–e Despite
extensive studies on the anticancer activity of metal thiosemi-
carbazone complexes7c–e and related Au(III) dithiocarbamate
(2.2782(15)–2.2873(14)
A
and 171.23(5)–174.84(6)1 for
Au(I);11a 2.4058(8) A and 1801 for Ag(I)11b). The nearest
Mꢀ ꢀ ꢀM distance in 1 and 2 (>3.2894(7) A) suggests the
absence of significant intermolecular metal–metal interactions.
Complexes 1–3 are all stable in the solid state in air and are
soluble in DMSO to form a 10 mM stock solution; they also
exhibit some solubility and considerable stability in aqueous
solution, and no precipitation occurred when the concentration
of their solutions in serum supplemented cell culture medium
reached up to 30 mM.
a Department of Chemistry and Open Laboratory of Chemical Biology
of the Institute of Molecular Technology for Drug Discovery and
Synthesis, The University of Hong Kong, Pokfulam Road,
Hong Kong, China. E-mail: cmche@hku.hk;
We examined the cytotoxity of 1–3 against a panel of cancer
cell lines. The observed half maximal inhibitory concentrations
(IC50), together with those observed for the free TU ligand and
the benchmark anticancer drug cisplatin, are listed in Table 1.
These IC50 values indicate that 1–3 exhibit similar or higher
potency compared with cisplatin, whereas the free TU ligand is
much less cytotoxic, suggesting that the biological activities of
1–3 are largely metal-mediated.
Fax: (+852) 2857-1586
b CNRS/UPPA, Laboratory of Bio-Inorganic Analytical and
Environmental Chemistry, UMR5254, Helioparc, 2 Av. Pr. Angot,
F-64053, Pau, France
w Electronic supplementary information (ESI) available: Synthesis of
1–3, X-ray crystallographic data for 1 and 2, experimental procedures
for cellular and biochemical assays and animal studies. CCDC
773697–773698. For ESI and crystallographic data in CIF or other
electronic format see DOI: 10.1039/c0cc01058h
ꢁc
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Table 1 Cytotoxicities (IC50 in mM, 72 h treatment) of 1–3 against
human cancer cell lines
HeLaa
HepG2b
SUNE1c
NCI-H460d
1
2
3
TU
14.6 ꢂ 0.7
7.2 ꢂ 0.7
12.7 ꢂ 0.9
>100
17.4 ꢂ 1.0
4.0 ꢂ 0.4
13.0 ꢂ 0.9
>100
10.8 ꢂ 0.2
8.8 ꢂ 1.0
8.5 ꢂ 1.0
>100
3.7 ꢂ 0.3
8.9 ꢂ 1.0
11.2 ꢂ 0.9
>100
Cisplatin
4.7 ꢂ 0.3
14.2 ꢂ 1.0
35.2 ꢂ 0.3
Human cervical epithelioid carcinoma. Human hepatocellular
38.6 ꢂ 0.4
a
b
c
d
Fig. 1 TrxR, GR and GPx activities of HeLa cells treated with 1 or 2
carcinoma. Human nasopharyngeal carcinoma. Human lung
carcinoma.
for 1 h.
showed that half maximal inhibition of TrxR (recombinant
rat TrxR1, 1 nM) was obtained using approximately equal
molar concentration of 1, suggestive of a tight-binding mode
of inhibition (ESI, Fig. S4 and S5w). This was further studied
by progress curve analysis15 (ESIw) of the inhibition of
TrxR by 1, performed by adding various amounts of excess
1 (3–100 nM) to a mixture containing 0.2 mM NADPH, 1 nM
TrxR and 3 mM disulfide substrate 5,50-dithiobis(2-nitro-
benzoic acid) (DTNB) in phosphate buffer (pH 7.4). The time
course of product concentrations at different concentrations of
1 is depicted in Fig. 2A. The progress curves are non-linear,
revealing two-phase equilibria typical of slow-onset tight-
binding inhibition. A plot of the apparent first-order rate
constants (kapp, determined by fit of the data in Fig. 2A)
against the concentrations of 1 follows a hyperbolic function
(Fig. 2B), indicative of a two-step, tight-binding inhibition
mechanism:15
It appears that 1–3 induced cell death through apoptosis, as
apoptotic cells were detected by cell permeable DNA
fluorescent stain Hoechst 33342 when HeLa cells were treated
with 1–3 (10 mM) for 24 h (ESI, Fig. S3w).
The metal uptake by cells upon treatment with 10 mM of 1
or 2 for 2 h was measured by inductively coupled plasma
mass spectrometry (ICP-MS), which revealed a Au content of
18 ꢂ 1 fg/cell and a Ag content of 119 ꢂ 3 fg/cell; the latter is
5-fold higher than the Ag content of 22 ꢂ 1 fg/cell in the cells
treated with 10 mM of AgNO3 under the same conditions.
Complex 2 also has nearly a 5-fold higher cytotoxicity
against HeLa cells (IC50 = 7.2 ꢂ 0.7 mM) than AgNO3
(IC50 = 32.1 ꢂ 1.2 mM). These results suggest that the TU
ligand serves as a lipophilic carrier of the metal ion to the cells.
The therapeutic potential of Au(I) complexes has long
attracted considerable interest. For example, aurothiomalate
(Au(I)–thiolates) and auranofin are disease modifying anti-
arthritic drugs and have been studied for their anticancer
properties.7d,12 We examined the in vivo anticancer activities
of 1 in mice inoculated with NCI-H460 non-small cell lung
cancer cells (ESIw, performed with approval from the
Committee on the Use of Live Animals for Teaching and
Research, The University of Hong Kong). Intraperitoneal
injection of 1 at 100 mg/kg body weight twice a week resulted
in reduction in tumor size by 38 ꢂ 11% (n = 5) compared to
vehicle control after a 28-day treatment (ESI, Table S2w).
The exact molecular mechanism of action of Au(I) complexes
has yet to be elucidated, but is generally related to their ability
to undergo facile ligand exchange with protein thiol groups,
particularly those with low pKa values.13 In this regard, TrxR
which involves rapid formation of an initial collision complex
(EI) that subsequently undergoes isomerization to the final
slow dissociating enzyme–inhibitor complex (EI*). According
to this mechanism, the inhibition of TrxR by 1 gave
k3 = 0.011 sꢃ1, k4 = 0.00014 sꢃ1 and Ki = 1.39 nM.
is
a
compelling molecular target enzyme of Au(I).4,14
We investigated the effect of 1–3 and the free TU ligand on
the cellular activities of TrxR, GPx (glutathione peroxidase,
another selenoenzyme) and GR. A one-hour treatment of
HeLa cancer cells with 1 resulted in an inhibition of the
cellular TrxR activity with an IC50 value of 50 nM; GPx was
also inhibited albeit at a higher concentration (IC50 = 1 mM),
but GR activity was not affected (Fig. 1). Complex 2 inhibited
TrxR and GPx with IC50 of 100 nM and 1 mM, respectively,
and also significantly suppressed GR activities when added at
50 mM (Fig. 1). Neither 3 nor the free TU ligand (added up to
100 mM) affected the activities of the three enzymes. These
data demonstrate that among the coinage metal [MI(TU)2]+
complexes, the Au(I) complex preferentially targets the
selenoenzymes.
Fig. 2 (A) Progress curves of TrxR in the absence or presence of 1
(3–100 nM). (B) Plot of kapp against concentrations of 1.
We consider that 1 is particularly useful for understanding
the Au(I) inhibition of TrxR. Our in vitro enzyme assays
Fig. 3 Probing the free –SH and –SeH site of TrxR treated with or
without 1 by BIAM labelling.
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Thus, the tight-binding inhibition is essentially irreversible.
Indeed, the enzyme activities could not be recovered after
removal of the free inhibitors by centrifugal ultrafiltration.
The overall inhibitory constant Ki* was determined to be 18 pM.
These inhibitory constants are close to the values Ki = 0.67 nM
and Ki* = 36 pM determined from the steady state rate law
established under conditions when EI* was preformed
(ESI, Fig. S4; details about the determination of the foregoing
kapp, k3, k4, Ki, Ki* values are described in ESIw). Complex 1 is
thus among the most potent TrxR inhibitors reported.5b,14a
The reduced TrxR has free –SH (Cys497) and –SeH
(Sec498) groups at the C-terminal active site, making them
vulnerable to attack by Au(I).5a,b,14a It has been shown that
biotinylated iodoacetamide (BIAM) alkylates both the –SH
and –SeH at pH 8.5, but at pH 6.5 alkylates only the –SeH; the
resulting adduct can be detected by western blot experiment
using streptavidin-linked horseradish peroxidase.14d,16
When NADPH-reduced TrxR (0.1 mM) was preincubated
with 1 (4 mM), the BIAM labelling at pH 8.5 and 6.5 (buffered
with 0.1 M Tris-HCl) was inhibited (Fig. 3), suggesting that
the Sec or additionally the Cys residue at the active site was
involved in the enzyme inactivation. Compared with NADPH-
reduced TrxR, the oxidized TrxR having the –S–Se– group,
instead of free –SH and –SeH groups, was much less efficiently
inhibited by 1 (ESI, Fig. S5w). Moreover, size exclusion
chromatography and ICP-MS analysis (SEC-ICP-MS) of the
trypsin digest of TrxR treated with 1 showed the appearance
of a peptide fraction co-eluted with Se and Au (ESI, Fig. S6w).
The formation of the tight enzyme–inhibitor complex (EI*) is
likely to involve modification of the redox active Sec/Cys
residue via coordination with Au(I). This may be congruent
with the report of Berners-Price and co-workers on the two-
step ligand exchange reactions of [AuI(NHC)2]+ with cysteine
and selenocysteine.6
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This work was supported by the Area of Excellence Scheme
(AoE/P-10/01) established under the University Grants
Committee of the Hong Kong SAR, P.R. China. Support of
the PROCORE project (French Ministry of Foreign Affairs,
Project No. 20637NA) to K.B. and Ryszard Lobinski is
acknowledged. We thank Z.-Y. Zhou, N.-Y. Zhu and S.-Y.
Chui for crystallography analysis. We also thank J.-S. Huang,
Ricky Ho and Raymond Sun for constructive comments.
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Chem. Commun., 2010, 46, 7691–7693 | 7693