Drug Repurposing of Haloperidol: Discovery of New Benzocyclane Derivatives as Potent Antifungal Agents against Cryptococcosis and Candidiasis

Article abstract of DOI:10.1021/acsinfecdis.9b00197

Despite the high morbidity and mortality of invasive fungal infections (IFIs), effective and safe antifungal agents are rather limited. Starting from antifungal lead compound haloperidol that was identified by drug repurposing, a series of novel benzocycl

Full text of DOI:10.1021/acsinfecdis.9b00197

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Article
Drug Repurposing of Haloperidol: Discovery of
New Benzocyclane Derivatives as Potent Antifungal
Agents against Cryptococcosis and Candidiasis
Changjin Ji, Na Liu, Jie Tu, Zhuang Li, Guiyan Han, Jian Li, and Chunquan Sheng
ACS Infect. Dis., Just Accepted Manuscript • DOI: 10.1021/acsinfecdis.9b00197 • Publication Date (Web): 24 Sep 2019
Downloaded from pubs.acs.org on September 26, 2019
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Drug Repurposing of Haloperidol: Discovery of New Benzocyclane
Derivatives as Potent Antifungal Agents against Cryptococcosis and
Candidiasis
Changjin Ji1,2, #, Na Liu , Jie Tu , Zhuang Li , Guiyan Han , Jian Li *, Chunquan
2, #
2
1,2
2
1,
2
,
Sheng *
1
School of Pharmacy, East China University of Science & Technology, 130 Meilong
Road, Shanghai 200237, People’s Republic of China
2
Department of Medicinal Chemistry, School of Pharmacy, Second Military Medical
University, 325 Guohe Road, Shanghai 200433, People’s Republic of China
*Corresponding Author: E-mail, shengcq@smmu.edu.cn (C. Sheng)
*
Corresponding Author: E-mail, jianli@ecust.edu.cn (J. Li)
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Despite the high morbidity and mortality of invasive fungal infections (IFIs), effective
and safe antifungal agents are rather limited. Starting from antifungal lead compound
haloperidol that was identified by drug repurposing, a series of novel benzocyclane
derivatives were designed, synthesized and assayed. Several compounds showed
improved antifungal potency and broader antifungal spectrum. Particularly,
compound B10 showed good inhibitory activities against a variety of fungal
pathogens, and was proven to be inhibitors of several virulence factors important for
drug resistance. In the in vivo cryptococcosis and candidiasis models, compound B10
could effectively reduce the brain fungal burden of Cryptococcus neoformans, and
synergize with fluconazole to treat resistant Candida albicans infections. Preliminary
antifungal mechanism studies revealed that compound B10 regained cell membrane
damage and down-regulated the overexpression of ERG11 and MDR1 genes when
used in combination with fluconazole. Taken together, haloperidol derivative B10
represent a promising lead compound for the development of new generation of
antifungal agents.
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KEYWORDS: Antifungal, haloperidol, drug repurposing, Cryptococcus neoformans,
drug
resistance,
Candida
albicans
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It is estimated that there are approximately 5 million species of fungi widely prevalent
in environment, out of which about 300 can cause human infections, but only 20-25 of
1
them are common in clinic. Invasive fungal infections (IFIs) are mostly caused by
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opportunistic pathogens and often occur in immunocompromised people, such as
HIV-infected and organ transplantation patients or people undergoing
chemotherapy.2-6 Candida, Cryptococcus, Aspergillus and Pneumocystis are the most
common fungal pathogens causing approximately 2 million infected people and 1
million deaths each year. 7-9
With rapid expansion of the immunodeficiency population, the morbidity of IFIs
increases each year, and the mortality remains high.10-17 For example, Cryptococcus
and Candida infect over 1,000,000 and 400,000 people each year, while the mortality
can be up to 20-70% or 46-75%, respectively.11 However, the development of
antifungal drugs has lagged far behind. Since 1950s, there have been only four classes
of antifungal agents (i.e. polyenes, azoles, echinocandins and flucytosine) are
available for the treatment of IFIs.18, 19 Even though, they can hardly achieve a fast
action and significant efficacy against severe IFIs such as cryptococcal meningitis
(
CM).20-22 In some cases, drug treatment has to last for several months or even
23
longer. Meanwhile, most antifungal agents have moderate to severe side effects,
especially polyenes.24-28 Although echinocandins are well tolerated by patients, they
are poorly effective against Cryptococcus.29, 30 Moreover, severe drug resistance has
been developed due to broad use of antifungal agents, especially that the resistance of
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Candida albicans towards azoles is becoming increasingly common.31-33 Therefore,
the development of new generation of antifungal agent is highly desirable.
Recently, drug repurposing (also called drug repositioning) has been proven to be
an promising approach to accelerate drug development not only in identifying new
uses for old drugs but also offering new lead compounds.34 A series of
“non-antifungal” drugs with antifungal activity were identified by screening libraries
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of marketed drugs. Among them, haloperidol, a butylbenzoic antipsychotics drug,
35
was reported to exhibit inhibitory effect against Candida albicans and a synergistic
3
6
effect against Malassezia . More recently, Garneau-Tsodikova et al. reported
bromperidol derivatives had synergistic effects with triazole antifungal agents to treat
Candida infections.37 Thus, haloperidol represents a good lead compound for the
development of novel antifungal agents. However, the structure-activity relationship
(SAR) of haloperidol is still unknown, and further improving the antifungal potency
and clarifying the antifungal mechanisms remains a highly challenging task.
Herein, a series of haloperidol derivatives were designed and synthesized using the
scaffold hopping approach (Figure 1). As compared with haloperidol, several
benzocyclane derivatives showed improved antifungal activity and broader antifungal
spectrum. In particular, compound B10 exhibited potent in vitro and in vivo antifungal
activity in treating cryptococcosis and candidiasis. Interestingly, compound B10
revealed excellent synergistic effects with fluconazole (FLC) against FLC-resistant C.
albicans. The synergistic mechanisms were preliminarily clarified by transmission
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electron microscopy and real-time RT-PCR analysis. Thus, compound B10 represents
a promising lead compound for the development of novel antifungal agents.
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Figure 1. Structural optimization process of antifungal lead compound haloperidol.
RESULTS AND DISCUSSION
Chemistry. Chemical synthesis of benzocyclane derivatives are described in
Schemes 1-5. Starting from compound 1, its carbonyl group was reduced by
Et SiH and trifluoroacetic acid (TFA), followed by Friedel-Crafts acylation with
3
4
-bromobutanoyl chloride (3b) to afford intermediate 4b, which was further
substituted with 4-(4-chlorophenyl)piperidin-4-ol (5) to give target compound A1
Scheme 1). Ester 7 was obtained by Friedel-Crafts acylation of intermediate 2 with
(
methyl 4-chloro-4-oxobutanoate (4), and then hydrolyzed to acid 8, which was treated
with intermediate 5 to afford target compound A2 (Scheme 2). After the reduction of
intermediate 7, the resulting compound 9 was hydrolyzed into acid 10, and then
condensed with intermediate 5 in the presence of HATU and Et N to afford target
3
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compound A3 (Scheme 2). By a similar procedure depicted in Schemes 1 and 2,
intermediates 11a-11e were prepared from compounds 2a-2c via acylation and
reduction reaction, and then were substituted with intermediate 5 to afford target
compounds B1-B7 (Scheme 3). Intermediate 3b was substituted by various piperazine
or piperidine derivatives (12a-12f) to give target compounds B8-B14 (Scheme 4). As
shown in Scheme 5, compounds 13a-13d were treated with compound 14 in the
presence of n-BuLi and THF to afford intermediates 15a-15d, then the benzyl group
was removed by hydrogenation, and finally reacted with intermediate 3b to afford
target compounds B15-B18.
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Scheme 1a
O
Br
O
O
Cl
3b
Br
a
b
1
2a
4b
Cl
Cl
OH
OH
O
N
HN
5
c
A1
a
o
Reagents and conditions: a) Et SiH, TFA, 60 C, N , 4 h, yield 65.9%, b) AlCl ,
3
2
3
o
o
CH Cl , 0 C, 1.5 h, yield 86.9%, c) DIPEA, DMF, 35 C, 16 h, yield 45.1%.
2
2
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Scheme 2a
O
O
b
c
Cl
CH3
O
O
1
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O
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CH3
O
a
2a
7
Cl
OH
O
O
N
OH
d
O
O
O
A2
8
Cl
OH
HN
O
OH
5
CH3
O
f
e
10
9
Cl
OH
N
O
A3
a
o
Reagents and conditions: a) AlCl , CH Cl , 0 C, 1.5 h, yield 69.1%, b) LiOH·H O,
3
2
2
2
o
THF/MeOH/H O, rt, 3 h, yield 84.4%, c) Et SiH, TFA, 60 C, N , 4 h, yield 86.3%, d)
2
3
2
HATU, Et N, DMF, rt 3 h, yield 80.1%, e) LiOH·H O, THF/MeOH/H O, rt, 3 h,
3
2
2
yield 79.6%, f) HATU, Et N, DMF, rt, 3 h, yield 79.8%.
3
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Scheme 3a
O
Cl
X
n
OH
Cl
X = Cl or Br
O
HN
0
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X
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X
n
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R
R-H
a-2c
R
a
b
11a-11g
c
2
4a-4g
B1 n = 2, R =
B2 n = 3, R =
B3 n = 4, R =
B4 n = 5, R =
B5 n = 6, R =
B6 n = 3, R =
B7 n = 3, R =
Cl
OH
HCl
N
n
R
B1-B7
a
o
Reagents and conditions: a) AlCl , CH Cl , 0 C, 1.5 h, yield 78.9-86.8%, b)
3
2
2
o
o
Et SiH, TFA, 60 C, N , 4 h, yield 71.5-86.4%, c) ① DIPEA, DMF, 60 C, 16 h ②
3
2
NH Cl, yield 37.1- 53.4 %.
4
Scheme 4a
R-H
Br
R
12a-12g
HCl
a
11b
B8-B14
OH
OH
B8 R =
B9 R =
B10 R =
B11 R =
N
N
N
N
Cl
Cl
F
B12 R =
B13 R =
B14 R =
N
N
N
CH3
OH
OH
OH
N
N
CF3
a
o
Reagents and conditions: a) DIPEA, DMF, 60 C, 16 h, yield 30.7-50.9%.
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Scheme 5a
Br
O
N
HO
R
14
11b
R-Br
1
1
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N
a
b
13a-13d
15a-15d
HO
B15 R = 4-CH O-Ph
B17 R =
B18 R =
R
3
N
HCl
B16 R =
N
O
B15-B18
a
o
Reagents and conditions: a) n-BuLi, THF, -78 C-rt, N , overnight, yield 48.1-
2
o
o
5
5.4%, b) ① H /Pa(OH) -C, MeOH, 30 C, 6 h, ② DIPEA, DMF, 60 C, 16 h,
2 2
yield 40.9-49.8%.
Antifungal Susceptibility Testing. The in vitro antifungal activity of the target
compounds were assayed against C. albicans SC5314, C. glabrata 8535, C.
neoformans H99 and C. neoformans ATCC34877 using the plate microdilution
3
8
method according to CLSI (M27-A) . Most of the compounds showed moderate to
excellent in vitro activities (Table 1). As compared with haloperidol, the replacement
of the fluorophenyl group with the 6,7,8,9-tetrahydro-5H-benzo[7]annulene scaffold
(
compound A1) led to slight increase of the antifungal activity. When compound A1
was added an additional carbonyl group in the linker, the antifungal activity was lost
compounds A2 and A3). In contrast, compound B2 showed much better activity than
(
compound A1 when the carbonyl group was removed. Variation of the linker length
had significant impact on the antifungal activity and antifungal spectrum. For example,
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among compounds B1-B5, compound B2 with a linker length of 4 carbon atoms
showed the best activity against C. neoformans (MIC = 8 g/mL), while compound
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B3 (linker length: 5 carbon atoms) exhibited the best activity against C. albicans
(MIC = 16 g/mL) and compound B1 (linker length: 3 carbon atoms) exhibited the
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0
best activity against C. glabrata (MIC = 4 g/mL). In contrast, the change of the
5
0
cycloalkane part had little effect on the antifungal activities because compounds B6
and B7 showed similar inhibitory activity against C. albicans, C. glabrata and C.
neoformans to compound B2. The replacement of the piperidine group to piperazine
(compound B8) led to loss of the antifungal activity. The removal of hydroxyl group
had little effect on the anti-Cryptococcus activity, whereas the anti-Candida potency
was decreased for compound B9.
The substituent groups on the terminal benzene ring were important for the
antifungal activity. Generally, the small and electron-withdrawing substituent groups
were favorable for the antifungal activity. 4-Fluoro derivative B10 showed the best
activity, whereas 4-methyl (B13) and 4-methoxyl (B15) derivatives were less potent.
The benzene/pyridine exchange was tolerated because compound B12 also showed
good activity. When the phenyl group was replaced or extended by more steric groups
(e.g. naphthyl, biphenyl), obvious decrease of the antifungal activity was observed.
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Table 1. In Vitro Antifungal Activity of Compounds against C. albicans SC5314,
C. glabrata 8535, C. neoformans H99 and C. neoformans ATCC34877 (MIC ,
5
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9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
μg/mL).
C.neo.
C.neo.
C. alb.
C. gla.
C.neo.
C. alb.
C. gla.
C.neo.
Cmpd.
ATCC
Cmpd.
ATCC
SC5314
8535
H99
SC5314
8535
H99
3
4877
32
128
128
8
34877
2
A1
A2
A3
B1
B2
B3
B4
B5
B6
B7
B8
B9
128
>128
>128
32
64
128
>128
4
32
>128
>128
16
B10
B11
16
16
4
4
2
4
4
B12
64
8
2
2
B13
>128
>128
64
16
>128
8
8
8
32
8
8
4
B14
8
16
16
16
8
16
8
16
8
B15
16
16
8
64
8
64
16
16
4
B16
>128
32
>128
8
64
2
8
B17
64
16
16
>128
32
8
B18
128
>128
0.25
>128
64
4
64
64
4
32
32
4
32
8
8
HALa
FLCa
>128
>128
>128
4
>128
16
aAbbreviations: FLC, Fluconazole, HAL, Haloperidol, C. alb., Candida albicans, C.
gla., Candida glabrata, C. neo., Cryptococcus neoformans.
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The Evaluation of Synergistic Inhibitory Effects against FLC-Resistant C.
albicans. The synergistic antifungal activity of benzocyclane derivatives against
FLC-resistant C. albicans clinical isolates (strains number: 0304103, 4108, 7781)
were also investigated. The synergistic effects were evaluated by the fractional
inhibitory concentration (FIC) index.39 Most of the target compounds exhibited
excellent synergistic inhibitory effect with C. albicans, with the FIC index (FICI)
ranging from 0.125 to 0.312. Considering the antifungal activity and synergistic
effects, compounds B10 and B12 were selected for further evaluations. In vitro
inhibitory activities against other Candida spp., including Candida tropicalis,
Candida parapsilosis and Candida krusei were assayed (Table 3). The results
indicated that they only showed moderate inhibitory activity agianst these strains, and
the activity of compound B10 was better than that of compound B12.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
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Table 2. Antifungal Activity and Synergistic Effects of Target Compounds with
FLC against FLC-Resistant C. albicans.
0304103
4108
7781
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Strains
(
MIC50, μg/mL)
(MIC50, μg/mL)
used in
(MIC50, μg/mL)
used in
used in
used alone
FIC
used alone
FIC
used alone
FIC
combination
combination
combination
Indexa
Indexa
Indexa
FLC Cmpd. FLC Cmpd.
FLC Cmpd. FLC Cmpd.
FLC Cmpd. FLC Cmpd.
A1
A2
A3
B1
> 64
> 64
> 64
> 64
> 64
> 64
> 64
> 64
> 64
> 64
> 64
> 64
> 64
> 64
> 64
> 64
> 64
> 64
> 64
32
2
32
> 64
> 64
16
8
0.504
>2
> 64
> 64
> 64
> 64
> 64
> 64
> 64
> 64
> 64
> 64
> 64
> 64
> 64
> 64
> 64
> 64
> 64
> 64
64
2
16
> 64
> 64
16
8
0.281
> 2
> 64
> 64
> 64
> 64
> 64
> 64
> 64
> 64
> 64
> 64
> 64
> 64
> 64
> 64
> 64
64
2
> 64
> 64
8
16
> 64
> 64
8
0.281
>2
> 64
> 64
> 64
> 64
> 64
> 64
> 64
> 64
64
> 64
>2
> 64
>2
>2
2
4
0.281
0.312
0.531
1.01
1
2
0.266
0.156
0.187
0.312
0.25
0.25
B2
64
2
8
0.156
0.250
0.258
0.504
0.187
0.187
>2
B3
16
2
8
64
4
8
8
8
B4
64
1
64
16
4
> 64
> 64
64
4
16
8
0.5
0.25
4
16
32
8
B5
16
1
1.02
8
B6
32
2
0.156
0.312
>2
4
8
0.187
0.312
>2
> 64
> 64
> 64
> 64
32
B7
32
4
8
64
4
16
> 64
8
4
8
B8
> 64
> 64
32
> 64
4
> 64
16
4
> 64
> 64
64
> 64
4
> 64
4
> 64
8
B9
0.312
0.187
0.187
0.156
0.187
0.125
0.187
0.156
0.187
0.187
0.250
0.156
B10
B11
B12
4
4
4
4
4
64
4
8
64
4
8
64
8
8
> 64
2
8
> 64
2
8
> 64
2
8
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1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
B13
B14
B15
B16
B17
B18
HAL
> 64
> 64
> 64
> 64
> 64
> 64
> 64
32
> 64
64
4
2
8
4
4
2
2
4
8
0.187
0.156
0.250
0.125
0.562
0.156
0.531
> 64
> 64
> 64
> 64
> 64
> 64
> 64
> 64
> 64
> 64
> 64
> 64
> 64
> 64
4
1
2
2
2
4
2
8
8
0.187
0.141
0.281
0.281
0.531
0.187
0.281
> 64
> 64
> 64
> 64
> 64
> 64
> 64
> 64
> 64
> 64
> 64
> 64
> 64
> 64
2
2
2
8
1
2
1
16
8
0.281
0.156
0.281
0.250
0.516
0.187
0.504
8
16
16
32
8
16
8
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
64
4
16
8
32
8
> 64
> 64
8
32
16
32
aFIC index is defined as the sum of MIC of each drug used in combination divided by
3
9
the MIC of the drug used alone. FICI < 0.5 = synergism, FICI ≥ 4 = antagonism, 0.5
FICI > 4 = indifferent.39
Table 3. In vitro antifungal activity of Haloperidol, compound B10 and B12 a
≤
Cmpd.
B10
C. tro. 3890
C. par. 20090
C. kru. 4996
32
32
64
0.5
32
64
32
64
64
64
B12
HAL a
FLC a
>128
2
a
Abbreviations: FLC, Fluconazole, HAL, Haloperidol, C. tro., Candida tropicalis, C.
par., Candida parapsilosis, C. kru., Candida krusei.
Growth Curve Assay. The efficacy of compound B10 against C. neoformans and
FLC-resistant C. albicans were further investigated by growth curve assay. C.
neoformans or C. albicans cells were treated with different concentrations of FLC
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or/and compound B10 for 48 h at 30 °C (Figure 2). It was found that compound B10
could strongly and dose-dependently inhibited the growth of C. neoformans, and the
effect is much stronger than that of FLC (Figure 2A). Compared with the untreated
group, the log CFU (colony-forming units) of cells treated with 4 μg/mL of
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
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4
5
6
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9
0
1
2
3
4
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7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
0
compound B10 was reduced from 7.82 to 6.96 at the terminal time 48 h. As shown in
Figure 2B, when used in combination with FLC, compound B10 could intensively
inhibit the growth of FLC-resisitant C. albicans. Rather than FLC, the inhibitory
effect was dependent on the concentration of compound B10. In particlular, when 32
μg/mL of compound B10 was used in combination with FLC, the growth of fungi was
almost completely inhibited (inhibition rate > 98%).
Figure 2. Growth curves assay of C. neoformans H99 and FLC-resistant C. albicans
(strain number: 7781) treated with different concentrations of FLC or/and compound
B10. (A) Growth curves of C. neoformans H99 cells treated with different
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concentrations of FLC or compound B10, and the statistic comparison of different
groups at 48 h. (B) Growth curves of C. albicans 7781 cells treated with different
concentrations of FLC or/and compound B10, and the statistic comparison of different
groups at 48 h. Statistical significance of groups was determined by Student’s t test
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
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8
9
0
1
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9
0
1
2
3
4
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0
1
2
3
4
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9
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(*** P < 0.001).
The Inhibitory Activity of Compounds B10 and B12 on the Virulence Factors
of C. neoformans and C. albicans. Virulence factors are produced by pathogens to
enhance the pathogenicity or drug resistance, by improving the ability of colonization,
invasiveness, immuno-evasion and immunosuppression, or the ability to resist
antifungal drugs.40 Targeting virulence factors is an attractive approach for the
development of new generation of antifungal agents.41, 42 Compound B10 exhibited
notable in vitro activity against C. neoformans and C. albicans. In order to further
investigate the antifungal activity, the effect of compound B10 against important
virulence factors, such as melanin, urease, capsule, filamentation and biofilm were
determined.
Melanin is an important virulence factor of Cryptococcus, which is mainly
43
produced by phenoloxidase and located on the inner surface of cell wall. It plays an
important role in maintaining cell wall integrity, antioxidant, resistance to lysozyme
and host immunity.44-46 As shown in Figure 3A, compound B10 significantly
inhibited the production of melanin at the concentration of 32 μg/mL, while FLC and
compound B12 were inactive. Therefore, by inhibiting the production of melanin,
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compound B10 may kill Cryptococcus indirectly because the survival ability of
Cryptococcus in the host was restricted.
Urease is a metalloenzyme catalyzing the hydrolysis of urea to ammonia and
carbamate, and plays a critical role in the transfer of C. neoformans cells from blood
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
4
7
into the brain. As shown in Figure 3B, compound B10 also inhibited the production
of urease at the concentration of 32 μg/mL. Since that meningitis/encephalitis is a
major lethal factor of C. neoformans infection, the inhibition of urease production
may reduce the morbidity of cryptococcosis and improve the survival rate of the
hosts.
As a major virulence factor of Cryptococcus, capsule plays a key role in the process
4
8
of inducing phagocytosis, dissemination and immune escape. Since the cupsule is
primarily composed of glucuronoxylomannan (GXM) and
glucuronoxylomannogalactan (GalXM), we evaluated the effect of compound B10 on
the production of these capsular polysaccharides. Compared with the untreated group,
compound B10 could significantly reduce the production of capsular polysaccharides
of each gram wet fungal (P < 0.05, Figure 3C). As a result, compound B10 might
reduce the pathogenicity of C. neoformans.
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1
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9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Figure 3. Inhibitory effects of compounds B10, B12 and FLC on the production of
melanin (A), urease (B) and capsular polysaccharides (C) of C. neoformans H99.
Statistical significance of groups was determined by Student’s t test (* P < 0.05).
Inhibitory Effect of Compound B10 on Filamentation of FLC-resisitent C.
albicans. Filamentation is a key virulence factor of C. albicans. It was reported that
hyphal C. albicans showed stronger ability of adhesion and invasion into the hosts
than normal cells, and also was more likely to escape from the attack of host immune
system.49 The activity of compound B10 on the yeast-to-hypha morphological
transition of C. albicans was evaluated by culturing C. albicans in the RPMI 1640
medium (Figure 4A) and solid spider medium (Figure 4B, Figure 4C). The results
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revealed that FLC couldn’t inhibit the formation of hyphae at the concentration of 32
μg/mL, while compound B10 was able to inhibit the morphological transition. When
treated with 4 μg/mL of compound B10, the formation of hyphae was completely
inhibited. As a result, the inhibition of filamentation by compound B10 would restrain
the pathogenicity of C. albicans.
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Figure 4. Inhibition of filamentation of C. albicans 7781. (A) Filamentation in liquid
RPMI 1640 medium; (B, C) Filamentation in solid spider medium.
Inhibitory Activity of Compound B10 on the Biofilm Formation of C.
neoformans H99 and FLC-resistant C. albicans. Fungal biofilm is a membrane-like
multicellular complex attaching to a host cavity or a surface of a biological material,
which is crucially important for the drug resistance to fungal pathogens.50, 51 The
effect of FLC and compound B10 on the biofilm formation of C. neoformans and C.
albicans was evaluated by the 2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-
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tetrazolium-5-carboxanilide (XTT) reduction assay. The results revealed that
compound B10 could significantly and dose-dependently inhibit the biofilm formation
of both C. neoformans and C. albicans (Figure 5).
0
1
2
3
4
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6
7
8
9
0
1
2
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1
2
3
4
5
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9
0
1
2
3
4
5
6
7
8
9
0
Figure 5. Inhibition of C. neoformans H99 biofilm formation by FLC (A) and
compound B10 (B) and C. albicans 7781 biofilm formation by FLC (C) and
compound B10 (D). Biofilm formation was evaluated by the XTT reduction assay,
and the results were presented as a percentage in comparison with the control groups
without compounds treatment. Biofilm formation results were represented as the
mean ± standard deviation for three independent experiments. Statistical significance
of groups was determined by Student’s t test. * P < 0.05, ** P < 0.01, *** P < 0.001
versus the control groups.
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In vivo Efficacy Evaluation of Compound B10. Prior to the in vivo studies,
metabolic stability of compound B10 was evaluated with mouse liver microsomes. As
shown in Table 4, compound B10 exhibited excellent metabolic stability with a
half-live of 106.65 min and an intrinsic clearance rate of 51.17 mL/min/mg.
Considering good in vitro activity and metabolic stability of compound B10, in vivo
efficacy of compound B10 towards C. neoformans and C. albicans was evaluated in a
murine model of systemic cryptococcosis and candidiasis, respectively (Figure 6A).
In the systemic cryptococcosis model, it was found that FLC and compound B10
could reduce the fungal burden of the brain from log CFU/g value of 7.63 to 7.37 (P
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
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0
<
0.001) and 7.29 (P < 0.001), respectively. A similar protocol was followded in the
systemic candidiasis model. Compared with the control group, compound B10 had
poor effect in reducing the fungal burden in the kidney when used alone at the dose of
1
0 mg/kg (P > 0.05). In contrast, FLC could effectively reduce the kidney fungal
burden from log10 CFU/g value of 6.35 to 5.70 (P < 0.001). Interestingly, the
combinational use of FLC and compound B10 had synergistic in vivo antifungal
effects, which significantly reduce the log CFU/g value to 4.76 (P < 0.001). The
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result revealed that compund B10 could effectively inhibit C. neoformans and
drug-resistant C. albicans in vivo, and showed potent therapeutic effects for the
treatment of CM and candidiasis.
In order to further evaluate the in vivo efficacy of compound B10, we investigated
the survival time of mice infected with C. neoformans and C. albicans when treated
with the tested compounds (Figure 6B). In the CM model, the median survival time
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(MST) of the mice treated with compound B10 was longer than that of control group
(8 days vs 6 days ), but there was no significant difference (P > 0.05). As shown in the
candidiasis model, the MST was significantly increased when treated with FLC (8
days vs 4 days, P < 0.05). And when FLC was used with compound B10 in
combination, the MST was significantly increased (10 days vs 4.0 days, P < 0.001),
which was also longer than that in the FLC treated groups (P < 0.05).
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The above results suggested that compund B10 could effectively inhibit C.
neoformans and drug-resistant C. albicans in vivo, and showed potent therapeutic
effects for the treatment of CM and candidiasis. On the basis of the results from the
in vivo models, we found that compound B10 exhibited better antifungal potency in
the candidiasis model than in the CM model, possibly due to its relatively poor ability
to penatrate the blood brain barrier (Table S1 in Supporting Information).
Table 4. Metabolic stability of compound B10 in mouse liver microsomes
T1/2a (min)
21.29
Clintb (mL/min/kg)
256.36
Ketanserin
B10
106.65
51.17
a
b
Half-life. Intrinsic Clearance.
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Figure 6. In vivo antifungal efficacy of compound B10 and FLC in the CM and
candidiasis model. (A) The fungal burden in the brain or kidney of infected mice
treated with saline, FLC and/or compound B10 in the CM or candidiasis model. (B)
The survival time of mice treated with saline, FLC and/or compound B10 in the CM
and candidiasis model. Statistical significance among groups was determined by
Log-rank (Mantel-Cox) Test. Data were represented as mean ± standard deviation (*P
< 0.05, *** P < 0.001).
Cell Cycle Analysis of C. neoformans H99 Treated with FLC and Compound
B10. As shown above, compound B10 exhibited potent in vitro and in vivo antifungal
activities against C. neoformans and drug-resistant C. albicans. In order to understand
how compound B10 exerted the antifungal effects, the mechanisms of action was
preliminary investigated. C.neoformans cells were treated with different
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concentrations of FLC or compound B10 for 24 h, the cell cycle was analyzed by flow
cytometry. After being treated with FLC at 2 μg/mL and 4 μg/mL, the ratio of cells in
the G1 phase were increased to 6.03% (Figure 7B) and 14.2% (Figure 7C),
respectively, compared with the ratio of 3.74% in the control group (Figure 7A).
When treated with 1 μg/mL and 2 μg/mL of compound B10, the ratio of cells in G1
phase were sharply increased to 83.7% (Figure 7D) and 87.5% (Figure 7E),
respectively. As shown in Figure 7F, compound B10 significantly arrested C.
neoformans H99 cells in the G1 phase (P < 0.001). The results indicated that
compound B10 had similar cell cycle arrest effect to FLC, but its effect was much
stronger than that of FLC.
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Figure 7. C. neoformans H99 cell cycle analysis by flow cytometry. (A) Untreated
control group. (B) Cells treated with 2 μg/mL of FLC for 24 h. (C) Cells treated with
4
μg/mL of FLC for 24 h. (D) Cells treated with 1 μg/mL of B10 for 24 h. (E) Cells
treated with 2 μg/mL of B10 for 24 h. (F) Mean percentages of cells in G1, S and G2.
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The percentages in different phases were calculated by flow cytometry software.
Statistical significance of groups was determined by Student’s t test. Data were
represented as mean ± standard deviation from three independent experiments. ** P <
0.01, *** P < 0.001 versus the control group.
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Transmission Electron Microscopy Analysis. The morphological changes of C.
neoformans cells after being treated with or without compound B10 were examined
with transmission electron microscopy (TEM). However, no obviously morphological
structure changes were found when treated with 4 μg/mL of compound B10 (Figure
8
A). The morphological changes of C. albicans were also examined (Figure 8B).
When treated with 16 μg/mL of FLC, the cell membrane of C. albicans was slightly
damaged. When treated with compound B10 and FLC in combination, the cells were
remarkably damaged in which the cell membrane was totally destroyed and the
cytoplasm leaked extensively. The results also revealed that compound B10 could not
damage FLC-resisitent C. albicans cells when used along. However, the cell
membrane was severely damaged when it was used in combination with FLC, leading
to the death of cells. Thus, the activity of FLC against the cell membrane of
drug-resistant fungi might be regained by the synergistic effects of compound B10.
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Figure 8. Transmission electron micrographs. The arrows indicated some stuctures as
follows: 1, cell wall, 2. cell membrane. (A) C. neoformans H99 cells treated with or
without compound B10. (B) Drug-resistant C. albicans cells (strain number: 0304103)
treated with DMSO, FLC or/and compound B10.
Evaluation of Efflux of Rhodamine 6G, Sterol Composition Analysis and
Real-time RT-PCR Analysis. In order to further explore the mechanism that how
compound B10 synergistically inhibited the drug-resistant fungi, real-time RT-PCR
analysis was performed. Lanosterol 14a-demethylase (CYP51), which is encoded by
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ERG11 gene, is the target of azoles in the ergosterol biosynthetic pathway. The
mutation and high expression of ERG11 gene in the ergosterol synthesis pathway was
considered to be one of the most important mechanisms of Candida’s resistance to
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azoles. When cells were treated with 16 μg/mL of FLC, the expression of ERG11
was up-regulated about 4-fold stimulated by the antifungal agent (Figure 9A).
However, when compound B10 was used in combination with FLC, the up-regulation
effect was reversed (Figure 9A). The results suggested that compound B10 could
reverse the overexpression of ERG11 (P < 0.01) when it was used in combination
with FLC, and reduced the resistance of C. albicans to azoles.
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The overexpression of efflux pumps is another important mechanism causing drug
resistance of Candida spp., and MDR1 is one of the efflux pumps that involved in
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drug resistance of C. albicans. As shown in Figure 9A, the expression of MDR1
was up-regulated by 2-fold when treated with 16 μg/mL of FLC. And when treated
with compound B10 and FLC in combination, the expression of MDR1 was
significantly down-regulated (P < 0.001 ， Figure 9A). Therefore, compound B10
could synergistically inhibit the efflux pumps and increase the drug concentration in
the cells, leading to the decline of resistance to azole antifungal agents.
In order to explore the effect of compound B10 on the efflux pump of
drug-resistant C. albicans, the fungal efflux efficiency of fluorescent compound
rhodamine 6G was evaluated. As shown in Figure 9B, when no glucose (GLC) was
added, the efflux of rhodamine 6G in the fungal cells was significantly increased after
the treatment with the combination of FLC and compound B10. The results were
consistent with the fact that the damage of cell membrane (Figure 9B) was able to
cause the exudation of intracellular contents. Also, it was found that when GLC was
added, there was no significant difference among the total efflux amounts of
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rhodamine 6G in the three groups (P > 0.05). The D-value between the groups with or
without GLC reflects the efficiency of ATP-depended efflux pump of fungal cells.
Comparing the D-value between the FLC and FLC/B10 group indicated that the
active ATP-depended efflux of rhodamine 6G was significantly reduced when treated
with FLC and compound B10 in combination (P < 0.01). Surprisingly, the active
ATP-depended efflux of rhodamine 6G was also reduced when the drug-resistant C.
albicans was treated with FLC alone, which might be caused by the damage of cell
membrane by high concentration of FLC (Figure 8B) because drug efflux pumps are
a class of proteins that exist on cell membrane.
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Figure 9. Real-time RT-PCR analysis of ERG11, MDR1 expression (A) and the
efflux of rhodamine 6G (B) of C. albicans (strain number: 0304103). Gene expression
is indicated as a fold change relative to ACT1. All gene expression levels and the
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efflux of rhodamine 6G were measured in triplicate, and the data were shown as mean
±
standard deviation. Statistical significance among groups was determined by
Student’s t test (** P < 0.01, *** P < 0.001).
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The sterol composition analysis (Table 5) revealed that when treated with 16
μg/mL of FLC, the production of ergosterol was reduced (from 68.79% to 24.14%),
and a high amount of obtusifoliol (14.64%) and eburicol (31.67%) were observed.
The results were consistent with the TEM analysis that high concentration of FLC
could slightly damage the cell membrane (Figure 8B). When 16 μg/mL of compound
B10 was used in combination with FLC, the production of ergosterol was further
reduced (13.45%), and the production of lanosterol and several unknown sterols were
increased. Moreover, when compound B10 was used alone, no obvious change was
observed comparing with the untreated group. These results indicated that compound
B10 could affect the ergosterol biosynthetic pathway of drug-resistant C. albicans
when used in combination with FLC.
Table 5. Sterol composition of untreated and FLC or/and compound B10 treated
C. albicans (strain number: 0304103). a
Sterol
Retention
time (min)
14.02
Groups
Control
68.79%
ND
B10
74.34%
ND
FLC
B10 + FLC
13.45%
15.63%
22.11%
27.88%
6.48%
Ergosterol (1)
Obtusifoliol (2)
Lanosterol (3)
24.14%
14.64%
15.44%
31.67%
1.59%
6.83%
2.70%
15.13
15.67
18.89%
ND
13.69%
ND
Eburicol (4)
16.49
Unknown sterol (5)
Unknown sterol (6)
Unknown sterol (7)
13.49
5.30%
ND
3.41%
ND
13.59
6.62%
14.48
7.02%
8.55%
7.82%
a
ND = Not Determined
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