Discovery of Carboline Derivatives as Potent Antifungal Agents for the Treatment of Cryptococcal Meningitis

Article abstract of DOI:10.1021/acs.jmedchem.8b01598

Clinical treatment of cryptococcal meningitis (CM) remains a significant challenge because of the lack of effective and safe drug therapies. Developing novel CM therapeutic agents with novel chemical scaffolds and new modes of action is of great importanc

Full text of DOI:10.1021/acs.jmedchem.8b01598

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Article
Discovery of Carboline Derivatives as Potent Antifungal
Agents for the Treatment of Cryptococcal Meningitis
Jie Tu, Zhuang Li, Yanjuan Jiang, Changjin Ji, Guiyan Han, Yan Wang, Na Liu, and Chunquan Sheng
J. Med. Chem., Just Accepted Manuscript • Publication Date (Web): 12 Feb 2019
Downloaded from http://pubs.acs.org on February 12, 2019
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Discovery of Carboline Derivatives as Potent Antifungal Agents for
the Treatment of Cryptococcal Meningitis
†
†
Jie Tu , Zhuang Li , Yanjuan Jiang, Changjin Ji, Guiyan Han, Yan Wang, Na Liu*,
Chunquan Sheng*
Department of Medicinal Chemistry, School of Pharmacy, Second Military Medical
University, 325 Guohe Road, Shanghai 200433, People’s Republic of China
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ABSTRACT
Clinical treatment of cryptococcal meningitis (CM) remains a significant challenge
due to the lack of effective and safe drug therapies. Developing novel CM therapeutic
agents with novel chemical scaffolds and new modes of action is of great importance.
Herein, new -hexahydrocarboline derivatives were shown to possess potent
anticryptococcal activities. In particular, compound A4 showed potent in vitro and in
vivo anticryptococcal activity with good metabolic stability and BBB permeability.
Compound A4 was orally active and could significantly reduce brain fungal burdens
in a murine model of CM. Moreover, compound A4 could inhibit several virulence
factors of C. neoformans and might act by a new mode of action. Preliminary
mechanistic studies revealed that compound A4 induced DNA double stranded breaks
and cell cycle arrest at the G2 phase by acting on the Cdc25c/CDK1/cyclin B
pathway. Taken together, -hexahydrocarboline A4 represents a promising lead
compound for the development of next-generation CM therapeutic agents.
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INTRODUCTION
The clinical treatment of life-threatening invasive fungal infections (IFIs) remains a
significant global challenge.1, 2 Species of Candida, Cryptococcus, Aspergillus, and
Pneumocystis are major fungal pathogens accounting for nearly 90% of lethal cases in
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humans. Cryptococcus neoformans (C. neoformans var. grubii and C. neoformans
var. neoformans) and Cryptococcus gattii are responsible for most cases of fungal
meningitis worldwide.4, 5 Cryptococcal meningitis (CM), one of the most important
lethal factors in HIV/AIDS patients, is always fatal if left untreated.5-7 Clinically,
antifungal agents for the treatment of CM mainly include polyenes (e.g., liposomal or
deoxycholate amphotericin B), nucleic acid analogues (e.g., 5-flucytosine), and azoles
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(e.g., fluconazole (FLC)). However, amphotericin B, the drug with the greatest early
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fungicidal activity (EFA), is associated with renal impairment. Toxic cytopenias can
occur during 5-flucytosine therapy, and thus regular full blood counts and therapeutic
drug monitoring (TDM) are advised. FLC is fungistatic rather than fungicidal in the
normal human dose range, and it is therefore often used as a maintenance therapy.4
The newly developed β-1,3-glucan synthase inhibitors (i.e., echinocandins) are
ineffective against Cryptococcus species.10, 11 The current “gold standard” for the
treatment of CM is still based on a combination of amphotericin B and 5-flucytosine
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that was developed more than 50 years ago. Currently, 5-fluorocytosine remains
unlicensed in most African and Asian countries.4, 12 Despite the increasing clinical
demands, the development of new drugs for the treatment of CM has been relatively
slow. In recent years, the search for anticryptococcal agents has mainly focused on
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drug repurposing and alternative preparations of the “gold standard” mixture.3, 13-16
The discovery of new molecules to treat cryptococcal infections remains quite
challenging. Therefore, developing novel CM therapeutic agents with novel chemical
scaffolds and new modes of action is of great importance.17
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Previously, our group identified γ-hexahydrocarboline scaffold 1 as a new
antifungal scaffold by screening against an in-house library.18 Further structural
optimization led to γ-tetrahydrocarboline 2 (C38), which showed improved antifungal
activity. Its activity was comparable to that of FLC but without toxicity to human
1
8
embryonic lung cells. In particular, carboline 2 showed features that suggested it
could overcome the drug resistance of FLC. For example, it exhibited good antifungal
activity against both FLC-sensitive and FLC-resistant C. albicans cells and had potent
inhibitory activity against C. albicans biofilm formation and hyphal growth.
Moreover, the anticryptococcal activity of compound 2 (C. neoformans, MIC = 1
8
0
μg/mL) was also better than that of compound 1 (C. neoformans, MIC = 8 μg/mL,
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Figure 1). However, based on further studies, the development of
γ-tetrahydrocarbolines was hampered by their poor in vivo potency.
A series of β-carboline derivatives based on γ-carboline 2 were designed and
synthesized, and they showed increased anticryptococcal activities (Figure 1 and
Figure S1 in the Supporting Information). In particular, compound A4 showed strong
in vitro and in vivo anticryptococcal activities, making it a promising lead compound
for the development of novel anticryptococcal agents. Preliminary studies on the
anticryptococcal mechanism of A4 indicated that it might act by a new mode of
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action.
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Figure 1. Discovery and structural optimization of carboline-based antifungal
compounds. The minimum inhibitory concentration (MIC) was measured by the
optical density at 600 nm (OD600), and the relative growth is displayed as a heatmap.
Abbreviations: C. alb. Candida albicans; C. par. Candida parapsilosis; C. neo.
Cryptococcus neoformans; C. gatti. Cryptococcus gattii; C. gla. Candida glabrata; A.
fum. Aspergillus fumigatus; T. rub. Trichophyton rubrum; M. gyp. Microsporum
gypseum.
CHEMISTRY
The chemical syntheses of the β-tetrahydrocarboline derivatives are depicted in
Schemes 1-4. Intermediates 5a-q were synthesized from commercially available
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substituted tryptamines via two steps according to the reported methods. Reacting
1
,3-dibromopropanes (3a-o) with the appropriately substituted phenols in the presence
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of K CO and ethanol gave intermediates 4a-o, which were reacted with intermediates
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a-q to form compounds 6. Finally, the Boc protecting groups were removed under
acidic conditions (trifluoroacetate (TFA)) to afford substituted β-tetrahydrocarboline
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derivatives A1-30 (Scheme 1).
Scheme 1
Br
Br
R2
R2
HO
Br
O
a
3a-o
4a-o
O
R2
3
3
, 4a R2 = 4'-Cl
, 4b R2 = 2'-F
3, 4i R2 = 3'-OCH3
3, 4j R2 = 4'-F
3, 4c R2 = 2'-Cl
3, 4k R2 = 4'-Br
3, 4l R2 = 4'-CH3
, 4e R2 = 2'-OCH3 3, 4m R2 = 4'-OCH3
N
3
3
3
3
3
, 4d R2 = 2'-Br
Boc
N
3
, 4n R2 = 4'-NO2
b
R1
c
, 4f R2 = 3'-F
, 4g R2 = 3'-Cl
, 4h R2 = 3'-Br
3, 4o R2 = 4'-Cl
6
5
5
5
5
a
b
c
d
R1 = 5-Br
R1 = 5-Cl
R1 = 5-F
R1 = 6-Cl
5j R1 = 7-Br
5k R1 = 7-CH3
5l R1 = 7-OCH3
5m R1 = 8-Cl
5n R1 = 8-Br
5o R1 = 8-CH3
H
N
Boc
N
R1
5e R1 = 6-Br
5a-q
5
5
5
5
f
R1 = 6-CH3
g
h
i
R1 = 6-OCH3 5p R1 = 8-OCH3
R1 = 7-F
R1 = 7-Cl
5q R1 = H
A1 R1 = 5-Br
A2 R1 = 5-Cl
A3 R1 = 5-F
A4 R1 = 6-Cl
A5 R1 = 6-Br
A6 R1 = 6-CH3
R2 = 4'-Cl
R2 = 4'-Cl
R2 = 4'-Cl
R2 = 4'-Cl
R2 = 4'-Cl
R2 = 4'-Cl
A16 R1 = 8-OCH3 R2 = 4'-Cl
A17 R1 = H
A18 R1 = H
A19 R1 = H
A20 R1 = H
A21 R1 = H
A22 R1 = H
A23 R1 = H
A24 R1 = H
A25 R1 = H
A26 R1 = H
A27 R1 = H
A28 R1 = H
A29 R1 = H
A30 R1 = H
R2 = 2'-F
R2 = 2'-Cl
R2 = 2'-Br
R2 = 2'-OCH3
R2 = 3'-F
2'
O
1'
3'
R2
4'
A7 R1 = 6-OCH3 R2 = 4'-Cl
R2 = 3'-Cl
8
N
1
A8 R1 = 7-F
A9 R1 = 7-Cl
A10 R1 = 7-Br
R2 = 4'-Cl
R2 = 4'-Cl
R2 = 4'-Cl
R2 = 4'-Cl
R2 = 3'-Br
R2 = 3'-OCH3
R2 = 4'-F
R2 = 4'-Br
R2 = 4'-CH3
R2 = 4'-OCH3
R2 = 4'-NO2
R2 = 4'-Cl
7
NH CF COOH
3
R1
2
6
3
5
4
A11 R1 = 7-CH3
A1-30
A12 R1 = 7-OCH3 R2 = 4'-Cl
A13 R1 = 8-Cl
A14 R1 = 8-Br
A15 R1 = 8-CH3
R2 = 4'-Cl
R2 = 4'-Cl
R2 = 4'-Cl
Reagents and conditions: (a) K CO , EtOH, 80°C, 4 h, yield 51-66%; (b) NaH,
2
3
DMF, rt, 1 h, yield 34-54%; (c) TFA, CH Cl , rt, 0.5 h, yield 71-85%.
2
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Compounds B1-16 were synthesized according to the procedures outlined in
Scheme 2. Excess 1,3-dibromopropane was reacted with intermediate 5q to give
compound 7. Compound 7 was reacted with various substituted anilines in the
presence of KI, K CO and MeCN to afford intermediates 8a-p, which were further
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deprotected in the presence of TFA to give target compounds B1-16. As shown in
Scheme 3, compounds 9a-l were obtained by reacting intermediate 7 with various
amines in DMF. The Boc groups of compounds 9a-l were removed under acidic
conditions (TFA), and then free amine C1-12 were prepared under alkaline
conditions. Using a similar procedure, compounds C13-14 were synthesized by
reacting intermediate 5q with 10a-b in the presence of NaOH and DMF followed by
Boc deprotection (Scheme 4).
Scheme 2
H
Br
N
R
H
N
N
R
N
Br
H
Br
H N
2
Boc
Boc
Boc
N
N
N
a
b
5q
7
8a-p
2
N 1
3
8
8
8
8
8
8
a, B1 R = 4-Cl
b, B2 R = 2-Cl
c, B3 R = 2-Br
d, B4 R = 2-OCH3
e, B5 R = 2-OCH CH
8i, B9 R = 3-OCH3
8j, B10 R = 3-OCH2CH3
8k, B11 R = 4-F
8l, B12 R = 2-F
8m, B13 R = 4-Br
8n, B14 R = 4-CH3
8o, B15 R = 4-OCH3
8p, B16 R = 4-OCH2CH3
R
4
c
N
NH CF3COOH
2
3
f, B6 R = 3-F
8g, B7 R = 3-Cl
8h, B8 R = 3-Br
B1-16
Reagents and conditions: (a) NaOH, DMF, rt, 1 h, yield 17-21%; (b) K CO , MeCN,
2
3
8
5°C, 1 h, yield 42-86%; (c) TFA, CH Cl , rt, 0.5 h, yield 37-80%.
2 2
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Scheme 3
Br
R
R
N
N
N
0
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RH
a
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Boc
N
N
NH
b
7
9a-l
R
C1-C12
R
R
N
N
N
N
N
9a, C1
9e, C5
9f, C6
9g, C7
9h, C8
9i, C9
N
N
O
N
N
9j, C10
9k, C11
9l, C12
9b, C2
O
N
9
c, C3
d, C4
N
N
O
N
H
N
N
N
O
9
Reagents and conditions: (a) DMF, rt, 4 h, yield 13-91%; (b) TFA, CH Cl , rt, 0.5 h,
2
2
yield 18-71%.
Scheme 4
R
R
H
N
N
N
Boc
R
N Boc
N
NH
O
Br
0a-b
a
b
1
11
C13-14
5q
1
0a, C13 R =
N
1
0b, C14 R =
N
Reagents and conditions: (a) NaOH, DMF, rt, 1 h, yield 15-21%; (b) TFA, CH Cl ,
2
2
rt, 0.5 h, yield 45-76%.
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RESULTS AND DISCUSSION
Anticryptococcal Activity and Structure−Activity Relationship. A series of new
carboline derivatives were designed and synthesized (Schemes 1-4). Their in vitro
anticryptococcal activities are summarized in Tables 1-3. The antifungal activities are
expressed as the MIC to achieve 80% inhibition of C. neoformans. The triazole-based
antifungal drug FLC, used as the first-line therapy in the treatment of candidiasis and
cryptococcosis, was chosen as the positive control. TFA itself has no antifungal
activity (MIC > 64 g/mL).
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First, various substituents on the benzene ring of the tetrahydrocarboline scaffold
(A1-16) and the terminal phenol group in the side chain (A17-30) were investigated.
Generally, derivatives A1-30 exhibited moderate to good antifungal activities against
the C. neoformans H99 strain. Substitution at the C6 position was more favorable than
at the C7, C8 and C5 positions, and a C6-chlorine group gave the best results. Among
the tested substituents on the terminal benzene ring, halogen substituents provided
better activity than methyl, alkoxyl and nitro substituents. Substitution at the C4’ and
C3’ positions provided better results than substitution at the C2’. Interestingly,
compound A4, the C6,C4’-dichloro derivative, showed excellent activity against C.
neoformans (MIC = 0.25 g/mL at 48 h; MIC = 0.5 g/mL at 72 h), and it was
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more potent than FLC.
Second, the importance of the oxygen atom in the side chain (B1-16) was
investigated. The replacement of the oxygen atom with a nitrogen atom led to a
decrease in the antifungal activity. Consistent with the SAR in series A, compounds
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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
with halogen substituents on the benzene ring of the side chain were more potent than
the corresponding methyl and alkoxyl derivatives. Finally, substituted benzene rings
were replaced by various aliphatic amines (C1-C14). However, an obvious decrease
in the antifungal activities was observed for all of the aliphatic amine derivatives.
Among the target compounds, compounds A4, A5 and A6 showed potent
anticryptococcal activities. Notably, the solubility assay revealed that A4 (solubility:
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
5
4.6 μg/mL) and A6 (solubility: 57.5 μg/mL) were significantly more soluble than A5
(solubility: 19.3 μg/mL). Therefore, compounds A4 and A6 were chosen for further
biological evaluations. An assay of the spectrum of the antifungal activity revealed
that compounds A4 and A6 could inhibit a wide range of fungal pathogens, including
Candida albicans, Candida parapsilosis, Candida glabrata, Cryptococcus gattii,
Aspergillus fumigates, Trichophyton rubrum and Microsporum gypseum (Table 4).
Moreover, both compounds showed high plasma protein binding and low toxicity to
human HUVECs (Tables S1 and S2 in Supporting Information).
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Table 1. In vitro antifungal activities of carboline derivatives A1-30 (MIC ,
8
0
µg/mL, 48 h)
2'
1'
O
3'
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
R2
4'
8
N
1
7
NH CF COOH
3
R1
2
6
3
5
4
Compds
R1
R2
C. neo.
Compds
R1
R2
C. neo.
A1
A2
5- Br
5-Cl
4’-Cl
4’-Cl
4’-Cl
4’-Cl
4’-Cl
4’-Cl
8
4
A17
A18
A19
A20
A21
A22
A23
A24
A25
A26
A27
A28
A29
A30
FLC
H
H
H
H
H
H
H
H
H
H
H
H
H
H
2’-F
2’-Cl
16
4
A3
5- F
4
2’-Br
8
A4
6-Cl
0.25
0.5
1
2’-OCH3
3’-F
32
8
A5
6-Br
6-CH3
A6
3’-Cl
4
A7
6-OCH3 4’-Cl
4
3’-Br
4
A8
7-F
7-Cl
4’-Cl
4’-Cl
4’-Cl
4’-Cl
2
3’-OCH3
4’- F
16
8
A9
2
A10
A11
A12
A13
A14
A15
7-Br
2
4’- Br
4’- CH3
4’- OCH3
4’-NO2
4’-Cl
4
7-CH3
2
8
7-OCH3 4’-Cl
2
16
16
4
8-Cl
8-Br
4’-Cl
4’-Cl
4’-Cl
2
4
8-CH3
2
4
1
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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
A16
8-OCH3 4’-Cl
4
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
Table 2. In vitro antifungal activities of the carboline derivatives B1-16 (MIC ,
8
0
µg/mL, 48 h)a
H
N
2
1
3
R
4
N
NH CF COOH
3
Compds
R
C. neo.
Compds
R
C. neo.
B1
B2
B3
B4
B5
B6
B7
B8
B9
4-Cl
2-Cl
8
8
B10
B11
B12
B13
B14
B15
B16
FLC
3-OCH CH
64
16
16
8
2
3
4-F
2-F
2-Br
8
2-OCH3
32
32
16
8
4-Br
2-OCH CH
4-CH3
4-OCH3
16
32
32
4
2
3
3-F
3-Cl
4-OCH CH
2
3
3-Br
8
3-OCH3
32
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Table 3. In vitro antifungal activities of carboline derivatives C1-14 (MIC ,
8
0
µg/mL, 48 h)a
R
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
n
N
NH
Compds
R
n
C. neo.
Compds
R
n
C. neo.
N
C1
N
2
64
C9
N
2
32
O
C2
C3
N
N
2
2
64
32
C10
C11
N
2
2
32
64
N
N
N
O
O
C4
C5
C6
2
2
2
64
64
64
C12
C13
C14
N
2
1
1
64
64
64
NH
N
O
N
N
N
N
O
C7
C8
N
2
2
64
32
FLC
4
O
N
2
3
4
5
6
7
8
9
Table 4. In vitro antifungal activities of compounds A4 and A6
MIC80, μg/mL, 48 h
MIC80, μg/mL, 72 h
0
M.
1 Compds
C. gla.
C. alb.
C. alb.
C. par.
A. fum.
T. rub.
C. neo.
C. neo.
C. gat.
gyp.
MZC
2
3
4
5
6
7
8
9
0
8
535
7781
0304103
5008
7544
TIA
H99
ATCC34877 ATCC14116
A4
1
2
4
4
4
2
4
2
8
16
8
4
4
2
8
8
0.5
2
0.5
2
1
2
4
A6
FLC
32
>64
32
>64
16
2
4
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4
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4
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5
5
5
5
5
6
Compounds A4 and A6 Showed Effective Inhibitory Activities against C.
neoformans. To further evaluate the antifungal profiles of compounds A4 and A6,
time-growth curves were prepared. Both compounds exhibited potent fungistatic
activities. Compounds A4 and A6 inhibited C. neoformans H99 growth in a
concentration-dependent manner and completely inhibited H99 cell growth at 8
μg/mL. In contrast, FLC did not obviously inhibit C. neoformans H99 growth in a
concentration-dependent manner (Figure 2).
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 2. Fungal growth curves of C. neoformans H99 obtained by using initial
inoculums containing 106 colony-forming units (CFU)/mL. CFUs/mL were
determined after 0, 4, 8, 12, 24 and 48 h of incubation. C. neoformans H99 was
treated with different concentrations of compounds A4 (A), A6 (B) and FLC (C).
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Compounds A4 and A6 Inhibited the Formation of C. neoformans Biofilms.
Fungal biofilm formation is closely related to drug resistance and repeated infection.20
20
The adhesion of fungi to a surface is the first stage in this process. Therefore, the
1
1
1
1
1
1
1
1
1
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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
effects of different concentrations of FLC and compounds A4 and A6 on the
adherence of C. neoformans H99 cells (90 min) and subsequent biofilm formation
2
1
were investigated using the XTT reduction assay. Compounds A4 and A6 could
significantly inhibit biofilm formation and were more active than FLC. At a
concentration of 16 μg/mL, compounds A4 and A6 almost completely disrupted the
formation of C. neoformans biofilms (Figure 3). The effects of FLC and compound
A4 on the cellular surface hydrophobicity (CSH) of C. neoformans H99 were also
investigated. Compound A4 significantly decreased the CSH of C. neoformans (P <
0
.001, Figure 4), whereas FLC was ineffective.
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8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Figure 3. Effects of different concentrations of DMSO (A), FLC (B), compound A4
(C) and compound A6 (D) on the adherence of C. neoformans H99 cells (90 min) and
subsequent biofilm formation using the XTT reduction assay. The values are the mean
absorbance values (optical density (OD)) at 490 nm and standard deviations (SDs) of
triplicate analyses. The results are presented as a percentage compared to the control
group, which was untreated. * P < 0.05, ** P < 0.01, *** P < 0.001 versus the control
group.
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2
2
2
2
2
2
2
2
2
2
3
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3
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3
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3
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3
4
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8
9
0
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2
3
4
5
6
7
8
9
0
Figure 4. Effects of FLC and compound A4 on the CSH of C. neoformans H99.
Relative CSH values were obtained by a water-hydrocarbon two-phase assay. SDs are
depicted and based on three independent experiments. *** P < 0.001.
Compounds A4 and A6 Inhibited the Production of Melanin without Affecting
that of Urease. Melanin and urease are specific virulence factors of C. neoformans
that play a significant role in pathogenic effects and immunity escape.22, 23 In this
study, specific inducing culture media were used to induce the production of melanin
2
4
and urease. The production of melanin and urease was identified by observing the
features of the colonies and chromogenic reactions. C. neoformans H99 strains were
treated with different concentrations of compounds A4 and A6 and then inoculated in
the corresponding media. Compounds A4 and A6 at 2.5 μg/mL could inhibit the
production of melanin, whereas FLC was inactive. However, A4 and A6 had no
obvious inhibitory effects on the production of urease even at high concentrations
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2
2
2
2
2
2
2
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3
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3
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3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
(Figure 5).
0
1
2
3
4
5
6
7
8
9
0
1
2
3
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5
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8
9
0
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8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Figure 5. Effects of A4, A6 and FLC on the production of melanin and urease in C.
neoformans H99 cells. (A) Melanin production assay. Strains treated with different
concentrations of the test compounds were grown on L-DOPA medium at 37°C. (B)
Strains were grown on the urea medium at 37°C for the detection of urease
production.
Compound A4 Displayed Potent in vivo Antifungal Effects in a Murine Model of
CM. Before investigating their in vivo antifungal activities, their microsomal
metabolic stabilities were tested. Compound A4 had a low clearance rate in the liver
microsomal metabolic stability test with a half-life (T ) of 70 min, indicating that it
1
/2
may have good metabolic stability. In contrast, compound A6 was unstable (T1/2 = 11
min) in the mouse liver microsomal stability assay (Table 5). Furthermore, the
permeability of the blood-brain barrier (BBB) to compound A4 was tested by the
parallel artificial membrane permeation assay (PAMPA). Compound A4 could easily
penetrate the BBB at a level comparable to that of caffeine (Table 6).25
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Table 5. Metabolic stabilities of compounds A4 and A6 in mouse liver microsomes
T1/2a
b
CL (mL/min/mg)
(min)
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
Midazolam
2
2996
78
A4
A6
70
11
477
a
b
Half-life. Clearance.
-6
Table 6. Permeability (P × 10 cm/s) and prediction of BBB penetration in the
e
PAMPA-BBB assay for compound A4.
a
-6
prediction b
P (10 cm/s)
e
Caffeine
A4
1.4 ± 0.03
1.8 ± 0.14
CNS±
CNS±
a
Values are expressed as the mean ± SD of at least three independent experiments.
b
-6
Compounds with Pe > 3.08 × 10 cm/s can cross the BBB by passive diffusion
−
6
(
CNS+). Compounds with Pe < 1.13 × 10 cm/s cannot cross the BBB (CNS−), and
−
6
−6
compounds with 1.13 × 10 cm/s < Pe < 3.08 × 10 cm/s show uncertain BBB
permeation (CNS±).
Considering the good metabolic stability and BBB penetration of compound A4,
we evaluated its in vivo antifungal activity using a CM model. Mice CM models were
established by injecting C. neoformans H99 cells into the tail veins of the animals.
Mice were assigned into three groups: the control group, the FLC treatment group and
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2
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5
6
the A4 treatment group. FLC and A4 treatment were initiated at 24 h postinfection by
oral administration. The treatments were continued for 5 consecutive days. The mice
were sacrificed 5 days postinfection, and their brain fungal burdens were determined
by measuring the number of CFU. Treatment with compound A4 significantly
reduced the fungal burden in the brain (P < 0.001), and it was more effective than
FLC (P < 0.001). (Figure 6). The brain fungal burden could also be significantly
reduced by intraperitoneal injection of A4 at a dosage of 10 mg/kg once daily (P <
0
1
2
3
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5
6
7
8
9
0
1
2
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5
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9
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5
6
7
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9
0
1
2
3
4
5
6
7
8
9
0
0
.001, Figure S2 in the Supporting Information). The results highlighted the potential
of compound A4 for the treatment of CM.
Figure 6. The therapeutic efficacies of compound A4 and FLC in the C. neoformans
H99 CM model. Brain burdens of fungi (in Log CFU/mL of brain) are shown for
1
0
mice treated with the indicated doses of A4 or FLC for 5 days after infection with C.
neoformans H99. Data are presented as the mean ± SD. * P < 0.05, ** P < 0.01, ***
P < 0.001, determined by ANOVA.
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Cell Wall and Cell Membrane Collapse, Chromosome Condensation and
Nuclear Pyknosis Effects of Compound A4 Contributed to the Inhibition C.
neoformans. To clarify the antifungal mechanism of compound A4, its influence on
the morphology of C. neoformans was monitored by transmission electron
microscopy (TEM). Normal C. neoformans cells and cells treated with FLC at 4
μg/mL for 24 h were used as controls. As shown in Figure 7A, normal C. neoformans
cells have a uniform central density and a regular, well-outlined cell wall. The cells
treated with FLC had damaged cell membranes and fragmented nuclei, but the cell
walls were intact (Figure 7B). In contrast, after treatment with A4 at 4 μg/mL for 24
h, the C. neoformans cells became abnormally shaped (Figure 7C). Cell wall collapse
and nuclear pyknosis could also be observed (Figure 7C). Thus, A4 and FLC may act
by different antifungal mechanisms.
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3
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4
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6
0
1
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5
6
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9
0
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8
9
0
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8
9
0
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9
0
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Figure 7. Transmission electron micrographs of C. neoformans H99 cells under
different conditions. The arrows indicate organelles as follows: 1, cell wall; 2, cell
membrane; 3, cell nucleus. (A) Normal C. neoformans cells; (B) fungal cells treated
with FLC at 4 μg/mL for 24 h; and (C) fungal cells treated with compound A4 at 4
μg/mL for 24 h.
The Cdc25c/CDK1/Cyclin B Pathway is Essential for A4-Induced G2/M phase
arrest. Inspired by the morphological changes to C. neoformans cells treated with A4
and FLC, the impacts of the two compounds on the cell cycle were further
investigated using flow cytometric analysis. Interestingly, after 24 h of exposure,
51.64% of the cells were in S phase, 44.50% of the cells were in G1 phase and 3.86%
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of the cells were in G2 phase in the control, compared to 48.17%, 50.86% and 0.96%,
respectively, in the sample treated with 4 μg/mL FLC. The results indicated that FLC
induced cell cycle arrest at the G1 phase. In contrast, compound A4 arrested the G2/M
phase of the cell cycle (53.21% in S phase, 36.45% in G1 phase and 10.34% in G2
phase, Figure 8). The Cdc25c/CDK1/cyclin B pathway is associated with G2/M
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phase control. To further investigate the mode of action of A4-induced G2/M phase
arrest, the expression levels of CDK1 and cyclin B were evaluated by Western blot.
As shown in Figure 9A and 9B, compound A4 upregulated the expression of CDK1
and Cdc25c in a dose-dependent manner, whereas FLC did not obviously influence
the expression of CDK1 and Cdc25c. The results revealed that compound A4 induced
cell cycle arrest at the G2 phase by acting on the Cdc25c/CDK1/cyclin B pathway.
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Figure 8. Flow cytometric analysis and cell cycle arrest by compound A4. (A) Cell
cycle analysis by flow cytometry. C. neoformans H99 cells were treated with different
concentrations of FLC and A4 for 24 h after staining with PI. Determinations were
performed in triplicate, and one representative experiment is shown. (B) Mean
percentages of cells in G , S, and G phases. The percentages in different phases were
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calculated by flow cytometry software. Data are presented as the mean ± SD from
three independent experiments. ** P < 0.01, *** P < 0.001, determined by ANOVA.
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Figure 9. Effects of compounds A4 and FLC on the expression levels of CDK1 (A),
cyclin B1 (A) and Cdc25C (B) in C. neoformans H99 cells. (C) Gray intensity
analysis of the Western blots. (D) C. neoformans H99 cells were treated with A4 at 2
μg/mL for 48 h and assayed for DNA damage by immunofluorescence staining with
Hoechst 33342 (blue) and γH2AX (green). * P < 0.05, ** P < 0.01, *** P < 0.001
versus the control group.
Compound A4 Induces DNA Damage and Increases Nuclear γ-H2AX Foci. In the
presence of DNA double stranded breaks (DSBs), cells arrest in the G2/M phase to
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allow time to repair the damage. In addition, histone H2AX is phosphorylated by
ataxia telangiectasia mutated (ATM), termed γ-H2AX, which is a major cascade.
Hence, γ-H2AX is a sensitive biomarker for DSBs.28, 29 In this study, the effects of
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treatment with A4 on DSBs in C. neoformans H99 cells were investigated. As shown
in Figure 9C, compound A4 induced γH2AX expression, which was colocalized with
Hoechst staining in C. neoformans H99. A4 increased the appearance of nuclear
γ-H2AX foci and induced DSBs.
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KEGG Pathways Analysis of DEGs was Consistent with the Induction of G2/M
Phase Arrest and DSBs by A4. To further understand the changes in biological
processes upon treatment with compound A4, RNA-sequencing (poly A) analysis of
C. neoformans H99 cells was performed. Kyoto encyclopedia of genes and genomes
(KEGG) enrichment analysis was performed to classify the functions of the
differentially expressed genes (DEGs, Figures S3 and S4 in the Supporting
Information).30-34 Notably, 12 DEGs were enriched in the cell cycle (cell cycle and the
yeast cell cycle), and 6 DEGs were enriched related to DNA replication. In the cell
cycle, the 12 upregulated DEGs included APC/C (CNAG_00320 and CNAG_02963),
PNAC (CNAG_06079), PLK1 (CNAG_01907), MCM (CNAG_03341,
CNAG_05825 and CNAG_06182), ORC (CNAG_06183), Mps1 (CNAG_06697),
Hsl7 (CNAG_02829), Ycg1 (CNAG_00681) and Mob1 (CNAG_05541). In DNA
replication, the 6 upregulated DEGs included Lig1 (CNAG_04278), DNA polymerase
α2 (CNAG_06142), PCNA and MCM (Figure 10, Table 7). These genes are closely
related to cell division, the regulation of mitotic metaphase/anaphase transitions and
DNA replication.
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Figure 10. C. neoformans H99 cells were treated with compound A4 at 2 μg/mL or
DMSO for 24 h, and RNA–seq analysis was performed. (A) Significantly enriched
pathway in terms of their differentially expressed genes based on KEGG analysis. (B)
Heatmaps showing selected gene panels involved in pathways related to those
described in (A); red, induced; blue, suppressed; log (RPKM)-based scale.
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Table 7. Signaling pathway enrichment analysis of differentially expressed genes.
KEGG Pathway
Count
P-Value
Genes
CNAG_00320 CNAG_01907 CNAG_02963
CNAG_03341 CNAG_05825 CNAG_06079
CNAG_06182 CNAG_06183 CNAG_06697
CNAG_00320 CNAG_00681 CNAG_02829
CNAG_02963 CNAG_03341 CNAG_05541
CNAG_05825 CNAG_06182 CNAG_06183
Cell cycle
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3.6 × 10-6
Cell cycle - yeast
10
1.7 × 10-6
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CNAG_06697
CNAG_00320 CNAG_02963 CNAG_03341
CNAG_05825 CNAG_06182 CNAG_06183
CNAG_03341 CNAG_04278 CNAG_05825
CNAG_06079 CNAG_06142 CNAG_06182
CNAG_02855 CNAG_04364 CNAG_04743
CNAG_06901
Meiosis - yeast
DNA replication
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2.4 × 10-4
N-Glycan biosynthesis
2.9 × 10-2
2.7 × 10-4
3.4 × 10-3
CNAG_00450 CNAG_02812 CNAG_05398
CNAG_05725 CNAG_05899 CNAG_06421
CNAG_06849 CNAG_07529
Biosynthesis of
amino acids
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Valine, leucine and
CNAG_00450 CNAG_05725 CNAG_06421
CNAG_07529
isoleucine biosynthesis
CONCLUSION
The discovery of novel lead compounds for the treatment of CM remains a significant
challenge. Herein, a series of new β-tetrahydrocarboline derivatives were shown to
possess potent anticryptococcal activities. In particular, compound A4 showed several
advantages as a promising lead compound for the development of next-generation
CM therapeutic agents. First, it showed potent in vitro and in vivo anticryptococcal
activities with good metabolic stability and BBB permeability. Compound A4
demonstrated potent fungistatic activity against C. neoformans and significantly
reduced the brain fungal burdens in a murine model of CM following oral
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administration. Second, compound A4 inhibited several virulence factors of C.
neoformans, such as biofilm formation and the production of melanin, whereas FLC
was completely inactive. Finally, compound A4 may act by a new mode of action.
Preliminary mechanistic studies revealed that compound A4 induced DSBs and cell
cycle arrest at G2 phase by acting on the Cdc25c/CDK1/cyclin B pathway. Cell wall
and cell membrane collapse, chromosome condensation and nuclear pyknosis were
observed by TEM after treating C. neoformans cells with compound A4. Further
structural optimization, mechanism and target verification studies are in progress.
EXPERIMENTAL SECTION
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Chemistry. General methods. H NMR and 13C NMR spectra were recorded on
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Bruker AVANCE300, AVANCE500 or AVANCE600 spectrometers (Bruker
Company, Germany) using TMS as an internal standard and CDCl or DMSO-d6 as
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solvents. Chemical shifts and coupling constants are given in ppm (δ values) and Hz
(J values). Elemental analyses were performed with a MOD-1106 instrument and
were consistent with theoretical values within 0.4%. The mass spectra were recorded
on an Esquire 3000 LC-MS mass spectrometer. Silica gel thin-layer chromatography
separations were performed on precoated plates GF-254 (Qingdao Haiyang Chemical,
China). All solvents and reagents were analytically pure, and no further purification
steps were needed. All starting materials were commercially available. The purities of
the compounds were determined by HPLC (Agilent 1260) using MeOH as the mobile
phase at a flow rate of 0.6 mL/min on a C18 column, and all final compounds
exhibited purities greater than 98%.
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