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Inhibition of the Ras/Raf interaction and repression of renal cancer xenografts in vivo by an enantiomeric iridium(iii) metal-based compound.
Showcasing research from Dr Chung-Hang Leung’s laboratory,
Institute of Chinese Medical Sciences, University of Macau,
Macau
As featured in:
Inhibition of the Ras/Raf interaction and repression of renal
cancer xenografts in vivo by an enantiomeric iridium(III)
metal-based compound
An iridium(III)-based compound exhibited potent inhibitory activity
against the H-Ras/Raf-1 interaction and its downstream pathways
both in vitro and in vivo. Intriguingly, the Δ-enantiomer showed
superior potency compared to the ^-enantiomer or the racemic
compound. The compound repressed tumor growth in a murine
xenograft model of renal cancer.
See Zongwei Cai,
Hui-Min David Wang, Dik-Lung Ma,
Chung-Hang Leung et al.,
Chem. Sci., 2017, 8, 4756.
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Inhibition of the Ras/Raf interaction and repression
of renal cancer xenografts in vivo by an
enantiomeric iridium(III) metal-based compound†
Li-Juan Liu,‡a Wanhe Wang,‡b Shi-Ying Huang,‡cd Yanjun Hong,‡e Guodong Li,a
Sheng Lin,b Jinglin Tian,e Zongwei Cai,*e Hui-Min David Wang, *f Dik-Lung Ma*b
and Chung-Hang Leung *a
Targeting protein–protein interactions (PPIs) offers tantalizing opportunities for therapeutic intervention for
the treatment of human diseases. Modulating PPI interfaces with organic small molecules has been found to
be exceptionally challenging, and few candidates have been successfully developed into clinical drugs.
Meanwhile, the striking array of distinctive properties exhibited by metal compounds renders them
attractive scaffolds for the development of bioactive leads. Here, we report the identification of iridium(III)
compounds as inhibitors of the H-Ras/Raf-1 PPI. The lead iridium(III) compound 1 exhibited potent
inhibitory activity against the H-Ras/Raf-1 interaction and its signaling pathway in vitro and in vivo, and
also directly engaged both H-Ras and Raf-1-RBD in cell lysates. Moreover, 1 repressed tumor growth in
Received 21st January 2017
Accepted 8th May 2017
a mouse renal xenograft tumor model. Intriguingly, the D-enantiomer of 1 showed superior potency in
DOI: 10.1039/c7sc00311k
the biological assays compared to L-1 or racemic 1. These compounds could potentially be used as
starting scaffolds for the development of more potent Ras/Raf PPI inhibitors for the treatment of kidney
rsc.li/chemical-science
cancer or other proliferative diseases.
Introduction
Protein–protein interactions (PPIs) are indispensable for
numerous biological processes such as cell signaling, gene
expression, cell division, immunity, and metabolism.1 Up to
a staggering 650 000 PPIs are estimated to exist in living
systems,2,3 with 367 527 unique PPIs already documented on the
BioGRID Database.4 Crucially, however, less than 0.01% of the
PPIs constituting the interactome have been targeted with an
inhibitor.2,5 Dysfunction of PPIs is implicated in oncogenesis,
for example, as PPIs mediate the homo-/heterodimerization of
a
State Key Laboratory of Quality Research in Chinese Medicine, Institute of Chinese
Medical Sciences, University of Macau, Macao, China. E-mail: duncanleung@umac.
mo
b
Department of Chemistry, Hong Kong Baptist University, Kowloon Tong, Hong Kong,
China. E-mail: edmondma@hkbu.edu.hk
c
College of Oceanology and Food Science, Quanzhou Normal University, Quanzhou
362000, China
d
Key Laboratory for the Development of Bioactive Material from Marine Algae,
Quanzhou 362000, China
e
Partner State Key Laboratory of Environmental and Biological Analysis, Department of
Chemistry, Hong Kong Baptist University, 224 Waterloo Road, Kowloon Tong, Hong
Kong SAR, P. R. China. E-mail: zwcai@hkbu.edu.hk
f
Graduate Institute of Biomedical Engineering, National Chung Hsing University,
Taichung 402, Taiwan. E-mail: davidw@dragon.nchu.edu.tw
† Electronic supplementary information (ESI) available: Synthetic methods,
characterization, and biological assays details. See DOI: 10.1039/c7sc00311k
‡ These authors contributed equally to this work.
4756 | Chem. Sci., 2017, 8, 4756–4763
receptor tyrosine kinases to initiate a relay of oncogenic signals
to enable cancer progression.6 PPIs also offer a viable target to
engage pathogenicities such as Alzheimer’s disease or viral
infections that lack conventional targets such as enzymes and
G-protein coupled receptors.2,7 As a consequence, targeting PPIs
offers tantalizing potential for therapeutic intervention for the
treatment of human disease. However, PPIs oen consist of
extended shallow and non-polar interfaces, thwarting the
identication of small-molecule inhibitors. In this context,
metal-based compounds have emerged as feasible alternatives
to small molecules for the development of PPI modulators.8–16
The distinctive but tunable characteristics of metal compounds,
including their molecular geometries, chemical reactivities, and
electronic properties, can allow them to adopt unique threedimensional architectures that can engage hitherto unexplored regions of the chemical space of proteins that are inaccessible to purely organic molecules.17,18
The Ras/Raf/mitogen-activated protein kinase (MEK)/
extracellular-signal-regulated kinase (ERK) (Ras/Raf/MEK/ERK)
signaling pathway transmits information from membrane
receptors to transcription factors,19–23 thereby regulating
apoptosis and cell cycle progression.24 Abnormal activation of
the Ras/Raf/MEK/ERK pathway enhances tumor initiation,
progression, and metastasis and is thus a frequent event in
proliferative disorders such as renal cell carcinoma (RCC).25 In
particular, constitutive activation of MAP kinases has been
implicated in renal cell carcinogenesis,26 while activated H-Ras
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activity has been detected in human renal cell carcinomas.27
These studies suggest that inhibition of the Ras/Raf/MEK/ERK
signaling pathway could be a potential therapeutic strategy to
treat RCC. For example, sorafenib is a small molecule inhibitor
of several kinases, including the Raf family kinases, which has
been approved for the clinical treatment of RCC, hepatocellular
carcinoma, and radioactive iodine-resistant advanced thyroid
carcinoma.28–30 Recently, rigosertib was identied as the rst
small molecule Ras/Raf PPI inhibitor that acts as a Ras-mimetic
by binding to the Ras-binding domains (RBDs) of Raf kinases,
resulting in the inhibition of Raf activation and perturbation of
the Ras/Raf/MEK/ERK pathway.31
In continuation of our previous efforts in developing metal
compounds to modulate different PPIs,32–36 we were inspired to
explore the application of group 9 metal compounds to target
the PPI between Ras and Raf as a potential therapeutic strategy
to treat renal cancer. In this work, we describe the identication
of an iridium(III)-based compound, 1, that blocks the H-Ras and
Raf-1 PPI and inhibits Ras/Raf/MEK/ERK signaling pathway
activation both in vitro and in vivo. Isolation and testing of both
enantiomers of 1 critically revealed that D-1 is the more potent
of the two enantiomers at inhibiting the H-Ras/Raf-1 PPI via
engaging H-Ras and Raf-1-RBD, and thus it contributes the
majority of the biological activity of racemic 1. Moreover, the
ability of both racemic 1 and D-1 to suppress tumor growth in
a murine xenogra model of renal cancer without causing overt
toxicity to mice was demonstrated. To our knowledge,
compound 1 is the rst substitutionally-inert organometallic
compound that has been reported to inhibit the Ras/Raf PPI.
Results and discussion
Discovery of an iridium-based compound as an inhibitor of
the H-Ras/Raf-1 interaction
An in-house library of 15 structurally diverse, potentially
biologically-privileged iridium-based and rhodium-based
compounds 1–15 (Fig. 1) were rst investigated for their
ability to inhibit the H-Ras/Raf-1 PPI using the biomolecular
uorescence complementation (BiFC) assay. In this assay,
HEK293T cells were transiently co-transfected with the BiFC
plasmid pair (pEGFP-VC-H-RasV12 and pEGFP-VN-Raf-1-RBD)
and exposed to 5 mM of the compounds for 6 h. In the
absence of the compounds, H-Ras could bind to the Rasbinding domain present in Raf-1 in cells, generating a strong
yellow uorescence that could be detected using confocal
imaging. Compounds that disrupt the H-Ras/Raf-1 PPI would be
expected to reduce the uorescence of the HEK293T cells, as
was observed with the positive control compound sulindac
sulphide.37 Among the library of 15 metal compounds, the iridium(III) compound 1 showed the greatest effect at disrupting
the H-Ras/Raf-1 interaction in cellulo (Fig. S1a and b†). Moreover, we determined that 1 was stable at room temperature for
over 48 h as there were no changes of absorbance of 1 in 80%
acetonitrile/20% Tris–HCl buffer or cell culture medium
(Fig. S2†) aer subtraction of the corresponding background
absorbance in UV spectroscopy, and no changes of 1H NMR
spectra of 1 in 90% DMSO-d6/10% D2O (Fig. S3†).
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Fig. 1 Chemical structures of the cyclometallated iridium(III) and
rhodium(III) compounds 1–34 that were synthesized and evaluated in
this study.
The iridium(III) compound 1 bears the 4,40 -dinonyl-2,20 bipyridine (dnbpy) N^N ligand and two 2-(p-tolyl)pyridine (tpy)
C^N ligands. To further explore the potential structure–activity
relationships (SAR) of these metal compounds against the HRas/Raf-1 PPI, a lead-like diverse collection of compounds
(16–34, Fig. 1) were designed and synthesized. Iridium(III)
compounds 16–19 contained the dnbpy N^N ligand of 1, but
varied in the nature of their C^N ligands, while conversely iridium(III) compounds 20–24 retained the tpy C^N ligands of 1
but possessed different N^N ligands. Compounds 25–29 were
rhodium(III) congeners of iridium(III) compounds 1, 16–19,
while compounds 30–34 were the rhodium(III) congeners of 19–
24. These compounds were subsequently tested at 5 mM for their
ability to disrupt the H-Ras/Raf-1 interaction using the BiFC
assay (Fig. S1c and d†). Encouragingly, compound 1 still
emerged as the most potent compound, inhibiting the H-Ras/
Raf-1 interaction by 80.2% inhibition at 5 mM.
Based on the BiFC results, a preliminary SAR analysis could
be performed. Compound 1 was more potent than compounds
16–19, indicating that the tpy C^N ligand was superior to the
other C^N ligands tested. Moreover, based on the fact that
compounds 20–24 were among the least potent compounds of
the library, the dnbpy N^N ligand of 1 was also deemed to be
crucial for H-Ras/Raf-1 inhibitory activity. The iridium(III) center
was also important for the activity of 1, as its rhodium(III)
congener 25 showed signicantly reduced activity. However, the
other rhodium(III) congeners showed comparable or even
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slightly higher potency than the iridium(III) series. Of particular
interest is the rhodium(III) compound 29, bearing the dnbpy
N^N ligand and 1-phenylisoquinoline (piq) C^N ligand, which
showed 60.9% inhibition of the H-Ras/Raf-1 PPI, and was the
second most active compound of the library. Potentially, the
rhodium(III) compound 29 could also be developed further as an
alternative scaffold class for H-Ras/Raf-1 PPI inhibitors. Taken
together, the results indicate that the nature of both the C^N
and N^N ligands as well as the character of the metal center are
important for the Ras/Raf inhibitory properties of 1. We also
checked whether 1 affected the expression of H-Ras and Raf-1 in
the transfected HEK293T cells. The results showed that 1 had
no signicant effect on H-Ras and Raf-1 protein expression in
transfected HEK293T cells aer incubation for 6 h (Fig. S4†).
These ndings indicate that 1 disrupts the H-Ras/Raf-1 interaction without affecting their expression levels.
Enantiomer D-1 is more potent than L-1 at disrupting the HRas/Raf-1 interaction
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However, no signicant differences in cellular distribution were
observed between the different compounds.
To further conrm the mechanism of H-Ras/Raf-1 interaction inhibition, a pull-down assay was performed. Aer overexpression of the Raf-1-Flag and H-Ras-Myc fusion proteins in
A498 human kidney carcinoma cells, racemic 1 or enantiomers
D-1 and L-1 (5 mM) were added and cells were incubated for 6 h.
In the presence of enantiomer D-1 and, to a lesser extent
racemic 1, the amount of H-Ras-Myc detected in Flag immunoprecipitates was signicantly decreased, suggesting that D-1
or racemic 1 could disrupt the H-Ras/Raf-1 interaction in cellulo
(Fig. 3a). However, L-1 did not inhibit the interaction between
H-Ras and Raf-1 in the pull-down assay. In the previous experiments, the G12V mutant of H-Ras was used. We were therefore
also interested to investigate whether 1 could also potentially
inhibit the interaction between wt-H-Ras and Raf-1. As shown in
Fig. S6,† D-1 or racemic 1 inhibited the interaction between wtH-Ras and Raf-1, but with less potency than against the HRasV12/Raf-1 interaction. We also examined the ability of 1 to
To further elucidate the mechanism of disruption of the H-Ras/
Raf-1 interaction by 1, the two enantiomers of 1 (L-1 and D-1)
were synthesized and tested side-by-side using the BiFC assay.
Intriguingly, enantiomer D-1 (5 mM) effectively suppressed the
interaction between H-Ras and Raf-1, whereas D-1 showed no
obvious effect at the same concentration (Fig. 2). This result
demonstrates that the inhibition of the H-Ras/Raf-1 interaction
by racemic 1 could be attributed mainly to the action of the D-1
enantiomer. In order to examine the accumulation of racemic 1
and its enantiomers in cells, an inductively-coupled plasma
mass spectrometry (ICP-MS) assay was performed (Fig. S5†).
The compounds tended to localize in the cytoplasm, where HRas interacts with Raf-1, and also in the mitochondria.
Racemic 1, L-1 and D-1 inhibit the H-Ras/Raf-1 PPI as determined by the BiFC assay. Racemic 1, L-1 and D-1 (5 mM) were incubated with A498 cells for 6 h and the fluorescence was monitored
using confocal imaging.
Fig. 2
4758 | Chem. Sci., 2017, 8, 4756–4763
Fig. 3 (a) Racemic 1, L-1 and D-1 (5 mM) disrupt the interaction of HRas-Myc and Raf-1-Flag in A498 cells as revealed by a pull-down
assay. Protein complexes were immunoprecipitated by the Flag antibody and analyzed with the Myc antibody. (b) Effects of racemic 1, L-1
and D-1 (5 mM) on inhibiting the H-Ras-Flag and Raf-1-RBD-his PPI in
A498 cells as determined by a pull-down assay. (c) Effects of racemic 1,
L-1 and D-1 on inhibiting the H-Ras-Flag or Raf-1-CRD-HA PPIs in
A498 cells as determined by the pull-down assay. Significantly
different from control at *p < 0.05, **p < 0.005, and ***p < 0.001.
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inhibit the interaction between H-Ras and other H-Ras effectors, such as RalGDS and PI3K. Slight inhibitions of the H-Ras/
RalGDS and H-Ras/PI3K interactions were observed aer treatment with 5 mM of D-1, but not aer treatment with racemic 1 or
L-1 in A498 cells (Fig. S7†).
As a critical Ras effector target, Raf-1 contains two Rasbinding sites for activation, the Ras-binding domain (RBD)
and the cysteine-rich domain (CRD).38 In order to elucidate the
possible mode of action of compound 1, we repeated the pulldown assay with the two separate Ras-binding domain
constructs, namely Raf-1-RBD-his and Raf-1-CRD-HA. The
results showed that the amount of Raf-1-RBD-his bound to HRas-Flag was obviously reduced aer incubation with D-1 or
racemic 1 (5 mM) Flag immunoprecipitates, with D-1 again
being more potent than racemic 1, while L-1 only had a slight
effect (Fig. 3b). However, neither racemic 1 nor L-1 (5 mM)
showed any inhibitory impact on the interaction of H-Ras-Flag/
Raf-1-CRD-HA in the treated cells, while D-1 only exhibited
moderate inhibition at the same concentration (Fig. 3c). Taken
together, these ndings indicate that D-1 and racemic 1 disrupt
the interaction of H-Ras and Raf-1 through selective targeting of
the Ras-binding domain of Raf-1, rather than the cysteine-rich
domain. Moreover, neither of the isolated ligands (tpy and
dnbpy) showed an observable effect on the H-Ras/Raf-1 interaction in cells in the BiFC assay (Fig. S8†), suggesting that the
coordination of the ligands to the iridium(III) center to form an
intact compound was required for the inhibition of the H-Ras/
Raf-1 interaction. Taken together, these ndings demonstrate
that the H-Ras/Raf-1 inhibitory potency of racemic 1 is largely
due to the activity of enantiomer D-1 rather than L-1. Additionally, these results provide indirect evidence that the interaction between 1 and H-Ras/Raf-1 is shape-driven rather than
being mediated through non-specic effects.
Drug target engagement of enantiomer D-1
Target engagement, which refers the ability of a small molecule
to interact with its intended protein target in a living system,39 is
an important parameter of drug efficacy. Therefore, we evaluated the H-Ras and Raf-1 binding ability of D-1 using the
cellular thermal shi assay (CETSA). Aer incubation of A498
cell lysates with D-1 (5 mM) for 30 min, aliquots were heated at
different temperatures ranging from 47 C to 68 C for 5 min
and then immunoblotted to quantitate the level of H-Ras or Raf1 remaining in the soluble fraction. A signicant increase of the
melting temperature of H-Ras from 50 C to 68 C and of Raf-1
from 59 C to 68 C was observed upon addition of D-1 to cell
lysates, indicating that D-1 could bind and stabilize H-Ras and
Raf-1 in the presence of complex cellular debris (Fig. S9a and
b†). On the other hand, D-1 had no effect on the thermal
stability of GAPDH (Fig. S9c†). Furthermore, to evaluate the
potential binding target of D-1 in the Raf-1 protein, two
domains of Raf-1, Raf-1-RBD and Raf-1-CRD, were transfected
into the cells (Fig. S9d and e†). As revealed by the result, D-1
showed more effectiveness for the stabilization of Raf-1-RBD by
targeting Raf-1-RBD than Raf-1-CRD under the same experimental conditions. Moreover, an in vitro thermal shi assay was
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performed to further evaluate the binding affinity of D-1
towards Raf-1 RBD and H-Ras. As shown in Fig. S10,† 10 mM of
D-1 shied the melt curves of both Raf-1 RBD (from 53.0 C to
58.1 C) and H-Ras (from 49.0 C to 53.3 C), suggesting that the
compound could stabilize both proteins. A dose experiment
with 0, 1, 3, 5, or 10 mM of D-1 allowed the determination of Kd
values for Raf-1-RBD and H-Ras of 4.40 and 2.88 mM, respectively. BSA was used as a negative control to evaluate the nonspecic binding of D-1 to hydrophobic surfaces, by detecting
the changes of UV absorption of D-1 (5 mM) at different
concentrations of BSA. BSA, having hydrophobic regions on its
surface, is widely used as an additive in biological experiments
to reduce the non-specic binding of other substances.40 As
shown in Fig. S11,† only a slight increase of the absorbance of
D-1 was observed in different concentrations of BSA ranging
from 0.67–6.67 mg mL1 in PBS buffer. Therefore, D-1 is
presumed to exhibit only weak non-specic binding to hydrophobic surfaces. Taking these results together, we hypothesize
that the inhibition of the H-Ras/Raf-1 PPI observed in the BiFC
and pull-down assays could be attributed to the ability of D-1 to
directly engage H-Ras and Raf-1-RBD in cells.
Iridium(III) compound 1 and enantiomer D-1 block the Rasdependent signal transduction
The Ras/Raf/MEK/ERK pathway is initiated when ligands bind
to cytokine receptors, activating Ras. Activated Ras then recruits
Raf-1 by binding the Ras-binding domain present on Raf-1.
Aer a multi-step phosphorylation and dephosphorylation
process, activated Raf-1 phosphorylates and activates MEK,
which in turn phosphorylates and activates ERK, nally culminating in the activation of transcriptional factor AP-1 which
translocates into the nucleus to regulate gene expression.41 We
also investigated the ability of 1 and its enantiomers to modulate the various stages of the Ras/Raf/MEK/ERK signaling
pathway in cells. Immunoblotting analysis revealed that the
phosphorylation of MEK and ERK was signicantly reduced
aer 6 h treatment with racemic 1, L-1, D-1, or sulindac sulde
(5 mM) in A498 cells (Fig. 4a). Notably, D-1 exhibited higher
potency at inhibiting phosphorylated MEK and ERK compared
to L-1, racemic 1, and sulindac sulde. Moreover, all of the
compounds had no impact on the expression of total MEK and
ERK. We further investigated the effect of D-1 on the transcriptional activity of AP-1 using a luciferase reporter assay.
Enantiomer D-1 antagonized AP-1 luciferase activity in a dosedependent manner with an IC50 value of ca. 1.6 mM, and was
more potent than racemic 1, which had an IC50 value of ca. 3.4
mM (Fig. 4b). Taken together, these results suggest that racemic
1 and D-1 could down-regulate the transcriptional activity of AP1, presumably via their inhibition of the H-Ras/Raf-1
interaction.
Compound 1 and D-1 inhibit the proliferation of kidney
cancer cells
We next tested the anti-proliferative activity of compound 1
towards a panel of cancer cell lines including human kidney
cancer cells (A498 and HEK293), human breast cancer cells
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1 PPI, however, we do not preclude the possibility that the
compound could also act via other modes of action.
Iridium(III) compound 1 and enantiomer D-1 suppress tumor
growth and inhibit oncogenic Ras-induced signaling in
a mouse renal xenogra tumor model
Given the promising anti-proliferative activity exhibited by
racemic 1 and D-1 in vitro, we investigated the biological efficacy
of those compounds in a mouse renal xenogra tumor model.
In a preliminary animal toxicity study, high concentrations of
compound 1 (140 or 280 mg kg1) caused signicant toxicity to
mice in terms of decreases in body weight and changes in the
liver and spleen indices (Fig. S12†). Female BALB/cAnN.CgFoxn1nu/CrlNarl were injected subcutaneously with A498
(human kidney cancer) cells and were treated four times a week
with an intraperitoneal (i.p.) injection of racemic 1 or D-1
(14 mg kg1) or with vehicle until sacrice at day 30. Encouragingly, treatment of mice with racemic 1 or D-1 resulted in
a signicant decrease in the estimated tumor volume from day
10 onwards compared to the control group, with D-1 being more
potent than racemic 1 (Fig. 5a and b). There were no signs of
gross toxicity or statistically signicant weight loss of the
treated mice over the course of the experiment (Fig. 5c).
Fig. 4 The effect of compound 1 and its enantiomers (D-1 and L-1) on
the Ras/Raf/MEK/ERK signaling pathway. (a) Compound 1 and its
enantiomers (L-1 and D-1) (5 mM) down-regulate the phosphorylation
of MEK and ERK, but have no significant effect on total MEK and ERK
content in A498 cells. (b) Racemic 1 and D-1 (5 mM) attenuate the
transcriptional activity of AP-1 in a dose-dependent manner. Significantly different from control at *p < 0.05, **p < 0.005, ***p < 0.001,
and ****p < 0.0001.
(MDA-MB-231, MCF7, T47D, and MCF10A), human lung cancer
cells (A549 and H1299), human melanoma cancer cells (A375),
human prostatic cancer cells (DU145), human ovarian cancer
cells (A2780), and human erythroleukemic cancer cells (K562)
using the MTT assay (Table S1†). We found that our lead
compound was most toxic towards A498 cells (IC50 of D-1: 9.2
1.1 mM) harboring mutant H-Ras and K-Ras oncogenes and
showed a similar toxicity towards H1299 cells (IC50 of D-1: 10.3
1.2 mM) harboring a mutant K-Ras oncogene. Additionally,
the lead compound exhibited moderate cytotoxicity (IC50 of D-1:
>18 mM) towards A431 cells harboring the activated H-Ras
oncogene, DU145 cells harboring the wild-type Ras, and
A2780 cells harboring K-Ras oncogenic activation. However, the
compound exhibited similarly modest cytotoxicity towards
T47D cells and A375 cells that do not have activated H-Ras
oncogenes. We reason that the anti-proliferative activity of D-1
might be due, at least in part, to the disruption of the H-Ras/Raf-
4760 | Chem. Sci., 2017, 8, 4756–4763
Fig. 5 The effects of racemic 1 and D-1 in a kidney cancer xenograft
model. Mice harboring A498 (human kidney cancer) tumors were
injected with vehicle or with 1 or D-1 (14 mg kg1) four times a week.
(a) Photographs of control and treatment mice after 30 days. (b)
Average tumor volumes of control group and treatment group over
the measurement period. (c) Changes of body weight over the
measurement period. Significantly different at *p < 0.05, **p < 0.005,
***p < 0.001, and ****p < 0.0001.
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Furthermore, both racemic 1 and D-1 inhibited the phosphorylation of MEK and ERK in vivo, without affecting their total
protein expression (Fig. 6a). Moreover, racemic 1 and D-1
induced the expression of the apoptotic markers caspase 3, 6,
and 9 in vivo, suggesting that they could induce apoptosis of
tumor tissues (Fig. 6b).
Conclusions
This study has identied the iridium(III) compound 1 as the
rst kinetically-inert organometallic inhibitor of the H-Ras/
Raf-1 PPI. Compound 1 was identied using a preliminary
BiFC screening campaign of an in-house library of 15 iridium(III) and rhodium(III) compounds (1–15). A further 19
compounds (16–34) were synthesized and tested to explore the
SAR surrounding compound 1, and this revealed that the
combination of the dnbpy N^N ligand and tpy C^N ligands
together with the iridium(III) center were important for
achieving effective inhibition of the H-Ras/Raf-1 PPI. Moreover, the two enantiomers (L and D) of compound 1 were
isolated for individual evaluation. In addition to using the
BiFC assay, we also performed a pull-down experiment to
demonstrate that racemic 1 and D-1 could target the H-Ras/
Raf-1 PPI in cells. Furthermore, the domain pull-down assay
and domain CETSA assay indicated that racemic 1 and D-1
showed selective binding to Raf-1-RBD over Raf-1-CRD. As the
H-Ras/Raf-1 PPI is a crucial signaling hub of the Ras/Raf/MEK/
ERK signaling pathway, disrupting this PPI can have knock-on
effects on downstream mediators of the pathway. In this study,
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we demonstrated that compound 1 inhibited the phosphorylation of MEK and ERK and also attenuated the transcriptional
activity of AP-1, which we presume is due to its inhibition of
the H-Ras/Raf-1 PPI. Furthermore, the specicity of compound
D-1 was highlighted by CETSA, which revealed that compound
D-1 directly engaged H-Ras and Raf-1-RBD even in the
complicated biological environment of cell lysates, whereas it
had no effect on GAPDH. Finally, we demonstrated the ability
of racemic 1 and D-1 to suppress tumor growth in a mouse
xenogra model of human kidney cancer without causing
weight loss or overt signs of toxicity to mice over 30 days.
Compound 1 also upregulated pro-apoptotic caspase activity
in vivo, thereby providing a probable mechanism for the anticancer activity of compound 1.
Overall, these ndings highlight the exciting potential of
iridium(III) compounds to be developed as effective Ras/Raf
PPI inhibitors for the treatment of kidney cancer. Future
work will explore the structural basis of Ras/Raf inhibition by 1
using techniques such as X-ray crystallography or molecular
modelling, and subsequent structure-based optimization
could drive the potency of the compounds from the micromolar down to the nanomolar range. Previous research groups
have shown that the two enantiomers of a metal complex can
show markedly different biological activities.42–44 However,
this work is the rst to demonstrate that two enantiomers of
iridium(III)-based metal compounds can show different biological activities both in vitro and in vivo, which is an important consideration for progressing organometallic compounds
towards drug-like status. We envisage that iridium(III)
compound 1, as the rst metal-based Ras/Raf inhibitor reported in the literature, could potentially be used as a starting
scaffold to develop more potent metal-based Ras/Raf PPI
inhibitors, including targeting the Ras-binding domain of Raf1, for the treatment of kidney cancer or other proliferative
diseases.
Experimental
Biomolecular uorescence complementation (BiFC) assay
The effect of racemic 1 and D-1 on the phosphorylation of MEK
and ERK and expression of caspase 3, 6, and 9 in a kidney cancer
xenograft model. Mice harboring A498 (human kidney cancer) tumors
were injected with vehicle or with 1 or D-1 (14 mg kg1) four times
a week. (a) Racemic 1 and its enantiomer D-1 inhibit the phosphorylation of MEK and ERK. (b) Racemic 1 and its enantiomer D-1 induce
caspase 3, 6, and 9 expression.
Fig. 6
This journal is © The Royal Society of Chemistry 2017
The BiFC assay was performed in order to investigate the
inhibition of the H-Ras/Raf-1 interaction by the compounds.
HEK293T and A498 cells co-transfected with pEGFP-VC-HRasV12 and pEGFP-VN-Raf-1-RBD by TurboFect transfection
reagent were seeded in a 6-well plate with a serum-free
DMEM medium and incubated for 6 h. The cells were then
plated in a complete medium for additional 24 h. Aer
formulating the concentration of each compound to 5 mM,
the cells were placed in 8-well chamber slides and treated with
the compounds for 6 h. Later, the cells were xed with 4%
paraformaldehyde (PFA) for 15 min at room temperature, and
were washed with phosphate-buffered saline (PBS) buffer
three times. Aer 5 min incubation with Hoechst 33342
(10 mg mL1, 1 : 2000 dilution) at room temperature, the
cells were imaged using a Leica TCS SP8 confocal microscope. Fluorescence intensity was determined using ImageJ
soware.
Chem. Sci., 2017, 8, 4756–4763 | 4761
View Article Online
Chemical Science
Cellular thermal shi assay (CETSA)
2 10 A489 cells were lysed and collected. 240 mg of cell lysates
were diluted and allocated in same aliquots (160 mL) with PBS.
Each lysate was treated with 5 mM of compounds for 30 min at
room temperature and was then divided into 8 aliquots of 20 mL
and placed in separate PCR tubes. The PCR tubes were heated
individually at different temperatures ranging from 47 C to
68 C (Applied Biosystems 7500, Life Technologies). The heated
lysates were then centrifuged for 5 min at 13 000g and the
supernatants were subjected to SDS-PAGE followed by immunoblotting with anti-Raf and anti-Ras antibodies (Abcam,
1 : 1000 dilution).
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6
Animal Materials
In this study, the use of animals complied with the Guiding
Principles in the Care and Use of Animals of the American
Physiology Society and was approved by the Animal Care and
Use Committee at the National Kaohsiung Medical University.
Female BALB/cAnN.Cg-Foxn1nu/CrlNarl (4–5 weeks) were
purchased from the BioLASCO Experimental Animal Center
(Taiwan Co., Ltd). The mice were housed in Plexiglas cages in
a temperature-controlled room (22 1 C), on a 12 h/12 h light/
dark schedule, and with free access to food and water. Aer one
week, the mice were randomly divided into control and treatment groups.
Xenogra tumor assay
Female BALB/cAnN.Cg-Foxn1nu/CrlNarl were housed and tested
at the animal center (Kaohsiung Medical University, Kaohsiung,
Taiwan). Mice were implanted subcutaneously with 1 107
A498 cells in 0.1 mL PBS. Aer the establishment of palpable
tumors (the mean tumor volume was around 150–200 mm3),
mice were treated four times a week with a intraperitoneal (i.p.)
injection of compounds (14 mg kg1) or vehicle (13% DMSO) in
0.05 mL PBS until sacrice at 30 day. The diameters of xenogra
tumors were measured at 3 day intervals with vernier calipers,
and the tumor volume (in mm3) was calculated using the
formula: volume ¼ length width2/2. The treatment and
control groups each contained 6 mice.
Additional information on materials, synthesis of
compounds, plasmid construction, pull-down assay, luciferase
reporter assay, immunoblot analysis, and MTT assay is provided
in the ESI.†
Acknowledgements
This work is supported by the Hong Kong Baptist University
(FRG2/15-16/002), the Health and Medical Research Fund
(HMRF/14130522), the Research Grants Council (HKBU/
12301115, HKBU/204612, and HKBU/201913), the National
Natural Science Foundation of China (21575121), the
Guangdong
Province
Natural
Science
Foundation
(2015A030313816), the Hong Kong Baptist University Century
Club Sponsorship Scheme 2016, the Interdisciplinary
Research Matching Scheme (RC-IRMS/15-16/03), the Science
and Technology Development Fund, Macao SAR (098/2014/A2),
4762 | Chem. Sci., 2017, 8, 4756–4763
Edge Article
the University of Macau (MYRG2015-00137-ICMS-QRCM,
MYRG2016-00151-ICMS-QRCM and MRG044/LCH/2015/ICMS),
the National Natural Science Foundation of China (21628502),
and Ministry of Science and Technology (MOST 105-2622-E005-006-CC2; MOST 104-2221-E-005-096-MY2; and MOST 1042628-E-005-004-MY3). We thank Prof. Kazumasa Ohashi of the
Department of Biomolecular Sciences, Graduate School of Life
Sciences for the gis of the BiFC plasmids.
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