An anticancer gold(III)-activated porphyrin scaffold that covalently modifies protein cysteine thiols

Article abstract of DOI:10.1073/pnas.1915202117

Cysteine thiols of many cancer-associated proteins are attractive targets of anticancer agents. Herein, we unequivocally demonstrate a distinct thiol-targeting property of gold(III) mesoporphyrin IX dimethyl ester (AuMesoIX) and its anticancer activities. While the binding of cysteine thiols with metal complexes usually occurs via M-S bond formation, AuMesoIX is unique in that the mesocarbon atom of the porphyrin ring is activated by the gold(III) ion to undergo nucleophilic aromatic substitution with thiols. AuMesoIX was shown to modify reactive cysteine residues and inhibit the activities of anticancer protein targets including thioredoxin, peroxiredoxin, and deubiquitinases. Treatment of cancer cells with AuMesoIX resulted in the formation of gold-bound sulfur-rich protein aggregates, oxidative stress-mediated cytotoxicity, and accumulation of ubiquitinated proteins. Importantly, AuMesoIX exhibited effective antitumor activity in mice. Our study has uncovered a gold(III)-induced ligand scaffold reactivity for thiol targeting that can be exploited for anticancer applications.

Full text of DOI:10.1073/pnas.1915202117

An anticancer gold(III)-activated porphyrin scaffold
that covalently modifies protein cysteine thiols
Ka-Chung Tonga,b,1, Chun-Nam Loka,b,1, Pui-Ki Wana,b, Di Hua,b, Yi Man Eva Funga,b, Xiao-Yong Changa, Song Huangc,

Haibo Jiangc, and Chi-Ming Chea,b,2
aState Key Laboratory of Synthetic Chemistry, Department of Chemistry, The University of Hong Kong, Hong Kong, China; bLaboratory for Synthetic
Chemistry and Chemical Biology, Health@InnoHK, Hong Kong, China; and cSchool of Molecular Sciences, The University of Western Australia, Perth, WA
6009, Australia
Contributed by Chi-Ming Che, December 3, 2019 (sent for review September 3, 2019; reviewed by Harry B. Gray, Yi Lu, and Peter J. Sadler)
Cysteine thiols of many cancer-associated proteins are attractive
targets of anticancer agents. Herein, we unequivocally demon-
strate a distinct thiol-targeting property of gold(III) mesoporphyrin
IX dimethyl ester (AuMesoIX) and its anticancer activities. While
the binding of cysteine thiols with metal complexes usually occurs
via M–S bond formation, AuMesoIX is unique in that the meso-
carbon atom of the porphyrin ring is activated by the gold(III) ion
to undergo nucleophilic aromatic substitution with thiols. AuMesoIX
was shown to modify reactive cysteine residues and inhibit the
activities of anticancer protein targets including thioredoxin,
peroxiredoxin, and deubiquitinases. Treatment of cancer cells with
AuMesoIX resulted in the formation of gold-bound sulfur-rich pro-
tein aggregates, oxidative stress-mediated cytotoxicity, and accu-
mulation of ubiquitinated proteins. Importantly, AuMesoIX exhibited
effective antitumor activity in mice. Our study has uncovered a
gold(III)-induced ligand scaffold reactivity for thiol targeting that
can be exploited for anticancer applications.
anticancer biomolecular targets. Nonetheless, such interaction has
not been observed. A less commonly known aspect is the activa-
tion of the reactivity of the porphyrin ligand of metalloporphyrins
for nucleophilic attack/substitution reactions, as revealed in early
works on the reactivity of rhodium(III) octaethylporphyrin (31)
and antimony(V) porphyrins (octaethylporphyrin and tetraphe-
nylporphyrin) (32) as well as the functionalization of gold(III)
meso-tetraphenylporphyrin (33) via nucleophilic reactions on the
meso-carbon atom of the porphyrin ligand. In a biological context,
the heme moiety in natural or engineered hemoproteins is in-
herently reactive through the iron center to mediate the formation
of covalent links of vinyl, methyl, or meso-carbons with nucleo-
philic amino acids (34). Given the strong inductive effect of the
gold(III) ion, we are intrigued by the possibility that meso-
unsubstituted gold(III) porphyrins can react with various nucleo-
philic substrates in biological systems, particularly those containing
thiol groups.
Cysteine thiols are nucleophiles that are abundant in cells and
play important roles in enzyme catalysis, signal transduction, and
cellular redox regulation (35, 36). Cellular thiols from protein
gold porphyrin ligand reactivity cysteine thiol conjugation
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antitumor agents
ransition metal complexes can be developed to display ef-
fective anticancer therapeutic properties by the judicious
Significance
T
choice/combination of metal ions and coordination ligands (1–8).
The mechanisms of cytotoxic action of many anticancer metal
complexes are usually attributable to 3 modes: 1) the coordination
of metal ions to biomolecules via ligand exchange reactions, with
notable examples being the formation of the platinum–DNA ad-
ducts of cisplatin (1) and gold–thiol/selenol adducts of gold(I)
phosphine compounds such as auranofin (9). 2) Metal complexes
with metal ions supported by strong donor ligands can also be used
as stable scaffolds to noncovalently interact with biomolecules.
Thus, substitutionally inert metal complexes of platinum (10), gold
(11), rhodium (12, 13), ruthenium, and iridium (14) have been
developed to target protein chaperones, transporters, cytoskele-
tons, protein kinases, and DNA as well as mismatched base pairs
via supramolecular noncovalent interactions. 3) Redox-active or-
ganometallic complexes of ruthenium, iridium, and osmium have
been developed to mediate anticancer cytotoxicity via intracellular
catalytic oxidation/reduction processes (15). Nonetheless, metal
complexes with coordinated ligands that covalently bind antican-
cer biomolecular targets and display favorable anticancer and
antitumor properties have not yet been described.
We have a longstanding interest in anticancer gold compounds
that have been historically known to display potent cytotoxicity
in vitro and in vivo (16–23). The gold(III) ion is generally unstable
in solution but can be stabilized by the use of strong σ-donor li-
gands, affording stable structures for specific biomolecular inter-
actions and targeting (11, 24–26). We have developed antitumor
gold(III) porphyrins, as exemplified by stable gold(III) meso-
tetraphenylporphyrin that specifically targets a mitochondrial
chaperone protein, likely through noncovalent interactions (24,
27–30). The initial objective of the use of a porphyrin ligand was to
stabilize the gold(III) ion and to allow Au(III)–S interaction with
A previously unknown biomolecular binding interaction of
metal complexes is uncovered. The periphery of coordinated
ligands is activated by the electrophilic metal center to selec-
tively form covalent adducts with cysteine thiols of cancer-
associated proteins. This is exemplified by gold(III) meso-
porphyrin IX, which we found to 1) form thiol adducts with
cysteines of thioredoxin, peroxiredoxin, and deubiquitinases
via the meso-carbon atom of the porphyrin ligand; 2) target
cellular thiol proteins, as revealed by correlative nanoSIMS and
EM imaging techniques as well as thermal proteome profiling;
and 3) display effective in vitro and in vivo anticancer activity.
It is envisaged that metal coordination complexes with ligand
periphery active toward nucleophilic substitution–addition can
serve as new platforms for anticancer molecular targeting.
Author contributions: C.-M.C. designed research; K.-C.T., P.-K.W., D.H., Y.M.E.F., X.-Y.C.,
and S.H. performed research; K.-C.T., C.-N.L., P.-K.W., D.H., Y.M.E.F., X.-Y.C., S.H., H.J., and
C.-M.C. analyzed data; and K.-C.T., C.-N.L., and C.-M.C. wrote the paper.
Reviewers: H.B.G., California Institute of Technology; Y.L., University of Illinois at Ur-
bana–Champaign; and P.J.S., University of Warwick.
The authors declare no competing interest.
Published under the PNAS license.
Data deposition: The X-ray crystallographic data have been deposited in the Cambridge
Crystallographic Data Centre (CCDC), https://www.ccdc.cam.ac.uk/ (accession nos. 1919615
[AuMesoIX] and 1919616 [AuOEP]). The mass spectrometry proteomics data have been
deposited to the ProteomeXchange Consortium via the PRIDE partner repository, https://
www.ebi.ac.uk/pride/archive/ (ID code PXD016875).
1K.-C.T. and C.-N.L. contributed equally to this work.
2To whom correspondence may be addressed. Email: cmche@hku.hk.
This article contains supporting information online at https://www.pnas.org/lookup/suppl/
doi:10.1073/pnas.1915202117/-/DCSupplemental.
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cysteines also represent a large redox-active pool directly in-
volved in the defense against oxidative stress, which may be
targeted to achieve cytotoxic effects (37, 38). Furthermore, the
functional cysteine thiols of many cancer-driving proteins are
appealing anticancer molecular targets (39). Herein, the in-
teractions with cellular thiols and the anticancer activities of
the gold(III) porphyrin complex AuMesoIX bearing a meso-
unsubstituted mesoporphyrin IX dimethyl ester ligand are de-
scribed. The electrophilic methine group of AuMesoIX is capa-
ble of forming covalent thiolato adducts with protein thiols
through nucleophilic aromatic substitution. These findings un-
cover a biochemical feature of metal complexes via metal-
induced ligand reactivity that could be harnessed for anticancer
applications.
with a distinct mass peak at m/z 1,094.3396 (z = +1) attributable to
[AuMesoIX–GSH]+ (Fig. 2A and SI Appendix, Fig. S5). Likewise,
a new species of m/z 1,034.3918 (z = +1) consistent with the
[AuOEP–GSH]+ formulation was observed upon incubation of
AuOEP with GSH (SI Appendix, Figs. S6 and S7). Formation of
[AuMesoIX–GSH]+ and [AuOEP–GSH]+ adducts was also con-
firmed by matching the isotopic patterns with those of the simu-
lated ones and by tandem-MS analysis of the fragment ions (SI
Appendix, Figs. S5, S7–S9). No GSH-adduct was found for the
AuTPP and MesoIX ligand (SI Appendix, Figs. S10 and S11). In-
dividual amino acids (cysteine, glutamic acid, or glycine) of GSH
were incubated with AuMesoIX or AuOEP and only the formation
of adducts (m/z 908.2755 or 848.3202; z = +1; SI Appendix, Figs.
S12 and S13) between the gold complexes and cysteine was ob-
served in MS analysis.
The chemical structure of the gold(III) porphyrin-thiol adduct
was also examined by NMR spectroscopy. Regarding the possi-
ble regioisomeric adducts that could be derived from AuMesoIX
at different methine groups, symmetric AuOEP and GSH were
used for the structural analysis (Fig. 2 B and C and SI Appendix,
Figs. S16–S20). The 1H signals at δ 8.8 to 9.9 ppm are indicative
of an asymmetric OEP porphyrin ring with 3 protons at different
meso-carbon atoms (Fig. 2B) and the 1H signals of α-CH and
β-CH2 of the cysteine residue were significantly shifted (SI Ap-
pendix, Fig. S16). Moreover, the 1H–13C heteronuclear multiple-
bond correlation spectroscopy (HMBC) spectrum showed cross-
peaks between the protons of β-CH2 of cysteine at 2.4 to 2.7 ppm
and one meso-carbon atom of OEP at 114 ppm (Fig. 2C). Taken
together, the findings reveal a covalent conjugation of the sulf-
hydryl group of the cysteine of GSH to the methine group of
octaethylporphyrin (Fig. 2E). The UV–vis absorption spectrum of
the [AuOEP–GSH]+ adduct shows a bathochromic shift (∼22 to
32 nm), weakening in intensity and band broadening of both the
Soret and Q bands when compared with that of AuOEP alone (SI
Appendix, Fig. S21). All of these spectral changes are indicative of
an increase in the distortion of the OEP macrocyclic ring.
The interaction between AuMesoIX and a small thiol protein,
thioredoxin (Trx), was studied by matrix-assisted laser desorption/
ionization (MALDI)–time-of-flight (TOF)–MS. Incubation of Trx
(molecular weight, 11,729.0 Da) with AuMesoIX resulted in the
emergence of a new mass peak at 12,515.7 Da (Fig. 2D and SI
Appendix, Fig. S22), consistent with formation of an adduct with a
mass of ∼787 Da comprising one AuMesoIX and Trx. Pretreat-
ment of Trx with the thiol-alkylating agent iodoacetamide prior to
incubation with AuMesoIX resulted in no adduct formation (SI
Appendix, Fig. S23). Purified and cellular Trx activities were
inhibited by AuMesoIX in a dose-dependent manner (SI Appendix,
Fig. S24).
Results
Synthesis and Characterization. Three gold(III) porphyrin complexes
[Au(Por)]Cl (Por = mesoporphyrin IX dimethyl ester [AuMesoIX],
octaethylporphyrin [AuOEP], and meso-tetraphenylporphyrin
[AuTPP]) were synthesized in 40 to 53% isolated yield (Fig. 1).
Experimental procedures and characterization data of the com-
plexes are detailed in SI Appendix. The crystal structures of
AuMesoIX and AuOEP show the average Au–N distances being
2.029 and 2.025 Å, respectively (SI Appendix, Fig. S1 and Table S1).
The porphyrin planes of both complexes do not show significant
ruffling or saddling deformation. The UV–vis absorption spectra of
AuMesoIX and AuOEP feature a hypsochromic shift of 10 to 30 nm
for both the Soret and Q bands compared with those of the ab-
sorption maxima of AuTPP (SI Appendix, Fig. S2A). A similar trend
was also observed for the zinc(II) complexes (SI Appendix, Fig.
S2B), which can be attributed to the smaller degree of porphyrin
macrocycle distortion in complexes with MesoIX and OEP ligands
compared to that in AuTPP and ZnTPP, the porphyrin scaffolds of
which have meso-phenyl substituents.
Reactivity of Meso-Unsubstituted Gold(III) Porphyrin with Cysteine
Thiols. The 3 gold(III) porphyrin complexes were stable in bi-
ological buffer solutions as revealed by no change in the absorp-
tion spectra for a day (SI Appendix, Fig. S3). However, upon
incubation with excess glutathione (GSH) for 24 h, a new ab-
sorption band at 430 nm was observed for AuMesoIX and AuOEP
but not for AuTPP (SI Appendix, Fig. S4). Ultraperformance liquid
chromatography (UPLC)–quadrupole time-of-flight (QTOF)–
mass spectrometry (MS) analysis showed that incubation of
AuMesoIX with GSH resulted in the formation of a new species
The UV–vis absorption spectroscopy, NMR, and MS data
allowed us to identify a nucleophilic aromatic substitution re-
action between the meso-carbon atom of meso-unsubstituted
gold(III) porphyrins and cysteine thiols (Fig. 2E).
In Vitro Anticancer Activity. All of the gold(III) porphyrin com-
plexes were remarkably cytotoxic in a panel of lung, colon, liver,
breast, and ovarian cancer cell lines (half-maximal inhibitory
concentration [IC50]: <1 μM; Table 1 and SI Appendix, Fig. S25).
The corresponding zinc(II) porphyrins were almost inactive
(IC50: >100 μM). AuMesoIX demonstrated a strong cytotoxic
effect against both cisplatin-resistant A2780cis ovarian cancer
cells (IC50: 0.12 μM) and the cisplatin-sensitive parental cells
(IC50: 0.06 μM). AuMesoIX displayed a higher cytotoxicity in
HCT116 colon cancer and NCI-H460 lung cancer cells compared
to noncancerous colon epithelial cells (NCM460) and normal
lung fibroblasts (CCD-19Lu) with 25- and 10-fold differences in
IC50 values, respectively. AuMesoIX, but not the other 2 gold
porphyrins, completely inhibited the clonogenic growth of A2780
ovarian cancer cells (Fig. 3A and SI Appendix, Fig. S26). AuMesoIX
Fig. 1. Chemical structures of gold(III) and zinc(II) porphyrin complexes.
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Fig. 2. Cysteine thiol conjugation by meso-unsubstituted gold(III) porphyrins. (A) UPLC-QTOF-MS analysis of the thiolato adduct for the parent ion of
[AuMesoIX–GSH)]+ at a retention time of 4.13 min. (B) 1H and (C) 1H–13C HMBC NMR spectra of [AuOEP–GSH)]+. (D) MALDI-TOF-MS spectra of thioredoxin
(Trx) before and after reaction with AuMesoIX. (E) Schematic reaction for the bioconjugation of meso-unsubstituted gold(III) porphyrins with GSH under
physiological conditions.
induced cell cycle arrest in the G0/G1 phase in A2780 cells (Fig. 3B)
and triggered apoptosis (Fig. 3C). Moreover, AuMesoIX exhibited
in vitro antiangiogenic activity (Fig. 3 D and E) and displayed
antimetastatic activity in breast cancer cells (Fig. 3F).
with m/z 775.2232 and an isotopic pattern matching that of
hydrolyzed AuMesoIX with monopropionic acid (SI Appendix,
Fig. S30), suggesting intracellular retention of AuMesoIX in the
carboxylated form.
To examine the interaction of AuMesoIX with cellular com-
ponents, nanoscale secondary ion mass spectrometry (nano-
SIMS) was employed to image the intracellular elemental
distribution (40, 41). NanoSIMS allows the simultaneous de-
tection of multiple elements inside cells at high spatial resolution
(50 nm). In combination with electron microscopy (EM), the
correlative imaging allows the subcellular localization of added
metal compounds to be visualized. As depicted in Fig. 4A and SI
Appendix, Fig. S31A, the 197Au map of AuMesoIX-treated A2780
ovarian cancer cells clearly showed the presence of gold in the
form of small aggregates in the cytosol. The region of 197Au
aggregates was also colocalized with signals with high 32S/12C14N
ratio, which could be attributed to sulfur-rich proteins. In con-
trast, the 197Au map of AuTPP-treated cells showed diffuse gold
signals in the cytosol, and the association between the Au and S
signals was not significant (SI Appendix, Fig. S31C). Scanning
electron microscopy images of the same AuMesoIX-treated cells
revealed the presence of small electron-dense aggregates in the
mitochondria (Fig. 4B and SI Appendix, Fig. S31B). In particular,
the mitochondria of AuMesoIX-treated cells displayed a disrupted
Cellular Uptake of AuMesoIX and In Cellulo Gold–Sulfur Interaction
Revealed by Nanoscale Secondary Ion Mass Spectrometry. AuMesoIX
demonstrated rapid time- and dose-dependent cellular accu-
mulation in A2780 cancer cells through determining the gold
content by using inductively coupled plasma–MS (SI Appendix,
Fig. S27 and Table S2). While AuTPP and AuOEP have similar
lipophilicities as AuMesoIX (SI Appendix, Table S2), the uptake
of gold for AuMesoIX-treated cells was markedly higher than
that of the other 2 complexes. Such a difference was also ob-
served in other cancer and noncancer cell lines (SI Appendix, Fig.
S28). Notably, a lower gold content was found in AuMesoIX-
treated noncancer cells (CCD-19Lu fibroblasts and NCM460
cells) than in cancer cells (NCI-H460 lung and HCT116 colon
cancer). The uptake of gold for AuMesoIX was temperature
dependent with a significant reduction at low temperature (4 °C)
compared to that at 37 °C, but those of AuTPP and AuOEP dis-
played only minor temperature-dependent differences (SI Ap-
pendix, Fig. S29). Liquid chromatography–MS analysis of
AuMesoIX-treated cells revealed the appearance of a species
Table 1. In vitro cytotoxicity of gold(III) and zinc(II) porphyrin complexes and cisplatin on human cancerous and normal cell lines
IC50 values, μM; 72 h
A2780
(ovarian
cancer)
A2780cis
(ovarian cancer,
cisplatin-resistant)
NCI-H460
(lung
cancer)
MDA-MB-231
(breast
HepG2
(liver
cancer)
HCT116
(colon
cancer)
NCM460
(colon
epithelial)
CCD-19Lu
(lung
fibroblast)
Complex
cancer)
AuMesoIX
AuTPP
AuOEP
ZnMesoIX
ZnTPP
0.06 0.01
0.2 0.02
0.3 0.03
32.3 6.9
>100
0.1 0.01
0.1 0.01
0.3 0.03
>100
0.07 0.02
0.2 0.03
0.2 0.04
15.5 1.4
>100
0.4 0.02
0.5 0.03
0.7 0.05
84.3 9.6
>100
0.8 0.06
0.3 0.02
0.5 0.04
>100
0.06 0.01
0.20 0.02
0.3 0.02
10.2 1.2
>100
1.5 0.15
0.4 0.03
1.4 0.12
>100
0.7 0.04
0.4 0.04
0.5 0.03
>100
>100
>100
>100
>100
ZnOEP
>100
>100
>100
>100
>100
>100
>100
>100
Cisplatin
0.7 0.1
26.2 1.9
3.7 0.7
35.5 1.5
22.3 3.2
10.1 2.2
93.8 8.9
30.8 4.9
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Fig. 3. In vitro anticancer activities of AuMesoIX. (A) Clonogenic assay of A2780 cells incubated with AuMesoIX (1 μM) for 2 h followed by incubation in fresh
growth medium for 10 d. Flow-cytometric analyses of (B) the cell cycle and (C) apoptotic cell death of A2780 cells treated with 1 μM AuMesoIX for 24 h. (D) Tube
formation assay of MS-1 endothelial cells treated with AuMesoIX (1 μM) for 3 h. (Scale bar, 100 μm.) (E) Wound-healing assay of MS-1 cells incubated with AuMesoIX
(0.5 μM) for 24 h. (Scale bar, 100 μm.) (F) Matrigel invasion assay of MDA-MB-231 breast cancer cells treated with AuMesoIX (0.5 μM) for 24 h. (Scale bar, 40 μm.)
morphology along with disintegrated cristae, compared to that of
the untreated control (Fig. 4 B and C and SI Appendix, Fig. S32).
For AuTPP-treated cells, electron-dense aggregates were hardly
observed in the SEM image (SI Appendix, Fig. S31D). Taken
together, the results of the nanoSIMS and EM analyses of
AuMesoIX-treated cancer cells revealed a possible tight asso-
ciation between the gold and sulfur-rich proteins in the form of
aggregates in the cytosol and disrupted mitochondria.
Interactions with Cancer-Associated Thiol-Containing Proteins.
Peroxiredoxin III. Thermal proteome profiling (TPP) was performed
to identify the proteins engaged by AuMesoIX in cancer cells (42).
Protein targets become more resistant to heat-induced denaturation
upon interaction with a small molecule compound. Measurement of
the protein levels in the soluble fraction (nondenatured) allows the
extent of target engagement by the compound to be determined.
In conjunction with MS-based quantitative proteomics analysis,
the thermal stability of the potential targets against a temperature
gradient can be profiled (SI Appendix, Table S3). Using this ap-
proach, peroxiredoxin III (PRDX3) was identified as one of the
proteins targeted by AuMesoIX; this was confirmed by an inde-
pendent cellular thermal shift assay (CETSA) using immuno-
blotting, which showed an increase in the melting temperature
(Tm) of PRDX3 by 2.6 °C following treatment of cells with
AuMesoIX (Fig. 5A). PRDX3 is a mitochondrial antioxidant thiol
enzyme that functions to decompose hydrogen peroxide via 2 cat-
alytic cysteine residues (43, 44). In this work, AuMesoIX was found
to react with PRDX3-derived peptides containing catalytic cys-
teines as revealed by electrospray ionization (ESI)-QTOF-MS
analysis (Fig. 5B). Further tandem-MS analysis of the observed
adducts (m/z 1,142.5017 and 1,143.4646) revealed that AuMesoIX
was conjugated to the cysteine residues (SI Appendix, Fig. S33 and
Tables S4 and S5). AuMesoIX reduced the peroxidase activity
of purified PRDX3 in a dose-dependent manner in which ∼40%
of the activity was inhibited by 10 μM AuMesoIX (Fig. 5C and
Fig. 4. NanoSIMS and EM analyses of AuMesoIX-treated cells. (A) NanoSIMS
images of the 197Au and 32S/12C14N secondary ion signals of A2780 cells
treated with AuMesoIX (3 μM) for 18 h. (Scale bar, 4 μm.) (B) SEM images of
A2780 cells treated with AuMesoIX (Left). The yellow arrows show the in-
tracellular electron-dense aggregates in degraded mitochondria (Right).
(Scale bar, 4 μm.) (C) TEM image shows the morphology of intact mito-
chondria in cells treated with DMSO vehicle for 18 h. (Scale bar, 0.5 μm.)
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Fig. 5. Peroxiredoxin III PRDX3 as a target of AuMesoIX. (A) Thermal stabilization of PRDX3 following treatment of A2780 ovarian cancer cells with
AuMesoIX as determined by CETSA. (B) Mass spectra of the adducts formed between AuMesoIX and peptides of PRDX3 containing different active-site
cysteines; red square indicates the modification site of AuMesoIX. (C) Dose-dependent effect of AuMesoIX on recombinant PRDX3 activity as measured by
NADPH-dependent hydrogen peroxide reduction via a coupled Trx/Trx reductase (TrxR) system. (D) Immunoblotting analysis of PRDX3 expression in cells
treated with indicated concentrations of AuMesoIX for 8 h. (E) Immunofluorescence staining of PRDX3 (green) and COXIV (red) in cells treated with
AuMesoIX or DMSO vehicle. Nuclei were stained with DAPI (blue). (F and G) Flow cytometry analysis of intracellular oxidants of cells treated with AuMesoIX
(3 μM) for 8 h as examined by (F) DCFH-DA or (G) MitoPY1 probe.
SI Appendix, Fig. S34). In immunoblot analysis, both the mono-
mer and disulfide-bonded dimer of mitochondrial PRDX3 were
observed in untreated control cells (Fig. 5D and SI Appendix,
Fig. S35). Intriguingly, expression levels of the PRDX3 mono-
mer and dimer were down-regulated upon prolonged treatment
with AuMesoIX. The level of the housekeeping cytochrome c
oxidase (COXIV), which resides in the mitochondrial inner
membrane, was unaffected. Immunofluorescence analysis showed
that AuMesoIX treatment resulted in a significant reduction in
overall PRDX3 staining, while the mitochondrial staining pat-
tern and intensity of COXIV remained unaffected (Fig. 5E).
These results are indicative of specific targeting of PRDX3 by
AuMesoIX.
Inhibition of antioxidant enzymes such as thioredoxins and
peroxiredoxins will lead to an imbalance in cellular redox status.
AuMesoIX treatment augmented the intracellular and mitochon-
drial oxidant levels as measured with the fluorogenic probes
DCFH-DA (Fig. 5F), MitoSOX (SI Appendix, Fig. S36), and
MitoPY1 (45) (Fig. 5G), the last of which is specific for mito-
chondrial hydrogen peroxide. The increases in cellular and mito-
chondrial oxidant levels induced by AuMesoIX could be blocked
by pretreatment of cells with the antioxidant N-acetylcysteine
(NAC). Notably, pretreatment of cells with NAC also attenu-
ated AuMesoIX-induced cytotoxicity, increasing the IC50 by 6.8-
fold from 0.07 to 0.48 μM (SI Appendix, Fig. S37A and Table S6),
whereas treatment with the GSH synthesis inhibitor L-buthionine
sulfoximine (BSO) potentiated the cytotoxic effect of AuMesoIX,
decreasing the IC50 by 3.5-fold to 0.02 μM (SI Appendix, Fig. S37B
and Table S6). These results suggest that AuMesoIX-induced cy-
totoxicity was in part mediated by oxidative stress.
Protein Deubiquitinases. Protein deubiquitinases (DUBs) catalyze
the removal of ubiquitin from ubiquitinated proteins that par-
ticipate in cancer growth (46, 47). Most of the DUBs are cysteine
proteases containing catalytic thiols, which are potential drug
targets for DUB inhibitors. Incidentally, our TPP analysis revealed
that AuMesoIX treatment increased the cellular thermal stability
of a DUB of the cysteine protease class, the ubiquitin carboxyl-
terminal hydrolase isozyme L3 (UCHL3) (SI Appendix, Table S3);
this was verified by CETSA in which the Tm increase was found to
be 1.8 °C (Fig. 6A). The interaction of AuMesoIX with peptides
derived from UCHL3 and several other DUBs containing catalytic
cysteines was examined using ESI-QTOF-MS. A number of
doubly charged adduct ions were found in the corresponding re-
action mixtures and the isotopic patterns were consistent with the
simulated AuMesoIX-peptide adducts (Fig. 6B and SI Appendix,
Figs. S38–S41). No adducts with peptides containing other cata-
lytic residues such as aspartic acid and histidine were found (SI
Appendix, Figs. S42 and S43). AuMesoIX inhibited the activities of
several purified DUBs as well as that in cell extracts (Fig. 6C). As
anticipated, treatment of cells with AuMesoIX resulted in accu-
mulation of high–molecular-weight, polyubiquitinated proteins
(Fig. 6D). We also found that AuMesoIX targets the cancer
chaperone heat shock protein 90 (HSP90) (SI Appendix, Fig. S44),
which mediates folding of tumor-promoting client proteins and
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Fig. 6. Protein deubiquitinase UCHL3 as a target of AuMesoIX. (A) Thermal stabilization of UCHL3 in AuMesoIX-treated A2780 cells as determined by CETSA.
(B) List of the m/z of adducts formed between AuMesoIX and peptides derived from DUBs containing the catalytic amino acid residues (cysteine, aspartic acid,
or histidine) as determined by ESI-QTOF-MS. (C) DUB inhibitory activity of AuMesoIX (0.5 μM) toward purified enzymes (UCHL1, 3 or 5; 0.4 μg) and A2780
cellular lysate (5 μg). (D) Immunoblot analysis of ubiquitinated protein accumulation in cells treated with AuMesoIX for 18 h. MG132 treatment (5 μM) was
used as a positive control.
contains functional cysteine residues (48, 49). AuMesoIX could (hydroxide ion) gave gold(III) hydroxyphlorin with functionalization
form adducts with HSP90-derived cysteines, inhibit both in vitro at the meso-carbon atom of the porphyrin scaffold (33). Nonethe-
and cellular HSP90 activity, and down-regulate the expression of less, addition/substitution reactions at the periphery of coordinated
chelating ligands with biological nucleophiles have not been
reported as a mechanism of action to account for the anticancer
properties of metal complexes. In this work, we uncovered a pre-
viously unknown mode of interactions between metal compounds
and biomolecules as illustrated by bioconjugation of cysteine thiol
via the porphyrin scaffold of AuMesoIX with unsubstituted meso-
carbons. The biological activities of many gold compounds are
usually proposed to involve Au–S or Au–Se coordination via li-
gand substitution with protein thiols/selenols (18, 22). Herein, a
direct Au–S interaction was not observed in the case of AuMesoIX;
rather, thiol-specific bioconjugation to the porphyrin scaffold was
demonstrated. This distinct thiol reactivity may account for the
cytotoxic mechanisms of the gold(III) complexes and could be
further exploited for anticancer applications.
The unsubstituted meso-positions of porphyrins are known to
be the most reactive sites for both electrophilic and nucleophilic
substitutions (50–52). In the literature, modification of the por-
phyrin scaffold at meso-positions has been reported on activated
porphyrin systems with aromatic π-cation radical/π-dications on an
oxidized porphyrin scaffold (51), electron-withdrawing substitu-
ents at meso-carbons (52), or high-valent metal centers (31–33).
By varying the inductive effect on the conjugated π-electron sys-
tem, the chemical reactivity of the porphyrin macrocycle can be
modulated. For instance, the central metal ion of metalloporphyrin,
HSP90 client proteins including several signaling proteins known
to drive cancer progression (SI Appendix, Figs. S45–S49).
In Vivo Inhibition of Tumor Growth, Biodistribution, and Metabolism.
Treatment of nude mice bearing human ovarian cancer A2780
xenografts with AuMesoIX (2 mg/kg) by intravenous (i.v.) in-
jection twice per week for 21 d resulted in suppression of tumor
growth by 80% compared to those treated with vehicle control
(Fig. 7A). Similar AuMesoIX treatment of mice bearing human
colon cancer HCT116 xenografts was shown to reduce tumor
growth by 72% (Fig. 7B). All mice survived, and no signs of
toxicity or body weight loss were observed. Biodistribution of the
gold(III) compounds in BALB/c mice following a single i.v. in-
jection was studied. In AuMesoIX-administered mice, the gold
content was found to be transient in the liver and kidney fol-
lowed by urinary excretion (Fig. 7C). By contrast, in AuTPP-
treated mice, the gold compounds accumulated in the liver and
kidney with little urinary excretion (SI Appendix, Fig. S50).
Possible metabolites of the gold(III) compounds were identified
using the rat liver microsome as a source of biotransformation
enzymes. Several metabolites of AuMesoIX including hydroxyl-
ated species and hydrolyzed products of the dimethyl ester
(monopropionic and dipropionic acids) were found (SI Appendix,
Fig. S51). However, metabolites of AuTPP were not detected
under the same experimental conditions (SI Appendix, Fig. S52). such as Sb(V) (32) or Au(III) (33) of meso-tetraphenylporphyrin,
with a high oxidation state and Lewis acidity strongly reduces
Discussion
the electron density at the porphyrin periphery and hence is
susceptible to nucleophilic meso-hydroxylation. On the other
hand, divalent metal ions, such as Zn(II) or Cd(II), is incapable
to activate the peripheral atoms of porphyrin toward nucleo-
philic agents (53). In our case, the strong Lewis acidity of
Au(III) rendered the porphyrin ring of AuMesoIX or AuOEP
more reactive toward bioconjugation with the cysteine thiol of
GSH compared with ZnMesoIX wherein only trace amount of
Conjugation of Coordinated Ligands to Biomolecules. For decades, it
has been well documented that the reactivity of coordinated
organic ligands toward nucleophilic attack/addition is significantly
enhanced attributable to the electron-withdrawing inductive effect
and/or π-back-bonding interaction involving the metal ion. In gold
porphyrin chemistry, it has also been reported that the reaction of
gold(III) meso-tetraphenylporphyrin (AuTPP) with a strong base
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1H and 1H–13C HMBC NMR spectroscopy data as well as the high-
resolution MS measurements (Fig. 2 and SI Appendix, Figs. S5–S9,
S12, S13, and S16). To date, only one report on gold(III) meso-
porphyrin IX, which was limited to the study of electrochemical
properties, has been published, in which the central Au(III) ion was
demonstrated to be electrochemically inactive upon coordination by
the mesoporphyrin IX ligand (54). By considering the steric hin-
drance and electron-withdrawing inductive effect of the gold(III)
ion, we propose that the delta-meso-carbon of the porphyrin of
AuMesoIX is activated for nucleophilic aromatic substitution with
thiols of peptides and proteins (Scheme 1).
Distinct Anticancer Properties of AuMesoIX. Targeting antioxidant
and oncogenic signaling proteins by blocking functional cysteine
thiols is an attractive anticancer strategy (37–39). Our studies show
that thiol-modifying properties of AuMesoIX may in part con-
tribute to its anticancer activities. In AuMesoIX-treated cells, a
tight association of gold with sulfur-rich proteins in mitochondria
was demonstrated by correlative nanoSIMS and EM imaging (Fig.
4), whereas in AuTPP-treated cells only weak correlation of gold
and sulfur signals was observed (SI Appendix, Fig. S31C). From
thermal proteome profiling and individual cellular experiments, we
identified 2 possible protein targets of AuMesoIX, mitochondrial
peroxiredoxin III (43, 44) and deubiquitinase UCHL3 (47), which
are thiol-dependent enzymes essential for cancer cell survival and
hence attractive anticancer molecular targets. AuMesoIX could
conjugate with active cysteine residues on peptides of these thiol-
based proteins and inhibit their biological activities, which may be
linked to its ability to induce accumulation of intracellular reactive
oxidants and polyubiquitinated proteins (Figs. 5 and 6). AuMesoIX
also forms a covalent adduct with thioredoxin (Fig. 2D) and in-
hibits thioredoxin activity (SI Appendix, Fig. S24), which may fur-
ther impose oxidative stress on cancer cells (55). The increases in
cellular oxidant levels, as well as the cytotoxicity induced by
AuMesoIX, could be attenuated by pretreatment of cells with the
antioxidant NAC and potentiated by treatment with the GSH
synthesis inhibitor BSO (Fig. 5 F and G and SI Appendix, Figs. S36
and S37 and Table S6). However, the cytotoxicity of AuTPP was
not significantly affected by these redox modulators. These results
suggest that AuMesoIX-induced cytotoxicity may be, at least in
part, mediated by oxidative stress attributable to the thiol-targeting
property of the compound. Importantly, AuMesoIX displayed ef-
fective in vivo antitumor activities with favorable metabolism,
biodistribution, and clearance conferred by the quasiphysiological
mesoporphyrin IX ligand (Fig. 7).
Fig. 7. Antitumor activity of AuMesoIX and biodistribution in mice. Tumor
volume and body weight of nude mice bearing (A) A2780 or (B) HCT116
xenografts following i.v. injection of AuMesoIX (2 mg/kg) or solvent control
twice per week. Data are represented as means
SEM (n = 5; Student’s
t test; *P < 0.05, compared with vehicle control group). (C) Biodistribution of
gold content in liver, kidney, and urine of BALB/C mice following i.v. in-
jection of a single dose of AuMesoIX (2 mg/kg; n = 3).
adduct was observed (SI Appendix, Fig. S15). The MesoIX free
ligand without the Au(III) ion also displayed no adduct formation
under similar reaction conditions (SI Appendix, Fig. S11), in-
dicating that thiol conjugation of the porphyrin scaffold of
AuMesoIX is mediated by the electrophilic Au(III) center. In
addition to the electronic effect, the meso-reactivity of porphyrin
can be masked by the presence of meso- or β-pyrrolic substituents
(50). For AuTPP with tetra-phenylated meso-substituents, no de-
tectable adduct in the reactions with GSH/cysteine was found in
MS analysis (SI Appendix, Figs. S10 and S14). Moreover, the lower
abundance of adduct formed from the reaction between AuOEP
and GSH compared to that with AuMesoIX (Fig. 2A and SI Ap-
pendix, Fig. S6A) can be explained by the lower meso-reactivity of
AuOEP due to the steric hindrance of β-ethyl substituents.
Implications of Metal-Induced Ligand Reactivity for Anticancer
Compound Design. Our findings demonstrate that anticancer
transition metal complexes can target biological thiols through
metal-induced ligand bioconjugation. In the literature, the acti-
vation of coordinative ligand reactivity by electron-deficient
metal ions is fundamental to organometallic chemistry for syn-
thetic applications. Different metal catalysts with electrophilic
metal ions such as gold, platinum, and palladium have been
reported for the activation of olefins toward nucleophilic reac-
tions and C–H bond functionalization of small organic com-
pounds via carbon–carbon and/or carbon–heteroatom bond
formation (56, 57). In addition, transition-metal–mediated bio-
conjugation for the modification of peptides and proteins has
recently been described (58). Although the high chemoselectivity
of thiol conjugation can be achieved by these metal catalysts,
their biocompatibility and pharmacological application remain
to be determined.
Thiol-Selective Conjugation of Gold(III) Meso-Unsubstituted
Porphyrins. The porphyrin ligand bioconjugation of gold(III)
meso-unsubstituted porphyrins was demonstrated to be cysteine-
selective in experiments with individual amino acids, different pep-
tides with or without cysteine, and protein with thiol blocking agent
(Fig. 2D and SI Appendix, Figs. S12, S13, S22, S23, and S38–S43).
This bioconjugation was confirmed to be nucleophilic aromatic
substitution between the thiol group of cysteine and the methine H
Protein thiol conjugation is usually mediated by organic Mi-
chael addition-type thiol alkylators involving the α,β-unsaturated
carbonyl group as a functional handle (59). Only the carbonyl
group adjacent to the alkene allows polarization of the Michael
atom of meso-unsubstituted gold(III) porphyrin, as verified by the acceptor for nucleophilic conjugation. By virtue of the versatility
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Scheme 1. Proposed reaction of AuMesoIX with thiol-containing biomolecules under physiological conditions.
Physical Measurements, MS, Proteomic Analysis, and Bioimaging. 1H, 13C, 1H–13C
of metal–ligand coordination, it is conceivable that transition
metal complexes can be a distinct platform for thiol conjugation
via adjustment of the electronic properties and optimization of
the ligand scaffold. We envision that by judicious selection of
Lewis acidic metal ions and electrophile-containing ligands such
as meso-unsubstituted porphyrin, specific targeting to cancer-
associated proteins with tunable bioconjugation reactivity can
be achieved.
heteronuclear single-quantum coherence, and 1H–13C HMBC NMR spectra
were recorded on a Bruker Avance DPX 400 or 500 spectrometer at 298 K
with chemical shift relative to tetramethylsilane (for CDCl3) or residual
nondeuterated solvent (for CD3OD). UV-vis spectra were recorded on
a
Hewlett-Packard 8452A diode array spectrophotometer. X-ray diffraction data
were collected on a Bruker Proteum X8 diffractometer with monochromated
Cu-Kα radiation (λ = 1.54178 Å). The gold contents in cells and mouse tissues
were determined using an inductively coupled plasma mass spectrometer
(Agilent 7500a). Mass spectra of metal compounds, peptides, and proteins
were recorded on a Q-TOF Premier quadrupole time-of-flight tandem mass
spectrometer (Waters Micromass) or MALDI-TOF-MS (Bruker ultrafleXtreme).
Thermal proteome profiling was performed by bottom-up proteomic analysis
of tandem mass tag (TMT)-labeled peptides from trypsin-digested proteins
using an Orbitrap Fusion Tribrid mass spectrometer (Thermo Scientific) (42).
Bioimaging of the elemental distribution in cells was performed on a nano-
scale secondary ion mass spectrometer (Cameca NanoSIMS 50) with correlative
electron microscopic imaging (40).
Conclusion
Our study showed a special mode of biomolecular interaction of
metal complexes, as illustrated by cysteine thiol conjugation to the
gold(III)-activated porphyrin scaffold of AuMesoIX through nu-
cleophilic aromatic substitution. AuMesoIX can covalently modify
the cysteine thiols of cancer-associated proteins and exhibit potent
cancer cell cytotoxicity as well as effective in vivo antitumor ac-
tivities, demonstrating an alternative modality for cysteine tar-
geting and for the development of cancer therapeutics.
Data Availability. The X-ray crystallographic data have been deposited in the
Cambridge Crystallographic Data Centre (CCDC) (accession nos. 1919615
[AuMesoIX] and 1919616 [AuOEP]). Thermal proteome profiling datasets are
available via ProteomeXchange Consortium with identifier PXD016875.
Detailed experimental procedures on general chemical and biological
analyses; analytical data including NMR and mass spectra, UPLC/MS and UV-
vis absorption spectra, NanoSIMS and EM images, thermal proteome pro-
filing data, and biological results are provided in SI Appendix.
Materials and Methods
Synthesis. The gold(III) porphyrin complexes were synthesized by reacting the
corresponding porphyrin ligands with KAuCl4 in glacial acetic acid following
Fleischer’s procedure (SI Appendix, Materials and Methods). The AuOEP-GSH
adduct was prepared by the reaction of AuOEP with GSH in ammonium bi-
carbonate buffer solution (pH 7.4) at 37 °C for 18 h and purified by flash column
chromatography (silica gel). Detailed procedures and characterization of all of the
synthesized compounds and the thiolato adduct are described in SI Appendix.
Reactivity of Gold(III) Meso-Unsubstituted Porphyrins with Cysteine Thiols.
Gold(III) porphyrin complexes were incubated with respective freshly pre-
pared GSH, cysteine-containing peptides derived from PRDX3, DUBs, and
HSP90, or Trx protein in ammonium bicarbonate buffer solution (pH 7.4) at
37 °C for 18 h. For the peptide adducts, the reaction mixtures were diluted
with methanol and subjected to ESI-QTOF-MS analysis. For the protein ad-
duct, samples were diluted with saturated sinapinic acid solution and
analyzed by MALDI-QTOF-MS.
ACKNOWLEDGMENTS. We thank Dr. Harry Gray for valuable comments. We
also thank Dr. Clive Yik-Sham Chung and Dr. Ka-Pan Shing for helpful
discussions. We acknowledge the Core Facility of Li Ka Shing Faculty of
Medicine for confocal imaging and flow cytometry. This work was supported
by the Innovation and Technology Fund (ITS/130/14FP and ITS/188/18) and
Health@InnoHK administered by Innovation and Technology Commission;
and General Research Fund (HKU 17300518 and 17160916) from the Re-
search Grants Council.
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