Lanosterol 14α-demethylase (CYP51)/histone deacetylase (HDAC) dual inhibitors for treatment of Candida tropicalis and Cryptococcus neoformans infections

Article abstract of DOI:10.1016/j.ejmech.2021.113524

Invasive fungal infections remain a challenge due to lack of effective antifungal agents and serious drug resistance. Discovery of antifungal agents with novel antifungal mechanism is important and urgent. Previously, we designed the first CYP51/HDAC dual

Full text of DOI:10.1016/j.ejmech.2021.113524

European Journal of Medicinal Chemistry 221 (2021) 113524
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Lanosterol 14
a-demethylase (CYP51)/histone deacetylase (HDAC) dual
inhibitors for treatment of Candida tropicalis and Cryptococcus
neoformans infections
,
b
,
,
,
, **
Tianbao Zhu a 1, Xi Chen c 1, Chenglan Li b, Jie Tu b, Na Liu b ***, Defeng Xu a
,
Chunquan Sheng b
,
*
a National & Local Joint Engineering Research Center for High-efficiency Refining and High-quality Utilization of Biomass, School of Pharmacy, 1 Gehu Road,
Changzhou University, Changzhou, 213164, China
b School of Pharmacy, Second Military Medical University, 325 Guohe Road, Shanghai, 200433, China
c Key Laboratory of Synthetic and Natural Functional Molecule of the Ministry of Education, College of Chemistry and Materials Science, Northwest
University, 1 Xuefu Avenue, Xi'an, 710127, China
a r t i c l e i n f o
a b s t r a c t
Article history:
Invasive fungal infections remain a challenge due to lack of effective antifungal agents and serious drug
resistance. Discovery of antifungal agents with novel antifungal mechanism is important and urgent.
Previously, we designed the first CYP51/HDAC dual inhibitors with potent activity against resistant
Candida albicans infections. To better understand the antifungal spectrum and synergistic mechanism,
herein new CYP51/HDAC dual inhibitors were designed which showed potent in vitro and in vivo anti-
fungal activity against C. neoformans and C. tropicalis infections. Antifungal mechanism studies revealed
that the CYP51/HDAC dual inhibitors acted by inhibiting various virulence factors of C. tropicalis and
C. neoformans and down-regulating resistance-associated genes. This study highlights the potential of
CYP51/HDAC dual inhibitors as a promising strategy for the discovery of novel broad-spectrum antifungal
agents.
Received 7 December 2020
Received in revised form
28 April 2021
Accepted 30 April 2021
Available online 7 May 2021
Keywords:
Antifungal
CYP51/HDAC dual Inhibitors
Virulence factors
Cryptococcus neoformans
Candida tropicalis
© 2021 Elsevier Masson SAS. All rights reserved.
1. Introduction
meningitis (CM) caused by C. neoformans frequently occurred in
AIDS patients with the mortality rate over 15% [7]. In addition, it is
In recent years, the morbidity and mortality of invasive fungal
infections (IFIs) are continuing to increase due the large population
of immunocompromised patients suffering from tumor, organ
transplantation, acquired immune deficiency syndrome (AIDS) and
so on [1,2]. Candida, Cryptococcus and Aspergillus are the major
pathogens in clinical IFIs. Among the Candida spp., Candida tropi-
calis (C. tropicalis), accounting for 45% of non-Candida albicans in-
fections, has become the second common species of Candida
estimated that more than 22 thousand individuals suffer from CM
annually [8]. Traditional antifungal drugs used to treat IFIs include:
polyenes, pyrimidines, azoles and echinocandins [9,10]. However,
due to severe drug resistance and side effects of clinically available
antifungal drugs, therapeutic effect of IFIs remains rather limited.
Therefore, there is an urgent need for the development of mecha-
nistically distinct antifungal agents.
Histone acetylation modification, including histone acetylation
and deacetylation, is an important component of epigenetic
research. As a series of enzymes with deacetylation function, his-
tone deacetylases (HDACs) account for an important role in the
bloodstream
infection
[3,4].
Cryptococcus
neoformans
(C. neoformans) is one of the most common fungi species with high
mortality rate [5,6]. Particularly, the incidence of cryptococcal
* Corresponding author.
** Corresponding author.
*** Corresponding author.
E-mail addresses: liuna@smmu.edu.cn (N. Liu), markxu@cczu.edu.cn (D. Xu), shengcq@smmu.edu.cn (C. Sheng).
These authors contributed equally to this work.
1
https://doi.org/10.1016/j.ejmech.2021.113524
0223-5234/© 2021 Elsevier Masson SAS. All rights reserved.

T. Zhu, X. Chen, C. Li et al.
European Journal of Medicinal Chemistry 221 (2021) 113524
chromosomes remodeling and regulation of gene expression
[11,12]. HDACs in fungi, such as Aspergillus fumigatus [13], Candida
albicans [14,15], Saccharomyces cerevisiae [16], and C. neoformans
HDACs [17,18] have been reported to participate in virulence-
related processes and morphological changes. Therefore, inhibi-
tion of fungal HDACs could be an effective strategy to treat fungal
infections. Recently, HDAC inhibitors have been proven to be syn-
ergized with azole antifungal drugs [19,20]. For example, HDAC
inhibitor MGCD290 synergized with fluconazole (FLC) against a
variety of clinical fungal isolates [21]. In addition, HDAC inhibitors
trichostatin A (TSA) and vorinostat (SAHA) synergized with itra-
conazole (ICA) and FLC against azole-resistant Candida albicans
(C. albicans) and C. tropicalis [22,23]. However, the synergistic
mechanism and in vivo antifungal activity of azoles synergized with
HDAC inhibitors are still unclear.
Clinically, specific drug combination is important for disease
control, whereas drug cocktails have the disadvantages of drug-
drug interaction, poor patient compliance and unpredictable
pharmacokinetic (PK) profiles [24]. Therefore, multi-targeting drug
is a feasible solution to retain the synergistic effect and overcome
these limitations. Previously, JAK/HDAC dual inhibitors were
designed by our group, which exhibited excellent synergistic
antifungal potency for the treatment of acute myeloid leukemia
(AML) and azole-resistant C. albicans infection [25]. In addition, we
2. Results and discussion
2.1. Introducing piperazine substituents to design CYP51/HDAC dual
inhibitors
Previously, we reported the first example of CYP51/HDAC dual
inhibitors [26]. It represents a novel strategy for the treatment of
azole-resistant Candidiasis. Herein, for further investigating the
structure-activity relationship (SAR) of CYP51/HDAC dual inhibitors
and developing more efficient broad-spectrum antifungal com-
pounds, a new series of CYP51/HDAC dual inhibitors containing the
piperazine linker were designed by the key pharmacophore fusion
of CYP51 and HDAC inhibitors. In general, HDAC inhibitors contain a
cap group, a variable linker and a zinc-binding group (ZBG). ZBG
(e.g. hydroxamic acid) is critical for the inhibition of zinc-
dependent HDACs, while diverse skeletons can be used as the cap
group (Fig. 1A). The key pharmacophores of typical azoles include a
triazole group, a 2,4-difluorophenyl group and a variable side chain
(Fig. 1B). On the basis of the pharmacophores and binding modes
[26], CYP51/HDAC dual inhibitors can be designed by the fusion of
triazole, 2,4-difluorophenyl group and ZBG. In our previous study,
side chains of the first generation CYP51/HDAC dual inhibitors are
mainly flexible structures. Herein, piperazine group was introduced
as a part of linker to increase structural rigidity and adjust the
physicochemical properties (Fig. 1C). Consequently, four types
CYP51/HDAC dual inhibitors with different linkers were designed:
alkoxybenzyl linker (benzene ring is linked to piperazine by
methylene, A1-A5), phenylalkoxyl linker (alkoxy is linked to
piperazine, A6-A8), 1,3-diphenoxypropane (A9), and triazolone
alkoxybenzyl (A10-A13, Fig. 1D).
also designed the first generation of lanosterol 14a-demethylase
(CYP51)/HDAC dual inhibitors, which exhibited excellent in vitro
and in vivo antifungal activity against azole-resistant C. albicans
infections [26]. These proof-of-concept studies offered a promising
way for the treatment of resistant fungal infections. However, it is
still unknown that whether the CYP51/HDAC dual inhibitors are
effective in treating non-C. albicans infections. In an attempt to
further investigate the antifungal spectrum and synergistic mech-
anism, herein new CYP51/HDAC dual inhibitors were designed
(Fig. 1). After in vitro and in vivo investigation of the antifungal
activity and mechanism, the dual inhibitors showed potent anti-
fungal activity against C. neoformans and azole-resistant C. tropicalis
infections by inhibiting virulence factors and down-regulating
resistance-associated genes.
2.2. Chemistry
The synthesis routes of target compounds A1-A13 are described
in Schemes 1e4. Starting materials 1, 2a-2e, 4, 9, 10a-10c are
commercially available. Intermediate oxirane 7 was synthesized
according to the reported methods [27]. Treating brominated
compounds 2a-2e with hydroxybenzaldehyde 1 in K2CO3 and
Fig. 1. Design strategies of CYP51/HDAC dual inhibitors. (A) Pharmacophoric model of HDAC inhibitors; (B) pharmacophoric model of CYP51 inhibitors; (C) Design of CYP51/HDAC
dual inhibitors; (D) Chemical structures of the target compounds.
2

T. Zhu, X. Chen, C. Li et al.
European Journal of Medicinal Chemistry 221 (2021) 113524
a
Scheme 1. Reagents and conditions: (a) EtOH, K2CO3, 85 ꢀC, reflux, 5 h; (b) (i) Ti(i-propoxyl)4, Et3N, MeOH, rt, 12 h, (ii) NaBH4, rt, 30 min; (c) TFA, CH2Cl2, rt, 1 h; (d) K2CO3,
anhydrous DMF, 85 ꢀC, 12 h; (e) NH2OH HCl, KOH, MeOH, rt, 5 h.
a
Scheme 2. Reagents and conditions: (a) (b) K2CO3, EtOH, 85 ꢀC, 12 h; (c) TFA, CH2Cl2, rt, 1 h; (d) K2CO3, anhydrous DMF, 85 ꢀC, 12 h; (e) NH2OH HCl, KOH, MeOH, rt, 5 h.
a
Scheme 3. Reagents and conditions: (a) K2CO3, EtOH, 85 ꢀC, 3 h; (b) (i) Ti(i-propoxyl)4, Et3N, MeOH, rt, 12 h; (ii) NaBH4, rt, 30 min; (c) TFA, CH2Cl2, rt, 1 h; (d) K2CO3, anhydrous
DMF, 85 ꢀC, 12 h; (e) NH2OH HCl, KOH, MeOH, rt, 5 h.
anhydrous EtOH to give intermediates 3a-3e, which further reacted
with Boc-protected piperazine 4 to afford intermediates 5a-5e.
Then, Boc-protecting group was removed by trifluoroacetic acid
(TFA) to afford intermediates 6a-6e, which further reacted with
oxirane 7 to give intermediates 8a-8e. Finally, intermediates 8a-8e
were reacted with freshly prepared NH2OH in CH3OH to obtain
target compounds A1-A5 (Scheme 1).
Target compounds A6-A8 were synthesized by the route out-
lined in Scheme 2. Substituted phenol 9 reacted with dibromo-
substituted alkanes 10a-10c to give intermediates 11a-11c, which
further reacted with Boc-protected piperazine 4 via Borch reduc-
tion to afford intermediates 12a-12c. Then, the Boc-protecting
group of intermediates 12a-12c in TFA afforded intermediates
13a-13c, which reacted with oxirane 7 to form intermediates 14a-
14c. After treating with fresh hydroxylamine, target compounds
A6-A8 were obtained (Scheme 2).
Intermediates 11a reacted with starting material 1 in the pres-
ence of K2CO3 and ethanol to give intermediate 15. By a similar
procedure depicted in Scheme 2, intermediate 18 was prepared
from intermediate 15 via Borch reduction, deprotection and ring
opening, which treating with fresh hydroxylamine to give target
compound A9 (Scheme 3).
Starting material 19 reacted with Boc-protected piperazine 4 by
Borch reduction to give intermediate 20, then nitro group was
3

T. Zhu, X. Chen, C. Li et al.
European Journal of Medicinal Chemistry 221 (2021) 113524
Scheme 4. aReagents and conditions:(a) Ti(i-propoxyl)4, Et3N, MeOH, rt, 12 h; (b) 95% EtOH, Fe, NH4Cl, reflux, 3 h; (c) methyl hydrazinecarboxylate, trimethoxymethane, MeOH,
70 ꢀC, 6 h; (d) anhydrous DMF, K2CO3, 80 ꢀC, 4 h; (e) TFA, CH2Cl2, rt, 1 h; (f) K2CO3, anhydrous DMF, 85 ꢀC, 12 h; (g) NH2OH HCl, KOH, MeOH, rt, 5 h.
reduced in the presence of Fe to afford intermediate 21. Interme-
diate 21 reacted with hydrazinecarboxylate to give intermediate 22,
which was substituted by brominated compounds 23a-23d to
afford intermediates 24a-24d. The Boc-protecting groups were
removed to give intermediates 25a-25d. Intermediates 26a-26d
were prepared via ring opening and substitution reaction, and
finally treated with fresh hydroxylamine to give target compounds
A10-A13 (Scheme 4).
against HUVEC (human umbilical vein endothelial cell line) with an
IC50 value of 5.9 g/mL (10.36 M), which was approximately 12
times higher than the MIC80 value of 0.5 g/mL. Preliminary
experimental results suggested that compound A5 had selective
antifungal activity with low toxicity to human normal cells.
m
m
m
2.4. Compounds A5 showed potent inhibitory activities against FLC-
resistant C. tropicalis and FLC-sensitive C. neoformans
The time-growth curves assay was performed to further eval-
uate the efficacy of compound A5 against C. tropicalis and
C. neoformans cells growth. The results indicated that compound A5
2.3. In vitro antifungal activity and SAR of CYP51/HDAC dual
inhibitors
at 8
m
g/mL inhibited the proliferation of C. tropicalis cells superior
The antifungal activity of the synthesized CYP51/HDAC dual
inhibitors was assayed against C. neoformans and azole-resistant
C. tropicalis, which was expressed by the MIC80 value. CYP51 in-
hibitors FLC, ICA, voriconazole (VRC) and the HDAC inhibitor SAHA
were used as positive controls. As shown in Table 1, compounds A1-
A5, featured as the piperazinylmethylenephenyloxyalkyl linker,
than FLC at a higher concentration of 32
compound A5 significantly inhibited the growth of C. neoformans
cells, whose activity was better than FLC (Fig. 2B).
m
g/mL (Fig. 2A). In addition,
2.5. Compound A5 inhibited the filamentation of C. tropicalis and
the biofilm formation of C. tropicalis and C. neoformans
showed moderate to good antifungal activity (MIC80 range: 0.5
mg/
mL ~ 2 g/mL). The antifungal activity of target compounds A1-A5
m
was increased with the extension of the alkoxy chains (3e7 carbon
atoms). Compound A5 (7 carbon atoms length) exhibited the best
inhibitory activity against both C. tropicalis and C. neoformans with
As a virulence factor of C. tropicalis, filamentation plays an
important role in pathogenicity and invasiveness [28,29]. Fungal
cells in the hyphal phase invade mucosal tissue easily and cause IFIs
[30,31]. Moreover, transition between yeast phase and hyphal
phase makes fungal cells easier to evade the host recognition or
host defense [32,33]. Therefore, the effects of compound A5 and FLC
at different concentrations in inhibiting C. tropicalis filamentation
were investigated by using the cell inverted imaging microscope. As
compared to the control group, compound A5 effectively inhibited
an MIC80 of 0.5 mg/mL. In particular, it showed excellent activity
towards azoles resistant C. tropicalis, whereas positive controls FLC,
VOR, ITR and SAHA were not totally inactive. When the direction of
alkoxybenzene was changed (alkoxy is linked to piperazine, A6-
A8), antifungal activity was decreased obviously. Compound A9
with the 1,3-diphenoxypropane linker showed moderate anti-
fungal activity (MIC80 range: 2
mg/mL ~ 32
m
g/mL). Among tri-
hyphae formation at 1
mg/mL, whereas FLC was poorly active even
azolone derivatives A10-A13, only compound A10 showed obvious
activity against both FLC-sensitive C. tropicalis and C. neoformans
at 8 g/mL (Fig. 3A). Fungal biofilm, a high-density structure, pro-
m
hibits antifungal drugs penetrating into the cells to exert antifungal
activity, which is closely related to drug resistance [34e37]. The
2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-
carboxanilide (XTT) reduction method was used to evaluate the
effects of compounds A5 on C. tropicalis and C. neoformans biofilms
with FLC as a positive control. The results indicated that compound
A5 inhibited biofilm formation of C. tropicalis and C. neoformans in a
dose-dependent manner. Especially, compound A5 showed better
activity than FLC against C. tropicalis, and disrupted biofilm for-
with an MIC80 of 2
antifungal effects.
mg/mL, while compounds A11-A13 lost the
In addition, the antifungal activity of compound A1-A13 against
C. albicans was also investigated. Different from our previously re-
ported CYP51/HDAC dual inhibitors, compound A1-A13 generally
exhibited weak to moderate inhibitory activity against azoles-
resistant C. albicans with an MIC80 value range from 1
64 g/mL. The inhibitory effects of compounds A1-A13 against FLC-
sensitive C. albicans were also inferior to FLC (Table 2).
mg/mL to
m
mation completely at 64
approximately blocked 70% of biofilm formation in C. neoformans at
a high concentration of 64 g/mL, whereas FLC exhibited the same
inhibitory effect at a concentration of 8 g/mL (Fig. 3B). FLC
mg/mL (Fig. 3B). Moreover, compound A5
Considering the excellent antifungal activity, compound A5 was
selected for further biological activity evaluation and mechanism
study. Initially. we investigated the cytotoxicity of compound A5
m
m
4

T. Zhu, X. Chen, C. Li et al.
European Journal of Medicinal Chemistry 221 (2021) 113524
Table 1
In vitro antifungal activity of compounds A1-A13 against C. tropicalis and C. neoformans.
Cpds
R1
R2
C. tropicalis
C. neoformans
5008
4
3890
4
H99
8
A1
-CH2-
A2
A3
-CH2-
-CH2-
4
1
2
2
8
8
A4
A5
-CH2-
-CH2-
0.5
0.5
2
2
0.5
0.5
A6
A7
-CH2-
-CH2-
-CH2-
>64
>64
32
4
32
32
A8
A9
4
2
2
16
4
-CH2-
-CH2-
32
A10
A11
16
2
2
8
-CH2-
>64
>64
A12
A13
-CH2-
-CH2-
>64
>64
>64
>64
64
32
FLC
e
e
e
e
e
e
e
e
>64
>64
>64
>64
0.5
2
>64
0.0625
0.125
SAHA
VOR
ITC
>64
>64
>64
Table 2
In vitro antifungal activity of compounds A1-A13 against C. albicans.
Cpds
C. alb SC5314
C. alb. 0304103
C. alb. 7781
C. alb. 4108
C. alb. 10061
C. alb. 9770
A1
A2
A3
A4
4
1
1
8
8
32
16
8
4
2
16
32
16
32
>64
>64
32
>64
>64
32
1
1
8
32
A5
8
8
1
8
16
16
A6
A7
A8
>64
32
16
32
>64
16
>64
>64
4
>64
>64
16
>64
>64
16
>64
>64
16
A9
4
2
32
2
4
2
32
4
>64
32
>64
NT
NT
>64
NT
>64
32
A10
A11
A12
A13
FLC
SAHA
VRC
ICZ
>64
>64
>64
0.5
NT
<0.125
0.5
>64
>64
64
>64
>64
>64
>64
16
32
64
>64
>64
>64
>64
>64
NT
NT
>64
>64
>64
>64
>64
>64
>64
>64
NT
NT
NT
NT
NT
aAbbreviations: C. alb., Candida. albicans; NT ¼ Not Tested.
5

T. Zhu, X. Chen, C. Li et al.
European Journal of Medicinal Chemistry 221 (2021) 113524
Fig. 2. The time-growth curves of FLC-resistant C. tropicalis 5008 cells (A) and FLC-sensitive C. neoformans H99 cells (B) treated with the various concentrations compound A5 as
well as FLC at different time periods.
Fig. 3. Inhibitory activity of compound A5 against filamentation and biofilm formation. (A) The inhibitory effects on the filamentation of C. tropicalis 5008 treated with compound
A5 (0.5e1 mg/mL) and FLC (8 m
g/mL) at 37 ꢀC for 7 h. (B) The effects of various concentrations of compound A5 and FLC on the biofilm formation of C. tropicalis 5008 and
C. neoformans H99 compared to no compound treated group. Three independent experiments were conducted and the data was expressed as the mean SD. The results were
presented as inhibition percentages compared to the untreated groups and the statistical significance was performed by t-test (*P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001).
performed better at inhibiting the biofilm formation in
C. neoformans than azole-resistant C. tropicalis. Conversely, com-
pound A5 exhibited better inhibitory effect on azole-resistant
C. tropicalis.
up-regulated, which led to the weak inhibitory effect on the capsule
formation. Thus, compound A5 could reduce the virulence of
C. neoformans through down-regulating capsule-associated genes
CAP10 and CAP60.
2.6. Compound A5 inhibited the formation of C. neoformans capsule
2.7. Analysis of sterol composition and resistance-associated genes
and down-regulated the capsule-associated genes
Ergosterol is the main sterol in yeast, which plays an indis-
pensable role in the maintenance of the cell membrane integrity
and function [41]. The target of azole antifungal drugs is CYP51,
encoded by ERG11 gene in fungi. The azoles exert antifungal activity
by blocking the synthesis of ergosterol and accumulating toxic
sterol intermediates [42,43]. Therefore, the influence of compound
A5 on the sterol composition in C. tropicalis and C. neoformans cell
membrane was evaluated by gas chromatography-mass spec-
trometry (Fig. S1 in Supporting Information). After treated with
As a specific virulence factor of C. neoformans, the capsule is a
major role in host infection, and protects C. neoformans cells from
immune attack [38,39]. As show in Fig. 4A and B, the capsule size
was reduced significantly after the treatment of compound A5
(P < 0.0001) without inhibitory effects on the growth of the fungi
body, whereas the inhibitory effect of FLC with the treatment of
equivalent concentration was relatively weak (P < 0.01). It is re-
ported that CAP genes (CAP10, CAP59, CAP60, CAP64) were essential
for capsule biosynthesis [38,40]. Therefore, real time RT-PCR was
conducted to evaluate the expression levels of four capsule-
associated genes after the treatment of compound A5. As shown
in Fig. 4C, capsule-associated genes CAP10 and CAP60 were signif-
compound A5 at a concentration of 4 mg/mL, the ergosterol content
of C. tropicalis was reduced from 84.91% to 1.51%, with high levels of
the obtusifoliol (16.02%) and eburicol (69.34%). As for C. neoformans,
the ergosterol content was reduced from 60.18% to 19.14% at the
same concentration of compound A5 (Table 3). These results indi-
cated that compound A5 significantly reduced the fungal ergosterol
content by inhibiting CYP51, whose mechanism is similar to that of
azole drugs.
icantly down-regulated by compound A5 at 2 mg/mL. In contrast,
expression of capsule-associated genes CAP59 and CAP64 did not
change obviously. When treated with equivalent concentration of
FLC, capsule-associated genes CAP10 and CAP59 were significantly
6

T. Zhu, X. Chen, C. Li et al.
European Journal of Medicinal Chemistry 221 (2021) 113524
Fig. 4. Effects of compound A5 on the formation of C. neoformans capsule and expression of the capsule-associated genes. (A) The microscopy images of the C. neoformans H99
capsule formation were photographed under the high concentrations of compound A5 or FLC. (B) The data of boxes represent whole cell size, capsule size and cell body size after the
treatment of different concentrations of compound A5 or FLC (measured with imageJ software). (C) Expression levels of four capsule associated-genes in C. neoformans H99 with
compound A5 or FLC at a concentration of 2 mg/mL. Three independent experiments of the capsule-associated genes expression were conducted and the data was expressed as the
mean SD. Statistical significance was performed by t-test (*P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001).
Table 3
pathway [47]. As shown in Fig. 5, compound A5 significantly down-
Sterol compositions of FLC-resistant C. tropicalis and FLC-sensitive C. neoformans
after the treatment of compound A5.
regulated the resistant-associated genes (ERG11, CDR1, MDR1) in
C. tropicalis with FLC group as the control. The results indicated that
compound A5 might inhibit the overexpression of efflux pump
genes, and down-regulated ERG11 gene in ergosterol biosynthetic
pathway, thus increased the activity against FLC-resistant
C. tropicalis.
Sterol
Retention
Time (min)
Sterol composition (%)
C. tropicalis 5008 C. neoformans
H99
no drug
A5
ND
1.51
1.31
16.02
ND
no drug
A5
ND
19.14
ND
ND
9.59
71.27
Unknown sterol (1)
Ergosterol (2)
Unknown sterol (3,4)
Obtusifoliol (5)
Lanosterol (6)
13.79
14.07
14.48,14.93
15.12
15.75
5.78
84.91
6.48
ND
2.81
ND
ND
60.18
ND
ND
ND
2.8. Compound A5 inhibited HDAC enzyme activity
According to our previously reported assay with optimized
protocols [26], fungal whole cell HDAC enzyme inhibitory activity
of compound A5 was evaluated. Initially, fungal cell wall was
lysated to obtain the protoplasts by using snailase. Then HDAC
substrate was added after the protoplasts were treated with com-
pound A5 and SAHA. Finally, inhibitory activity was assessed by
monitoring the fluorescence intensity of substrate decomposition.
In C. tropicalis cells, compound A5 exhibited stronger HDAC inhib-
Eburicol (7)
16.69
69.34
39.82
Up to date, azoles resistance have become a serious problem in
clinical treatment of IFIs [44]. One major cause is the increased
efflux pump proteins (e.g. ATP-binding cassette transporters Cdr1p
and Mdr1p), which leads to reduced drug concentration in fungal
cells [45,46]. Another essential factor of resistance is up-regulation
or mutations of the ERG11 gene in the ergosterol biosynthetic
itory activity (IC50 ¼ 2.38
m
M) than SAHA (IC50 ¼ 3.56
mM). How-
ever, it was less active in C. neoformans cells (Table 4).
Fig. 5. Different concentrations of compound A5 stimulated the transcription of the azoles resistant-associated genes ERG11, CDR1, MDR1 in C. tropicalis 5008 with 4
the control. The results are shown by the means SD (n ¼ 3 independent assay). **P < 0.01, ***P < 0.001, determined by Student's t-test.
mg/mL FLC as
7

T. Zhu, X. Chen, C. Li et al.
European Journal of Medicinal Chemistry 221 (2021) 113524
2.9. Compound A5 down-regulated HDAC genes of C. neoformans
Fungal HDACs control cellular processes associated with viru-
lence [48], such as capsule formation, host-pathogen interaction,
and melanin synthesis. The “classical” fungal HDAC family proteins
of C. neoformans divide into two major classes: class I (e.g. Rpd3,
Hos1, Hos2, Clr61, and Clr62) and class II (Hda1 and Hos3) [48].
Thus, the influence of compound A5 on the expression of seven
HDAC genes of C. neoformans was investigated. As shown in Fig. 6,
compound A5 significantly reduced the expression of seven HDAC
genes at 2 mg/mL, indicating that down-regulation of HDAC genes
by compound A5 could contribute to reduce pathogenicity of
C. neoformans.
Fig. 6. Expression levels of seven HDAC genes in C. neoformans H99 with the treatment
of compound A5 and SAHA at a concentration of 2
means SD (n ¼ 3 independent assay).
mg/mL. The data was shown as
2.10. In vivo antifungal efficacy of compound A5
CH3OH/H2O (v/v, 7/3) at a flow rate of 0.5 mL/min on an Agilent C18.
The high-resolution mass spectra (HRMS) were recorded on an
Agilent UPLC-QTOF/MS mass spectrometer. All final compounds
exhibited purities greater than 95%.
Due to excellent in vitro antifungal activity of compound A5
against C. tropicalis and C. neoformans, its in vivo antifungal potency
was further evaluated in two IFIs mouse models. Mouse models
were established by injection with C. tropicalis and C. neoformans
via tail vein with FLC group as positive control. Compound A5 and
FLC were administered orally (20 mg/kg) for 5 consecutive days
after 24 h post infection. As shown in Fig. 7A, compared to the
saline group, compound A5 treated group significantly reduced
kidney fungal burden of FLC-resistant C. tropicalis by about 0.7 log
(P < 0.0001). In the C. neoformans infection model, compound A5
treated group also effectively reduced brain fungal burden
(P < 0.0001, Fig. 7B), which was superior to FLC (P < 0.001). These
results confirmed that compound A5 exhibited potent therapeutic
effects for both tropical candidiasis and CM.
4.1.1. 4-(4-((4-(2-(2,4-difluorophenyl)-2-hydroxy-3-(1H-1,2,4-
triazol-1-yl)propyl) piper-azin-1-yl)methyl)phenoxy)-N-
hydroxybutanamide (A1)
The freshly prepared NH2OH solution in CH3OH was added into
intermediate 8a (1.77 g, 3.34 mmoL, 1.0 equiv) and the mixture was
stirred at room temperature for 5 h[26]. Then 2 M HCl was added to
the mixture to adjust the PH to 3.0 and the CH3OH solvent was
removed under reduced pressure. The crude product was diluted
with water (50 mL) and extracted with EtOAc (50 mL ꢁ 2), the
combined organic layers dried with Na2SO4, filtered, and concen-
trated under reduced pressure. The residual mixture was purified
via silica gel column chromatography (CH2Cl2/CH3OH, 25/1, v/v) to
afford target compound A1 (0.92 g, 52% yield) as a white solid. 1H
3. Conclusion
NMR (600 MHz, CD3OD)
d 8.32 (s, 1H), 7.72 (s, 1H), 7.48-7.41 (m,
In summary, a series of new fungal CYP51/HDAC dual inhibitors
were designed and synthesized for the treatment of C. neoformans
and C. tropicalis infections. Among them, compound A5 showed
excellent in vitro antifungal activity and significantly reduced
fungal burden in two IFIs mouse models. As a CYP51/HDAC dual
inhibitor, compound A5 blocked the biosynthesis of ergosterol and
showed potent whole cell HDAC enzyme inhibitory activity. It
effectively inhibited important virulence factors (e.g. hypha, biofilm
and capsule) of C. neoformans and C. tropicalis, and down-regulated
resistance-associated genes. Taken together, this study offered an
effective strategy for treatment of CM and FLC-resistant C. tropicalis
infections.
1H), 7.16 (d, J ¼ 8.6 Hz, 2H), 6.92-6.87 (m, 1H), 6.85-6.80 (m, 3H),
4.66 (d, J ¼ 14.4 Hz, 1H), 4.57 (d, J ¼ 14.3 Hz, 1H), 3.96 (t, J ¼ 6.2 Hz,
2H), 3.38 (d, J ¼ 2.3 Hz, 2H), 2.96 (dd, J ¼ 13.8, 1.2 Hz, 1H), 2.77 (d,
J ¼ 13.8 Hz, 1H), 2.502.41 (m, 4H), 2.35 (bs, 4H), 2.27 (t, J ¼ 7.4 Hz,
2H), 2.07-2.02 (m, 2H). 13C NMR (151 MHz, CD3OD)
d 170.59, 162.39
(dd, J ¼ 247.7, 12.2 Hz), 158.93 (dd, J ¼ 246.5, 12.2 Hz), 158.52,
149.38, 144.30, 130.72, 129.13 (d, J ¼ 8.7 Hz), 126.22, 125.24 (d,
J ¼ 10.5 Hz), 113.78, 110.26 (d, J ¼ 21.0 Hz), 103.11 (t, J ¼ 26.9 Hz),
73.51 (d, J ¼ 4.9 Hz), 66.29, 62.51, 60.83, 55.65, 52.73, 51.76, 28.54,
24.65.
Target compounds A2-A13 were synthesized according to the
synthetic method of target compound A1.
4. Experimental section
4.1.2. 8-(4-((4-(2-(2,4-difluorophenyl)-2-hydroxy-3-(1H-1,2,4-
triazol-1yl)propyl)piperazin-1-yl)methyl)phenoxy)-N-
hydroxyoctanamide (A5)
4.1. Chemistry
General Methods. 1H NMR and 13C NMR spectra were recorded
on Bruker AVANCE 300, or AVANCE 600 spectrometers (Bruker
Company, Germany) using TMS as an internal standard and CD3OD,
CDCl3 or dimethyl sulfoxide (DMSO)-d6 as solvents. Thin-layer
chromatography analysis was performed by using silica gel plates
GF254. The chemical purities were analyzed by RP-HPLC with
White solid, 55% yield. 1H NMR (600 MHz, CD3OD)
d 8.34 (s, 1H),
7.75 (s, 1H), 7.46 (dd, J ¼ 6.7 Hz, 1H), 7.17 (d, J ¼ 8.5 Hz, 2H), 6.96-
6.87 (m, 1H), 6.85-6.81 (m, 3H), 4.67 (d, J ¼ 14.3 Hz, 1H), 4.59 (d,
J ¼ 14.3 Hz, 1H), 3.93 (t, J ¼ 6.4 Hz, 2H), 3.43-3.37 (m, 2H), 2.98 (d,
J ¼ 13.8 Hz, 1H), 2.78 (d, J ¼ 13.8 Hz, 1H), 2.47 (bs, 4H), 2.37 (bs, 4H),
2.10 (t, J ¼ 7.4 Hz, 2H), 1.78-1.71 (m, 2H), 1.67-1.59 (m, 2H), 1.50-1.42
(m, 2H), 1.42-1.31 (m, 4H). 13C NMR (151 MHz, CD3OD)
d 171.54,
162.70 (dd, J ¼ 247.8, 12.2 Hz), 159.28 (dd, J ¼ 246.7, 11.9 Hz), 158.59,
149.73, 144.67, 130.62, 129.43 (d, J ¼ 14.8 Hz), 128.32, 125.83 (d,
J ¼ 9.9 Hz), 113.91, 110.65 (d, J ¼ 20.9 Hz), 103.52 (t, J ¼ 26.8 Hz),
73.50 (d, J ¼ 5.2 Hz), 67.56, 63.03, 61.78, 56.13 (d, J ¼ 4.2 Hz), 53.84,
52.46, 32.39, 28.93, 28.69, 25.61, 25.29. HRMS (ESI) m/z: calcd for
Table 4
HDAC enzyme inhibition activities (IC50, mM).
Compounds
C. tropicalis
Whole Cell HDAC
C. neoformans
Whole Cell HDAC
C
30H40F2N6O4 [M þ H]þ, 587.3157; found 587.3156. HPLC purity:
A5
SAHA
2.38
3.56
12.59
3.11
96.4%.
8

T. Zhu, X. Chen, C. Li et al.
European Journal of Medicinal Chemistry 221 (2021) 113524
Fig. 7. In vivo antifungal activity of compound A5. (A) C. tropicalis 5008 mouse model; (B) C. neoformans H99 mouse model. Cyclophosphamide was injected intraperitoneally 24 h
before the fungal infection in order to destroy the immune system. The experiment was divided into 3 groups: saline group, compound A5 oral (PO) administration group (20 mg/
kg) and FLC oral (PO) administration group (20 mg/kg). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, determined by Student's t-test.
4.1.3. 2-(4-((5-(4-(2-(2,4-difluorophenyl)-2-hydroxy-3-(1H-1,2,4-
triazol-1-yl)propyl) piperazin-1-yl)pentyl)oxy)phenyl)-N-
hydroxyacetamide (A7)
collected and resuspended in 3 mL of fresh RPMI 1640 medium
with a concentration of 1 ꢁ 105 cells/mL, 1 ꢁ 106 cells/mL respec-
tively. FLC and compound A5 at different concentrations were
added to treat the fungal cells with constant shaking cultured
(240 rpm/min) at 30 ꢀC, then the numbers of the fungal cells at the
specified times (0, 6, 12, 24, 36, 48 h) were measured via OD630. The
experiment was conducted triply.
White solid, 51% yield. 1H NMR (600 MHz, CD3OD)
d 8.33 (s, 1H),
7.74 (s, 1H), 7.49-7.42 (m, 1H), 7.17 (d, J ¼ 8.6 Hz, 2H), 6.93-6.89 (m,
1H), 6.85-6.79 (m, 3H), 4.67 (d, J ¼ 14.3 Hz, 1H), 4.59 (d, J ¼ 14.3 Hz,
1H), 3.92 (t, J ¼ 6.3 Hz, 2H), 3.30 (s, 2H), 2.98 (d, J ¼ 13.8, 1.1 Hz, 1H),
2.78 (d, J ¼ 13.8 Hz, 1H), 2.47 (bs, 4H), 2.38 (bs, 4H), 2.32-2.28 (m,
2H), 1.78-1.72 (m, 2H), 1.55-1.49 (m, 2H), 1.47-1.41 (m, 2H). 13C NMR
4.2.3. In vitro biofilm formation assay
(151 MHz, CD3OD)
d
169.80, 162.81 (dd, J ¼ 247.9, 12.3 Hz), 161.74,
According to the previously reported protocols [50], the biofilm
formation assay of compound A5 and FLC against the FLC-resistant
C. tropicalis 5008 and C. neoformans H99 were performed. The
fungal cells suspensions were prepared from the exponential
growth stage of C. tropicalis 5008 or C. neoformans H99 cells in YEPD
medium and diluted with RPMI 1640 medium to 1 ꢁ 106 CFU/mL,
respectively. Then the fungal cells suspensions were added to a 96-
well plate and incubated at 37 ꢀC for 1.5 h. After that, the upper
RPMI 1640 medium liquid of each well was absorbed and removed,
159.29 (dd, J ¼ 246.6,12.0 Hz),158.09,149.90,144.71,129.69,127.09,
125.03 (d, J ¼ 12.8 Hz), 114.21, 110.76 (d, J ¼ 20.9 Hz), 103.56 (t,
J ¼ 27.0 Hz), 74.90 (d, J ¼ 5.0 Hz), 67.12 (d, J ¼ 10.1 Hz), 62.49, 56.44,
55.79, 51.81, 51.04, 38.32, 28.31, 23.29, 22.88. HRMS (ESI) m/z: calcd
for C28H36F2N6O4 [M þ H]þ, 559.2844; found 559.2852.
4.1.4. 7-(4-(4-((4-(2-(2,4-difluorophenyl)-2-hydroxy-3-(1H-1,2,4-
triazol-1-yl)propyl)piperazin-1-yl)methyl)phenyl)-5-oxo-4,5-
dihydro-1H-1,2,4-triazol-1-yl)-N-hydroxyheptanamide (A12)
and washed with 100 mL PBS to remove the nonadherent fungal
White solid, 53% yield. 1H NMR (600 MHz, CD3OD)
d
8.32 (s, 1H),
cells. Compound A5 was added at different concentrations with FLC
as positive control, and the plate was incubated in at 37 ꢀC for 24 h.
Finally, a semiquantitative determination of biofilm formation was
performed using the XTT reduction method.
8.13 (s, 1H), 7.73 (s, 1H), 7.54 (d, J ¼ 8.3 Hz, 2H), 7.47-7.42 (m, 3H),
6.92-6.88 (m, 1H), 6.84-6.80 (m, 1H), 4.66 (d, J ¼ 14.3 Hz, 1H), 4.58
(d, J ¼ 14.3 Hz, 1H), 3.82 (t, J ¼ 6.9 Hz, 2H), 3.50 (s, 2H), 2.98 (d, 1H),
2.77 (d, J ¼ 13.8 Hz, 1H), 2.46 (m, 4H), 2.38 (bs, 4H), 2.08 (t,
J ¼ 7.3 Hz, 2H), 1.80-1.75 (m, 2H), 1.64-1.58 (m, 2H), 1.38-1.35 (m,
4.2.4. In vitro hyphal formation assay
4H). 13C NMR (151 MHz, CD3OD)
d
171.37, 162.74 (dd, J ¼ 247.6,
The exponential growth stage of C. tropicalis 5008 cells in YEPD
medium and diluted with 1.5 mL spider medium to 1 ꢁ 105 CFU/mL.
The fungal cells suspension was added to a 12-well plate, and
compound A5 was added at different concentrations with FLC as
positive drug. After the fungal cell incubation at 37 ꢀC for 7 h, the
difference in morphology between drug-treated group and no drug
group were recorded on the live cell imaging inverted microscope.
12.3 Hz), 159.31 (dd, J ¼ 246.5, 11.8 Hz), 152.21, 149.72, 144.69,
137.03, 134.84, 132.93, 130.34, 129.47 (d, J ¼ 6.1 Hz), 125.82 (d,
J ¼ 9.7 Hz), 122.05, 110.63 (d, J ¼ 21.0 Hz), 103.49 (t, J ¼ 26.8 Hz),
73.55 (d, J ¼ 5.3 Hz), 63.06, 61.63, 56.12, 53.98, 52.61, 45.01, 28.14,
27.99, 25.74, 25.19. HPLC purity: 96.6%.
4.2. Biological activity
4.2.5. In vitro capsule formation assay
4.2.1. MIC80 determination
The capsule formation assay was performed according to pre-
viously reported methods with a few optimizations [51]. Expo-
nentially growing C. neoformans H99 cells were incubated into
YEPD medium, collected and resuspended in 2 mL of PBS at a
The antifungal activity of the target compounds in vitro was
determined by plate microdilution method according to CLSI
(M27A3). Experiment was performed on the 96-well plated and
fungal cells (1 ꢁ 103 cells per well) in RMPI 1640 medium, the test
compounds were added in two-fold serial dilutions at 35 ꢀC for
48 h (C. tropicalis) or 72 h (C. neoformans). Then the optical density
OD at 630 nm (OD630) was monitored to evaluate the growth of
fungi and calculate the inhibition rate of each well. The minimum
concentration with inhibition over 80% of each compound was used
concentration of 1 ꢁ 107 cells/mL. Then the 100
mL fungal cells
suspension was added into a 12-well culture plate, 200
mL FBS and
1.7 mL DMEM were added in each well with different concentra-
tions of compound A5, no drug treated group as black control. The
12-well culture plate was placed in 37 ꢀC with 10% CO2 atmosphere
for 24 h. For capsule imaging, 100
ml of fungal cells suspension was
as MIC80
.
mixed with 10 L India Ink (Kingmorn, Shanghai) and observed
m
under Laser scanning Confocal Microscopy at a magnification of
63ꢁ (Leica SP5, Germany). The 10 different fields were randomly
chosen to photographed. For the calculation of capsule size, the
diameter of whole fungal cell and the diameter of fungal cell body
were measured by using ImageJ software.
4.2.2. Time-growth curve assay
The assay was performed according to our previously reported
method [49]. Exponentially growing C. tropicalis 5008 cells and
C. neoformans H99 cells were incubated into YEPD medium,
9

T. Zhu, X. Chen, C. Li et al.
European Journal of Medicinal Chemistry 221 (2021) 113524
4.2.6. Analysis of fungal sterol composition assay
24 h in advance and intravenously injected 1 ꢁ 105 CFU C. tropicalis
5008 cells on day 0. The murine model of CM was established by
intravenously injected 2 ꢁ 105 CFU C. neoformans H99 cells on day
0. Compound A5 (20 mg/kg) and FLC (20 mg/kg) were given orally
once a day at regular time, and the mice were administered with
drug solutions for 5 consecutive days with saline as blank control.
On day 6, the mice were euthanized, then kidney for C. tropicalis
infective mice and brain for C. neoformans infective mice were
picked aseptically. The picked infective organ was homogenized
and the homogenates in appropriate concentration were inoculated
to Sabouraud's Dextrose Agar (SDA) medium at 35 ꢀC for 48 h.
Finally, counted the number of single colonies on each SDA me-
dium, and further calculated the fungal burden of the kidney tissue
or brain tissue. ANOVA was used to analyze the differences among
the groups.
After treated with compound A5 with different concentrations
in YEPD medium, the C. tropicalis 5008 and C. neoformans H99 cells
were collected (wet weight about 1.0 g). Then the mixtures were
mixed with 10 mL saponifier (90% ethanol solution containing 15%
NaOH) at 80 ꢀC for 1 h. After that, 10 mL of petroleum ether (boiling
range: 30e60 ꢀC) was added to extract the sterols of each groups for
3 times. The extracted solutions were evaporated under reduced
pressure, and 1 mL cyclohexane was added to dissolve the residue.
Finally, the sterols composition of each compound group was
analyzed by using GC-MS, and the identification of sterols was
achieved by using the molecular fragments in each peak of GC-MS
chromatograms to match the corresponding sterol compound in
the NIST (the National Institute of Standards and Technology)
reference database.
4.2.7. Whole fungal cell HDACs enzymatic assay
Declaration of competing interest
Referring to the previously reported fungal HDAC enzymatic
method and with a few modifications[26]. The exponential growth
phase of C. tropicalis 5008 and C. neoformans H99 cells were
collected with the wet weight at 100 mg. Then they were treated by
The authors declare that they have no known competing
financial interests or personal relationships that could have
appeared to influence the work reported in this paper.
snailase (3 mg) and 2-mercapto-ethanol (12
mL) in the snailase
reaction buffer (3 mL) to prepare fungal protoplast. The protoplast
was dispersed in PBS (20 mL) and added into 96 well plate with
Acknowledgment
100 mL per well. Then the different concentrations of compounds
This work was supported by the National Natural Science
Foundation of China (Grant 81725020 to C.S. and 81973175 to N.L.),
the Innovation Program of Shanghai Municipal Education Com-
mission (Grant 2019-01-07-00-07-E00073 to C.S.), and the Science
and Technology Commission of Shanghai Municipality (Grant
20S11900400 to C.S.).
were added to and further incubated at 35 ꢀC for 12 h. The HDAC
substrate Boc-Lys(Ac)-AMC was added and adjusted the substrate
concentration to 30
37 ꢀC for 6 h. Subsequently, 100
solution were added and incubated at 37 ꢀC for 2 h. Finally, 100
mM in each well, then the plate incubated at
mL of HDAC enzymatic assay stop
mL
culture of each well was added to 96-well plate to monitor the
fluorescence intensity (Ex ¼ 360 nm, Em ¼ 460 nm), which was
used to calculate the HDAC inhibition rate.
Appendix A. Supplementary data
Supplementary data to this article can be found online at
https://doi.org/10.1016/j.ejmech.2021.113524.
4.2.8. RT-qPCR analysis
Exponentially growing C. tropicalis 5008 cells were used to
inoculate in 10 mL YPD with different concentrations of compound
A5 and DMSO as a blank control. After the cultures were incubated
at 35 ꢀC for 24 h, Total RNA of each group was isolated according to
the manufacture's protocol (Column Fungal RNAOUT, TIANDZ,
China), then the RNA was converted to cDNA by a reverse tran-
scription kit (TaKaRa, Biotechnology). Quantitative real-time RT-
PCR analysis were performed by using the StepOne System Fast
real-time PCR system (Applied Biosystems) with ACT1 was used as
an internal control gene. The relative fold change of the gene
expression levels for each group was calculated using formula
References
[1] G.D. Brown, D.W. Denning, S.M. Levitz, Tackling human fungal infections,
Science 336 (2012) 647.
[2] M.C. Fisher, N.J. Hawkins, D. Sanglard, S.J. Gurr, Worldwide emergence of
resistance to antifungal drugs challenges human health and food security,
Science 360 (2018) 739e742.
[3] L.Y. Chai, D.W. Denning, P. Warn, Candida tropicalis in human disease, Crit.
Rev. Microbiol. 36 (2010) 282e298.
[4] M. Negri, S. Silva, M. Henriques, R. Oliveira, Insights into Candida tropicalis
nosocomial infections and virulence factors, Eur. J. Clin. Microbiol. Infect. Dis.
31 (2012) 1399e1412.
[5] H. Xin, Effects of immune suppression in murine models of disseminated
Candida glabrata and Candida tropicalis infection and utility of a synthetic
peptide vaccine, Med. Mycol. 57 (2019) 745e756.
[6] C.C. Lai, C.K. Tan, Y.T. Huang, P.L. Shao, P.R. Hsueh, Current challenges in the
management of invasive fungal infections, J. Infect. Chemother. 14 (2008)
77e85.
[7] R. Rajasingham, R.M. Smith, B.J. Park, J.N. Jarvis, N.P. Govender, T.M. Chiller,
D.W. Denning, A. Loyse, D.R. Boulware, Global burden of disease of HIV-
associated cryptococcal meningitis: an updated analysis, Lancet, Inf. Disp. 17
(2017) 873e881.
[8] F.L. Mayer, J.W. Kronstad, Disarming fungal pathogens: Bacillus safensis in-
hibits virulence factor production and biofilm formation by Cryptococcus
neoformans and Candida albicans, mBio 8 (2017) e01517.
DD
2(ꢂ CT). Real-time PCR reaction for each cDNA was performed in
triplicate. The Gene-specific primers used in the assay are shown in
Table S1.
The C. neoformans H99 cells RNA isolation was different from
C. tropicalis 5008 cells. Initially, exponentially growing
C. neoformans H99 cells were used to inoculate in 50 mL YPD with
different concentrations of compound A5. After incubated at 35 ꢀC
for 24 h, the cultures grind thoroughly in liquid nitrogen for using.
Total RNA of each group was isolated according to the manufac-
ture's protocol (RNeasy Plant Mini kit, QIAGEN), the RNA reverse
transcription and RT-PCR analysis were similar to C. tropicalis. The
Gene-specific primers used in the assay are shown in Table S1.
[9] F.C. Odds, Genomics, molecular targets and the discovery of antifungal drugs,
Rev. Iberoam. Micol. 22 (2005) 229e237.
[10] F.C. Odds, A.J. Brown, N.A. Gow, Antifungal agents: mechanisms of action,
Trends Microbiol. 11 (2003) 272e279.
[11] J. Kim, J.E. Lee, J.S. Lee, Histone deacetylase-mediated morphological transition
in Candida albicans, J. Microbiol. 53 (2015) 805e811.
[12] L. Cui, J. Miao, T. Furuya, Q. Fan, X. Li, P.K. Rathod, X.Z. Su, L. Cui, Histone
acetyltransferase inhibitor anacardic acid causes changes in global gene
expression during in vitro Plasmodium falciparum development, Eukaryot.
Cell 7 (2008) 1200e1210.
4.2.9. In vivo antifungal assay
Female ICR mice (weight: 18e20 g, age: 4e6 weeks) were used
to build the in vivo experimental model, which purchased from
Shanghai Experimental Animal Center, Chinese Academy of Sci-
ences. The invasive C. tropicalis infective mice model was estab-
lished by intraperitoneally injected cyclophosphamide (100 mg/kg)
[13] I. Lee, J.H. Oh, E.K. Shwab, T.R. Dagenais, D. Andes, N.P. Keller, HdaA, a class 2
histone deacetylase of Aspergillus fumigatus, affects germination and
10

T. Zhu, X. Chen, C. Li et al.
European Journal of Medicinal Chemistry 221 (2021) 113524
secondary metabolite production, Fungal Genet. Biol. 46 (2009) 782e790.
[14] M.B. Lohse, A.D. Johnson, Temporal anatomy of an epigenetic switch in cell
programming: the white-opaque transition of C. albicans, Mol. Microbiol. 78
(2010) 331e343.
[32] J.A. Romo, C.G. Pierce, A.K. Chaturvedi, A.L. Lazzell, S.F. Mchardy, S.P. Saville,
J.L. Lopez-Ribot, J. Heitman, Development of anti-virulence approaches for
candidiasis via a novel series of small-molecule inhibitors of Candida albicans
filamentation, mBio 8 (2017) e01991.
[15] D. Hnisz, T. Schwarzmuller, K. Kuchler, Transcriptional loops meet chromatin:
a dual-layer network controls white-opaque switching in Candida albicans,
Mol. Microbiol. 74 (2009) 1e15.
[16] N. Robbins, M.D. Leach, L.E. Cowen, Lysine deacetylases Hda1 and Rpd3
regulate Hsp90 function thereby governing fungal drug resistance, Cell Rep. 2
(2012) 878e888.
[17] O.W. Liu, C.D. Chun, E.D. Chow, C. Chen, H.D. Madhani, S.M. Noble, Systematic
genetic analysis of virulence in the human fungal pathogen Cryptococcus
neoformans, Cell 135 (2008) 174e188.
[18] P.A. Dumesic, C.M. Homer, J.J. Moresco, L.R. Pack, E.K. Shanle, S.M. Coyle,
B.D. Strahl, D.G. Fujimori, J.R. Yates 3rd, H.D. Madhani, Product binding en-
forces the genomic specificity of a yeast polycomb repressive complex, Cell
160 (2015) 204e218.
[19] L. Kmetzsch, Histone deacetylases: targets for antifungal drug development,
Virulence 6 (2015) 535e536.
[20] S. Su, X. Li, X. Yang, Y. Li, X. Chen, S. Sun, S. Jia, Histone acetylation/deacety-
lation in Candida albicans and their potential as antifungal targets, Future
Microbiol. 15 (2020) 1075e1090.
[21] M.A. Pfaller, S.A. Messer, N. Georgopapadakou, L.A. Martell, J.M. Besterman,
D.J. Diekema, Activity of MGCD290, a Hos2 histone deacetylase inhibitor, in
combination with azole antifungals against opportunistic fungal pathogens,
J. Clin. Microbiol. 47 (2009) 3797e3804.
[33] J.A. Romo, C.G. Pierce, M. Esqueda, C.Y. Hung, S.P. Saville, J.L. Lopez-Ribot,
In vitro characterization of a biaryl amide anti-virulence compound targeting
Candida albicans filamentation and biofilm formation, Front. Cell Infect.
Microbiol. 8 (2018) 227.
[34] N. Liu, H. Zhong, J. Tu, Z. Jiang, Y. Jiang, Y. Jiang, Y. Jiang, J. Li, W. Zhang,
Y. Wang, C. Sheng, Discovery of simplified sampangine derivatives as novel
fungal biofilm inhibitors, Eur. J. Med. Chem. 143 (2018) 1510e1523.
[35] R.M. Donlan, J.W. Costerton, Biofilms: survival mechanisms of clinically rele-
vant microorganisms, Clin. Microbiol. Rev. 15 (2002) 167e193.
[36] D.D. Li, L.X. Zhao, E. Mylonakis, G.H. Hu, Y. Zou, T.K. Huang, L. Yan, Y. Wang,
Y.Y. Jiang, In vitro and in vivo activities of pterostilbene against Candida
albicans biofilms, Antimicrob. Agents Chemother. 58 (2014) 2344e2355.
[37] S. Wu, Y. Wang, N. Liu, G. Dong, C. Sheng, Tackling fungal resistance by biofilm
inhibitors, J. Med. Chem. 60 (2017) 2193e2211.
[38] T.R. O'Meara, J.A. Alspaugh, The Cryptococcus neoformans capsule: a sword
and a shield, Clin. Microbiol. Rev. 25 (2012) 387e408.
[39] A. Casadevall, C. Coelho, R.J.B. Cordero, Q. Dragotakes, E. Jung, R. Vij,
M.P. Wear, The capsule of Cryptococcus neoformans, Virulence 10 (2019)
822e831.
[40] K. Okabayashi, R. Kano, S. Watanabe, A. Hasegawa, Expression of capsule-
associated genes of Cryptococcus neoformans, Mycopathologia 160 (2005)
1e7.
[22] W.L. Smith, T.D. Edlind, Histone deacetylase inhibitors enhance Candida
albicans sensitivity to azoles and related antifungals: correlation with
reduction in CDR and ERG upregulation, Antimicrob. Agents Chemother. 46
(2002) 3532e3539.
[23] A. Mai, D. Rotili, S. Massa, G. Brosch, G. Simonetti, C. Passariello, A.T. Palamara,
Discovery of uracil-based histone deacetylase inhibitors able to reduce ac-
quired antifungal resistance and trailing growth in Candida albicans, Bioorg.
Med. Chem. Lett 17 (2007) 1221e1225.
[24] A. Anighoro, J. Bajorath, G. Rastelli, Polypharmacology: challenges and op-
portunities in drug discovery, J. Med. Chem. 57 (2014) 7874e7887.
[25] Y. Huang, G. Dong, H. Li, N. Liu, W. Zhang, C. Sheng, Discovery of janus kinase 2
(JAK2) and histone deacetylase (HDAC) dual inhibitors as a novel strategy for
the combinational treatment of leukemia and invasive fungal infections,
J. Med. Chem. 61 (2018) 6056e6074.
[41] F. Abe, K. Usui, T. Hiraki, Fluconazole modulates membrane rigidity, hetero-
geneity, and water penetration into the plasma membrane in Saccharomyces
cerevisiae, Biochemistry 48 (2009) 8494e8504.
[42] J.R. Perfect, The antifungal pipeline: a reality check, Nat. Rev. Drug Discov. 16
(2017) 603e616.
[43] N. Robbins, G.D. Wright, L.E. Cowen, Antifungal drugs: the current arma-
mentarium and development of new agents, Microbiol. Spectr. 4 (2016) 1e20.
[44] Y. Lee, E. Puumala, N. Robbins, L.E. Cowen, Antifungal drug resistance: mo-
lecular mechanisms in Candida albicans and beyond, Chem. Rev. (2020),
https://doi.org/10.1021/acs.chemrev.0c00199.
[45] S. Schubert, P.D. Rogers, J. Morschhauser, Gain-of-function mutations in the
transcription factor MRR1 are responsible for overexpression of the MDR1
efflux pump in fluconazole-resistant Candida dubliniensis strains, Antimicrob.
Agents Chemother. 52 (2008) 4274e4280.
[26] G. Han, N. Liu, C. Li, J. Tu, Z. Li, C. Sheng, Discovery of novel fungal lanosterol
14alpha-demethylase (CYP51)/histone deacetylase dual inhibitors to treat
azole-resistant candidiasis, J. Med. Chem. 63 (2020) 5341e5359.
[27] Z. Jiang, Y. Wang, W. Wang, S. Wang, B. Xu, G. Fan, G. Dong, Y. Liu, J. Yao,
Z. Miao, W. Zhang, C. Sheng, Discovery of highly potent triazole antifungal
derivatives by heterocycle-benzene bioisosteric replacement, Eur. J. Med.
Chem. 64 (2013) 16e22.
[28] S. Silva, M. Negri, M. Henriques, R. Oliveira, D.W. Williams, J. Azeredo, Candida
glabrata, Candida parapsilosis and Candida tropicalis: biology, epidemiology,
pathogenicity and antifungal resistance, FEMS Microbiol. Rev. 36 (2012)
288e305.
[29] K. Diba, K. Makhdoomi, E. Nasri, A. Vaezi, J. Javidnia, D.J. Gharabagh,
N.H. Jazani, A. Reza Chavshin, P. Badiee, H. Badali, H. Fakhim, Emerging
Candida species isolated from renal transplant recipients: species distribution
and susceptibility profiles, Microb. Pathog. 125 (2018) 240e245.
[30] S.M. Noble, B.A. Gianetti, J.N. Witchley, Candida albicans cell-type switching
and functional plasticity in the mammalian host, Nat. Rev. Microbiol. 15
(2017) 96e108.
[31] S.M. Noble, S. French, L.A. Kohn, V. Chen, A.D. Johnson, Systematic screens of a
Candida albicans homozygous deletion library decouple morphogenetic
switching and pathogenicity, Nat. Genet. 42 (2010) 590e598.
[46] C. Jiang, D. Dong, B. Yu, G. Cai, X. Wang, Y. Ji, Y. Peng, Mechanisms of azole
resistance in 52 clinical isolates of Candida tropicalis in China, J. Antimicrob.
Chemother. 68 (2013) 778e785.
[47] T. Noel, The cellular and molecular defense mechanisms of the Candida yeasts
against azole antifungal drugs, J. Mycol. Med. 22 (2012) 173e178.
[48] F. Brandao, S.K. Esher, K.S. Ost, K. Pianalto, C.B. Nichols, L. Fernandes,
A.L. Bocca, M.J. Pocas-Fonseca, J.A. Alspaugh, HDAC genes play distinct and
redundant roles in Cryptococcus neoformans virulence, Sci. Rep. 8 (2018)
5209.
[49] S. Wang, Y. Wang, W. Liu, N. Liu, Y. Zhang, G. Dong, Y. Liu, Z. Li, X. He, Z. Miao,
J. Yao, J. Li, W. Zhang, C. Sheng, Novel carboline derivatives as potent anti-
fungal lead compounds: design, synthesis, and biological evaluation, ACS Med.
Chem. Lett. 5 (2014) 506e511.
[50] Z. Jiang, N. Liu, D. Hu, G. Dong, Z. Miao, J. Yao, H. He, Y. Jiang, W. Zhang,
Y. Wang, C. Sheng, The discovery of novel antifungal scaffolds by structural
simplification of the natural product sampangine, Chem. Commun. 51 (2015)
14648e14651.
[51] F.A. Brandao, L.S. Derengowski, P. Albuquerque, A.M. Nicola, I. Silva-Pereira,
M.J. Pocas-Fonseca, Histone deacetylases inhibitors effects on Cryptococcus
neoformans major virulence phenotypes, Virulence 6 (2015) 618e630.
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