A silver complex of N,N′-disubstituted cyclic thiourea as an anti-inflammatory inhibitor of IκB kinase

Article abstract of DOI:10.1039/c3cc00063j

A silver complex of N,N′-disubstituted cyclic thiourea inhibits inflammatory cytokine-stimulated NF-κB activity via IκB kinase inactivation. The Royal Society of Chemistry 2013.

Full text of DOI:10.1039/c3cc00063j

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A silver complex of N,N0-disubstituted cyclic thiourea
as an anti-inflammatory inhibitor of IjB kinase†
Cite this: Chem. Commun., 2013,
49, 3297
Iris Wing-Shan Lin, Chun-Nam Lok, Kun Yan and Chi-Ming Che*
Received 4th January 2013,
Accepted 27th February 2013
DOI: 10.1039/c3cc00063j
www.rsc.org/chemcomm
A silver complex of N,N0-disubstituted cyclic thiourea inhibits inflam- an inhibitory constant at the nanomolar level, and was found to show
matory cytokine-stimulated NF-jB activity via IjB kinase inactivation.
moderate in vivo anti-tumour activities in mice. In this work,
we report that the silver thiourea complex AgTU (Fig. 1) displays
The prominent biomedical applications of silver are owing to its anti-inflammatory properties by effective and potent inhibition of the
extraordinary antimicrobial properties and its relatively low toxicity to inflammatory cytokine-stimulated activity of the transcription factors
humans when compared to that normally associated with many heavy NF-kB, which control the expression of diversified genes involved in
metals.1 Thus, silver based materials are used as some of the most inflammatory response and cancer progression.5 In many tumor
effective biocidal agents in burn wound treatment (e.g. silver sulfadi- cells, NF-kB is active and blockade of NF-kB leads to inhibition of cell
azine, silver dressing) and antimicrobial coatings of critical bio- proliferation, cell death or sensitization to cytotoxic agents. In the
medical materials (e.g. silver impregnated catheters, bone cements literature, studies of the effects of metal compounds on NF-kB
and surgical sutures). Associated with the antimicrobial activities of activities are sparse.6 Auranofin6a and cyclometalated platinum(II)
silver compounds are their intriguing anti-inflammatory properties complexes6b have been shown to inhibit NF-kB activities. In this
that are favorable for wound healing and topical uses;2 however, these work, we have demonstrated for the first time the inhibition of NF-kB
long claimed properties of silver are not well understood and explored. via inactivation of the cellular IkB kinase by silver ions which are
The biological activities of silver compounds are mostly attributed delivered to cells via coordination ligands.
to the release of Ag+ ions which readily bind to thiol and imidazole
We have previously demonstrated that cellular uptake of
moieties. While Ag+ ions are susceptible to precipitation, nucleophilic silver in HeLa cancer cells mediated by AgTU is five times
attack and reduction in physiological media, the stability and activity higher than that by AgNO3 solution,4 suggesting that the TU
of Ag+ ions can be modulated by using appropriate auxiliary ligands. ligand serves as a lipophilic carrier of the metal ion to the cells.
In the literature, phosphines, carboxylates, coumarins, thiolates and Once inside the cells, one of the fates of Ag+ ions is determined
N-heterocyclic carbenes have been employed to develop bioactive by their susceptibility to nucleophilic attack by reactive thiols
silver complexes that display cytotoxicities to microbial or cancer such as reduced glutathione (GSH). The reactivity of AgTU in
cells.3 Some silver carbene complexes have been shown to be active in the presence of GSH was investigated by ESI-MS. In a 50 mM
mice models of cancer3c and bacterial infection.3b We have recently ammonium bicarbonate solution (pH 8.0), mixing of 100 mM
developed bioactive homoleptic coinage metal thiourea complexes AgTU (m/z = 737.0 for Ag(TU)2+ and m/z = 423.2 for Ag(TU)+) with
([AuI(TU)2]Cl (AuTU), [AgI(TU)2]OTf (AgTU) and [CuI(TU)2]PF6 (CuTU), a five-fold excess of GSH (m/z = 308.1) did not result in
where TU = N,N0-disubstituted imidazolidine-2-thione) with mono- appearance of new species in the mass spectrum up to 3 h,
dentate S-donor ligands.4 The TU ligand serves as a lipophilic carrier suggesting that AgTU is reasonably stable toward GSH (Fig. S1A
of the metal ion to cells and does not exert cytotoxicity. We have and S1B, ESI†). In comparison, mixing of the gold thiourea
demonstrated that the AuTU complex exhibits potent tight-binding complex AuTU with GSH under similar conditions resulted in
ꢀ
inhibition of the anti-cancer drug target thioredoxin reductase with the ready formation of Au(GS)2 (m/z = 809.3) (Fig. S1C, ESI†).
Department of Chemistry, State Key Laboratory of Synthetic Chemistry, HKU
Shenzhen Institute of Research and Innovation, 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; Fax: +852 2857 1586; Tel: +852 2857 2154
† Electronic supplementary information (ESI) available: Synthesis and character-
ization of silver complexes; detailed biological experimental procedures; supple-
Fig. 1 Chemical structure of AgTU.
mental figures and tables. See DOI: 10.1039/c3cc00063j
c
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Fig. 2 Inhibition of TNF-a stimulated NF-kB activity by AgTU. (A) HeLa cells trans-
fected with the NF-kB-luciferase reporter gene were treated with AgTU or AgNO3 for
2 h, followed by TNF-a stimulation for 4 h and the cellular luciferase activities were
determined. (B) The cells were treated with 10 mM AuTU, AgTU, CuTU or TU for 2 h,
followed by TNF-a stimulation and the cellular luciferase activities were determined.
Fig. 3 The NF-kB pathway and inhibition of IKK by AgTU.
The nuclear factor kappa B (NF-kB) transcription factors control
the expression of genes involved in cell survival, proliferation and
inflammation.5 Thus, NF-kB is an important anti-inflammatory and
anticancer drug target. In this work, we have investigated the effects of
AgTU on NF-kB transcriptional activity using HeLa cells stably
transfected with the luciferase reporter gene of NF-kB (experimental
procedures described in ESI†). Upon stimulation by TNF-a, the NF-kB
transcriptional activity as measured by luciferase assay was increased
by at least B8 fold (Fig. 2A). Pretreatment of cells with AgTU for 2 h
markedly inhibited the TNF-a stimulated NF-kB activity with an IC50
of about 5 mM and exerted maximal inhibition of 80% at 10 mM
(Fig. 2A). For comparison, several metal complexes and ligands were
also tested. Under identical assay conditions, the free TU ligand, AuTU
and CuTU added at 10 mM did not show a significant effect on the
TNF-a-stimulated NF-kB activity (Fig. 2B). A homoleptic Ag complex of
the N-heterocyclic carbene complex ([AgI(NHC)2]OTf, where NHC =
1,3-dimethylimidazol-2-ylidene) shows a weak inhibitory effect on the
NF-kB activity (Fig. S2A, ESI†). It is notable that treatment of cells with
AgNO3 only moderately inhibited the stimulated NF-kB activity by at
most 50% at concentration up to 100 mM (Fig. 2A). Thus, AgTU
potently inhibits the cytokine-stimulated NF-kB activity, presumably
via the TU ligand-mediated delivery of Ag+. NF-kB activity can be
stimulated by a number of cytokines and tumor promoters via
Fig. 4 Inhibition of TNF-a-stimulated IkBa phosphorylation, IkBa degradation
and NF-kB p65 phosphorylation by AgTU. HeLa cells were treated with or
without 10 mM AgTU for 2 h followed by TNF-a stimulation for 10 min as
indicated. The phosphorylation of IkBa (A) and NF-kB p65 (S536) (B) and
expression of IkBa were determined by immunoblot analysis. (C) Nuclear trans-
location of p65 was determined by immunofluorescence.
different upstream cell receptor signaling. In addition to inhibition phosphorylation at S536, which could also be mediated by IKK
of the NF-kB activity stimulated by TNF-a, AgTU treatment also (Fig. 4B).5c Thus these data altogether suggest that AgTU effectively
effectively inhibited the NF-kB activity induced by the tumor promoter blocks the NF-kB activity mainly by inhibiting IKK. A role of direct
phorbol ester (Fig. S3, ESI†). This implies that AgTU may act on a inhibition of proteasomal degradation of IkB seems less likely, as
converging molecular component downstream to the receptor AgTU was found to be a relatively weak inhibitor of the ubiquitin–
stimulation in the NF-kB pathway.
AschemeoftheNF-kB pathway is depicted in Fig. 3.5c Acriticalstep ylation even in the presence of a proteasome inhibitor (Fig. S5, ESI†).
in the activation of NF-kB is the phosphorylation of IkB by the IkB The consequence of AgTU inhibited IKK-mediated IkB degrada-
proteasome system7 (Fig. S4, ESI†) and it inhibited the IkB phosphor-
kinase (IKK) complex. This process results in proteasomal degrada- tion has also been revealed by the following experiments: treatment
tion of IkB and release of NF-kB to the nucleus, thereby inducing of cells with AgTU blocked the translocation of p65 from the
inflammatory gene transcription. In this work, the effect of AgTU on cytoplasm to the nucleus triggered by TNF-a (Fig. 4C). AgTU also
the IKK activation has been investigated. As depicted in Fig. 4A, TNF-a suppressed the TNF-a-stimulated transcriptional upregulation of
markedly stimulated the phosphorylation of IkB, which is in con- inflammatory cytokines such as IL-6, IL-8 and TNF-a (Fig. S6, ESI†).
comitance with decrease of IkB levels. Treatment of cells with AgTU It was noted that treatment of cells with AgNO3 (50–100 mM) did not
markedly inhibited the stimulated IkB phosphorylation and pre- result in significant inhibition of TNF-a stimulated responses includ-
vented the reduction of IkB levels (Fig. 4A). The observed concen- ing IkB phosphorylation and degradation, p65 phosphorylation and
tration dependence of the AgTU blockade of IkB degradation (Fig. 4B) nuclear translocation, and cytokine induction (Fig. S6–S8, ESI†).
is also in accord with that of inhibition of stimulated NF-kB
The effect of AgTU on the IKK was also investigated by an in vitro
activity (Fig. 2). AgTU treatment also resulted in decreased p65 kinase assay (Fig. 5). The IKK complex was immunoprecipitated
c
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also investigated whether additional mechanisms of IKK inhi-
bition by AgTU treatment may operate at the IKK complex
assembly stage (Fig. S9, ESI†). In IKKb transfected cells, AgTU
treatment resulted in distinct appearance of a high-molecular-
weight protein band (200 kDa) that could be attributed to an
IKKb dimer as indicated by its mobility on the SDS–polyacrylamide
gel (Fig. S9A, ESI†). Furthermore, when cells were treated with
AgTU and the IKK complexes were immunoprecipitated followed
by immunoblot detection of the IKK subunits, a loss of the
heteromeric assembly of the three IKK subunits was observed
(Fig. S9B, ESI†). Taken together, AgTU inhibits IKK kinase activity
via intervention of the IKK complexation.
In summary, we have identified AgTU to be a potent NF-kB
inhibitor acting through suppression of IKK activity via thiol
modifications and interference of the IKK complex assembly.
Our study demonstrated that the inhibitory effect of Ag+ ions on
an anti-inflammatory and anti-cancer drug target could be
effectively delivered via a non-toxic thiourea ligand.
Fig. 5 Inhibition of in vitro IKK activities by AgTU and AgNO3. The immunopre-
cipitated IKK complex from HeLa cells was treated with AgTU or AgNO3 in the
presence or absence of 1 mM GSH, and the kinase activities were assayed using
IkBa–GST as a substrate.
with IKKg antibody and the kinase activity was assayed with IkB–
GST fusion protein as the phosphorylation substrate. Incubation of
the kinase with 1–10 mM AgTU resulted in concentration-dependent
inhibition of the phosphorylation of the IkB substrate. To examine
the effect of the thiol moiety, which is abundant in the intracellular
environment, the assays were performed in the presence of 1 mM
GSH buffered in 10 mM Tris–HCl buffer (pH 8.0). The presence of
GSH partially reduced the inhibition of the IKK activity by AgTU but
significant inhibition was still observed when the GSH was 100-fold
excess than AgTU (10 mM). Under the same assay conditions, AgNO3
also inhibited the in vitro IKK activity and the inhibition was
partially blocked by excess GSH (Fig. 5). As AgNO3 did not show
inhibition of cellular IKK activity (Fig. S7, ESI†), it is likely that AgTU
delivers otherwise less cell-permeable Ag+ ions to mediate specific
inhibition of IKK.
AgTU may inhibit the IKK presumably via inactivation of func-
tionally important cysteine residues of IKK.8 To examine whether
cysteine residues are involved in the AgTU mediated inhibition of
IKK, the free cysteines of the IKK complex after treatment with or
without AgTU were probed with biotinylated iodoacetamide (BIAM)
using previously described procedures (Fig. 6).4,8b,c The results show
that the IKK subunits (a, b, g) can be efficiently labelled by BIAM.
The labelling was more efficient at pH 8.5 than at pH 6.5, owing to
the fact that typical pKa values of most protein cysteine sulfhydryl
residues are near the alkaline range.8c Upon treatment of IKK with
AgTU or AgNO3, a marked inhibition of the BIAM labelling of the
IKKb subunit was observed (Fig. 6). These results suggest that Ag+
ion modifies the sensitive cysteine residues of IKK.
This work was supported by the Innovation and Technology
Fund (ITF-Tier 2, ITS/134/09FP) administered by the Innovation
and Technology Commission, HKSAR, and the National Key Basic
Research Program of China (No. 2013CB834802), P.R. China.
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Fig. 6 Probing the sensitive cysteines of IKK. The IKK complex isolated by
immunoprecipitation was treated with 10 mM AgTU or 10 mM AgNO3 and the
sensitive cysteine residues were detected by BIAM labeling at pH 6.5 and pH 8.5.
The IKK subunits were detected as loading control by immunoblot.
c
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Chem. Commun., 2013, 49, 3297--3299 3299