Article

Cite This: ACS Infect. Dis. 2019, 5, 1376−1384 pubs.acs.org/journal/aidcbc

Discovery of Simplified Sampangine Derivatives with Potent

Antifungal Activities against Cryptococcal Meningitis
Zhuang Li,†,‡,∥ Na Liu,†,∥ Jie Tu,† Changjin Ji,† Guiyan Han,† and Chunquan Sheng*,†,‡
†
School of Pharmacy, Second Military Medical University, 325 Guohe Road, Shanghai 200433, People’s Republic of China
‡
School of Pharmacy, Fujian University of Traditional Chinese Medicine, 1 Qiuyang Road, Fuzhou, Fujian 350122, People’s
Republic of China
*
S Supporting Information
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ABSTRACT: Cryptococcal meningitis (CM) is associated

with high morbidity and mortality. Current antifungal drug
Downloaded via NOTTINGHAM TRENT UNIV on August 13, 2019 at 04:06:05 (UTC).

therapy for CM has the following challenges: limited efficacy,

significant side effects, emerging drug resistance, and
unavailability in highly needed countries. There is an urgent
need to develop novel CM therapeutic agents with a new
mode of action. On the basis of the antifungal natural product
sampangine, herein, novel simplified isoxazole derivatives were
identified to possess excellent inhibitory activity against
Cryptococcus neoformans (C. neoformans). Particularly, com-
pound 9a was highly active (the minimum inhibitory
concentration of 80% inhibition, MIC80 = 0.031 μg/mL)
and significantly inhibited biofilm formation, melanin, and urease production of C. neoformans. 9a had good blood−brain barrier
(BBB) permeability and effectively reduced the brain fungal burden in a murine model of cryptococcosis. The antifungal
mechanism of compound 9a was preliminarily investigated by transmission electron microscopy and flow cytometry. It was able
to cause necrocytosis of C. neoformans cells and cell cycle arrest in the G1/S phase. Isoxazole compound 9a represents a
promising lead compound for the development of novel CM therapeutic agents.
KEYWORDS: cryptococcal meningitis, sampangine derivatives, anticryptococcal activity, biofilm

L ife-threatening invasive fungal infections (IFIs) are mainly

caused by species of Candida, Cryptococcus, Aspergillus, and
Pneumocystis.1 Recently, the incidence and mortality of IFIs have
been increased rapidly due to the growing number of
immunosuppressive patients and limitations of available
antifungal therapy.2,3 In the central nervous system (CNS),
Cryptococcus neoformans (C. neoformans) is the most common
cause of fungal infection among the HIV-1/AIDS populations,
leading to cryptococcal meningitis (CM). It is estimated that
there are about 1,000,000 cases of CM in AIDS patients each
year with over 600,000 annual deaths.4−6 The high mortality of
CM is associated with the lack of effective antifungal
treatment.7,8 Clinically, only three drugs, namely, amphotericin
B (AMB), flucytosine, and fluconazole (FLC), are available for
the induction and maintenance regimens of CM.7,8 The
combination of AMB and 5-flucytosine is the “gold standard”
for the treatment of CM. However, AMB has limitations due to
significant adverse effects (e.g., renal toxicity), and 5-flucytosine
has not been marketed in most African and Asian countries,
where the morbidity of CM is high.8 FLC acts by a fungistatic
mechanism and is limited by severe drug resistance and
hepatotoxicity. The newly developed echinocandin antifungal
agents are poorly effective against Cryptococcus species.9,10 Thus,
there is an urgent need to develop novel anticryptococcal
agents.11,12
Screening of compound libraries13 and drug repurposing14
have been used to identify small molecules against the
C. neoformans infection. Alternatively, the discovery of novel
antifungal lead compounds from natural products represents an
effective strategy.15 Two important classes of antifungal agents,
polyenes and echinocandins, were originally discovered from
natural products.15 In our previous studies, a series of novel
antifungal lead compounds were identified from the natural
product sampangine by structural simplification and structural
optimization.16−18 As compared with sampangine, the simplified
new compounds showed improved antifungal potency and more
favorable physicochemical properties.16−18 Interestingly, they
revealed promising features to overcome drug resistance of
Candida albicans (C. albicans) in that compound ZG-20-7 was
fungicidal against FLC-resistant strains and significantly
inhibited biofilm formation and yeast-to-hypha morphological
transitions.16−18
Herein, the potential applications of sampangine derivatives
for the treatment of CM were investigated. After structural
optimization of compound ZG-20-7, novel tricyclic isoxazole
derivatives were identified to possess excellent activity against
C. neoformans. Particularly, compound 9a was highly active
Received: March 2, 2019
Published: May 9, 2019

© 2019 American Chemical Society 1376 DOI: 10.1021/acsinfecdis.9b00086

ACS Infect. Dis. 2019, 5, 1376−1384
ACS Infectious Diseases Article

Figure 1. Discovery of compound 9a as a novel antifungal agent.

Table 1. In Vitro Antifungal Activity of Tricyclic Isoxazole Derivatives (MIC80, μg/mL, 48 h)a

a
Abbreviations: C. neo., Cryptococcus neoformans; C. alb., Candida albicans.

(C. neoformans with a minimum inhibitory concentration of 80%

inhibition, MIC80, of 0.031 μg/mL, Figure 1) and showed potent
efficacy in the murine model of CM, representing a promising
lead compound for the development of novel CM therapeutic
agents.
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■ RESULTS
In Vitro Antifungal Activities and Structure−Activity
Relationship of the Tricyclic Isoxazole Derivatives.
Scaffold hopping of ZG-20-7 by isoxazole/pyridine replacement
was proven to be an effective strategy to improve the antifungal
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Table 2. In Vitro Antifungal Activity of Part of the Target Compounds (MIC80, μg/mL)a
48 h 72 h
compds C. gla. 8535 C. alb. 4108 C. alb. 0304103 C. tro. 5008 C. neo. ATCC 34877 C. gat. ATCC 14116 C. neo. H99
9a 1 1 1 1 0.031 0.25 0.031
9c 2 2 2 2 0.12 0.25 0.062
9d 4 4 4 8 0.5 0.5 0.25
9f 2 2 0.5 16 1 2 0.5
9h 16 8 16 16 0.5 1 1
FLC 8 >64 >64 >64 4 2 2
VRC 0.25 >64 >64 >64 0.062 0.031 0.12
a
Abbreviations: C. gla., Candida glabrata; C. tro., Candida tropicalis; C. gat., Cryptococcus gattii.

activity against C. neoformans.19 However, our previously
reported isoxazole derivatives were inactive in the in vivo CM
model (Figure S1). To further explore the structure−activity
relationship (SAR) and identify new antifungal lead compounds
with potent in vivo activity toward CM, derivatives 9a−h were
designed and synthesized (see the Supporting Information for
chemical synthesis and structural characterization). Their in
vitro antifungal activities were assayed against C. albicans
SC5314 and C. neoformans H99. FLC and voriconazole
(VRC) were used as the positive controls. Most compounds
showed better inhibitory activity against C. neoformans than
C. albicans (Table 1). Compounds 9a and 9c showed the best
antifungal activity against C. neoformans H99 (MIC80 = 0.031
μg/mL), which was significantly more potent than FLC and
VRC.
SAR revealed that the isoxazole/pyridine replacement led to
substantial improvement of the anti-C. neoformans activity. The
change of the molecular scaffold also resulted in different SAR
on the substitutions. The nitro group on the thiophene ring was
not essential for the antifungal activity, which was different from
that observed in the tricyclic pyridine derivatives.16−18 The
bromo substitution (9b) was found to have a negative impact on
the antifungal activity. When the quinone group was reduced
(9e) or replaced with pyranone (9g), an obvious decrease of the
antifungal activity was observed. The replacement of thiophene
with furan, benzene, or a pyridine ring was also unfavorable for
the antifungal activity.
On the basis of the antifungal activity, compounds 9a, 9c, 9d,
9f, and 9h were further assayed for the inhibitory activity against
drug resistant Candida species, such as C. glabrata, C. tropicalis,
and C. albicans, and other Cryptococcus species, such as
C. neoformans var. grubii and C. gattii (Table 2). These
compounds showed an MIC80 value ranging from 0.031 to 16
μg/mL. Among them, compound 9a was more potent than FLC
and comparable to VRC (Table 2). To further investigate
whether compound 9a has broad-spectrum antifungal activity,
A. f umigates, T. rubrum, and M. gypseum strains were assayed.
The result showed that compound 9a could inhibit a wide range
of fungal pathogens (Table 3). Due to its excellent
anticryptococcal activity, compound 9a was selected for further
biological evaluations.
Time-growth curves were assayed to further evaluate the
fungistatic activity of compound 9a. As shown in Figure 2A,
different concentrations of compound 9a (0.0078−0.062 μg/
mL) were used in the time-growth curve assay. The results
showed that compound 9a could inhibit C. neoformans H99 cell
growth in a concentration-dependent manner and completely
inhibited cell growth at 0.12 μg/mL. The time-kill curve assay
was used to test whether compound 9a had fungicidal activity.
The drug-free group, FLC group (4 and 16 μg/mL), and AMB
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Table 3. In Vitro Antifungal Activity of Compound 9a on
Mycete and Dermatophytes (MIC80, μg/mL)a
compds A. f um. 7544 T. rub. TIA M. gyp. MZC
9a 1 0.12 0.25
FLC >64 2 16
VRC 0.031 0.031 0.062
a
Abbreviations: A. f um., Aspergillus f umigutus; T. rub., Trichophyton
rubrum; M. gyp., Microsporum gypseum.
group (4 and 16 μg/mL) were used as controls. As shown in
Figure 2B, compound 9a had excellent fungicidal activity against
C. neoformans, which could completely kill fungi cells at 1 μg/mL
within 48 h. In contrast, FLC did not show fungicidal activity at
16 μg/mL. Compound 9a also showed excellent fungicidal
activity to drug resistant C. albicans (Figure S2).
Inhibitory Effect of Compound 9a on the Virulence
Factors of C. neoformans. Biofilm is an important virulence
factor contributing to antifungal drug resistance, which often
leads to repeated fungal infections in the clinic and represents a
significant therapeutic challenge.20 Thus, the effect of
compound 9a on biofilm formation of C. neoformans was
evaluated using AMB as the positive control. The results showed
that it significantly inhibited the biofilm formation with an
inhibition rate of more than 80% at the concentration of 0.062
μg/mL (Figure 3), which was more potent than AMB.
Melanin and urease are two important virulence factors
during C. neoformans infection. Melanin contributes to virulence
by protecting fungal cells against attack by multiple toxic insults
and immune effector cells.21 Urease in C. neoformans plays an
important role in the survival of C. neoformans in phagolyso-
somes and the dissemination to the brain causing meningoen-
cephalitis.22 Thus, various induction mediums were used to
determine the effects of compound 9a on the production of
melanin and urease. As shown in Figure 4A, the melanin
production could be determined; colonies containing melanin
turn black when exposed to melanin induction medium. The
results revealed that compound 9a inhibited the production of
melanin at the concentration of 8 μg/mL. Similarly, if the cells
produced urease, the medium would appear red. Compound 9a
also inhibited the production of urease at the concentration of
32 μg/mL (Figure 4B).
Compound 9a Induced Morphological Changes in
C. neoformans Cells. In order to investigate the fungicidal
mechanism of compound 9a, its influence on the morphology of
C. neoformans H99 was monitored by transmission electron
microscopy (TEM). C. neoformans H99 cells were treated with
compound 9a at the concentrations of 0.062 and 2 μg/mL for 8
h, and cells treated with dimethyl sulfoxide (DMSO) were used
as a control group. As shown in Figure 5A, normal C. neoformans
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Figure 2. Compound 9a inhibited and killed C. neoformans H99 cells. (A) Time-growth curves of C. neoformans H99 obtained by using initial
inoculums of 2 × 106 colony-forming units (CFU)/mL. The concentration of cells was determined after 0, 4, 8, 12, 24, and 48 h of incubation.
C. neoformans H99 cells were treated with different concentrations of compound 9a. (B) Time-kill curves of C. neoformans H99 obtained by using an
initial inoculum of 2 × 106 CFU/mL. The number of viable cells was measured after 12, 24, 36, and 48 h of incubation. Cells were treated with
compound 9a (0.062 and 1 μg/mL), FLC (4 and 16 μg/mL), and AMB (4 and 16 μg/mL). The number of fungal cells was counted at different times
and to the base 10 logarithm.

Figure 3. Effects of different concentrations of compound 9a and AMB on biofilm formation. C. neoformans H99 cells were treated with compound 9a
and AMB (0.062−8 μg/mL) for 24 h. The results of biofilm formation were represented as the means ± standard deviations for three independent
experiments. Statistical differences among groups were determined by ANOVA. The comparison between the two components was completed by the
t-test. *P < 0.05 compared to the control group; **P < 0.01 compared to the control group; ***P < 0.001 compared to the control group; ****P <
0.0001 compared to the control group.

cells revealed an intact cell wall and membrane, dense capsule,
uniform central density, and regular cell morphology. For the
cells treated with compound 9a at 0.062 μg/mL, a sparse capsule
and decomposed organelles could be observed (Figure 5B). At
the same time, the cell membrane is separated from the cell wall.
After the cells were treated with compound 9a at 2 μg/mL, large
vacuoles could be observed (Figure 5C), which is characteristic
of necrocytosis. TEM results indicated that compound 9a was
able to cause significant damage to C. neoformans cells, both at
low and high concentrations.
Compound 9a Caused Necrocytosis of C. neoformans
Cells and Induced G1/S Phase Arrest. Necrocytosis of
C. neoformans H99 treated with compound 9a was further
investigated by flow cytometry. The percentage of necrotic cells
in the control group was 2.83%. After treating with compound
9a at 0.12, 0.5, and 2 μg/mL for 24 h, the percentages of necrotic
cells were 10.1%, 34.9%, and 46.9%, respectively (Figure 6). The
results suggested that compound 9a caused a remarkable
necrotic effect instead of apoptosis, which was different from the
results of early apoptosis caused by FLC (percentage of
apoptosis cells: 11.5%).
The effect of compound 9a on the cell cycle was investigated
using flow cytometric analysis to further clarify the antifungal
mechanism. The results showed that 10.04% of cells treated with
DMSO were in the G1 phase. The ratios in the G1 phase were
49.19%, 37.68%, and 45.92% after being treated with compound
9a at 0.5, 1, and 2 μg/mL, respectively (Figure 7A). The results
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indicated that compound 9a could significantly increase the
subpopulation of cells in the G1 phase (P < 0.001) and reduce
the number of cells in the G2 phase (P < 0.001) (Figure 7B),
resulting in cell cycle arrest at the G1/S phase. Similarly, FLC
was proven to cause the accumulation of G1 and S phase cells in
our previous studies.20
Compound 9a Showed Potent Activity in a Murine
Model of CM. The blood−brain barrier (BBB) permeability of
compound 9a was tested by the parallel artificial membrane
permeation assay (PAMPA) prior to the investigation of in vivo
antifungal activity. The results indicated that compound 9a had
good BBB permeability and was superior to clonidine (Table 4).
Moreover, compound 9a showed good metabolic stability in
mouse liver microsomes (T1/2 > 120 min). Due to the excellent
in vitro antifungal activity, good ability to cross the BBB, and
metabolic stability, compound 9a was chosen for the evaluation
of in vivo anti-cryptococcus potency. Mice CM models were
established by injecting C. neoformans H99 cells into the veins of
the tails. Mice were assigned into four groups: the control group,
the FLC treatment group, and two 9a treatment groups (5 and
10 mg/kg). FLC and 9a treatments were initiated at 2 h
postinfection. Such treatments were continued for 5 consecutive
days. The treatment with compound 9a significantly reduced the
fungal burden in the brain at 5 mg/kg (P < 0.05) and 10 mg/kg
(P < 0.01). The results highlighted the promising features of
compound 9a as a lead compound for the treatment of CM
(Figure 8, Table S1).
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Figure 4. Effects of compound 9a on melanin and urease production by
C. neoformans H99 cells. (A) C. neoformans H99 cells grown on L-
DOPA medium were treated with different concentrations of the
compounds for 48 h to determine melanin production. (B) Strains
grown on urea medium for 72 h were used to determine the production
of urease.
Figure 5. TEM of C. neoformans H99 under different concentrations of
compound 9a for 8 h. (A) Cells treated with DMSO. (B) Cells treated
with compound 9a at 0.062 μg/mL. (C) Cells treated with compound
9a at 2 μg/mL. The organelles indicated by the arrows are as follows: 1,
capsules present in the outer layers of the cell wall; 2, cell well; 3, cell
membrane; 4, decomposed organelles; 5, wrinkled cell membrane; 6,
significantly increased vacuoles.
■ DISCUSSION
CM is a serious disease with high incidence and mortality in
HIV-positive individuals.7,8 However, current antifungal treat-
ment of CM remains a big challenge mainly due to poor efficacy
of existing drugs, significant side effects, emerging drug
resistance, and unavailability in highly needed countries.7,8
Thus, novel anti-CM agents with excellent anticryptococcal
activity, favorable BBB penetration, and good safety profiles are
highly desirable. As compared with the optimization of known
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antifungal agents (e.g., triazole derivative VT-159823), the
discovery of anticryptococcal molecules with new chemical
scaffolds and a new mode of action is a challenging task.
Natural products are an important source of antifungal agent
(e.g., AMB and echinocandins). Compound 9a was derived
from structural simplification and structural optimization of the
antifungal natural product sampangine. Although sampangine
showed potent in vitro antifungal activity, its further develop-
ment was terminated due to the lack of in vivo potency.16−18 As
compared with sampangine, the anticryptococcal activity of
compound 9a was greatly improved, which was also more potent
than triazole antifungal agents FLC and VRC. Moreover, an
antifungal agent with fungicidal activity is preferred more than a
drug only inhibiting fungal growth. Unlike FLC, compound 9a
revealed excellent fungicidal activity and completely killed
C. neoformans cells at 1 μg/mL. Interestingly, there is a
significant increase in dead cells from 36 to 48 h. Obvious
cellular damage can be observed in the TEM images after
treatment with compound 9a. The sustained damage caused by
a high concentration of drugs may lead to massive death of
fungal cells at a certain point in time.
Virulence factors that contribute to promoting disease or
damaging the host are produced by fungal pathogens.
Antivirulence agents are expected to decrease evolutionary
pressure for the development of drug resistance.24 Interestingly,
compound 9a could significantly inhibit several important
virulence factors of C. neoformans. The formation of biofilms is a
kind of survival mechanism of fungi, which is associated with
high-level drug resistance.25,26 Compound 9a showed excellent
activity to inhibit C. neoformans biofilm formation (>80%
inhibition at 0.062 μg/mL), highlighting its potential to treat
biofilm-related infections. Moreover, compound 9a also
effectively inhibited the production of melanin and urease in
C. neoformans cells. Melanin has antioxidant properties and
protects fungal cells against the oxidative stress responses, which
is generally understood to be the first line of defense against
reactive oxygen species (ROS) formation.27,28 Several com-
pounds (e.g., o-vanillin) with fungicidal activity were reported to
cause mitochondrial dysfunction by triggering oxidative stress
responses.29 Thus, it is suggested that compound 9a might
induce the oxidative stress in fungal cells, leading to the
inhibition of melanin production and fungicidal effect.
Although several new anticryptococcal molecules have been
identified, it is still a big challenge to translate in vitro activity into
therapeutic efficacy due to unfavorable physicochemical proper-
ties and pharmacokinetic profiles.11,12,14 For example, our
previously reported alkyl analogues of compound 9a lacked in
vivo anti-CM potency (Figure S1).19 In contrast, compound 9a
had good BBB permeability and metabolic stability and
significantly reduced the brain fungal burden in a murine
model of cryptococcosis. Thus, it represents a promising lead
compound for the development of novel therapeutic agents for
CM. However, the water solubility of compound 9a (7.72 μg/
mL, Table S2) was relatively poor, which remains to be
improved to achieve better pharmacokinetic profiles as well as
the in vivo antifungal potency.
As an important step in drug discovery and development,
clarifying the mode of action of compound 9a is highly required.
Herein, preliminary antifungal mechanisms of compound 9a
were investigated. The results revealed that compound 9a
caused necrocytosis of C. neoformans cells and cell cycle arrest in
the G1/S phase, which is totally different from FLC and acts by a
new mode of action. It has been reported that large doses of
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Figure 6. Compound 9a caused C. neoformans H99 cells necrosis. Cells treated with compound 9a at 0.12, 0.5, and 2 μg/mL and FLC at 16 μg/mL for
24 h. Necrosis was detected by flow cytometry (n = 3). Representative images from three independent experiments are displayed.
acetic acid (120 mM) could cause necrosis of C. albicans and
oxygen consumption in necrotic cells was negligible.30 The
morphological changes associated with ROS-induced death
caused by acetic acid in C. albicans also suggested that the
necrosis induced by compound 9a might be related to oxidative
stress. The detailed antifungal mechanisms and molecular
targets of compound 9a remain to be further explored. Also, the
SAR and structural optimizations of compound 9a are ongoing
to find more potent compounds for the treatment of CM.
■
METHODS
In vitro Antifungal Activity Assay. In vitro antifungal
activity was measured by the serial dilution method in 96-well
microtest plates. C. albicans strain SC5314 and C. neoformans
strain H99 were used in this study. MIC was analyzed according
to the recommendations of the Clinical and Laboratory
Standards Institute (CLSI, M27). RPMI 1640 (Gibco BRL)
was buffered with 0.165 M MOPS (Sigma), NaHCO3 (0.2%),
and NaOH (0.27%) as the test medium. All strains were
routinely grown in YPD with 1% yeast extract (BD), 2% peptone
(BD), and 2% D-glucose (Sangon Biotech) liquid medium at 30
°C in a shaking incubator. Briefly, the initial concentration of
fungal suspensions in RPMI 1640 medium was 103 CFU/mL,
and tested compounds were dissolved in DMSO serially diluted
in growth medium. The yeast strains were incubated at 35 °C for
48 or 76 h.
Growth Curve Assay. The growth curve assay was
performed according to our reported method with a few
modifications.31 Briefly, exponentially growing C. neoformans
H99 cells were harvested and resuspended in 5 mL of fresh
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RPMI 1640 medium to a concentration of 2 × 106 cells/mL.
Various concentrations of compound 9a were added. The
samples were cultured at 30 °C under constant shaking (200
rpm). A portion of the cell suspension was removed and counted
at the indicated time points (2, 4, 6, 9, 12, 24, and 48 h). DMSO
was added into the control group. Three independent
experiments were performed.
Fungicidal Activity Assay. Time-kill curves were used to
evaluate the fungicidal activity of compound 9a. The
experimental operation was carried out according to the
reported method.32 Exponentially growing C. neoformans H99
cells were washed with PBS (3 × 1 mL) and then resuspended
with RPMI 1640 medium to 2 × 106 cells/mL and divided into
different bottles. Different concentrations of the compound
were added into the bottles, and the suspension was cultured at
30 °C under constant shaking (200 rpm). A portion of the cell
suspension was removed at the indicated time points (12, 24, 36,
and 48 h) and diluted to appropriate folds. The diluent cell
suspension was plated on SDA agar medium to determine the
concentration after incubation for 48 h at 30 °C.
In vitro Biofilm Formation Assay. The experimental
procedure was performed according to the reported method
with a few modifications.32 Suspensions of C. neoformans H99
cells (1.0 × 106 CFU/mL in RPMI 1640 medium) were added
into a 96-well tissue culture plate (Corning, USA) and allowed
to incubate at 37 °C for 90 min. After that, the upper culture
medium and the nonadherent cells were removed. The adherent
cells were washed with PBS three times. Fresh RPMI 1640
medium with or without drugs was then added to adherent cells.
The concentration for AMB was 64 μg/mL, and a drug-free
sample served as a control. Concentrations for compound 9a
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Figure 7. Effect of compound 9a on cell cycle in C. neoformans H99
cells. (A) Cell cycle analysis by flow cytometry. C. neoformans H99 cells
were treated for 24 h with different concentrations of compound 9a.
(B) Mean percentages of cells in G1 and G2. The percentages in
different phases were calculated by flow cytometry software. Statistical
significance among groups was determined by analyses of variance.
Comparison between the drug-free treatment group and the compound
9a treatment group was performed by the Student’s t-test. The data
were shown as means ± SDs from three independent experiments. **P
< 0.01, compared with control; ***P < 0.001, compared with control.
Table 4. Permeability (Pe × 10−6 cm/s) and Prediction of
BBB Penetration of Compound 9a
compound Pea (10−6 cm/s) predictionb
clonidine 3.71 ± 0.33 CNS+
9a 7.91 ± 0.01 CNS+
a
Values are expressed as the mean ± SD of at least three independent
experiments. bCompounds with Pe > 3.08 × 10−6 cm/s could cross
the BBB by passive diffusion (CNS+). Compounds with Pe < 1.13 ×
10−6 cm/s could not cross the BBB (CNS−), and compounds with
1.13 × 10−6 cm/s < Pe < 3.08 × 10−6 cm/s show uncertain BBB
permeation (CNS±).
were 0.062−8 μg/mL. Then, the plates were incubated for a
further 24 h at 37 °C. A semiquantitative measure of biofilm
formation was calculated by using an XTT reduction assay.
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Figure 8. Therapeutic efficacy of compound 9a and FLC in mouse
models of cryptococcosis. C. neoformans brain burdens (in CFU/g of
brain) are shown for animals treated with the indicated dose of 9a and
FLC for 5 days after tail vein inoculation with 2 × 105 CFU/animal. P
values are from ANOVA. *P < 0.05, **P < 0.01, and ***P < 0.001.
Optical density at 490 nm (OD490) was determined using a
microtiter plate reader.
Melanin and Urease Production Assay. Exponentially
growing C. neoformans H99 cells were harvested, washed three
times with PBS, and resuspended in RPMI 1640 medium at 1 ×
106 cells/mL. Different concentrations of compound 9a and
FLC were added into the suspension. Cell suspensions
containing different concentrations of drugs were dropped
onto the L-DOPA or urea medium and incubated at 37 °C to
detect the production of melanin and urease.
Transmission Electron Microscopy. Transmission elec-
tron microscopy images of C. neoformans H99 cells were
obtained according to the previous protocol with a few
modifications.32 In the presence of compound 9a (0.062 and
2 μg/mL), C. neoformans H99 cells were collected after 8 h of
growth in liquid YPD medium, washed three times with PBS
solution, and fixed at 4 °C for 24 h in 1 mL of 4%
paraformaldehyde solution. The samples were then washed
with saline and fixed for 90 min with 1% phosphotungstic acid.
The fixed cells were dehydrated through a graded series of
ethanol and embedded with EPON-812. Ultrathin sections were
prepared and observed after double staining with uranium under
a transmission electron microscope (HITACHI H-800, Japan)
with 1 × 104 magnification.
Cell Necrosis Detection by Flow Cytometry. The
experimental procedure was performed according to the
reported method with a few modifications.33 Exponentially
growing C. neoformans H99 cells were harvested, washed twice
with PBS, and resuspended in RPMI 1640 medium in 20 mL at 5
× 105 cells/mL. Different concentrations of compound 9a were
added into the suspension. Each sample was centrifuged and
washed with PBS three times. Each 50 mg of cells (wet weight)
was resuspended with 100 μL of PBS (containing 0.24%
snailase) and then incubated for 1 h at 30 °C to break the cell
wall. The sample was washed with PBS twice and fixed with 70%
ethanol overnight. Then, the ethanol was removed. After the
sample was resuspended in 400 μL of binding buffer, it was then
added to 5 μL of annexin V-FITC and incubated at room
temperature for 15 min. Then, 10 μL of propidium iodide (PI)
was added into the suspensions and incubated at room
temperature for another 15 min in the dark. The stained cells
were analyzed by a flow cytometer (Becton, Dickinson, San Jose,
CA).
DOI: 10.1021/acsinfecdis.9b00086
ACS Infect. Dis. 2019, 5, 1376−1384
ACS Infectious Diseases
Cell Cycle Analysis by Flow Cytometry. The preparation
procedure of the sample treated with different concentrations of
compound 9a is similar to the step of the cell necrosis assay. The
cells were then stained with 50 μg/mL of PI at 4 °C for 30 min.
The samples were sonicated to obtain separate cells. Data were
collected using a FACSCalibur cytometer (Becton, Dickinson,
San Jose, CA) and analyzed with Cell Quest 3.0 software.
In Vitro Blood−Brain Barrier Permeation Assay. BBB
penetration of compound 9a was performed using a parallel
artificial membrane permeation assay (PAMPA) according to a
previous protocol.34 The acceptor 96-well microplate was filled
with 300 μL of a PBS/EtOH (7/3) solution, and the filter
membrane was impregnated with 4 μL of porcine brain lipid
(PBL) in dodecane (20 mg/mL). The compound was diluted to
100 μg/mL with PBS/EtOH (7/3), and then, 200 μL was added
to a donor well (PVDF membrane, pore size 0.45 mm). The
acceptor 96-well microplate filled with 300 μL of PBS/EtOH
(7/3) was placed on the donor plate to form an interlayer, which
was allowed to stand at 25 °C for 12 h. After the incubation was
complete, the donor plate was removed and the concentration of
compounds in the acceptor wells was determined by using a UV
plate reader (SpectraMax i3). Three replicates were performed
for each group.
In Vivo Antifungal Potency Assay. Female ICR mice were
purchased from JOINN Laboratories and were 20 to 25 g at the
time of experimentation. An inoculum of 2 × 105 CFU of
C. neoformans in 0.2 mL of normal saline (NS) was used for each
mouse. FLC (2 mg/kg in NS) and compound 9a (10 mg/kg
suspended in NS with 1.5% glycerin and 0.5% Tween 80) were
injected intraperitoneally 2 h after inoculation. The compound
was administered continuously for 5 days. Groups of mice were
sacrificed 5 days after continuous administration. The brains
were removed and homogenized. Serial 10 000-fold dilutions
were plated on YPD agar supplemented with chloramphenicol
and incubated in air at 30 °C for at least 48 h to enumerate the
total fungal burden. The control group consisted of mice treated
with NS. The concentration (CFU/mL) of each brain was
calculated, and the differences between groups were analyzed by
analysis of variance (ANOVA).
■
ASSOCIATED CONTENT
*
S Supporting Information
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acsinfec-
dis.9b00086.
Potency of compound 8a; time-kill curves and water
solubility of compound 9a; supplementary in vivo results;
detailed procedure for chemical synthesis and spectral
data of targets compounds; HPLC spectra of representa-
tive compounds (PDF)
■
AUTHOR INFORMATION
Corresponding Author
*E-mail: shengcq@smmu.edu.cn (C.S.).
ORCID
Chunquan Sheng: 0000-0001-9489-804X
Author Contributions
∥ (18) Liu, N., Zhong, H., Tu, J., Jiang, Z., Jiang, Y., Jiang, Y., Jiang, Y., Li,
Z.L. and N.L. contributed equally to this work.
Notes
The authors declare no competing financial interest.
1383
■
Article
ACKNOWLEDGMENTS
This work was supported by the National Natural Science
Foundation of China (Grant 81725020 to C.S.), the Innovation
Program of Shanghai Municipal Education Commission (Grant
2019-01-07-00-07-E00073 to C.S.), and the Science and
Technology Commission of Shanghai Municipality (Grant
17XD1404700 to C.S.).
■
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1384 DOI: 10.1021/acsinfecdis.9b00086

ACS Infect. Dis. 2019, 5, 1376−1384