Cyclometalated Gold(III) Complexes Containing N-Heterocyclic Carbene Ligands Engage Multiple Anti-Cancer Molecular Targets

Article abstract of DOI:10.1002/anie.201612583

Metal N-heterocyclic carbene (NHC) complexes are a promising class of anti-cancer agents displaying potent in vitro and in vivo activities. Taking a multi-faceted approach employing two clickable photoaffinity probes, herein we report the identification of multiple molecular targets for anti-cancer active pincer gold(III) NHC complexes. These complexes display potent and selective cytotoxicity against cultured cancer cells and in vivo anti-tumor activities in mice bearing xenografts of human cervical and lung cancers. Our experiments revealed the specific engagement of the gold(III) complexes with multiple cellular targets, including HSP60, vimentin, nucleophosmin, and YB-1, accompanied by expected downstream mechanisms of action. Additionally, Pt^II and Pd^II analogues can also bind the cellular proteins targeted by the gold(III) complexes, uncovering a distinct pincer cyclometalated metal–NHC scaffold in the design of anti-cancer metal medicines with multiple molecular targets.

Full text of DOI:10.1002/anie.201612583

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International Edition: DOI: 10.1002/anie.201612583
German Edition: DOI: 10.1002/ange.201612583
Gold Anti-Cancer Agents Very Important Paper
Cyclometalated Gold(III) Complexes Containing N-Heterocyclic
Carbene Ligands Engage Multiple Anti-Cancer Molecular Targets
Sin Ki Fung+, Taotao Zou+, Bei Cao, Pui-Yan Lee, Yi Man Eva Fung, Di Hu, Chun-Nam Lok,
and Chi-Ming Che*
Abstract: Metal N-heterocyclic carbene (NHC) complexes are
a promising class of anti-cancer agents displaying potent
in vitro and in vivo activities. Taking a multi-faceted approach
employing two clickable photoaffinity probes, herein we report
the identification of multiple molecular targets for anti-cancer
active pincer gold(III) NHC complexes. These complexes
display potent and selective cytotoxicity against cultured cancer
cells and in vivo anti-tumor activities in mice bearing xeno-
grafts of human cervical and lung cancers. Our experiments
revealed the specific engagement of the gold(III) complexes
with multiple cellular targets, including HSP60, vimentin,
nucleophosmin, and YB-1, accompanied by expected down-
stream mechanisms of action. Additionally, PtII and PdII
analogues can also bind the cellular proteins targeted by the
gold(III) complexes, uncovering a distinct pincer cyclometa-
lated metal–NHC scaffold in the design of anti-cancer metal
medicines with multiple molecular targets.
Figure 1) that is highly cytotoxic to cancer cells and is
effective to inhibit tumor growth in a mouse model of human
liver cancer.[5] The pincer-type PtII and PdII complexes
containing mono-anionic C^N^N (HC^N^N = 6-phenyl-
2,2’-bipyridine) or N^C^N (N^CH^N = 1,3-di(pyridin-2-
yl)benzene) ligands and ancillary NHC ligands, which are
structural analogues of [AuIII(C^N^C)(NHC)]+ complexes,
also display comparable cytotoxicity towards cancer cells and
exhibit potent in vivo anti-tumor activities.[6]
T
he success in treating certain cancers by cisplatin has
stimulated tremendous efforts to develop next generation,
metal-based anti-cancer drugs with improved efficacy but
lower side effects.[1] In this regard, other metal medicines
besides cisplatin including carboplatin, oxaliplatin, satrapla-
tin, NAMI-A, KP1019 and auranofin, have been reported to
display potent anti-tumor activities, with some of them now in
clinical trials or clinical use. In particular, gold complexes
have been demonstrated to potentially overcome cisplatin
resistance through their distinctly different anti-cancer mech-
anisms.[2] However, the low physiological stability of both
gold(I) and gold(III) complexes hampers their general
therapeutic utility in vivo.[3] By taking advantage of the
strong s-donor strength of N-heterocyclic carbene (NHC)
ligand and the ease of ligand modification,[4] we have reported
a stable pincer-type cyclometalated gold(III) complex con-
taining methyl-substituted NHC ligand, [AuIII(C^N^C)-
Figure 1. Chemical structure of gold(III) NHC complexes 1a–1g and
the chemical probes for target identification; green: photoaffinity unit;
blue: clickable moiety.
Target identification utilizing photoaffinity groups
together with clickable moieties recently emerged as
a useful strategy to identify macromolecular binding partners
for small organic molecules.[7] Nevertheless, this approach has
seldom been used for target identification of anti-cancer
metal complexes,[8] and only one example, combing benzo-
phenone-based photoaffinity labelling and click chemistry for
target identification of gold(III)porphyrin has been repor-
ted.[8f] To circumvent the possible interference of the bulky
benzophenone-containing functional groups to the interac-
tions with bio-macromolecules,[8f] herein, we introduced
a small photoaffinity diazirine group and a clickable alkyne
moiety on NHC to generate a chemical probe (probe-2) for
target identification of [AuIII(C^N^C)(NHC)]OTf (Figure 1;
the benzophenone analogue probe-3 was prepared for
comparison). By using these clickable photoaffinity probes,
we found that a two-butyl substituted gold(III) NHC com-
plex, which strongly inhibits tumor growth in multiple mouse
models, is a multi-target, anti-cancer agent that can specifi-
cally bind six cellular proteins, all having been reported to be
potential anti-cancer targets. The PtII and PdII analogues
having the same structural geometry were also found to target
these intracellular proteins.
(NHC2Me)]OTf
(1a,
H2C^N^C = 2,6-diphenylpyridine;
[*] S. K. Fung,[+] Dr. T. Zou,[+] Dr. B. Cao, Dr. P.-Y. Lee, Dr. Y. M. E. Fung,
Dr. D. Hu, Dr. C.-N. Lok, Prof. Dr. C.-M. Che
State Key Laboratory of Synthetic Chemistry, Institute of Molecular
Functional Materials, Chemical Biology Centre 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)
[+] These authors contributed equally to this work.
Supporting information for this article can be found under:
http://dx.doi.org/10.1002/anie.201612583.
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We synthesized and examined the cytotoxic-
ities of a series of gold(III) NHC complexes
containing different substituents on their NHC
ligands (Figure 1). As shown in Table S1, the
increase of alkyl chain length on NHC resulted in
enhanced cytotoxicity against HeLa (cervical),
HCT116 (colon), and NCI-H460 (lung) cells; the
IC50 gradually decreased from 2.04 mm (1a) to
0.36 mm (1d) for HeLa cells, with the same trend
for HCT116 and NCI-H460 cells after a 72 h
treatment. Probe-2 and probe-3 also showed
potent cytotoxicity with IC50 being 0.23–0.68 mm,
indicating that the modification on NHC did not
adversely affect the bioactivity. Importantly, all
the gold(III) NHC complexes showed much
higher cytotoxicity to HCT116 and NCI-H460
than to immortalized normal human hepatocyte
(MIHA) cells. Complex 1d, containing two n-
butyl groups, displayed the highest selective
cytotoxicity against cancer cells (> 30-fold lower
cytotoxicity against normal MIHA cells). We also
tested the cytotoxicity of 1d against 3D cancer
cell spheroids which are more representative of
malignant tumor tissues. While treatment of
Figure 3. a) Schematic of procedure to identify cellular protein targets with probe-2.
b) Detection of photoaffinity-labelled proteins in 1) HeLa, 2) NCI-H460, and
3) HCT116 cells treated with 5 mm probe-2. c) Fluorescence scanning of 2D gels of
lysates from HeLa cells treated with probe-2, and clicked with azide-Cy5. MALDI-
TOF indicates that each spot is HSP60 (1), vimentin (2), NDKA (3), nucleophos-
min (4), YB-1 (5) and PRDX1 (6). d) Protein blotting of photoaffinity-labelled pro-
teins in HeLa cells treated with 5 mm probe-2 for 1 h or 5 mm probe-2+10 mm of 1d
or 1a, followed by irradiation and click reaction with biotin azide.
spheroids developed from HeLa cells with cisplatin at
100 mm caused only around 30% inhibition of spheroid
growth after 72 h, 1d treatment disintegrated the spheroids
in a dose dependent manner and reduced the spheroid growth
by about 70% at 40 mm (Figure S1 in the Supporting
Information). Treatment of mice bearing xenografts of
HeLa cancer with 3 mgkgÀ1 of 1d inhibited tumor growth
by 71% without affecting survival and significant influence on
body weight (Figure 2a,b). Treatment of NCI-H460 xeno-
grafts with the same dosage of 1d also resulted in a 53%
tumor growth inhibition (p < 0.05) without negative effect on
body weight (Figure S2).
about six photoaffinity-labelled (biotinylated) proteins that
were identified in the one-dimensional (1D) protein blot of
the HeLa cell lysates. These bands were also observable in the
protein samples from NCI-H460 and HCT116 cells treated
with probe-2. When azido-Cy5 (a highly fluorescent cyanine
dye) was used as the reporter (Figure 3c), six distinct
fluorescent spots were detected in the two-dimensional
(2D) gel electrophoresis of the cellular proteins from HeLa
cells and identified to be mitochondrial heat shock protein 60
(HSP60), vimentin (VIM), nucleoside diphosphate kinase A
(NDKA), nucleophosmin (NPM), nuclease-sensitive element
binding protein (Y box binding protein, YB-1), and peroxir-
edoxin 1 (PRDX1) by tandem mass spectrometric analysis
using MALDI-TOF mass spectrometry (Tables S2–S7). It is
noteworthy that co-incubation of HeLa cells with probe-2 and
two-fold of 1d or 1a significantly reduced the intensity of
photoaffinity-labelled protein signals (Figure 3d). The pro-
teins bound to probe-2 were also identified by HPLC-LTQ-
Orbitrap MS analysis of the trypsin digests of the biotinylated
proteins pulled down by streptavidin beads. Again, HSP60,
vimentin, NDKA, nucleophosmin, and PRDX1 can be
repeatedly identified as the proteins bound to probe-2 with
high confidence (Table S8). Probe-3, which contains benzo-
phenone instead of diazirine, displays lower photoaffinity
towards cellular proteins compared to probe-2 (Figure S3a),
but can still label four proteins, namely, vimentin, HSP60,
nucleophosmin, and PRDX1 in HeLa cells (Figure S3b).
Cellular imaging experiments were carried out to examine
the subcellular distribution of probe-2. HeLa cells were
treated with probe-2, UV irradiation, and a click reaction with
Alexa Fluor 488. As shown in the first column in Figure S4,
probe-2 mainly localized to the cytoplasm with a small
portion in the nucleus. This is consistent with the 2D gel
results identifying vimentin, which is localized in the cyto-
plasm, as a major portion of the emissive spots, with the
Figure 2. Anti-tumor activities of 1d. a) Tumor volume (V) and b) body
weight (W) of mice bearing HeLa xenografts after treatment with
3 mgkgÀ1 of 1d (n=3) or solvent control (n=4). * p<0.05.
We used probe-2, which contains a photo-activatable
diazirine group that enables the gold(III) NHC complex to
form covalent adducts with interacting proteins upon light
irradiation and a clickable alkyne for linking to a reporter
(e.g., fluorophore, biotin). HeLa cells were treated with
probe-2 for 1 h and then irradiated at l = 365 nm UV light,
followed by a CuI-catalyzed click reaction with azido-biotin
which was subsequently detected with a streptavidin-perox-
idase conjugate (Figure 3a). As shown in Figure 3b, there are
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nuclear nucleophosmin spot less emissive. We further inves-
the HeLa cells transfected with a GFP-expressing construct
tigated the co-localization of probe-2 with vimentin, HSP60,
nucleophosmin and YB-1 by immunofluorescence. Figure S4
shows that all the protein immunofluorescence could be
merged with the Alexa Fluor 488 fluorescence of probe-2 to
a reasonable extent.
(EGFP) with no organelle-targeting property did not show
obvious changes in fluorescence upon treatment with 0.5 or
1 mm of 1d, the fluorescence intensity in HeLa cells trans-
fected with Mito-EGFP was markedly reduced upon 1d
treatment in a dose dependent manner. In a typical HSP60
chaperone assay using denatured malate dehydrogenase
(MDH) as the refolding substrate, HSP60 in combination
with HSP10 can recover approximately 25% of MDH
activity, while HSP60/HSP10 in the presence of 1d at 10
and 20 mm can only recover about 10% and about 3% of the
MDH activity (Figure 4c). Although not to the same extent as
1d, complex 1a, probe-2 and probe-3 also strongly inhibited
HSP60/HSP10 activity (Figure 4c).
Vimentin, an intermediate filament protein, is part of the
cytoskeleton with a mesenchymal origin and a high expres-
sion in various cancers.[9c] It has been demonstrated that direct
interaction of anti-cancer agents (e.g., withaferin A) with
vimentin can induce degradation of this protein and apoptotic
cell death.[11] HeLa cells were treated with increasing
concentrations (0, 1, 3, and 5 mm) of 1d for 24 h; western
blot analysis revealed a dose-dependent decrease in full-
length vimentin levels and an increase in the expression of
a vimentin degradation product (Figure 4d). This degradation
process is accompanied by gradually increased cleavage of
poly (ADP-ribose) polymerase (PARP), caspase-9, and
caspase-3 (Figure 4d), which suggests that apoptosis is
triggered.
It should be emphasized that all six identified binding
proteins have been described as potential anti-cancer tar-
gets.[9] In the current study, we singled out HSP60, vimentin,
nucleophosmin and YB-1 as proteins of interest and used the
highly potent 1d to test whether the binding interactions are
associated with the anti-cancer activity. We first enriched
probe-2 binding proteins using biotin affinity pull-down
followed by western blotting and detection with specific
antibodies. As shown in Figure 4a, the enriched probe-2
binding proteins can be detected by anti-HSP60, -vimentin,
-nucleophosmin, and -YB-1 antibodies on the western blot.
Importantly, 1d at a 2-fold excess concentration can signifi-
cantly attenuate the binding of probe-2 to HSP60, vimentin,
nucleophosmin, and YB-1 (Figure 4a).
We subsequently examined whether the specific binding
interactions could interfere the respective protein functions in
cancer cells. HSP60 is a chaperone protein responsible for the
transport of proteins into mitochondria and protein folding in
the mitochondrial matrix.[9d] We transfected a mitochondria-
targeting green fluorescent protein (Mito-EGFP) that is
known to be fluorescent only when it is properly folded by
HSP60 in mitochondria.[10] As presented in Figure 4b, while
Nucleophosmin is
a
nucleolar protein
involved in DNA replication and ribosome bio-
genesis.[12] This protein is upregulated in cancer
cells and can interact with p14ARF, a positive
regulator of tumor suppressor p53. As shown in
Figure 4e, treatment of HeLa cells with 1–5 mm
1d disrupted nucleophosmin oligomers (func-
tional forms) in a dose-dependent manner, in
association with a gradual formation of nucleo-
phosmin monomers.[9b] Meanwhile, an increased
expression of both p53 protein levels and its Ser15
phosphorylation status was observed.
YB-1 is an oncogenic transcription factor that
is overexpressed in many cancers.[9a] Its expres-
sion is closely associated with drug resistance and
YB-1 has been shown to control multidrug
resistance 1 (MDR1) gene expression. Also, YB-
1 is capable of promoting cell growth via tran-
scriptional upregulation of epidermal growth
factor receptor (EGFR). Knockdown of YB-
1 expression by shRNA has been reported to
inhibit tumor growth in vitro and in vivo.[13] We
found that treatment of HeLa cells with 1–5 mm of
1d for 24 h significantly decreased YB-1 levels
(Figure 4 f). At the same time, the expression of
EGFR was also significantly inhibited after treat-
ment of HeLa cells with 1d (Figure 4 f), which is
consistent with a previous observation that RNA
silencing of YB-1 down-regulated EGFR expres-
sion.[14]
Figure 4. a) Western blot analysis of the cell lysates from cells labelled with or
without probe-2 (5 mm) or with probe-2 (5 mm) in the presence of 1d (10 mm)
followed by click reaction with azido-biotin and pull-down with streptavidin beads.
Nucleophosmin (NPM), vimentin (VIM), HSP60, or YB-1 antibodies were used for
detection of the pull-down proteins. b) Inhibition of HSP60 activity in HeLa cells
treated with 1d. Left: representative fluorescence imaging of HeLa cells transfected
with EGFP or Mito-EGFP followed by treatment with different concentrations of 1d.
Right: dose-dependent effects of 1d on the EGFP and Mito-EGFP fluorescence
intensities (I). c) Inhibition of chaperone activity of Hsp60 in MDH refolding. d)–
f) Western blot analysis of HeLa cells treated with 1d (0–5 mm) for 24 h. The cell
lysates were resolved by polyacrylamide gel electrophoresis under denaturing
conditions, except for nucleophosmin which was performed under native conditions,
followed by western blot analysis of the indicated proteins.
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Molecular docking and hybrid quantum mechanics/
molecular mechanics (QM/MM) studies revealed that 1d
preferentially binds to the aromatic amino acid residues,
including Tyr, Phe and Trp via p–p interactions with the
[Au(C^N^C)] moiety (Figure S5a), in association with hydro-
phobic interactions (Figure S5b,c) with all the six binding
proteins. Taken together, our data indicate that the direct
binding interaction of 1d with HSP60, vimentin, nucleophos-
min, and YB-1 may result in the inhibition of HSP60 activity,
degradation of vimentin, disruption of nucleophosmin oligo-
merization and downregulation of YB-1 in cancer cells,
respectively. These processes either individually or synergisti-
cally lead to subsequent anti-cancer effects involving sup-
pression of EGFR, induction of tumor suppressor p53 and
activation of apoptotic caspases in cell death.
Figure 6. a) The chemical structure of PtII and PdII complexes.
b) Immunoblot analysis of photoaffinity-labelled proteins in HeLa cells
treated with probe-2 and a twofold excess of a different Pt, Pd, or Au
complex, followed by UV irradiation and click reaction with biotin
azide.
To obtain a holistic insight, proteomic analysis of the
effects of 1d on protein expression profiles in HeLa cells
using HPLC-LTQ-Orbitrap MS was performed. A bioinfor-
matics analysis of the proteomic data showed that the
eukaryotic initiation factor 2 (eIF2) signaling pathway was
predominantly modulated in HeLa cells treated with 1d
(5 mm) for 6 h with high statistical significance (Table S9).
eIF2 is an essential factor of the protein translation initiation
complex in protein synthesis. The alpha subunit of elF2
(eIF2a) is subjected to inhibitory regulation by specific
protein kinases via phosphorylation at Ser-51 under cellular
stress.[15] Three of the protein targets of 1d identified by using
probe-2, namely, vimentin, nucleophosmin, and YB-1, have
been reported to directly or indirectly inhibit eIF2a kinases,
resulting in dephosphorylation of eIF2a and the ensuing
protein synthesis under normal conditions.[16] Western blot
analysis (Figure S6) showed that there was a significant time-
dependent up-regulation of phosphorylated eIF2a in the
HeLa cells treated with 1d (5 mm), consistent with down-
regulation of the eIF2 signaling pathway as revealed by the
proteomic analysis. Thus the possibility exists that vimentin
degradation, disruption of NMP oligomers and down-regu-
lation of YB-1, all caused by 1d treatment (Figure 4d–f), may
result in inhibition (i.e. phosphorylation) of eIF2a, leading to
a global reduction in protein synthesis and hence anti-
proliferative activities (Figure 5).[15]
displayed comparable cytotoxicities against cancer cells as the
gold(III)–NHC analogues.[6] In a competition experiment, co-
incubating HeLa cells with probe-2 (5 mm) and a 2-fold excess
of any of the Pt or Pd complexes, Pt-1a, Pt-1b, Pt-2a, Pt-2b,
or Pd-1, markedly decreased the band intensity as effectively
as 1d and 1a (Figure 6b). However, Pt-3, which has one more
positive charge, did not attenuate the band intensity. Taken
together, the square planar mono-cationic Pt2+, Pd2+, and
Au3+ complexes containing pincer type ligand and NHC
ligand may share similar anti-cancer molecular targets in
cancer cells.
In summary, the [Au(C^N^C)(NHC)]+ complexes (as
exemplified by 1d) are a type of multi-target, anti-cancer
agent, exhibiting potent in vitro and in vivo anti-cancer
activities. We identified a number of intracellular proteins
as the molecular targets of the gold(III) NHC complexes
using clickable photoaffinity probes, and verified the ensuing
anti-cancer effects. Interestingly, the PtII and PdII NHC
analogues were found to compete with the gold(III) NHC
complexes in binding these proteins, highlighting the struc-
tural importance of pincer-type metal complexes containing
NHC ligands in their anti-cancer activities. Structural scaf-
folds targeting protein kinases using inert octahedral metal
complexes have been developed by Meggers and co-work-
ers.[17] Since drug resistance is usually encountered for single-
target anti-cancer agents as a result of naturally occurring
genetic mutations,[18] anti-cancer agents with multiple anti-
cancer molecular targets could minimize drug resistance. For
example, the FDA-approved Sorafenib and Sunitinib exhibit
cumulative effects of targeting multiple tyrosine kinases and
are superior to single-target drugs.[19] The pincer-type gold-
(III), PtII, and PdII NHC complexes are promising candidate
scaffolds to be developed as multi-target, anti-cancer agents
with potentially low drug resistance.
The analogue complexes [Pt(C^N^N)(NHC)]+, [Pt-
(N^C^N)(NHC)]+ and [Pd(C^N^N)(NHC)]+ (Pt-1a, Pt-1b,
Pt-2a, Pt-2b, Pd-1, Figure 6a) are highly stable, have the
same cationic charge, share similar structural scaffold, and
Acknowledgements
This work was supported by the Innovation and Technology
Fund (ITF-Tier 2 ITS/130/14FP), General Research Fund of
Research Grants Council (HKU17300614), and a Special
Equipment Grant of University Grants Committee
(SEG_HKU02).
Figure 5. Proposed anti-cancer pathway induced by 1d, according to
proteomics data and western blot analyses.
4
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The authors declare no conflict of interest.
Keywords: anti-cancer mechanisms · gold · metal medicines ·
N-heterocyclic carbene · target identification
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Manuscript received: December 28, 2016
Final Article published: && &&, &&&&
Angew. Chem. Int. Ed. 2017, 56, 1 – 6
ꢀ 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.angewandte.org
5
These are not the final page numbers!

Angewandte
Communications
Chemie
Communications
Gold Anti-Cancer Agents
On target: Anti-cancer active pincer
cyclometalated gold(III) complexes con-
taining N-heterocyclic carbene ligands
have been identified to be a type of multi-
target, anti-cancer agent by using a com-
bined photoaffinity labelling and click
chemistry approach. The PdII and PtII
analogues can competitively bind to the
same cellular targets.
S. K. Fung, T. Zou, B. Cao, P.-Y. Lee,
Y. M. E. Fung, D. Hu, C.-N. Lok,
C.-M. Che*
&&&& — &&&&
Cyclometalated Gold(III) Complexes
Containing N-Heterocyclic Carbene
Ligands Engage Multiple Anti-Cancer
Molecular Targets
6
www.angewandte.org
ꢀ 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Angew. Chem. Int. Ed. 2017, 56, 1 – 6
These are not the final page numbers!