Distorted Gold(I)-Phosphine Complexes as Antifungal Agents

Article abstract of DOI:10.1021/acs.jmedchem.9b01436

Fungi cause serious nosocomial infections including candidiasis and aspergillosis, some of which display reduced susceptibility to current antifungals. Inorganic compounds have been found to be beneficial against various medical ailments but have yet to be applied to fungal infections. Here, we explore the activity of linear and square-planar gold(I)-phosphine complexes against a panel of 28 fungal strains including Candida spp., Cryptococcus spp., Aspergillus spp., and Fusarium spp. Notably, two square-planar gold(I) complexes with excellent broad-spectrum activity display potent antifungal effects against strains of Candida auris, an emerging multidrug-resistant fungus that presents a serious global health threat. To characterize the biological activity of these gold(I) complexes, we used a series of time-kill studies, cytotoxicity and hemolysis assays, as well as whole-cell uptake and development of resistance studies.

Full text of DOI:10.1021/acs.jmedchem.9b01436

Article
pubs.acs.org/jmc
Cite This: J. Med. Chem. XXXX, XXX, XXX−XXX
Distorted Gold(I)−Phosphine Complexes as Antifungal Agents
Emily K. Dennis,† Jong Hyun Kim,‡ Sean Parkin,‡ Samuel G. Awuah,*,‡
and Sylvie Garneau-Tsodikova*,†
†Department of Pharmaceutical Sciences, College of Pharmacy, University of Kentucky, 789 South Limestone Street, Lexington,
Kentucky 40536-0596, United States
‡Department of Chemistry, College of Arts and Sciences, University of Kentucky, 505 Rose Street, Lexington, Kentucky 40506-0055,
United States
S
* Supporting Information
ABSTRACT: Fungi cause serious nosocomial infections including
candidiasis and aspergillosis, some of which display reduced
susceptibility to current antifungals. Inorganic compounds have been
found to be beneficial against various medical ailments but have yet to
be applied to fungal infections. Here, we explore the activity of linear
and square-planar gold(I)−phosphine complexes against a panel of 28
fungal strains including Candida spp., Cryptococcus spp., Aspergillus spp.,
and Fusarium spp. Notably, two square-planar gold(I) complexes with
excellent broad-spectrum activity display potent antifungal effects
against strains of Candida auris, an emerging multidrug-resistant fungus
that presents a serious global health threat. To characterize the
biological activity of these gold(I) complexes, we used a series of time−
kill studies, cytotoxicity and hemolysis assays, as well as whole-cell uptake and development of resistance studies.
agents during treatment. With a limited armament of
antifungal agents, there is a need for new classes of agents.
In agriculture, metal salts (e.g., copper salts11) are widely
used as fungicides to improve food production. As medicines,
inorganic compounds have been predominantly developed as
anticancer agents (e.g., cisplatin).12 These metal complexes
typically consist of either platinum, ruthenium, silver, or
copper. As anticancer agents, these compounds have been
successful but typically have problems with toxicity13 and
associated acquired resistance.14 More recently gold(I)−
phosphine and gold(III) complexes have gained attention as
anticancer agents15 as well as antimicrobials.16,17
INTRODUCTION
■
Fungal infections are deadly for patients with conditions that
weaken the immune system,1 as demonstrated by mortality
rates exceeding 50% for systemic fungal infections.2 Those
most affected include patients (i) with acquired immune
deficiency syndrome, (ii) having received recent chemo-
therapy, (iii) having had an organ transplant, and (iv) with
underlying lung disease such as chronic obstructive pulmonary
disorder and asthma.1,2 These systemic fungal infections are
primarily caused by only a few fungal genera, specifically
Candida3 and Aspergillus.4
For treatment of fungal infections, there are three classes of
antifungal agents that can be used. One class, the polyenes,
includes the widely used antifungal therapy, amphotericin B
(AmB). While effective in treating a broad spectrum of
infections, treatment is often associated with severe side effects.
The second class, the azoles, specifically fluconazole (FLC)
and voriconazole (VRC), is a first line of defense against fungal
infections but can cause drug−drug interactions. The third
class is the echinocandins, which includes caspofungin (CAS).
The echinocandins are narrow-spectrum and can only be
administered by intravenous catheters. What is of concern is
the ability of fungi to be intrinsically resistant to antifungal
agents. Examples include Candida glabrata5 to the echino-
candins and the emerging pathogen, Candida auris, which in
some cases is resistant to all three drug classes.6,7 C. auris is
currently attracting attention due to recent outbreaks of
resistant C. auris infections in the U.S.8−10 In addition,
infections can develop decreased susceptibility to antifungal
The arthritis drug, auranofin (Figure 1), is an exemplary
gold complex that has been used in the clinic since 1983. It can
be administered orally and has been shown to be well-tolerated
Figure 1. Structure of auranofin.
Special Issue: Women in Medicinal Chemistry
Received: August 30, 2019
© XXXX American Chemical Society
A
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Scheme 1. Synthetic Schemes Showing the Preparation of the Au Complexes 1−6
Figure 2. X-ray crystal structures of compounds (A) 3, (B) 4, (C) 5, and (D) 6. Ellipsoids are drawn at the 50% probability level. Hydrogen atoms
bound to carbon atoms are omitted for clarity. For compounds 4 and 6, the molecules cocrystallized with a molecule of CHCl3.
at a 6 mg daily dose in patients (www.fda.gov/drugsatfda).
Auranofin is believed to block inflammation in arthritis by
regulating the secretion levels of various cytokines.18 In recent
years, reports looking at repurposing auranofin as an
antimicrobial agent against bacteria19−22 and fungi have been
published.23−26 In fact, auranofin is currently in clinical trials
for cancer, HIV, amoebiasis, and tuberculosis.27,28 As an
anticancer and antimicrobial agent, auranofin acts to inhibit
thioredoxin reductase.29,30 With no known inorganic anti-
fungals on the market, auranofin speaks to the promise of using
gold scaffolds to investigate and develop novel antifungal
agents.
Our group has a long-standing interest in the development
of antifungal agents. In the past, we have used different
B
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a
Table 1. MIC Values in μg/mL for Compounds 1−6, Auranofin, and AmB against Various Fungal Strains
a
MIC values are also provided in μM in parentheses.
strategies to develop antifungals, including the development of
azole analogues,31−33 combinations of antifungal drugs,34−36
and synthesis and biological evaluation of new scaffolds.37−41
We previously developed gold complexes as potential
anticancer agents.42,43 We were intrigued to see if the
applications of gold complexes could be expanded to include
antifungal activity. Herein, we present the antifungal activity of
six distorted gold(I)−phosphine complexes, 1−6 (Scheme 1),
not derived or related in structure to auranofin, against yeast,
molds, and yeast biofilms. We then confirm the activity of the
two best complexes, 4 and 6, in time−kill studies. To evaluate
the efficacy of complexes 4 and 6, we use both cytotoxicity
studies against four mammalian cell lines as well as hemolysis
assay with both murine and human red blood cells. We also
present whole-cell uptake assays and development of resistance
studies. The gold complexes with square-planar geometry
appear to show great promise for future development as
antifungal agents. As there are currently no metal complexes
that have been thoroughly investigated for antifungal activity,
our distorted gold(I)−phosphine complexes are innovative in
the field of antifungal development.
RESULTS AND DISCUSSION
■
Design, Chemical Synthesis, and X-ray Crystallog-
raphy. For this study we wanted to create gold(I) complexes
that could be easily prepared in a single synthetic step (Scheme
1). The reaction of three commercially available phosphorus
ligands with AuCl(THT) (prepared by the reaction of
tetrahydrothiophene and tetrachloroauric acid
(HAuCl4•3H2O))44 in chloroform at room temperature
afforded mixtures of linear gold(I) complexes 1, 3, and 5
and their square-planar gold(I) complex counterparts 2, 4, and
6 in 23−37% yield, which could be easily separated by silica gel
flash chromatography. To expand the availability of chiral
gold(I) complexes, which are limited and underexplored for
biological applications, we used both the achiral bis-
(diphenylphosphino)benzene ligand and chiral ligands such
as the 1,2-bis[(2S,5S)-2,5-dimethylphospholano]benzene and
1,2-bis[(2R,5R)-2,5-dimethylphospholano]benzene. The struc-
tures of compounds 1−6 were confirmed by H, 13C, and 31P
1
NMR spectroscopy, mass spectrometry, as well as RP-HPLC
for purity determination. Additionally, the structures of
compounds 3, 4, 5, and 6 were confirmed by X-ray
crystallography. Single crystals of complexes were grown by
C
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a
Table 2. MIC Values in μg/mL for Compounds 4, 6, AmB, CAS, FLC, and VRC against a Panel of C. auris Strains
a
MIC values are also provided in μM in parentheses.
vapor diffusion (Figure 2). Crystal structures for the known
compounds 145 and 246 had already been solved. The
structures of complexes 3 and 5 were consistent with linear
geometry for classical gold(I) complexes. Furthermore,
complexes 4 and 6 were characterized by a distorted square-
planar arrangement around the gold(I) center as observed in
gold complexes with bisphosphine ligands. In all cases, the
gold(I) center is coordinated to bidentate ligands with
phosphorus donors; 3 and 5 have one chloride ion bound to
the gold(I) center, while 4 and 6 have all donors as
phosphorus atoms. Typically, Au−P distances vary from
2.229 to 2.239 Å and Au−Cl distances are in the range of
2.286−2.292 Å.
Determination of Minimum Inhibitory Concentration
(MIC) Values of Compounds 1−6 against 28 Fungal
Strains. For all biological studies, we used auranofin as a
control as it is one of the only metal complexes that is an FDA-
approved drug, is well-tolerated in patients, has some reported
antimicrobial activity, and may have a similar cellular target as
the gold(I)−phosphine complexes. As there are currently no
metal complexes that have been thoroughly investigated for
antifungal activity, our distorted gold(I)−phosphine complexes
are innovative in the field of antifungal development. For most
biological assays, we also used the current FDA-approved
antifungal AmB as a positive control.
infections. Furthermore, this panel includes many (five out of
seven) C. albicans strains designated as FLC-resistant by the
American Type Culture Collection (ATCC, see legend of
Table 1). We observed that auranofin had no antifungal
activity against Candida spp., while it displayed MIC values of
0.06−7.8 μg/mL against all four C. neoformans and two of the
three Aspergillus strains tested, which agrees with other reports
of its activity.24 We found that compounds 4 and 6 displayed
excellent activity against Candida spp. and Cryptococcus spp.
with MIC values against 17 strains in the range of 0.06−1.95
μg/mL, which were generally better than MIC values for AmB.
Compounds 4 and 6 also displayed good to excellent activity
(MIC values of 1.95−7.8 μg/mL) against all filamentous fungi,
the Aspergillus spp. and Fusarium spp., which was much better
than that of AmB (MIC values of 7.8−31.3 μg/mL).
Compound 3, on the other hand, was found to be completely
inactive against all fungal strains tested except for C.
neoformans (strain M). Compounds 1, 2, and 5 were inactive
against both Aspergillus spp. and Fusarium spp. and most of the
non-albicans Candida strains tested, whereas they displayed
some activity in the range of 0.12−7.8 μg/mL against a few
strains of C. albicans and C. neoformans. From these data, we
concluded that linear gold(I) complexes (i.e., 1, 3, and 5) and
achiral square-planar gold(I) complexes (i.e., 2) were poor
antifungals that should not be further pursued, whereas the
chiral square-planar gold(I) complexes (i.e., 4 and 6) showed
great promise as antifungals and deserved further investigation.
As C. auris is an emerging drug-resistant pathogen, we
included two C. auris strains in our initial panel (strains K and
L) (Table 1). As compounds 4 and 6 displayed good and
excellent activity against these two C. auris strains, respectively,
we expanded our panel and tested compounds 4 and 6 as well
as the control antifungals AmB, CAS, FLC, and VRC with an
additional eight C. auris strains (strains I−VIII) from the
Compounds 1−6 were first tested in MIC value
determination assays against a panel of 20 fungal strains
(Table 1). The panel consisted of seven Candida albicans
(strains A−G), five non-albicans Candida (one C. glabrata
(strain H), one C. krusei (strain I), one C. parapsilosis (strain
J), and two C. auris (strains K and L)), four Cryptococcus
neoformans (strains M−P), three Aspergillus (strains Q−S), as
well as one Fusarium graminearum (strain T). These strains
were chosen as they represent pathogens causing systemic
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Figure 3. Representative time−kill curves for compounds 4, 6, and AmB against (A) C. albicans ATCC 10231 (strain B), (B) C. glabrata ATCC
2001 (strain H), (C) C. auris AR bank no. 0384 (strain K), and (D) C. auris AR bank no. 0390 (strain L). Fungal strains were treated with no drug
(black circles), compound 4 at 1× MIC (inverted black triangle), compound 4 at 2× MIC (white triangle), compound 6 at 1× MIC (black square),
compound 6 at 2× MIC (white square), and AmB at 1× MIC (white circle). At the 24-h endpoint, resazurin was added to the cultures to
qualitatively measure the CFU/mL. Resazurin, which is a blue-purple color, is metabolized by viable cells to produce resorufin, which has a pink-
orange color. Cultures with little to no cells remain a blue-purple color, while dense cultures appear pink or orange.
Centers for Disease Control (CDC) Antibiotic Resistance
Bank47 (Table 2). We observed that both compounds 4 and 6
had excellent antifungal activity (MIC values of 0.98−1.95 μg/
mL) against almost all C. auris strains.
failed to reach the limit of detection by 24 h, but compound 6
at 1× MIC reached the limit at 6 h. However, with C. auris
(strain L) both compounds 4 and 6 at 1× MIC reached the
limit of detection by 3 h and remained under the limit of
detection, while AmB reached the limit of detection at 9 h
before the CFU/mL began to return to the original yeast cell
concentration.
Prevention of Biofilm Formation and Disruption of
Preformed Biofilm Assays for Compounds 4 and 6.
Biofilms are well-known in the world of bacteria to cause
difficult to treat and reoccurring infections by a multitude of
species including Staphylococcus aureus, Escherichia coli, and
Pseudomonas aeruginosa.48,49 There is an extensive number of
fungal strains known to form biofilms, but biofilm formation
on catheters, prostheses, and other medical devices in
healthcare associated infections is mainly limited to Candida
spp.50−53 The ability of compounds 4 and 6 to both prevent
and disrupt biofilm formation is important for prophylactic
treatment and to stop the spread of a fungal infection.
However, it is regarded that it is more challenging to disrupt a
Time−Kill Assays for Compounds 4 and 6. With the
very promising antifungal activity results for compounds 4 and
6, we next examined their killing kinetics. Time−kill assays
were done with four representative Candida strains, one C.
albicans (strain B), one C. glabrata (strain H), and two C. auris
(strains K and L) (Figure 3). Compounds 4 and 6 were tested
at both their 1× and 2× MIC values, and AmB at 1× MIC was
used as a known fungicidal control. Both compounds
significantly decreased fungal colony forming units (CFU) by
102 CFU/mL by the 3 h time point and did not increase over
the 24 h time period, which indicated that compounds 4 and 6
are fungicidal. This pattern was very similar to AmB. With C.
albicans (strain B), compound 4 at 1× MIC reached the limit
of detection at 9 h and compound 6 at 2× MIC at 3 h. With C.
glabrata (strain H), both compounds at 2× MIC were at the
limit of detection by 24 h. For C. auris (strain K), compound 4
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preformed biofilm as the large, sugary extracellular matrix that
is the key characteristic of biofilms can prevent many drugs
from reaching the fungal cells.54 Furthermore, in biofilms,
fungal cells can upregulate efflux pumps to prevent the action
of any drugs that do reach the fungal cells through the
extracellular matrix.55 We measured the ability of compounds 4
and 6, auranofin, and AmB both to prevent biofilm formation
and to disrupt preformed biofilms of C. albicans (strain B), C.
glabrata (strain H), and C. auris (strains K and L) (Table 3 and
the currently used antifungal agents, AmB and echinocandins
have the best efficacy with biofilms with SMIC90 in the range of
0.5−128 μg/mL (4- to 128-fold increase from MIC) and
0.03−8 μg/mL (2- to 16-fold increase from MIC),
respectively. For the azoles, itraconazole and posaconazole
have some efficacy against biofilms with 1- to 256-fold
increases in MIC against biofilms. However, VRC and FLC
have no efficacy with SMIC90 exceeding 512 μg/mL.
Additionally, new investigational antifungal molecules that
have been reported to be active against C. albicans biofilms
include azole derivatives and benzimidazole containing
compounds. For the azole derivatives, seven econazole
derivatives were reported with minimum biofilm inhibiting
concentrations at or near 8 μg/mL 57 (2- to 16-fold increase in
MIC) and alkylated azole derivatives displayed SMIC80 values
of 15.6−31.3 μg/mL.32 Other investigational molecules with
activity against biofilms included three neomycin B−
benzimidazole hybrid molecules with SMIC80 values of 7.8−
15.6 μg/mL 32,58 (2- to 4-fold increase in MIC). Candida
biofilms are known to be key virulence factors in mucosal
membrane infections (i.e., thrush and vulvovaginal infec-
tions),59−61 and Candida clinical isolates from bloodstream
infections can form biofilms as well. Of the bloodstream
isolates, it is estimated that approximately 20% of C. albicans
strains were able to form biofilm in vitro, with that percentage
increasing to near 70% for non-albicans Candida.62−64 With
few other antifungals displaying antibiofilm activity, the 4-fold
difference that we observed is highly promising.
Table 3. Prevention of Biofilm Formation and Disruption of
a Preformed Biofilm by Compounds 4, 6, Auranofin, and
a
AmB against Four Fungal Strains
biofilm prevention
SMIC90 (μg/mL)
preformed biofilm
SMIC90 (μg/mL)
strain
compd
B
4
6
3.9
0.98
7.8
7.8
3.9
7.8
auranofin
AmB
4
0.98
3.9
31.3
7.8
H
K
L
6
1.95
31.3
0.98
3.9
3.9
>31.3
7.8
7.8
7.8
>31.3
1.95
15.6
7.8
auranofin
AmB
4
6
auranofin
AmB
4
3.9
>31.3
1.95
3.9
Mammalian Cytotoxicity Assays for Compounds 4
and 6. For the gold complexes to progress further into the
drug development process, the gold complex activity should be
specific to fungal cells and not be toxic to mammalian cells.
Therefore, we tested compounds 4 and 6 as well as the control
auranofin against four mammalian cell lines: human
adenocarcinoma (A549), bronchial epithelial (BEAS-2B),
human embryonic kidney (HEK-293), and murine macro-
phage (J774A.1) (Table 4, Figure 4). Excluding auranofin with
6
3.9
auranofin
AmB
>31.3
1.95
>31.3
7.8
a
Strains: B = C. albicans ATCC 10231, H = C. glabrata ATCC 2001,
K = C. auris AR bank no. 0384, L = C. auris AR bank no. 0390.
Figures S31 and S32). As the biofilm assay is a colorimetric
assay and it is difficult to achieve 100% disruption of biofilms,
we report the sessile MIC90 (SMIC90), which is the
concentration of compound at which there is a 90% decrease
in metabolic activity as compared to untreated biofilm. Both
compounds 4 and 6 showed similar results with all four fungal
strains tested. SMIC90 values in prevention of biofilm
formation assays were 1- to 2-fold higher than with planktonic
cells. When tested against a preformed biofilm, compound 4
had a SMIC90 of 7.8−15.6 μg/mL with all four strains and
compound 6 had a SMIC90 of 3.9−7.8 μg/mL with all four
strains. These SMIC90 results were 4- to 8-fold higher than the
MIC results for the same Candida strains with planktonic cells.
Interestingly, auranofin with C. albicans (strain B) achieved the
same SMIC90 as compound 4 but was inactive against C.
glabrata (strain H) and the two C. auris (strains K and L)
tested. These values for auranofin are similar to a value
reported for auranofin against the biofilm of one C. albicans
strain.23 In contrast, AmB had SMIC90 values of 1.95 μg/mL
(C. auris, strain K) 7.8 μg/mL (C. auris, strain L; C. glabrata,
strain H), and 31.3 μg/mL (C. albicans, strain B), which were
1- and 16-fold higher than its MIC values against the same
strains in liquid culture.
Table 4. IC50 (μM) for Mammalian Cell Lines
compd
A549
BEAS-2B
HEK-293
J774A.1
4
6
4.5 0.6
2.5 0.2
0.5 0.1
2.0 0.3
4.9 0.3
1.3 0.1
1.9 0.7
5.7 0.5
3.0 0.2
1.5 0.1
1.5 0.1
16.2 0.9
auranofin
J774A.1, we observed <50% cell survival at 7.8 μg/mL with no
cell survival at 15.6 μg/mL for both compounds 4 and 6 and
auranofin. Auranofin displayed IC50 values of 0.5−3.0 μM
against A549, BEAS-2B, and HEK-293 cell lines, which agrees
with other published values against cisplatin-sensitive cell
lines.42 For J774A.1, the IC50 value for auranofin was
significantly higher at 16.2 μM. As J774A.1 is a macrophage
cell line, auranofin may have had an anti-inflammatory effect
that stimulated cell metabolism, which could account for the
higher IC50 value. There is interest in repurposing auranofin as
an antimicrobial; however, auranofin does not appear to be
promising as an antifungal. Auranofin displayed poor activity
against Candida spp. (MIC > 31.3 μg/mL) and only good
activity against two Aspergillus spp. (MIC = 3.9 and 7.8 μg/
mL). For compound 4, IC50 values were very similar for BEAS-
2B, HEK-293, and J774A.1 (1.5−2.0 μM) and somewhat
higher for A549 (4.5 μM). The MIC values for compounds 4
and 6 against 18 of the Candida spp. are in the range of 0.49−
It is promising that both compounds 4 and 6 have good
activity against biofilms of Candida spp. There have been
reports that compared planktonic and sessile MIC values of
other FDA-approved antifungal agents, which demonstrate the
reduced susceptibility of biofilms to antifungal agents.52,56 Of
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Figure 4. Evaluation of cytotoxicity for compound 4 (orange), compound 6 (blue), and auranofin (white) with (A) A549, (B) BEAS-2B, (C)
HEK-293, and (D) J774A.1 cell lines. Controls include treatment with Triton-X (TX, 1% v/v, positive control) and 0.5% DMSO (negative
control). Values of >100% were normalized to 100%.
1.95 μg/mL, which are concentrations at which there is toxicity
observed for the mammalian cells. Overall, compounds 4 and 6
displayed somewhat better selectivity to kill fungi over
mammalian cells than the FDA approved drug, auranofin.
Despite this result, there is room to improve these gold
complexes to increase the therapeutic window by reducing
mammalian cell toxicity.
murine and human red blood cells (RBCs) to evaluate the
hemolytic activity of compounds 4 and 6 as compared to both
auranofin and AmB, as well as to the detergent, Triton-X
(positive control) (Figure 5). Some drugs, especially those
containing both hydrophobic and hydrophilic components, can
disrupt cell membranes to cause hemolysis.66 Examples of
drugs that are known to be hemolytic include AmB as well as
cisplatin. With AmB, to minimize hemolytic activity a lipid
formulation has been developed.67 The results are similar for
both murine and human RBCs; however, the murine RBCs
appear more prone to hemolysis as compounds 4 and 6
displayed 13% and 60% hemolysis, respectively, at 3.9 μg/mL
and less than 5% with human RBCs. We observed that both
compounds 4 and 6 displayed hemolytic activity at 7.8 μg/mL.
AmB exhibited somewhat better values with 30−60%
hemolysis at 7.8 μg/mL and 100% hemolysis at 15.6 μg/mL.
In contrast, auranofin displayed no hemolytic activity at 15.6
μg/mL. With MIC values for compounds 4 and 6 typically in
the range of 0.49−1.95 μg/mL for Candida spp., there is a 1- to
2-fold therapeutic window, which is not perfect when
comparing to the desired 10-fold therapeutic window.
Whole-Cell Uptake Assay for Compounds 4 and 6. To
gain some insight into whether compounds 4 and 6 have an
intracellular or extracellular target, the uptake of gold into the
cell was measured using inductively coupled plasma optical
emission spectroscopy (ICP-OES, Figure 6). Uptake was
measured with 100 million yeast cells (Note: this is 2−3×
more cells than in MIC and time-kill studies) after 30 min
treatment with 10 μM (∼5× MIC for compounds 4 and 6
against strain B and H, respectively; ∼10× MIC for
compounds 4 and 6 against strain H and B, respectively)
compound. These conditions were chosen to have a significant
Reports have suggested that gold complexes bind to
thioredoxin reductase in bacteria and mammalian cells,29,30
but there is some evidence to suggest that gold complexes
could inhibit mitochondrial function in fungi.26 Future studies
for the gold complexes, out of scope for this proof-of-concept
work, should seek to answer whether these square-planar gold
complexes bind thioredoxin reductase or mitochondrial
enzymes, which if so, could lead to more in depth
structure−activity studies to decrease cytotoxicity. For other
reported gold complexes that were investigated for anticancer
activity, IC50 values for complexes comprising (1R,2R)-
(+)-1,2-diaminocyclohexane ligands ranged from 1.2 to 14.8
μM against cancer cell lines and were >100 μM against a
human normal lung fibroblast cell line, MRC5.42 Another
square-planar gold(I) diphosphine complex displayed IC50
values of 0.3−9.2 μM.65 In this report, IC50 values ranged
from 0.55 to 0.83 μM against two cancer cell lines for both
compounds 4 and 6. Interestingly, an achiral version of these
complexes was reported to be insoluble. Furthermore, in a
preliminary study with a xenograft model, compounds 4 and 6
were tolerated in mice at a dose of 2 mg/kg (100% survival) or
8 mg/kg (83% and 67% survival, respectively), which suggests
an acceptable level of toxicity at lower doses.
Measurement of Hemolysis for Compounds 4 and 6.
To expand upon the cytotoxicity results, we obtained both
G
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the pmol/million cells range, there appears to be a relatively
low amount of gold uptake per cell, but there are no reports of
similar uptake studies in yeast to compare to. However, we do
observe uptake and these values correspond to approximately
15% and 20% of total gold content for compounds 4 and 6,
respectively. It is possible that by the 30 min endpoint there is
some lysis of the fungal cells, especially with 10× MIC, which
would decrease uptake values. These results do suggest that the
compounds enter the yeast cell by facilitated diffusion or active
transport as with passive diffusion higher dosing of compound
(e.g., saturating amount) corresponds to greater cell uptake.
We previously published gold(III) complexes that we
investigated as anticancer agents, where we measured gold
uptake in OVCAR8 cells.42 We found that gold(III) complexes
that included a single chloride anion had improved uptake over
similar complexes with perchlorate anions, with relative uptake
of ∼300−400 and ∼200 pmol/million cells, respectively.
These values were significantly lower than the ∼1300 pmol/
million cells uptake of auranofin in the OVCAR8 cell. The
uptake values for compounds 4 and 6 in fungi appear
significantly lower than the values measured for other
complexes with the mammalian cells but are similar when
the difference in cell volume and incubation time between
yeast and mammalian cells is considered.68,69 Therefore, it is
still unclear but within reason for the gold complexes to have
an intracellular target. Conceivably, the structurally complex
cell wall of fungus composed of chitin, glucans, and
glycoproteins may contribute to the limited uptake of the
cationic gold complexes investigated. Further studies will focus
on developing neutral complexes and complexes that benefit
from active transport.
Development of Fungal Resistance for Compounds 4
and 6. Fungal drug resistance can be caused by mutation of
the target protein (observed with azoles and echinocan-
dins),70,71 overexpression of the target protein (observed with
azoles),72,73 the use of efflux pumps (observed with
azoles),74−78 or increased filamentation to decrease drug
uptake (observed with AmB).79,80 In order to assess the
potential for the development of fungal resistance, we
determined MIC values of compounds 4 and 6 as well as
AmB as a control over 15 serial passages with C. albicans
(strain B) and C. glabrata (strain H) (Figure S34). We
observed no significant changes in MIC values for our
compounds over the course of this study. The gold complexes
are likely to display different mechanisms of action in fungi
that can circumvent resistance pathways.
Figure 5. Hemolytic activity of compound 4 (orange), compound 6
(blue), auranofin (white), and AmB (gray) against (A) human and
(B) murine red blood cells. Positive control is Triton-X (TX, 1% v/v).
CONCLUSIONS
■
In summary, we synthesized three linear gold(I) phosphine
complexes and three corresponding square-planar gold(I)
complexes and explored their antifungal activity. Two square-
planar complexes, 4 and 6, displayed excellent antifungal
activity against a panel of 21 Candida strains which included C.
albicans, C. glabrata, C. krusei, C. parapsilosis, and C. auris as
well as four C. neoformans. Furthermore, these square-planar
complexes displayed good activity against four filamentous
strains of Aspergillus spp. and Fusarium spp. In addition,
compounds 4 and 6 displayed good activity against Candida
spp. biofilms. When tested against mammalian cells, the gold
complexes displayed limited improvement in selectivity index
over the FDA-approved drugs AmB and auranofin. Finally, by
development of resistance studies of compounds 4 and 6 in
Candida spp., it was found that Candida spp. has a low chance
Figure 6. Whole-cell uptake of 10 μM compound 4 (orange) and
compound 6 (blue) by C. albicans ATCC 10231 (strain B) and C.
glabrata ATCC 2001 (strain H) after 30 min treatment.
number of cells for analysis, a saturating amount of compound
(note that 10 μM was required to achieve saturation) and at a
time-point within the doubling time of the yeast. Both
compounds exhibited very similar uptake by C. albicans (strain
B) and C. glabrata (strain H) of ∼17 pmol/million cells.
However, the uptake when 5× MIC was used was higher than
when 10× MIC was used. With the values for gold uptake in
H
DOI: 10.1021/acs.jmedchem.9b01436
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Journal of Medicinal Chemistry
Article
was achieved via flash chromatography using CombiFlash Rf+ Lumen
with 5:95 MeOH:CH2Cl2.
of developing resistance to these gold complexes. Future
studies will work to decrease the toxic effect to mammalian
cells and to substantiate the mechanism of action of the gold
complexes in fungi.
Characterization of Compound 1. White solid (37 mg, 23%); Rf =
0.8 in 5:95 MeOH:CH2Cl2; 1H NMR (400 MHz, CDCl3, Figure S1)
δ 7.56−7.46 (m, 6H), 7.46−7.35 (m, 16H), 7.25−7.16 (m, 2H); 13C
NMR (101 MHz, CDCl3, Figure S2) δ 137.12, 137.05, 136.97,
134.81, 134.74, 134.67, 132.35, 131.90, 131.87, 131.84, 129.60,
129.54, 129.48, 128.91, 128.60, 128.28; 31P NMR (162 MHz, CDCl3,
Figure S3) δ 24.60. HRMS (ESI) (m/z): calcd for C30H24Au2Cl2P2
[M − Cl]+, 875.0373; found, 875.0408 Δ = 3.9998 (Figure S4).
Purity was demonstrated to be 97% by RP-HPLC: tR = 8.82 min using
the following method. Flow rate: 1 mL/min; λ = 260 nm; eluent A =
H2O with 0.1% TFA; eluent B = MeCN with 0.05% formic acid.
Elution program: 0 to 100% B over 10 min followed by 100 to 0% B
over 5 min and 4 additional min at 0% B (Figure S5).
EXPERIMENTAL SECTION
■
Chemistry. Materials and Instrumentation. Tetrahydrothio-
phene (THT) was from Sigma-Aldrich and used without further
purification or drying. Tetrachloroauric acid (HAuCl4•3H2O) was
purchased from Oakwood and used as received. THT and
HAuCl4•3H2O were used to prepare AuCl(THT) as previously
reported.44 All phosphorus ligands used, 1,2-bis(diphenylphosphino)-
benzene, 1,2-bis[(2S,5S)-2,5-dimethylphospholano]benzene, and 1,2-
bis[(2R,5R)-2,5-dimethylphospholano]benzene, were purchased from
Sigma-Aldrich and used as received. ACS grade solvents were
purchased from Pharmco-Aaper and used without further purification
or drying. Deuterated solvents were purchased from Cambridge
Isotope Laboratories and used as received. Silica gel for column
chromatography (Silicycle, P/N R10030B SiliaFlashF60, size 40−63
μm, Canada) was purchased from Silicycle. Aluminum backed silica-
gel plates (20 × 20 cm2) were purchased from Silicycle (TLA-
R10011B-323) and utilized for analytical thin-layer chromatography
(TLC).
Characterization of Compound 2. Yellow solid (68 mg, 36%); Rf
1
= 0.2 in 5:95 MeOH:CH2Cl2; H NMR (400 MHz, CDCl3, Figure
S6) δ 7.57−7.25 (m, 20H), 7.13−6.87 (m, 28H); 13C NMR (101
MHz, CDCl3, Figure S7) δ 142.13, 134.52, 134.37, 132.56, 132.44,
132.37, 132.33, 132.29, 132.25, 132.21, 132.18, 131.72, 130.40,
129.13, 129.11, 129.08, 129.06, 129.03, 129.01, 128.89, 128.86,
128.74; 31P NMR (162 MHz, CDCl3, Figure S8) δ 21.17. HRMS
(ESI) (m/z): calcd for C60H48AuClP4 [M − Cl]+, 1089.2372; found,
1089.2357 Δ = 1.3771 (Figure S9). Purity was demonstrated to be
100% by RP-HPLC: tR = 10.78 min using the following method. Flow
rate: 1 mL/min; λ = 260 nm; eluent A = H2O with 0.1% TFA; eluent
B = MeOH with 0.1% TFA. Elution program: 0 to 100% B over 5
min, stay at 100% B for 10 min, followed by 100 to 0% B over 4 min
(Figure S10).
All reactions were insensitive to air or moisture; as a result, they
were carried out under standard atmospheric conditions without air-
sensitive techniques or drying agents. Reactions were carried out in
round-bottom flasks or scintillation vials equipped with Teflon-coated
magnetic stir bars for stirring nonhomogenous reaction mixtures.
Reactions were monitored by NMR and TLC, and the TLC plates
were visualized under short-wavelength light (254 nm) or stained
with iodine on silica. All compound purification was performed using
silica-gel chromatography, employing CombiFlash Rf+ Lumen,
Teledyne ISCO. Filtrations were carried out using medium-porosity
ceramic funnels. Removal of solvents in vacuo was performed using a
Synthesis of [1,2-Bis[(2S,5S)-2,5-dimethylphospholano]-
benzene]digold(I) (3) and Bis-[1,2-bis[(2S,5S)-2,5-
dimethylphospholano]benzene]gold(I) (4). Compounds 3 and
4 were synthesized and separated following the procedure described
for the preparation of compounds 1 and 2 using AuCl(THT) (64.6
mg, 0.202 mmol) and 1,2-bis[(2S,5S)-2,5-dimethyl-1-phospholano]-
benzene (58.6 mg, 0.191 mmol).
Characterization of Compound 3. White solid (47 mg, 32%); Rf =
0.8 in 5:95 MeOH:CH2Cl2; 1H NMR (400 MHz, CDCl3, Figure S11)
δ 7.73−7.64 (m, 4H), 3.60 (sextet, J = 7.6 Hz, 2H), 2.99−2.85 (m,
2H), 2.53−2.38 (m, 2H), 2.30−2.13 (m, 2H), 1.92−1.77 (m, 2H),
1.57−1.44 (m, 2H), 1.37 (dd, J = 20.6, 6.7 Hz, 6H), 1.06 (dd, J =
17.2, 7.2 Hz, 6H); 13C NMR (101 MHz, CDCl3, Figure S12) δ
134.45, 134.39, 134.33, 132.19, 131.61, 131.58, 131.56, 37.20, 37.03,
37.01, 36.97, 36.84, 36.79, 36.61, 35.56, 35.54, 33.99, 33.92, 33.85,
19.59, 19.56, 19.52, 19.47, 19.42; 31P NMR (162 MHz, CDCl3, Figure
S13) δ 43.96. HRMS (ESI) (m/z): calcd for C18H28Au2Cl2P2 [M −
Cl]+, 735.0686; found, 735.0671 Δ = 2.0406 (Figure S14). Purity was
demonstrated to be 97% by RP-HPLC: tR = 7.94 min using the
following method. Flow rate: 1 mL/min; λ = 260 nm; eluent A = H2O
with 0.1% TFA; eluent B = MeCN with 0.05% formic acid. Elution
program: 0 to 100% B over 10 min followed by 100 to 0% B over 5
min and 4 additional min at 0% B (Figure S15).
Buchi rotary evaporator, and further drying was achieved by Schlenk
̈
line at ∼120 mTorr using a dynamic vacuum pump.
1H, 13C (1H-decoupled), and 31P (1H-decoupled) NMR spectra
were recorded on a Varian Unity 400 MHz NMR spectrometer with a
Spectro Spin superconducting magnet at the University of Kentucky
1
NMR facility in the Department of Chemistry. Chemical shifts in H
and 13C NMR spectra were internally referenced to solvent signals
(1H NMR, CDCl3 at δ = 7.26 ppm; 13C NMR, CDCl3 at δ = 77.16
ppm), and those in 31P NMR spectra, which were run in CDCl3, were
externally referenced to 85% H3PO4 in D2O at δ = 0 ppm.
High-resolution mass spectra (HRMS) were obtained using a direct
flow injection (injection volume = 1 μL) method with electrospray
ionization (ESI) on a Waters Q-TOF Premier instrument in the
positive mode. The optimized conditions were as follows: capillary =
3000 kV, cone = 35, source temperature = 120 °C, and desolvation
temperature = 350 °C. Mass spectrometry experiments and analysis
were conducted at the Chemical Instrumentation Center at Boston
University.
Characterization of Compound 4. Yellow solid (44 mg, 27%); Rf
1
= 0.4 in 5:95 MeOH:CH2Cl2; H NMR (400 MHz, CDCl3, Figure
In addition to spectroscopic characterization, the purity of all
compounds was assessed by RP-HPLC using an Agilent Technologies
1100 series HPLC instrument and an Agilent Phase Eclipse Plus C18
column (4.6 mm × 100 mm; 3.5 μm particle size). All compounds
were found to be ≥97% pure.
Synthesis and Characterization of Compounds 1−6. Syn-
t h e s i s o f t h e K n o w n C o m p o u n d s [ 1 , 2 - B i s -
(diphenylphosphino)benzene]digold(I) (1)45 and Bis-[1,2-bis-
(diphenylphosphino)benzene]gold(I) (2).46 Under normal at-
mospheric conditions, in a 25 mL round-bottom flask was placed
AuCl(THT) (58.7 mg, 0.183 mmol). CHCl3 (10.0 mL) was added,
and the solution (white suspension) was stirred at room temperature
for 2−3 min. To the solution was added 1,2-bis(diphenylphosphino)-
benzene (80.2 mg, 0.180 mmol). The solution turned yellow instantly.
The solution was stirred for about 1 h and monitored by TLC using
5:95 MeOH:CH2Cl2 as an eluent. Separation of compounds 1 and 2
S16) δ 7.75−7.67 (m, 4H), 7.60−7.54 (m, 4H), 2.71−2.46 (m, 8H),
2.35−2.13 (m, 8H), 1.87−1.72 (m, 4H), 1.62−1.49 (m, 4H), 1.19
(td, J = 10.6, 6.9 Hz, 12H), 0.81−0.71 (m, 12H); 13C NMR (101
MHz, CDCl3, Figure S17) δ 142.52, 142.36, 142.20, 133.34, 133.31,
133.29, 130.67, 40.56, 40.48, 40.40, 37.71, 37.65, 37.59, 36.06, 35.84,
21.47, 21.41, 21.36, 14.49; 31P NMR (162 MHz, CDCl3, Figure S18)
δ 38.34. HRMS (ESI) (m/z): calcd for C36H56AuClP4 [M − Cl]+,
809.2998; found, 809.3016 Δ = 2.2241 (Figure S19). Purity was
demonstrated to be 98% by RP-HPLC: tR = 10.81 min using the
following method. Flow rate: 1 mL/min; λ = 260 nm; eluent A = H2O
with 0.1% TFA; eluent B = MeCN with 0.05% formic acid. Elution
program: 0 to 100% B over 10 min followed by 100 to 0% B over 5
min and 4 additional min at 0% B (Figure S20).
Synthesis of the Known Compounds [1,2-Bis[(2R,5R)-2,5-
dimethylphospholano]benzene]digold(I) (5)81 and Bis-[1,2-
bis[(2R,5R)-2,5-dimethylphospholano]benzene]gold(I) (6).
I
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Journal of Medicinal Chemistry
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Compounds 5 and 6 were synthesized and separated following the
procedure described for the preparation of compounds 1 and 2 using
AuCl(THT) (61.9 mg, 0.193 mmol) and 1,2-bis[(2R,5R)-2,5-
dimethylphospholano]benzene (60.1 mg, 0.196 mmol).
Manassas, VA, USA). A panel of Candida auris strains were acquired
from the CDC & FDA Antibiotic Resistance Isolate Bank (CDC,
Atlanta, GA, USA), which included C. auris AR bank no. 0381-0390
(strains K, L, and I−VIII). C. neoformans clinical isolates CN1-CN3
(strains N−P) were generously provided by Dr. Nathan Wiederhold
(University of Texas, San Antonio, TX, USA). The filamentous fungi
Aspergillus nidulans ATCC 38163 (strain Q) and Fusarium
graminearum 053 (strain T) were kind gifts from Prof. Jon S.
Thorson (University of Kentucky, Lexington, KY) and Prof. Lisa
Vaillancourt (University of Kentucky, Lexington, KY, USA), while the
Aspergillus terreus ATCC MYA-3633 (strain R) and Aspergillus flavus
ATCC MYA-3631 (strain S) were purchased from the ATCC. Yeast
strains were cultured at 35 °C in yeast extract peptone dextrose
(YEPD) broth. Aspergillus spp. strains were cultured on potato
dextrose agar (PDA, catalog no. 110130, EMD Millipore, Billerica,
MA, USA) at 28 °C before the spores were harvested. All fungal
experiments were carried out in RPMI 1640 medium (catalog no.
R6504, Sigma-Aldrich, St. Louis, MO, USA) buffered to pH 7.0 with
0.165 M MOPS buffer (Sigma-Aldrich, St. Louis, MO, USA).
For cytotoxicity assays, the human embryonic kidney cell line
(HEK-293) was purchased from the ATCC. The human bronchial
epithelial cell line (BEAS-2B), the human lung carcinoma cell line
(A549), and the mouse macrophage cell line (J774A.1) were generous
gifts from Prof. David K. Orren (University of Kentucky, Lexington,
KY), Prof. Markos Leggas (University of Kentucky, Lexington, KY),
and Prof. David J. Feola (University of Kentucky, Lexington, KY),
respectively. A549, HEK-293, and BEAS-2B cells were cultured in
Dulbecco’s modified Eagle’s medium (DMEM, catalog no.
VWRL0100, VWR, Chicago, IL) supplemented with 10% fetal bovine
serum (FBS; from ATCC) and 1% penicillin/streptomycin (from
ATCC) at 37 °C with 5% CO2. The J774A.1 cells were cultured in
DMEM (catalog no. 30-2002, ATCC, Manassas, VA), which was also
supplemented with FBS and antibiotics and grown at 37 °C with 5%
CO2.
Characterization of Compound 5. White powder (35 mg, 35%);
Rf = 0.8 in 5:95 MeOH:CH2Cl2; 1H NMR (400 MHz, CDCl3, Figure
S21) δ 7.73−7.64 (m, 4H), 3.61 (sextet, J = 7.7 Hz, 2H), 2.99−2.86
(m, 2H), 2.54−2.38 (m, 2H), 2.30−2.12 (m, 2H), 1.92−1.76 (m,
2H), 1.56−1.44 (m, 2H), 1.37 (dd, J = 20.6, 6.8 Hz, 6H), 1.06 (dd, J
= 17.2, 7.2 Hz, 6H); 13C NMR (101 MHz, CDCl3, Figure S22) δ
134.62, 134.56, 134.49, 131.75, 131.72, 131.70, 37.37, 37.20, 37.19,
37.16, 37.02, 36.99, 36.81, 35.73, 35.71, 35.69, 34.17, 34.10, 34.03,
19.73, 19.71, 19.68, 19.63, 19.57; 31P NMR (162 MHz, CDCl3, Figure
S23) δ 43.96. HRMS (ESI) (m/z): calcd for C18H28Au2Cl2P2 [M −
Cl]+, 735.0686; found, 735.0697 Δ = 1.4965 (Figure S24). Purity was
demonstrated to be 97% by RP-HPLC: tR = 7.86 min using the
following method. Flow rate: 1 mL/min; λ = 260 nm; eluent A = H2O
with 0.1% TFA; eluent B = MeCN with 0.05% formic acid. Elution
program: 0 to 100% B over 10 min followed by 100 to 0% B over 5
min and 4 additional min at 0% B (Figure S25).
Characterization of Compound 6. Yellow powder (71 mg, 37%);
Rf = 0.2 in 5:95 MeOH:CH2Cl2; 1H NMR (400 MHz, CDCl3, Figure
S26) δ 7.74−7.66 (m, 4H), 7.60−7.53 (m, 4H), 2.71−2.44 (m, 8H),
2.35−2.13 (m, 8H), 1.86−1.72 (m, 4H), 1.61−1.48 (m, 4H), 1.18
(td, J = 10.5, 6.8 Hz, 12H), 0.80−0.71 (m, 12H); 13C NMR (101
MHz, CDCl3, Figure S27) δ 142.34, 133.32, 133.29, 133.27, 130.66,
40.54, 40.46, 40.39, 37.69, 37.63, 37.56, 36.04, 35.82, 21.45, 21.39,
21.34, 14.47; 31P NMR (162 MHz, CDCl3, Figure S28) δ 38.24.
HRMS (ESI) (m/z): calcd for C36H56AuClP4 [M − Cl]+, 809.2998;
found, 809.3025 Δ = 3.3362 (Figure S29). Purity was demonstrated
to be 97% by RP-HPLC: tR = 10.81 min using the following method.
Flow rate: 1 mL/min; λ = 260 nm; eluent A = H2O with 0.1% TFA;
eluent B = MeCN with 0.05% formic acid. Elution program: 0 to
100% B over 10 min followed by 100 to 0% B over 5 min and 4
additional min at 0% B (Figure S30).
Instruments for fungal assays with yeast were the V-1200
spectrophotometer (VWR, Radnor, PA, USA) and the SpectraMax
M5 plate reader (Molecular Devices, Sunnyvale, CA, USA) for
biofilm, cytotoxicity, and hemolysis assays. For the whole-cell uptake
assay, an inductively coupled plasma optical emission spectroscopy
instrument was used (ICP-OES, Agilent, Santa Clara, CA, USA). The
known antifungal drugs, amphotericin B (AmB, VWR, Chicago, IL,
USA), caspofungin (CAS, Sigma-Aldrich, St. Louis, MO, USA),
fluconazole (FLC, AK Scientific, Union City, CA, USA), voriconazole
(VRC, AK Scientific, Union City, CA, USA), and the antirheumatic
drug, auranofin (Santa Cruz Biotechnology, Dallas, TX, USA) were
used as positive controls.
Determination of Minimum Inhibitory Concentration (MIC)
Values of Compounds 1−6. The individual minimum inhibitory
concentration (MIC) values of compounds 1−6 were measured for
each fungal strain. The MIC values were determined using the broth
microdilution method88 in sterile 96-well plates. Concentrations of
compound tested were 0.06−31.3 μg/mL. For testing, compounds
were dissolved in DMSO at a concentration of 5 mg/mL allowing the
highest concentration of DMSO to be 0.63% in the assay. Serial 2-fold
dilutions of compound were made horizontally across the plate in 100
μL of RPMI 1640 medium. For yeast, the overnight culture was
diluted into RPMI 1640 (25 μL of a fungal stock with OD600 of 0.12−
0.15 into 10 mL of RPMI 1640 medium, resulting in final inoculum
size around (1−5) × 103 CFU/mL) and added to the plate (100 μL
per well), making a final volume of 200 μL total per well. Similarly, for
Aspergillus spp. and F. graminearum 053, spores were diluted in RPMI
1640 to 5 × 105 spores/mL and then 100 μL of stock was seeded in
each well.89 The MIC-0 value of each compound was determined by
visual inspection, and MIC-2 values were measured via optical density
reading at 600 nm. For Candida spp., plates were incubated for 48 h at
35 °C. For Cryptococcus spp. and Aspergillus spp. plates were
incubated for 72 h at 35 °C, and F. graminearum 053 the plate was
incubated at room temperature for 5 days. MIC values for CAS were
read at 24 h (Tables 1 and 2).
X-ray Crystallography of Compounds 3−6. The single crystal
of compound 3 was grown at 4 °C by vapor diffusion of Et2O into a
CH2Cl2 solution, and compounds 4, 5, and 6 were grown at room
temperature by vapor diffusion of Et2O into CDCl3 solutions. Suitable
crystals were selected by microscopic examination through crossed
polarizers, mounted on a fine glass fiber in polyisobutene oil, and
cooled to 90 K under a stream of nitrogen. A Bruker D8 Venture
diffractometer with graded-multilayer focused MoKα X-rays (λ =
0.710 73 Å) was used to collect the diffraction data from the crystals.
The raw data were integrated, scaled, merged, and corrected for
Lorentz polarization effects using the APEX3 package.82,83 Space
group determination and structure solution and refinement were
carried out with SHELXT and SHELXL,84,85 respectively. All non-
hydrogen atoms were refined with anisotropic displacement
parameters. Hydrogen atoms were placed at calculated positions
and refined using a riding model with their isotropic displacement
parameters (Uiso) set to either 1.2Uiso or 1.5Uiso of the atom to which
they were attached. The structures, deposited in the Cambridge
Structural Database (deposition number = 1889869 (3), 1889576
(4), 1889577 (5), and 1916580 (6)), were checked for missed higher
symmetry, twinning, and overall quality with PLATON,86 an R-
tensor,87 and finally validated using CheckCIF.86 The X-ray structures
of compounds 3−6 are presented in Figure 2 and the corresponding
structure refinement data in Table S1.
Biochemistry and Microbiology. Biochemical/Biological
Reagents and Instrumentation. The American Type Culture
Collection (ATCC) Candida albicans strains, including 10231 (strain
B), MYA-2876 (strain E), and 64124 (strain F), were a generous gift
from Dr. Jon Y. Takemoto (Utah State University, Logan, UT, USA).
The rest of the C. albicans strains, including MYA-1003 (strain A),
MYA-1237 (strain C), MYA-2310 (strain D), 90819 (strain G), and
the non-albicans Candida fungi C. glabrata ATCC 2001 (strain H), C.
krusei ATCC 6258 (strain I), C. parapsilosis ATCC 22019 (strain J),
and Cryptococcus neoformans ATCC MYA-85 (strain M) were
purchased from the American Type Culture Collection (ATCC,
J
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Time−Kill Assays for Compounds 4 and 6. To assess the time-
dependent inhibition of compounds 4 and 6 against four yeast strains,
C. albicans ATCC 10231 (strain B), C. glabrata ATCC 2001 (strain
H), C. auris AR bank no. 0384 (strain K), and C. auris AR bank no.
0390 (strain L), we performed time−kill assays. The protocol for
time−kill assays followed methods previously described with minor
modifications.34,90 Overnight cultures were grown in YEPD medium
at 35 °C with shaking at 200 rpm. The overnight cultures were diluted
in RPMI 1640 medium to an OD600 of 0.125 (∼1 × 106 CFU/mL).
Then, an amount of 200 μL of cells was added to 4.8 mL of RPMI
1640 medium in sterile culture tubes to afford a fungal cell
concentration of ∼1 × 105 CFU/mL. Compounds were then added
to the fungal cells. The treatment conditions included sterile control
(negative control), growth control, compound 4 at 1× MIC, 4 at 2×
MIC, 6 at 1× MIC, 6 at 2× MIC, as well as AmB at 1× MIC as a
positive control. Treated fungal cultures were incubated in the culture
tubes at 35 °C with 200 rpm shaking for 24 h. Samples were aliquoted
from the different treatments at regular time points (0, 3, 6, 9, 12, and
24 h) and plated in duplicate onto PDA plates. For each time point,
cultures were vortexed, 100 μL of culture was aspirated, and 10-fold
serial dilutions were made in sterile ddH2O. From the appropriate
dilutions, 100 μL of fungal suspension was spread on agar plates and
incubated at 35 °C for 48 h before colony counts were determined.
Only plates containing between 30 and 300 colonies were counted,
making 30 CFU/mL the limit of detection. At 24 h, 50 μL of sterile 2
mM resazurin in phosphate buffered saline (PBS) was added to the
treatments and incubated at 35 °C with 200 rpm shaking for 2 h in
the dark for visual inspection. As resazurin (blue-purple) is
metabolized by the cells to produce resorufin (pink-orange), the
addition of resazurin is used as a qualitative measure to confirm the
relative growth of the fungal cells in the different treatment conditions
(Figure 3).
Prevention of Biofilm Formation and Disruption of
Preformed Biofilm Assays for Compounds 4 and 6. To evaluate
the ability of the gold complexes to prevent formation of biofilms and
also their ability to disrupt preformed biofilms, we conducted assays
for compounds 4 and 6 against sessile yeast cells for four
representative yeast strains, C. albicans ATCC 10231 (strain B), C.
glabrata ATCC 2001 (strain H), C. auris AR bank no. 0384 (strain
K), and C. auris AR bank no. 0390 (strain L). All biofilm assays were
performed in 96-well plates using XTT [2,3-bis(2-methoxy-4-nitro-5-
sulfophenyl)-2H-tetrazolium-5-carboxanilide] to measure the viability
of the biofilm as previously described.91,92 An overnight culture of
yeast was grown at 35 °C in YEPD medium with shaking at 200 rpm
before dilution in RPMI 1640 medium to an OD600 between 0.12 and
0.15. For biofilm prevention assays, serial dilutions of compounds
were made in 100 μL of RPMI as in the MIC assays and 100 μL of
fungal suspension with OD600 of 0.12−0.15 was added. For assays
with a preformed biofilm, 100 μL of fungal cells were aliquoted in a
96-well plate, leaving one column empty for the sterile control. After
24 h incubation at 37 °C, visible biofilms had formed in the well. The
biofilm was washed three times with 100 μL of PBS. After washing,
RPMI 1640 medium and compound were added to the plate, in a
similar fashion to that described for the MIC values. All compounds
were tested in the concentration range of 0.06−31.3 μg/mL, and
AmB and auranofin were included as controls. Plates were incubated
at 37 °C for 24 h. Finally, the plates were washed three times with
PBS before adding 100 μL of XTT dye. The XTT was prepared by
dissolving XTT at 0.5 mg/mL concentration in sterile PBS. Before
addition to a plate, 1 μL of 10 mM menadione in acetone was added
to 10 mL of the 0.5 mg/mL solution of XTT. After addition of XTT
(containing menadione), the plates were incubated for 3 h at 37 °C in
the dark. 80 μL of liquid from each well was transferred to a new plate
and then with the plate reader for absorbance at 450 nm. For these
experiments, we determined the sessile MIC (SMIC90), which is
defined as the compound concentration required to inhibit the
metabolic activity of biofilm by 90% compared to the growth control
(Table 3). The plates used to determine the SMIC90 values are
provided in Figure S31 (prevention of biofilm formation) and Figure
S32 (disruption of preformed biofilm). Each assay was performed in
duplicate.
Mammalian Cytotoxicity Assays for Compounds 4 and 6.
To examine whether the compounds are safe for human cells,
cytotoxicity assays were done against four mammalian cell lines: HEK-
293, A549, BEAS-2B, and J774A.1 cells. Compounds 4 and 6 as well
as auranofin were tested against each cell line to measure their
cytotoxic effect by using a resazurin cell viability assay as previously
described with minor modifications.35,93 The assays were done in 96-
well plates, and cell counts were made using a hemacytometer. HEK-
293 and J774A.1 cells were plated at 1 × 104 cells/mL, while A549
and BEAS-2B were plated at 3 × 103 cells/mL. Compounds were
tested in concentrations ranging from 0.06 to 15.6 μg/mL with final
concentration of DMSO at 0.5% (Figure 4). It is important to note
that testing xenobiotics at sub-IC50 concentrations can result in
increase in cell growth, resulting in >100% cell survival in the
treatment groups.94−98 In instances where >100% cell survival was
observed, we displayed the data as 100% cell survival in Figure 4. We
are providing the data with observed % in Figure S33. All assays were
done in quadruplicate.
Measurement of Hemolysis for Compounds 4 and 6. To
extend on the cytotoxicity results, compounds 4 and 6 along with
auranofin and AmB were tested for their ability to lyse red blood cells
(RBCs). Both human and murine RBCs were provided in a citrate-
treated tube on ice, and the hemolysis assay was done as previously
described with minor modifications and in similar fashion to
cytotoxicity assays.39,99,100 The RBCs were washed three times in
PBS by resuspending 0.5 mL of RBCs in 5 mL of PBS and pelleting at
1000 rpm for 7 min. The RBCs were resuspended in PBS to achieve a
cell concentration on the order of 107 cells/mL. Compounds were
dissolved at concentration of 3.14 mg/mL (200×) in DMSO. Serial
double dilutions were made in DMSO. A 1:100 dilution of compound
in PBS was added to 100 μL of RBCs in a 96-well plate (total volume
of 200 μL). Compounds were tested in the range of 0.06−15.6 μg/
mL in quadruplicate with 0.5% DMSO and ∼5 × 106 RBCs per tube.
The RBCs were also treated with 1% Triton-X (positive control) and
PBS (negative control). The RBCs were treated for 30 min at 37 °C,
and the absorbance was read at 595 nm. Hemolysis is visually
observed by a decrease in optical density of the wells (turbid, dark red
to transparent pink). Percent hemolysis (Figure 5) was calculated
using this equation after subtraction of the background absorbance
(positive control):
absorbance of sample
absorbance of RBC + PBS (negative control)
% hemolysis =
× 100
Whole-Cell Uptake Assay for Compounds 4 and 6. To gain
insight into the mechanism of action of these compounds, we
measured the uptake of the gold-containing compounds into the yeast
cells. Compounds 4 and 6 were each tested with C. albicans ATCC
10231 (strain B) and C. glabrata ATCC 2001 (strain H) in
independent triplicates following protocols for whole-cell uptake
assays as previously described with minor modifications.42,101,102
A
single colony was used to inoculate 3 mL of YEPD, which was grown
overnight at 35 °C with 200 rpm shaking. Overnight culture was
diluted into 100 mL of YEPD to an OD600 of ∼0.075 and grown at 35
°C with 200 rpm shaking for 4−6 h until the culture reached an
OD600 of ∼0.3 indicating logarithmic phase growth. The cells were
pelleted by centrifugation at 500g for 5 min at room temperature and
diluted in RPMI to 108 cells/mL in RPMI 1640 medium as
determined by counting with a hemacytometer. 1 mL of fungal
suspension was aliquoted into a 12 mL culture tube. Treatment
conditions included 10 μM compound, growth control (no
compound), medium with compound (no cells), and 10 μM (8.5
μL) compound for ICP-OES analysis (100% signal). Each treatment
was tested in duplicate at 35 °C with 200 rpm shaking. After 30 min
of treatment, cells were pelleted by centrifugation at 3000 rpm
(∼1000g) for 5 min. Cell pellets were washed twice with 1 mL of ice-
cold PBS. Cell pellets were digested in 0.5 mL of concentrated HCl
and added to 4.5 mL of ddH2O (10% final concentration of HCl).
Samples were analyzed for gold content using ICP-OES. Data
K
DOI: 10.1021/acs.jmedchem.9b01436
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
presented (Figure 6) show values for 10 μM compound after
subtraction of values for media with compound.
ABBREVIATIONS USED
■
AmB, amphotericin B; ATCC, American Type Culture
Collection; CAS, caspofungin; CFU, colony forming unit;
ESI, electrospray ionization; FLC, fluconazole; HRMS, high
resolution mass spectrometry; ICP-OES, inductively coupled
plasma optical emission spectroscopy; MIC, minimal inhib-
itory concentration; PBS, phosphate buffered saline; RBC, red
blood cell; RP-HPLC, reverse-phase high-performance liquid
chromatography; SMIC, sessile minimal inhibitory concen-
tration; THT, tetrahydrothiophene; TLC, thin layer chroma-
tography; VRC, voriconazole
Development of Fungal Resistance for Compounds 4 and
6. To assess the rate at which fungal strains can develop resistance to
the gold compounds, fungal cells were repeatedly exposed to
subinhibitory amounts of compound and the MIC values for each
subculture were monitored. The procedure for the development of
resistance assay was modified for fungal cells following the reported
method.99 MIC assays were done as described above for compounds
4, 6, and AmB against C. albicans ATCC 10231 (strain B) and C.
glabrata ATCC 2001 (strain H). Overnight cultures were inoculated
1
from fungal cells exposed to
/ the MIC concentration for each
2
compound. This was repeated for 15 subcultures (Figure S34).
ASSOCIATED CONTENT
REFERENCES
■
■
S
* Supporting Information
The Supporting Information is available free of charge at
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AUTHOR INFORMATION
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*S.G.A.: e-mail, awuah@uky.edu.
*S.G.-T.: e-mail, sylviegtsodikova@uky.edu.
ORCID
Samuel G. Awuah: 0000-0003-4947-7283
Sylvie Garneau-Tsodikova: 0000-0002-7961-5555
Author Contributions
E.K.D. and S.G.-T. designed all the biochemical and biological
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Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
This work was supported in part by a University of Kentucky
Igniting Research Collaborations pilot grant, funded by the
Vice President for Research and College Deans (to S.G.-T. and
S.G.A.), and startup funds from the University of Kentucky (to
S.G.-T. and S.G.A.). We also thank the University of
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