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

Article abstract of DOI:10.1021/acs.jmedchem.8b00393

Clinically, leukemia patients often suffer from the limited efficacy of chemotherapy and high risks of infection by invasive fungal pathogens. Herein, a novel therapeutic strategy was developed in which a small molecule can simultaneously treat leukemia and invasive fungal infections (IFIs). Novel Janus kinase 2 (JAK2) and histone deacetylase (HDAC) dual inhibitors were identified to possess potent anti-proliferative activity toward hematological cell lines and excellent synergistic effects with fluconazole to treat resistant Candida albicans infections. In particular, compound 20a, a highly active and selective JAK2/HDAC6 dual inhibitor, showed excellent in vivo antitumor efficacy in several acute myeloid leukemia (AML) models and synergized with fluconazole for the treatment of resistant C. albicans infections. This study highlights the therapeutic potential of JAK2/HDAC dual inhibitors in treating AML and IFIs and provides an efficient strategy for multitargeting drug discovery.

Full text of DOI:10.1021/acs.jmedchem.8b00393

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Article
Discovery of Janus Kinase 2 (JAK2) and Histone Deacetylase
HDAC) Dual Inhibitors as a Novel Strategy for Combinational
Treatment of Leukemia and Invasive Fungal Infections
(
Yahui Huang, Guoqiang Dong, Huanqiu Li, Na Liu, Wannian Zhang, and Chunquan Sheng
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.8b00393 • Publication Date (Web): 25 Jun 2018
Downloaded from http://pubs.acs.org on June 25, 2018
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Discovery of Janus Kinase 2 (JAK2) and Histone Deacetylase (HDAC)
Dual Inhibitors as a Novel Strategy for Combinational Treatment of
Leukemia and Invasive Fungal Infections
1
,†
1,†
2,†
1
1
Yahui Huang , Guoqiang Dong , Huanqiu Li , Na Liu , Wannian Zhang ,
1
,*
Chunquan Sheng
1
School of Pharmacy, Second Military Medical University, 325 Guohe Road,
Shanghai 200433, People’s Republic of China
2
College of Pharmaceutical Science, Soochow University, Suzhou 215123, PR China
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ABSTRACT
Clinically, leukemia patients often suffer from limited efficacy of chemotherapy and
high risks to be infected by invasive fungal pathogens. Herein a novel therapeutic
strategy was developed that a small molecule can simultaneously treat leukemia and
invasive fungal infections (IFIs). Novel Janus kinase 2 (JAK2) and histone
deacetylase (HDAC) dual inhibitors were identified to possess potent antiproliferative
activity toward hematological cell lines and excellent synergistic effects with
flcuonazole to treat resistant Candida albicans infections. In particular, compound
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20a, a highly active and selective JAK2/HDAC6 dual inhibitor, showed excellent in
vivo antitumor efficacy in several acute myeloid leukemia (AML) models, and
synergized with fluconazole for the treatment of resistant Candida albicans infections.
This study highlights the therapeutic potential of JAK2/HDAC dual inhibitors in
treating AML and IFIs and provides an efficient strategy for multiꢀtargeting drug
discovery.
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INTRODUCTION
Invasive fungal infections (IFIs) are lifeꢀthreatening and often occur in
immunocompromised hosts. Leukemia patients undergoing chemotherapy,
hematopoietic stem cell transplantation (HSCT) or immunosuppressive therapy are
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highly vulnerable to IFIs. According to NCCN guidelines (national comprehensive
cancer network clinical practice guidelines in oncology), primary antifungal
prophylaxis (azoles, amphotericin B or echinocandins) should be used in highꢀrisk
patients, especially those with acute myeloid leukemia (AML) and graft versus host
disease (GVHD). However, severe drug resistance has been observed for antifungal
agents. In recipients of HSCT, the mortality associated with IFIs still remains high.
2
Tyrosine kinase inhibitors have been widely used in the targeted therapy of leukemia.
Despite the success, clinically available therapeutics generally suffered from limited
efficacy and drug resistance. Thus, the development new generation of kinase
inhibitors for targeted therapy remains an active research area. Especially, a kinase
inhibitor that can synergize with antifungal agents and improve their potency to treat
resistant IFIs is highly desirable. However, the discovery of such a diꢀfunctional
molecule is challenging.
The Janus kinases (JAK1, JAK2, JAK3, and TYK2) belong to the family of
intracellular protein tyrosine kinases, which play essential roles in the signaling of a
3
variety of cytokines. Activation of JAKs by different cytokines results in
phosphorylation and dimerization of the STAT (signal transducers and activators of
transcription) proteins, which further translocate to the nucleus and activate gene
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transcription. The JAK/STAT signaling axis is associated with diverse biological
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, 6
functions including inflammation, immune function and hematopoiesis.
JAK
inhibitors have been extensively investigated as new therapeutics for the treatment of
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immuneꢀinflammatory diseases (e.g. rheumatoid arthritis, RA) and cancer (e.g.
6ꢀ8
leukemia, lymphoma and solid tumor). Currently, a number of panꢀJAK and
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selective JAK inhibitors have been discovered. PanꢀJAK inhibitor tofacitinib was
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approved for the treatment of RA, and JAK1/2 inhibitor ruxolitinib (Figure 1) was
marketed to treat myelofibrosis and polycythemia vera. Among the four JAK subtypes,
JAK2 was proven to be critical for the growth and progression tumor. JAK2 inhibitors
have been evaluated in clinical trials for the treatment of a wide spectrum of
11
hematological malignancies as well as solid tumors. However, resistance of JAK2
12, 13
inhibitors has been observed.
To improve the therapeutic effects and reduce the
1
4ꢀ16
risk of resistance, combinational drug therapy
or development of JAK2ꢀbased
17, 18
multiꢀtargeting antitumor agents
offered new opportunities.
As a family of epigenetic enzymes, histone deacetylases (HDACs) remove acetyl
groups from lysine on histones and other proteins, which play an essential role on
various cellular functions including gene regulation, transcription, and cell
proliferation, differentiation and death. Overexpression of HDACs are observed in
different human cancers, and thus they are regarded as promising antitumor drug
19
targets. Up to date, four HDAC inhibitors, namely vorinostat (SAHA, 2),
romidepsin, belinostat and panobinostat, have been approved for the treatment of
20ꢀ23
hematologic cancer.
Similar to JAK2, HDACꢀbased multiꢀtargeting antitumor
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agents have achieved great success in achieving higher efficacy, broader antitumor
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spectrum and better safety profiles.
Clinically, specific drug combinations have been widely used in cancer therapy.
However, drug cocktails often suffer from complicated dose (or schedule) design as
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well as poor patient compliance. In contrast, a single multiꢀtargeting molecule has
obvious advantages in terms of synergistic antitumor effects, more predictable and
favorable pharmacokinetic (PK) profiles, reduced compliance difficulties and
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drugꢀdrug interactions. Recent evidence suggested that the design of dual
JAK2/HDAC inhibitors could be a promising strategy for the treatment of leukemia,
2
7
solid tumor and other related indications. Both JAK2 and HDAC inhibitors
demonstrated similar clinical efficacy, which have synergistic effects and can be used
27
in combination therapy. Dymock’s group further confirmed that dual JAKꢀHDAC
28, 29
pathway inhibition achieved with a single molecule.
Despite these advancements,
the therapeutic value of dual JAKꢀHADC inhibitors remains to be further explored.
Particularly, in vivo potency has not been achieved by the reported dual JAK2ꢀHDAC
28, 29
inhibitors due to unfavorable PK properties.
Herein in vivo therapeutic values of JAK2ꢀHADC dual inhibitors were confirmed
for the first time. Novel JAK2ꢀHADC dual inhibitors were identified, which showed
excellent potency in combinational treatment of AML and resistant Candida albicans
infections, providing a new strategy for clinical therapy of leukemia along with IFIs.
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Figure 1. (A) Chemical structures and pharmacophore of JAK2 inhibitors; (B)
Chemical structure and pharmacophore of HDAC inhibitors; (C) Design of
JAK2ꢀHDAC dual inhibitors.
RESULTS AND DISCUSSION
Structure-based Design of JAK2/HDAC Dual Inhibitors. Inspired by our previous
30ꢀ32
efforts in multiꢀtargeting drug design,
a series of novel JAK2ꢀHADC inhibitors
27
were designed according to synergistic effect between the two targets . Generally, the
pharmacophore of JAK2 inhibitors consists of a hinge region binder (typically an
aminopyrimidine), two hydrophobic groups (phenyl group) and a solvent exposed part
33
(
Figure 1A), which serves as a template in designing dual inhibitors. Since the
aminopyrimidine of JAK2 inhibitor CYTꢀ387 (1) has been proven to be responsible
for the JAK2 inhibitory activity, the motif was maintained in designing the dual
inhibitors. HDAC inhibitors (Figure 1B) generally consist of a hydrophobic cap
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group, a linker (alkyl, alkenyl or aryl) and a zincꢀbinding group (ZBG, hydroxamic
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acid or benzamide). Given that the cap group is flexible and can be replaced by
other moieties to generate HDAC inhibitors, the first series of dual inhibitors (8, 10
and 12a-b) were designed by merging the core group of 1 with the ZBG motif
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(hydroxamic acid or benzamide) through various linkers (Figure 1C). Moreover, on
the basis of the fact that Nꢀphenylmethanesulfonamide derivatives of compound 1
35
exhibited similar JAK2 inhibitory, the second series of JAK2ꢀHDAC inhibitors
(compounds
16,
18
and
20a-h)
were
designed
with
the
Nꢀcyanomethylbenzamide/Nꢀphenylmethanesulfonamide
replacement.
Finally,
considering that a minor substitution in the hydrophobic domain of JAK2 inhibitor 1
would be tolerated, hydroxamic acid or benzamide was introduced at this position to
achieve dual inhibition toward both targets (compounds 24 and 26).
Chemistry. Chemical synthesis of JAK2ꢀHDAC dual inhibitors are depicted in
Schemes 1-3. Starting from 2,4ꢀdichloropyrimidine (3), intermediate 7 was prepared
via regioselective Suzuki reaction and aniline displacement reaction according to the
35
reported methods (Scheme 1). Treatment of compound 7 with freshly prepared
hydroxylamine methanol solution gave target compound 8 or with LiOH yielded acid
9
. Condensation of 9 with oꢀphenylenediamine afforded target compound 10, or with
different aminoꢀesters yielded esters 11a-b, which were subsequently reacted with
hydroxylamine methanol solution to give target compounds 12a-b. Using a similar
protocol, Nꢀphenylmethanesulfonamide analogues (16, 18 and 20a-h) were obtained
according to the procedures outlined in Scheme 2. As shown in Scheme 3, target
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compounds 24 and 26 were prepared using the conditions similar to those for
compound 8 and 10, respectively.
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Reagents and conditions: (a) Pd(PPh
3
)
4
, toluene/nꢀPrOH, 2 M aq Na
2
CO
3
, 100 C,
o
55%; (b) 6, TsOH·H
2
O, 120 C, 3 days, 82%; (c) NH
2
OK, anhydrous CH
3
OH, 4 h,
56%; (d) LiOH, THF/H
2
O, reflux, 4 h, 85%; (e) oꢀphenylenediamine, HATU, DIPEA,
DMF, rt, 4 h, 63%; (f) various aminoꢀesters, HATU, DIPEA, DMF, rt, 4 h, 58%.
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Reagents and conditions: (a) Pd(PPh
3
)
4
, toluene/nꢀPrOH, 2 M aq Na , 100 C,
2
CO
3
o
51%; (b) 6, TsOH·H
2
O, 120 C, 3 days, 77%; (c) NH
2
OK, anhydrous CH OH, 4 h,
3
48%; (d) LiOH, THF/H O, reflux, 4 h, 82%; (e) oꢀphenylenediamine, HATU, DIPEA,
2
DMF, rt, 4 h, 56%; (f) various aminoꢀesters, HATU, DIPEA, DMF, rt, 4 h, 57%.
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3
)
4
, toluene/nꢀPrOH, 2 M aq Na
2
CO
3
, 100 C,
OK,
O, reflux, 4 h, 86%; (e)
o
48%; (b) 4ꢀmorpholinoaniline, TsOH·H
2
O, 120 C, 3 days, 73%; (c) NH
2
anhydrous CH
3
OH, 4 h, 62%; (d) LiOH, THF/H
2
oꢀphenylenediamine, HATU, DIPEA, DMF, rt, 4 h, 51%.
Enzyme Inhibitory Activities and in vitro Antitumor Potency of JAK2-HDAC
Dual Inhibitors. Initially, the JAKꢀHDAC dual inhibitors were assayed against JAK2
and HDAC1, using compounds 1 and 2 as reference drugs. As shown in Table 1, the
first series of dual inhibitors (compounds 8, 10 and 12a-b) exhibited excellent
inhibitory activity against JAK2 (IC50 range: 4ꢀ16 nM), which were better than
compound 1 (IC50 = 41 nM). However, their activity against HDAC1 was moderate
(IC50 range: 1.7ꢀ23.6 ꢁM). Then, their inhibitory activities against three human
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leukemia cellꢀlines HL60, K562 and HEL were evaluated using the CCKꢀ8 assay.
However, only compound 10 demonstrated moderate inhibitory activity against HEL
cell line (IC50 = 6.6 ꢁM).
1
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Table 1. Enzyme inhibition and in vitro antitumor activity of target compounds 8, 10
and 12a-b
IC (µM)
5
0
Compd.
R
JAK2
HDAC1
HL60
K562
HEL
8
0.004 ± 0.0006
2.7 ± 0.25
> 50
> 50
> 50
10
0.008 ± 0.0007
0.015 ± 0.008
1.7 ± 0.14
5.1 ± 0.37
> 50
>50
6.6 ± 0.65
> 50
12a
> 50
> 50
> 50
12b
0.016 ± 0.009
0.041 ± 0.005
24 ± 2.1
> 50
> 50
a
1
2
NT
8.3 ± 0.79
1.5 ± 0.13
6.5 ± 0.58
7.7 ± 0.64
2.3 ± 0.14
a
NT
0.04 ± 0.009
0.86 ± 0.069
1
+ 2
a
a
NT
NT
1.24 ± 0.17
1.81 ± 0.22
1.51 ± 0.26
(1:1)
a
NT = not tested.
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1
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1
1
2
2
2
2
2
2
2
2
2
2
3
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3
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5
6
Next, the nitrile carboxamide group of the first series of compounds was replaced
by Nꢀphenylmethanesulfonamide. As a result, compounds 16, 18 and 20a-c generally
displayed improved HDAC1 inhibitory activity (IC50 range: 0.25ꢀ2.9 ꢁM), while
excellent JAK2 inhibitory activity was retained. More importantly, their antitumor
activities were also increased. For the ZBG, hydroxamic acid derivative 20a showed
better antitumor activity than benzamide derivative 18. Thus, more derivatives
containing various alkyl linkers were investigated (compounds 20d-h). Enzyme
assays showed that their JAK2 and HDAC1 inhibitory activities were further
improved (JAK2 IC range: 1ꢀ3 nM; HDAC1 IC range: 0.018ꢀ9 ꢁM). SAR studies
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
0
50
indicated that the HDAC1 inhibitory activity increased along with the elongation of
the linker (20h > 20g > 20f > 20e > 20d). Among the second series of dual inhibitors,
compound 20a exhibited the best inhibitory activity against HEL cells (IC50 = 0.34
ꢁM), which was more potent than JAK2 inhibitor 1 (IC50 = 2.3 ꢁM) and HDAC
inhibitor 2 (IC = 0.86 ꢁM), although its JAK2 and HDAC inhibition activities were
5
0
less potent than the two inhibitors. The combination of 1 and 2 were also evaluated to
investigate the synergic effect between JAK and HDAC inhibitors. Compounds 1 and
2
(1:1) showed improved antitumor potency than 1 and 2 dosed alone against HL60
and K562 cells. Thus, JAK and HDAC inhibitors showed synergic effect in cancer
cells. Moreover, compound 20a showed better HEL inhibitory activities than the
combination of 1 and 2 (IC50 = 1.51 ꢁM). These results indicated that the cytotoxicity
might be the consequence of the synergism of the JAK2 and HDAC inhibition.
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Table 2. Enzyme inhibition and in vitro antitumor activity of target compounds 16, 18
and 20a-h
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
Compd
.
IC50 (µM)
R
JAK2
HDAC1
HL60
K562
HEL
16
0.001 ± 0.0003
2.9 ± 0.15
> 50
18 ± 1.8
> 50
18
0.002 ± 0.0004
0.008 ± 0.0007
2.1 ± 0.12
16 ± 2.3
9.5 ± 0.86
8.7 ± 0.71
3.2 ± 0.25
20a
0.25 ± 0.025
1.5 ± 0.12
0.34 ± 0.025
20b
0.002 ± 0.0003
0.38 ± 0.06
11 ± 1.2
8.6 ± 0.72
2.0 ± 0.16
20c
0.018 ± 0.0007
0.001 ± 0.0004
0.001 ±0.0002
0.003 ± 0.0006
1.1 ± 0.16
9.2 ± 0.78
2.2 ± 0.18
0.35 ± 0.026
> 50
> 50
10 ± 2.2
12 ± 2.1
11 ± 1.8
10 ± 1.3
6.3 ± 0.58
12 ± 2.6
20d
20e
> 50
8.1 ± 0.76
17 ± 1.6
20f
37 ± 3.5
20g
0.002 ± 0.0004
0.002 ± 0.0003
0.25 ± 0.021
0.018 ± 0.006
12 ± 1.3
> 50
8.9 ± 0.78
5.9 ± 0.48
3.9 ± 0.25
1.8 ± 0.12
20h
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1
1
2
2
2
2
2
2
2
2
2
2
3
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6
a
1
ꢀ
ꢀ
0.041 ± 0.005
NT
8.3 ± 0.79
1.5 ± 0.13
6.5 ± 0.58
7.7 ± 0.64
2.3 ± 0.14
a
2
NT
0.04 ± 0.009
0.86 ± 0.069
1
+ 2
a
a
NT
NT
1.24 ± 0.17
1.81 ± 0.22
1.51 ± 0.26
(
1:1)
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
a
NT = not tested.
Finally, ZBGs (hydroxamic acid or benzamide) were used to replace the
Nꢀcyanomethylbenzamide group in JAK2 inhibitor 1 (compounds 24 and 26). As
listed in Table 3, the ZBGs were important for the inhibition of enzyme and leukemia
cell lines. Compound 24 containing hydroxamic acid as the ZBG displayed good
inhibitory activity toward HDAC1 (IC = 0.16 ꢁM) and JAK2 (IC = 0.085 ꢁM),
50
50
and the antitumor activities were comparable to lead compounds 1 and 2. In contrast,
benzamide derivative 26 only exhibited moderate JAK2 inhibitory activity (IC50
.11 ꢁM), and was totally inactive in inhibiting HDAC1 and cell proliferation.
=
0
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Table 3. Enzyme inhibition and in vitro antitumor activity of the target compounds 24
and 26
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
IC50 (µM)
Compd.
R
JAK2
HDAC1
HL60
K562
HEL
2
2
4
6
0.085 ± 0.006
0.16 ± 0.03
6.1 ± 0.36
4.0 ± 0.32
3.5 ± 0.26
0.11 ± 0.02
> 50
> 50
> 50
> 50
a
1
ꢀ
ꢀ
0.041 ± 0.005
NT
8.3 ± 0.79
1.5 ± 0.13
6.5 ± 0.58
7.7 ± 0.64
2.3 ± 0.14
0.86 ± 0.069
a
2
NT
0.04 ±0.009
1 + 2
a
a
NT
NT
1.24 ± 0.17
1.81 ± 0.22
1.51 ± 0.26
(
1:1)
a
NT = not tested.
JAK2/HDAC1 Dual Inhibitors Showed Excellent Synergistic Effect with FLC
against Resistant C. albicans Isolates. HDACs are important regulators of heat
shock protein 90 (Hsp90), which is essential for fungal survival and is associated with
36
antifungal drug resistance. HDAC inhibitors were reported to enhance the potency
of antifungal agents (e.g. azoles and echinocandins) against various fungal pathogens
37ꢀ39
including resistant isolates.
Thus, the antifungal activity and synergistic effects of
JAK2/HDAC dual inhibitors were evaluated against FLCꢀresistant C. albicans
isolates 0304103, 100 and 071922. The antifungal activity was expressed as the
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2
2
2
2
2
2
2
2
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5
5
6
minimum inhibitory concentration that achieves 50% of inhibition (MIC50) using the
checkerboard microdilution assay according to the methods of the CLSI (formerly
40
NCCLS)(M27ꢀA). The synergistic effects were evaluated by the fractional inhibitory
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
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5
6
7
8
9
0
1
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7
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9
0
1
2
3
4
5
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7
8
9
0
41
concentration (FIC) index and the synergism was defined by FIC index of ≤ 0.5. As
shown in Table 4, most of the target compounds were inactive to three FLCꢀresistant
C. albicans isolates when used alone (MIC50 > 64 ꢁg/mL). Only compounds 20d-f
showed moderate antifungal activity against FLCꢀresistant C. albicans 0304103
(MIC50 = 32 ꢁg/mL). When used in combination with FLC, a majority of compounds
(8, 12a, 16, 20a, 20c-h and 24) exhibited excellent synergistic effects against
FLCꢀresistant C. albicans strain 0304103 with the FIC index ranging from 0.078 to
0.50. SAR studies indicated that compounds containing the benzamide ZBG (e.g.
compounds 10, 18 and 26) were devoid of the synergistic antifungal effects. HDAC
inhibitor 2 and the combination of 1 and 2 also showed synergistic effects against C.
albicans 0304103 strain, whereas the synergism was lost for strains 100 and 0710922.
In contrast, obvious synergistic effect (FIC range: 0.039 to 0.5) was retained for dual
inhibitors 8, 16, 20a, 20c-e, 20g-h and 24 against these resistant strains. Compounds
12a, 20a, 20g-h and 24 exhibited good synergism (FIC index range: 0.14 to 0.5).
Considering the potent antitumor activity and promising antifungal profile of
compounds 20a, 20h and 24, they were subjected for further evaluations.
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Table 4. Antifungal activity and synergistic effects of target compounds with FLC against resistant C. albicans isolates by the microdilution
assay
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
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
0
304103
100
(MIC50, µg/mL)
used in
0710922
(MIC50, µg/mL)
used in
Strains
(
MIC50, µg/mL)
used in
used alone
FIC
used alone
FIC
used alone
FIC
combination
combination
combination
a
Index
Index
Index
FLC Cmpd. FLC Cmpd.
FLC Cmpd. FLC Cmpd.
FLC Cmpd. FLC Cmpd.
8
> 64
> 64
> 64
> 64
> 64
> 64
> 64
> 64
> 64
> 64
> 64
> 64
> 64
> 64
> 64
> 64
> 64
> 64
> 64
32
0.25
> 64
1
8
> 64
4
0.13
> 2
> 64
> 64
> 64
> 64
> 64
> 64
> 64
> 64
> 64
> 64
> 64
> 64
> 64
> 64
> 64
> 64
> 64
> 64
> 64
> 64
0.5
> 64
1
2
> 64
64
64
8
0.039
> 2
> 64
> 64
> 64
> 64
> 64
> 64
> 64
> 64
> 64
> 64
> 64
> 64
> 64
> 64
> 64
> 64
> 64
> 64
> 64
> 64
1
64
64
1.02
2
10
64
12a
0.078
1.01
0.07
> 2
1.02
1.03
0.14
2
2
16
0.31
> 2
1
2b
0.5
0.5
> 64
0.5
> 64
2
64
4
2
> 64
0.25
> 64
1
> 64
32
16
1
0.51
> 2
18
> 64
4
64
0.5
16
1
64
4
> 64
8
20a
0.07
> 2
0.07
1.25
0.078
0.14
0.14
> 2
2
0b
0c
0d
> 64
8
64
4
> 64
0.5
0.5
> 64
64
2
0.15
0.31
1.01
0.51
2
4
8
1
8
32
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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
20e
0f
0g
0h
> 64
> 64
> 64
> 64
> 64
> 64
> 64
> 64
32
0.5
0.5
16
16
8
0.50
0.50
0.13
0.13
0.07
> 2
> 64
> 64
> 64
> 64
> 64
> 64
> 64
> 64
> 64
32
0.5
0.25
0.25
0.5
16
16
8
0.26
0.50
0.13
0.13
0.13
1.01
> 2
> 64
> 64
> 64
> 64
> 64
> 64
> 64
> 64
> 64
> 64
> 64
> 64
> 64
> 64
> 64
> 64
0.5
64
32
64
0.51
2
2
32
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
2
> 64
> 64
> 64
> 64
> 64
> 64
0.5
> 64
> 64
> 64
> 64
> 64
> 64
0.25
0.25
1
32
0.50
0.26
0.27
> 2
2
2
0.25
0.5
8
8
16
24
4
0.5
8
16
26
1
> 64
> 64
0.25
> 64
> 64
16
0.5
64
> 64
32
> 64
64
> 64
64
> 2
> 64
0.5
2
0.25
0.51
1
64
1.02
1
+ 2
>
64
> 64
0.5
16
0.26
> 64
> 64
1
64
1.01
> 64
> 64
4
64
1.06
(1:1)
a
FIC index is defined as the sum of MIC of each drug used in combination divided by the MIC of the drug used alone. Synergy and antagonism were defined by FIC
index of ≤ 0.5 and > 4, respectively. An FIC index between 0.5 and 4 was considered indifferent.
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Binding Mode of the JAK2-HDAC Dual Inhibitors. To investigate the binding
mode of compounds 20a, 20h and 24 with JAK2 and HDAC1, molecular docking
42
studies were performed. Compound 20a bound with HDAC1 (PDB ID: 4BKX )
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
mainly through the linker and ZBG (Figure 2A). The hydroxamic acid of 20a
2+
coordinated to the catalytic Zn of HDAC1 and formed two hydrogen bonds with
Gly146 and His178, respectively. The hydrophobic cinnamamide group formed πꢀπ
stacking interactions with His178, Phe150 and Phe205. Moreover, the
Nꢀphenylmethanesulfonamide moiety formed three additional hydrogen bonds with
Glu146, Lys143 and Ser148. Similarly, the ZBG of compound 20h was engaged with
a hydrogen boding network with Gly138, Asp176 and Tyr303 (Figure 2B). The
Nꢀphenylmethanesulfonamide group interacted with Glu146 through a hydrogen bond.
For compound 24, the hydroxamic acid moiety formed coordination interaction and a
2
+
hydrogen bond with Zn and Gly149, respectively (Figure 2C). In addition, its
aromatic linker and phenyl group in the cap formed πꢀπ stacking interactions with
Phe205. In contrast, the terminal Nꢀ(2ꢀaminophenyl)benzamide of compound 26
forced the whole molecule located at the outside of the active site, which might be the
reason why 26 was totally inactive toward HDAC1 (Figure S1 in Supporting
Information).
4
3
In JAK2 (PDB ID: 4AQC ), the aminopyrimidine scaffold of compound 20a
formed hydrogen bonds to the backbone of Leu932 in the hinge region (Figure 2D).
Its Nꢀphenylmethanesulfonamide group formed an additional hydrogen bond with
Asp994. Moreover, the linker and ZBG extended out of the binding site and formed
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9
1
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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
hydrogen bonds with Asp939 and Arg980, respectively. As shown in Figure 3E-F, the
binding modes of compounds 20h and 24 with JAK2 were similar to that of 20a
where hydrogen bonds between animopyrimidine and the backbone of Leu932 were
observed. The Nꢀphenylmethanesulfonamide and hydroxamic acid moiety of
compound 20h formed hydrogen bonds with Asp994 and Lys857, respectively.
Moreover, an additional hydrogen bond was found between the hydroxamic acid
group of compound 24 and Asn981.
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. Proposed binding mode of compounds 20a, 20h and 24 in the active site of
HDAC1 (PDB ID: 4BKX, A-C) and in the ATPꢀbinding site of JAK2 (PDB ID:
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AQC, D-F). The carbons of 20a, 20h and 24 are colored in yellow, pink and orange,
respectively. Oxygen atoms are colored in red and nitrogen atoms in blue. Hydrogen
bonds are indicated with dashed lines. The figure was generated using PyMol
(http://www.pymol.org/).
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
In vitro JAK and HDAC Isoform Selectivity of Compounds 20a, 20h and 24. To
obtain evidence for the JAK and HDAC isoform selectivity, compounds 20a, 20h and
24 were evaluated against four JAK isoforms (JAK1, JAK2, JAK3, TYK2) and five
HDAC isoforms (HDAC1, HDAC2, HDAC3, HDAC6 and HDAC8). As depicted in
Table 5, three compounds exhibited remarkable selectivity for JAK2 over other
isoforms. For HDACs, surprisingly, three compounds displayed different selective
profiles (Table 6, Table S1 and Table S2 in Supporting Information). Compound
20a showed good selectivity for HDAC6 (IC = 46 nM), whereas compound 24 was
5
0
observed to be a HDAC3 selective inhibitor (IC50 = 45 nM). In contrast, compound
20h showed similar inhibitory activity for HDAC1 and HDAC3 with an IC value of
50
18 nM and 17 nM, respectively.
Table 5. JAK isoform profiling of compounds 20a, 20h and 24
IC50 (nM)
20a
20h
24
JAK1
JAK2
JAK3
TYK2
359 ± 22
8.4 ± 0.7
121 ± 13
46 ± 1.7
76 ± 3.5
2.2 ± 0.3
144 ± 14
74 ± 6.4
69 ± 4.1
85 ± 6.0
517 ± 27
419 ± 71
Table 6. HDAC isoform profiling of compounds 20a, 20h and 24
IC (nM)
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HDAC1
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1100 ± 140
7472 ± 856
234 ± 31
18 ± 6.0
132 ± 28
17 ± 1.2
74 ± 6.4
572 ± 49
160 ± 32
1704 ± 115
45 ± 3.3
419 ± 71
609 ± 85
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46 ± 1.7
6065 ± 529
Due to HDAC selective profile of compounds 20a and 24, selective HDAC6
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inhibitors Tubastatin A (27) and ACY1215 (28) and selective HDAC3 inhibitor
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6
RGFP966 (29) were further assayed (chemical structures see Figure S4 in
Supporting Information). As listed in Table S3, HDAC6 inhibitors 27 and 28
exhibited moderate inhibitory activities against HL60, K562 and HEL cell lines (IC50
range: 3.75ꢀ23.88 ꢁM), which were less active than compound 20a. For HDAC3
inhibitor 29, it only showed marginal activity (IC50 range: 21.71ꢀ33.58 ꢁM). The
combinations of JAK2 inhibitor 1 with three selected HDAC inhibitors were also
investigated for antiproliferative activities. When compound 1 was used in
combination with HDAC6 inhibitor 27 or 28, the inhibitory activities were not
significantly changed (IC50 range: 2.54ꢀ8.55 ꢁM), indicating that there was no
synergistic effect between them. In contrast, when compound 1 was used in
combination with HDAC3 inhibitor 29, improved antitumor activity was observed
(IC50 range: 1.64ꢀ4.19 ꢁM). For the antifungal activity, compounds 27ꢀ29 were
inactive to three FLCꢀresistant C. albicans isolates (MIC50 > 64 ꢁg/mL). When used
in combination, only compound 28 showed synergistic effects with FLC against C.
albicans 0304103 strain (Table S4 in Supporting Information).
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Compounds 20a, 20h and 24 Induced Apoptosis and Cell Cycle Arrest. To
investigate the effect of the selected dual inhibitors on the induction of apoptosis,
compounds 20a, 20h and 24 were evaluated by annexin VFTIC/propidium iodide (PI)
assay. HEL cells were incubated with vehicle alone or tested compounds at 5 ꢁM and
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10 ꢁM for 48 h. As shown in Figure 3, compounds 20a, 20h and 24 caused a
doseꢀdependent induction of apoptosis in HEL cells. The percentages of apoptotic
cells for compounds 20a, 20h and 24 at the concentration of 10 ꢁM were 70.62%,
89.2% and 86.2%, respectively, which were significantly higher than JAK2 inhibitor 1
(25.9% apoptotic cells at 10 ꢁM, P < 0.001).
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Figure 3. Cell apoptosis induced by compounds 20a, 20h and 24. HEL cells were
incubated with the indicated concentrations of 1, 20a, 20h and 24 for 48 h. Cells
treated with DMSO were used for comparison. Data were represented as mean ±
standard deviation from three independent experiments. * P < 0.05, ** P < 0.01, ***
P < 0.001, determined with Student’s t test.
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To probe the effect of compounds 20a, 20h and 24 on various phases of cell cycle
progression, flow cytometry method was performed. HEL cells were treated with
vehicle alone or compound 20a, 20h and 24 at different concentrations (Figure 4).
After treating with compound 20a at 1 ꢁM and 5 ꢁM for 48 h, the ratios in G /M
2
phase of cell cycle were 11.75% and 28.14%, respectively. While the ratios of cells
untreated in G
Interestingly, the ratios of cells incubated with compound 20h at 1 ꢁM and 5 ꢁM in
/M phase of cell cycle were not dramatically changed (8.23% and 8.12%,
2
/M phase of the cell cycle were 9.32% and 9.31%, respectively.
G
2
respectively), but the ratios in S phase of cell cycle were 25.56% and 34.37%,
respectively. Similarly, the ratios of cells treated with compound 24 at 5 ꢁM and 10
ꢁM in the S phase of the cell cycle were 18.90% and 48.68%, respectively. Taken
together, compounds 20a arrested HEL cells mainly at the G /M phase (P < 0.001),
2
and compounds 20h and 24 could significantly arrest HEL cells at the S phase (P <
0.001).
47
Compound 2 arrested HEL cells mainly in G
induced cell cycle arrest at the G
inhibitors (27 and 28) and HDAC3 selective inhibitor 29 were further evaluated on
1
phase, while JAK2 inhibitors
48
2
/M phase in HEL cells. HDAC6 selective
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the various phases of cell cycle progression (Figure S5 in Supporting Information).
Surprisingly, all the three compounds arrested HEL cells at G
which was different with compounds 20a, 20h and 24. These data suggest that
compound 20a arrested HEL cells at G /M phase possibly because of the inhibition of
1
phase (P < 0.05),
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JAK2. The underlying mechanisms why compounds 20h and 24 arrested HEL cells at
S phase remained to be further investigated.
Figure 4. Cell cycle analysis after 48 h of treatment. HEL cells treated with 20a, 20h
and 24 at different concentrations for 48 h were assayed by flow cytometry after
staining with PI. Data were represented as mean ± standard deviation from three
independent experiments. * P < 0.05, ** P < 0.01, *** P < 0.001, determined with
Student’s t test.
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Compounds 20a, 20h and 24 Inhibited JAK and HDAC in HEL Cells. To
determine whether the three selected compounds inhibited both the JAKꢀSTAT and
HDAC pathway in cells, western blot assay was performed to evaluate the effect of
compounds 20a, 20h and 24 on the acetylation level of histone 3, histone 4 and
phosphorylation of STAT5. HEL cells were exposed to them at 0.2 ꢁM, 1 ꢁM and 5
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ꢁM for 24 h. As shown in Figure 5, all the three compounds caused a dramatic
increase in acetylꢀhistone 3 and acetylꢀhistone 4 in a dose dependent manner. On the
other hand, exposure of HEL cells to compounds 20a, 20h and 24 resulted in
doseꢀdependent decreases in STAT5 phosphorylation. Taken together, the results
clearly confirmed that the designed JAK2/HDAC inhibitors inhibited both targets in
HEL cells.
Figure 5. Effects of compounds 20a, 20h and 24 on the levels of acetylꢀH3, acetylꢀH4
and STAT5 phosphorylation in HEL cells. * P < 0.05, ** P < 0.01, *** P < 0.001
versus the control group.
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Synergistic Dose-response Relationships against FLC-resistant C. albicans cells.
The synergistic doseꢀresponse relationship of compounds 20a, 20h and 24 against
FLCꢀresistant C. albicans strain 0304103 was further investigated. The cells were
treated with different concentrations of FLC (1 ꢁg/mL to 32 ꢁg/mL) and compounds
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20a, 20h and 24 (2 ꢁg/mL to 32 ꢁg/mL) using the growth curves assay (Figure 6).
High dosage of target compounds (32 ꢁg/mL) or 1 ꢁg/mL of FLC alone exhibited no
antifungal effect, but the antifungal effect was significantly improved when they were
used together (Figure 6AꢀC). Interestingly, the results also indicated that the
synergistic effect mainly depended on the concentration of the dual inhibitors instead
of FLC (Figure 6DꢀF).
Figure 6. Synergistic doseꢀresponse relationships of the drug combination. Growth
curves of C. albicans 0304103 cells incubated with 1 ꢁg/mL of FLC and different
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concentration of compounds 20a (A), 20h (B) and 24 (C) for 24 h. Growth curves of
C. albicans 0304103 cells treated with 32 ꢁg/mL of compounds 20a (D), 20h (E) and
24 (F) and different concentration of FLC for 24 h.
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Compounds 20a and 24 Inhibited the Biofilm Formation of FLC-resistant C.
albicans Cells. C. albicans biofilms are complex communities that form on tissues
and implanted medical devices, causing drug resistance and repeated infections in
4
9, 50
clinics.
The effect of compounds 20a, 20h and 24 on FLCꢀresistant C. albicans
(strain number: 0304103) biofilm formation was evaluated by the XTT reduction
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assay.
1 × 10 cells of FLCꢀresistant C. albicans strain and 64 ꢁg/mL of FLC were
incubated in 96ꢀwell plate for 90 min, and then treated with different concentrations
of compounds 20a, 20h and 24 for 24 h. An equivalent volume of DMSO was added
as the control. Interestingly, they inhibited the biofilm formation in a doseꢀdependent
manner (Figure 8). More specifically, 16 ꢁg/mL of compound 20a or 24 inhibited
biofilm formation by about 30% (P < 0.05), and the antiꢀbiofilm effect increased with
the increasing of the concentration. However, compound 20h was less active.
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Figure 7. Inhibition of FLCꢀresistant C. albicans biofilm formation by DMSO (A)
compounds 20a (B), 20h (C) and 24 (D) using the XTT reduction assay. The results
were presented as a percentage compared to the control biofilms formed without
compound treatment. * P < 0.05, ** P < 0.01, *** P < 0.001 versus the control group.
Compounds 20a, 20h and 24 Showed Potent in vivo Antitumor Activity in AML
Xenograft Models. To investigate the in vivo antitumor effect, xenograft models of
AML were prepared. First, compounds 20a, 20h and 24 were tested in the HEL
6
xenograft model. HEL cells (5 × 10 ) were subcutaneously implanted in the right
flanks of the female nude mice. When implanted tumor reached a volume of 100ꢀ300
3
mm , compounds 20a, 20h and 24 were administered intraperitoneally (i.p) at 10
mg/kg twice a day for 21 consecutive days. Compounds 1 and 2 were used as the
positive controls. As shown in Figure 8A and 8B, treatment with three compounds
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