Small Molecules Simultaneously Inhibiting p53-Murine Double Minute 2 (MDM2) Interaction and Histone Deacetylases (HDACs): Discovery of Novel Multitargeting Antitumor Agents

Article abstract of DOI:10.1021/acs.jmedchem.8b00664

p53-Murine double minute 2 (MDM2) interaction and histone deacetylases (HDACs) are important targets in antitumor drug development. Inspired by the synergistic effects between MDM2 and HDACs, the first MDM2/HDACs dual inhibitors were identified, which sho

Full text of DOI:10.1021/acs.jmedchem.8b00664

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Article
Small Molecules Simultaneously Inhibiting p53-Murine Double
Minute 2 (MDM2) Interaction and Histone Deacetylases
(HDACs): Discovery of Novel Multi-targeting Antitumor Agents
Shipeng He, Guoqiang Dong, Shanchao Wu, Kun Fang, Zhenyuan Miao, Wei Wang, and Chunquan Sheng
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.8b00664 • Publication Date (Web): 25 Jul 2018
Downloaded from http://pubs.acs.org on July 26, 2018
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Small Molecules Simultaneously Inhibiting p53ꢀMurine
Double Minute 2 (MDM2) Interaction and Histone
Deacetylases (HDACs): Discovery of Novel Multiꢀtargeting
Antitumor Agents
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,†
2,†
2,†
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2,*
Shipeng He , Guoqiang Dong , Shanchao Wu , Kun Fang , Zhenyuan Miao ,
1
,3,*
2,*
Wei Wang , Chunquan Sheng
1
School of Pharmacy, East China University of Science and Technology, Shanghai
00237, P. R. China
2
2
Department of Medicinal Chemistry, School of Pharmacy, Second Military Medical
University, 325 Guohe Road, Shanghai 200433, People’s Republic of China
3
Department of Chemistry and Chemical Biology, University of New Mexico, MSC03
2060, Albuquerque, NM 87131ꢀ0001, USA.
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ABSTRACT
p53ꢀmurine double minute 2 (MDM2) interaction and histone deacetylases (HDACs)
are important targets in antitumor drug development. Inspired by the synergistic
effects between MDM2 and HDACs, the first MDM2/HDACs dual inhibitors were
identified, which showed excellent activities against both targets. In particular,
compound 14d was proven to be a potent and orally active MDM2/HDAC dual
inhibitor, whose antitumor mechanisms were validated in cancer cells. Compound
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14d showed excellent in vivo antitumor potency in the A549 xenograft model,
providing a promising lead compound for the development of novel antitumor agents.
Also, this proofꢀofꢀconcept study offers a novel and efficient strategy for
multiꢀtargeting antitumor drug discovery.
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INTRODUCTION
p53 is a transcriptional factor that plays a key role in preventing tumor development.
1
,
2
3
Approximately 50% of human cancers are related to the inactivation of p53. Its
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interaction with human murine double minute 2 (MDM2) protein is the main
inhibitory mechanism of the inactivation of transcriptional activity and tumor
4
ꢀ8
suppressor function of p53. Blocking the p53ꢀMDM2 proteinꢀprotein interaction
with smallꢀmolecule inhibitors can reꢀactivate the function of p53 and is emerging as
9
a promising strategy in cancer therapy. In the past few years, numerous
smallꢀmolecule p53ꢀMDM2 inhibitors were reported and several of them are currently
10ꢀ14
being evaluated in preclinical or clinical trials.
However, clinical studies indicated
that thrombocytopenia was the key adverse effect of MDM2 inhibitors, especially for
15ꢀ17
daily administration.
Reducing the dose frequency is an effective strategy to avoid
thrombocytopenia, and thus it is highly desirable to develop more efficient MDM2
inhibitors with prolonged dosing interval. Alternatively, simultaneous modulation of
multiple targets by a single compound may generate superior efficacy and fewer side
18, 19
effects.
For example, our group designed smallꢀmolecule inhibitors
simultaneously targeting the MDM2 and NFꢀκB pathway, which achieved excellent in
2
0, 21
vitro and in vivo antitumor potency.
Developing MDM2ꢀbased multiꢀtargeting
antitumor agents could serve as a promising strategy for novel antitumor drug
development.
Histone deacetylases (HDACs), a class of epigenetic enzymes, play a crucial role in
22, 23
regulating the expression of tumor suppressor genes.
In recent years, several
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HDAC inhibitors, such as vorinostat (SAHA, 1) and romidepsin, were approved by
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the FDA for the treatment of cancer.
However, clinical application of the
marketed HDAC inhibitors is generally restricted in the treatment of hematological
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malignancies, whose efficacy against solid tumors is poor.
Moreover, most
HDAC inhibitors are clinically used in combination with other antitumor agents to
28ꢀ31
achieve synergistic effects.
Although drug combinations have the advantage of
higher antitumor efficacy, they also suffered from enhanced adverse effects,
32
unpredictable pharmacokinetic profiles, drugꢀdrug interactions and so on. In contrast,
singleꢀmolecule with multiꢀtargeting profiles are recently emerging as a promising
3
3
alternative to combination therapy. Our previous work and other studies have
confirmed the effectiveness of HDACꢀbased multiꢀtargeting antitumor agents, which
34ꢀ36
showed improved antitumor activities and low toxicities.
Recent studies indicated that HDAC inhibitors could synergistically enhance the
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antitumor effects of a variety of antitumor agents, such as DNA damage reagents
39
and tubulin modulators . In particular, HDAC inhibitors were capable of synergizing
4
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with MDM2 inhibitors in the suppression of tumor cells proliferation. As shown in
Figure 1, p53ꢀMDM2 interaction acts by the autoꢀregulatory feedback loop
mechanism in the physiological conditions. MDM2 is involved in inhibiting the
2
, 41ꢀ43
function of p53 as a tumor suppressor by direct binding with p53 protein.
Blocking p53ꢀMDM2 interaction could release p53 from MDM2, thus restoring the
tumor suppressor function of p53. On the other hand, functional inactivation of the
4
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tumor suppressor p53 is a fundamental step in tumorigenesis. p53 acetylated at
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Lys382 is one of the activated forms of p53 tumor suppressor and the acetylation of
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p53 is mediated by HDAC inhibitors. Inhibition of HDACs or/and MDM2 may lead
to the accumulation of activated p53. HDAC inhibitors could synergistically improve
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the antitumor activity of MDM2 inhibitors by modifying the hyperacetylation of
40, 46
p53.
These evidence suggests that designing a singleꢀmolecule inhibitor
simultaneously targeting HDAC and MDM2 could be an effective strategy in cancer
40
therapy.
Figure 1. The relationship of MDM2 and HDACs in the signaling pathway.
Herein, the first MDM2/HDAC dual inhibitors were rationally designed, which
showed excellent inhibitory activities against the both targets. Particularly, dual
inhibitor 14d showed excellent in vivo antitumor efficacy in the A549 xenograft
model, highlighting the therapeutic value of this novel multiꢀtargeting antitumor drug
design strategy.
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Figure 2. Chemical structures of HDAC and MDM2 inhibitors.
RESULTS AND DISCUSSION
Structureꢀbased Design of Novel MDM2/HDAC Dual Inhibitors. Nutlins represent
47
a classic class of MDM2 inhibitors (e.g., compounds 3 and 4, Figure 2). Compound
3
showed good MDM2 inhibitory activity and has been widely used as a tool
4
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molecule or positive control to investigate the biological function of MDM2. The
optimization of Nutlins led to the discovery of compound 4 (RG7112) in the Phase 1
4
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clinical trial. The pharmacophore model of HDAC inhibitors can be divided into
three parts: the cap group that interacts with the surface residues of HDAC, the linker
2
+
and the zinc binding group (ZBG) that chelates with the active site Zn ion (Figure
50
3
). Due to the presence of large hydrophobic domain at the HDACs surface, it is
conceivable that appropriate substitution of the cap group of HDAC inhibitors by a
chemical scaffold (or pharmacophore) from other type of anticancer agents could
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generate new classes of multiꢀtargeting molecules.
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According to the binding models of known HDACs and MDM2 inhibitors,
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compounds 1, 2 and nutlins were selected as the templets to design dual
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MDM2/HDAC inhibitors. The binding mode of compound 3 with MDM2 revealed
that three functional groups on the imidazole scaffold mimicked Phe19, Trp23 and
Leu26 residues in p53, which were projected into the binding pocket in MDM2
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(
Figure 3). The N3 substitution of compound 3 was not buried inside the subpockets
of MDM2 and directly exposed to the solvent, which was suitable to introduce a
linker and the ZBG of HDAC inhibitors to obtain MDM2/HDAC dual inhibitors. Also,
a polar side chain attached to the N3 position of compound 3 was favorable to adjust
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the physiochemical properties (e.g. aqueous solubility).
Figure 3. Design of MDM2ꢀHDAC dual inhibitors. (A) The binding modes of
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MDM2 and HDAC inhibitors; (B) Design rationale of MDM2ꢀHDAC dual inhibitors.
Chemistry. As outlined in Scheme 1, treatment of intermediates 5 and 6 in the
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presence of B(OH) and xylenes afforded imidazole 7, which was further reacted
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with triphosgene to obtain acyl chloride 8.
α
Scheme 1
α
o
Reagents and conditions: (a) B(OH)
3
(0.12 eq), xylenes, 145 C, 6 h, yield 80%; (b)
BTC (0.34 eq), rt, CH Cl , yield 82%.
2
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Then, in the presence of DIPEA, intermediate 8 was condensed with commercially
available aminoalkylesters 9aꢀd to give amides 10aꢀd, which was subsequently
hydrolyzed to corresponding acids and condensed with orthoꢀphenylenediamine to
afford target compounds 11aꢀd (Scheme 2). Also, treatment of intermediates 10aꢀd
with freshly prepared hydroxylamine methanol solution yielded hydroximic acids
12aꢀd. Similarly, target compound 13 was prepared from intermediate 8 via the
condensation and ammonolysis reaction.
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α
Scheme 2
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α
Reagents and conditions: (a) DIPEA (1.2 eq), CH Cl , rt, 1.5 h, yield 70ꢀ90%; (b)
2
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LiOH (2.5 eq), THF: MeOH: H
2
O = 3: 2: 1, rt, 2 h, yield 80ꢀ95%; (c)
OrthoꢀPhenylenediamine (1.2 eq), HBTU (2.0 eq), TEA (2.0 eq), DMF, rt, 1ꢀ2 h, yield
5
2
5ꢀ70%; (d) NH OH (14 eq), MeOH, rt, 0.5 h, yield 88ꢀ95%.
A similar strategy was applied for the synthesis of piperazineꢀbased conjugates
4aꢀe (Scheme 3). Piperazines 19aꢀe were reacted with 8 in the presence of Et N and
Cl to give esters 20aꢀe in good yield. Then, the synthesis of target compounds
4aꢀe was similar to that of compounds 12aꢀd. Compounds 15a and 15b were
1
3
CH
2
2
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synthesized via the condensation and ammonolysis reaction from intermediate 8.
Starting from piperidine intermediates 22aꢀb and 23, target compounds 16aꢀb
(Scheme 4) were obtained by a similar protocol described in Scheme 1.
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α
Reagents and conditions: (a) Et
3 2 2
N (1.2 eq), CH Cl , rt, 1 h, yield 70ꢀ92%; (b)
NH
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OH (14 eq), MeOH, rt, 45 min, yield 88ꢀ92%.
α
Scheme 4
α
Reagents and conditions: (a) DIPEA (1.2 eq), CH Cl , rt, 1 h, yield 90ꢀ95%; (b)
2
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NH OH (14 eq), MeOH, rt, 1 h, yield 85ꢀ95%.
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Biochemical Evaluations and Structureꢀactivity Relationships. MDM2 and
HDACs inhibitory activities were evaluated by the fluorescence polarization binding
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assay and fluorescent activity assay , respectively, using compounds 1ꢀ3 as the
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positive controls. Initially, orthoꢀaminoanilide was used as the ZBG (11aꢀd). These
compounds generally showed good inhibitory activity against MDM2 (K
.96~1.43 ꢁM). However, compounds 11a and 11b were inactive against HDAC1.
The extension of the linker length led to improved activity against HDAC1 (e.g.
compound 11d, IC50 = 3.59 M). Interestingly, the replacement of the
i
range:
0
ꢀ
orthoꢀaminoanilide by hydroxamate acid (12aꢀd) resulted in enhanced activities
against both MDM2 and HDAC1 (Table 1). The linker length had little impact on the
i
MDM2 inhibitory activity and compounds 12aꢀd had a K value ranging from 0.28
ꢀ
M to 0.34 M. In contrast, with the increase of the linker length, the HDAC1
ꢀ
inhibitory activity was improved accordingly, indicating that a long alkyl linker might
be beneficial to HDAC1 binding. Among them, compound 12d showed balanced
activity towards both MDM2 (K
Moreover, the flexible alkyl linker was replaced by a rigid aromaticꢀbased chain
compound 13). Unfortunately, its MDM2/HDAC1 inhibitory activity was decreased.
i
= 0.34 ꢀM) and HDAC1 (IC50 = 0.27 ꢀM).
(
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Table 1. MDM2 Binding Affinity and HDAC Inhibitory Activities of the Target
Compounds
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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
MDM2
HDAC1
Compound
R
1
α
α
K (ꢁM)
i
IC (ꢁM)
50
1
1a
0.96 ± 0.032
1.43 ± 0.311
0.62 ± 0.107
1.02 ± 0.223
0.33 ± 0.029
＞20
＞20
11b
11c
6.07 ± 0.88
3.59 ± 0.67
2.92 ± 0.72
11d
12a
1
2b
0.28 ± 0.034
0.32 ± 0.026
0.34 ± 0.041
1.13 ± 0.56
0.89 ± 0.09
0.27 ± 0.04
1
2c
1
2d
1
3
0.58 ± 0.042
>20
3.70 ± 0.06
1
0.056 ± 0.015
1.09 ± 0.31
b
2
NT
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b
3
0.14 ± 0.04
NT
a
b
Values represent a mean ± SD of at least two independent experiments. NT = Not
tested.
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
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7
8
9
0
1
2
3
4
5
6
7
8
9
0
Next, piperazine or piperidine was incorporated into the linker to improve the
binding affinities toward both targets as well as the water solubility (Table S1 in
Supporting Information). Generally, piperazine derivatives 14aꢀe showed better
MDM2 inhibitory activities than compounds 12aꢀd (Table 2). In particular,
i
compound 14a had excellent MDM2 binding affinity (K = 0.07 ꢀM). Meanwhile,
HDAC1 inhibitory activities of compounds 14aꢀe were improved accordingly. Similar
to compounds 12aꢀd, a long alkyl linker was helpful to enhance HDAC1 inhibitory
activities. Among them, compounds 14bꢀe generally showed balanced inhibition
towards MDM2/HDAC1. When aromatic groups were introduced into the linker,
decreased HDAC1 binding activity was observed for phenyl derivative 15a.
Pyrimidine derivative 15b showed improved activity against HDAC1, whereas its
MDM2 binding affinity was obviously decreased. For the piperidine derivatives
(
i
16aꢀb, 17), compound 17 showed the best MDM2 binding affinity (K = 0.06 ꢀM)
among all the synthesized compounds, whereas its HDAC1 inhibitory activity was
poor. Further incorporation of the aromatic groups into the linker (compound 16b)
was beneficial for the HDAC1 inhibitory activity, but the activities for MDM2 were
significantly decreased.
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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
Table 2. MDM2 Binding Affinity and HDAC Inhibitory Activities of Target
Compounds
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
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7
8
9
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2
3
4
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8
9
0
MDM2
HDAC1
Compound
X
R
2
α
α
K (ꢁM)
i
IC (ꢁM)
50
1
4a
4b
A
A
A
A
A
0.07 ± 0.01
0.17 ± 0.02
0.17 ± 0.03
0.11 ± 0.03
0.29 ± 0.04
4.20 ± 0.59
0.98 ± 0.08
0.97 ± 0.13
0.82 ± 0.04
0.41 ± 0.05
1
1
4c
1
4d
1
4e
1
5a
A
A
0.26 ± 0.03
1.05 ± 0.43
3.51 ± 0.71
0.31 ± 0.07
15b
1
6a
6b
B
0.46 ± 0.034
2.65 ± 0.79
1.01 ± 0.71
0.97 ± 0.07
O
OH
O
N
1
B
B
H
N
H
1
7
0.057 ± 0.028
>20
9.72 ± 1.11
1
0.056 ± 0.015
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b
2
3
NT
1.09 ± 0.31
b
0.14 ± 0.04
NT
a
b
Values represent a mean ± SD of at least two independent experiements. NT = Not
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
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9
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1
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0
tested.
In vitro Antiproliferative Assay. Given the potent enzyme inhibitory activities, the
antiproliferative activities of the MDM2/HDAC dual inhibitors were investigated
against p53ꢀwild A549 (human lung cancer), HCTꢀ116 (human colon cancer) and
MCFꢀ7 (human breast cancer) cell lines by the CCK8 assay. As shown in Table 3,
most compounds showed good growth inhibition activities against the three human
cancer cells. Moreover, they were more effective against the A549 and HCT116 cells
than MCFꢀ7 cells. The in vitro antitumor activities were generally consistent with the
inhibitory activities towards the two targets. For example, compounds 14aꢀe with
better MDM2/HDAC1 inhibitory activity were also more potent than compounds
12aꢀd. Among them, compound 14d exhibited the best antiproliferative activity,
particularly for A549 cell line (IC50 = 0.91 ꢁM), which was more potent than the three
positive drugs. On the other hand, the improvement of solubility by introducing polar
5
7
group may be one of the reasons for the increasing the cellular activity. For
example, compound 14d had better solubility than 12c after introducing the
piperazine group (Table S1 in Supporting Information), and thus the
antiproliferative activity of compound 14d was superior to that of compound 12c. In
addition, the inhibitory activities against the p53ꢀdeleted NCIꢀH1299 cells (human
1
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1
1
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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
nonꢀsmall cell lung cancer) were tested. The results revealed most compounds had
relatively poor inhibitory activities towards NCIꢀH1299 cells compared with those of
p53ꢀwild A549 cells (Table 3). For example, compound 14d showed an IC50 value of
4.16 ꢁM and 0.91ꢁM against NCIꢀH1299 and A549 cells, respectively. Considering
the molecular and cellular activities, compound 14d was subjected to further
evaluations.
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 3. Antiproliferative Activity of Target Compounds against Four Solid
a
Tumor Cell Lines (IC50, ꢁM)
Compound
1a
1b
1c
11d
2a
12b
2c
2d
A549
HCT116
13.86 ± 0.68
21.80 ± 1.11
10.13 ± 2.03
9.91 ± 1.07
10.70 ± 0.98
7.15 ± 1.03
7.09 ± 0.64
5.03 ± 0.75
9.10 ± 0.81
5.88 ± 0.84
5.21 ± 0.69
4.03 ± 1.02
1.08 ± 0.46
MCFꢀ7
29.14 ± 1.82
>100
NCIꢀH1299
15.63 ± 2.70
19.10 ± 1.68
17.51 ± 2.92
19.58 ± 2.20
16.22 ± 1.36
10.38 ± 0.68
7.65 ± 0.83
9.06 ± 1.09
7.71 ± 0.57
11.54 ± 2.36
12.93 ± 2.07
9.39 ± 0.72
4.16 ± 1.13
1
18.35 ± 0.63
20.27 ± 1.71
15.97 ± 0.77
15.16 ± 0.83
9.34 ± 1.27
5.72 ± 0.76
5.97 ± 0.25
4.85 ± 0.89
7.64 ± 1.18
4.32 ± 0.66
3.78 ± 0.82
2.91 ± 0.32
0.91 ± 0.09
1
1
>100
20.26 ± 3.27
12.77 ± 1.31
10.55 ± 1.25
7.23 ± 0.92
7.92 ± 1.48
13.31 ± 2.73
11.67 ± 1.30
9.80 ± 0.76
7.26 ± 1.13
4.34 ± 0.83
1
1
1
13
14a
1
4b
4c
14d
1
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4e
15a
5b
16a
6b
2.01 ± 0.82
2.17 ± 0.98
1.97 ± 0.79
3.11 ± 0.48
7.59 ± 1.05
2.99 ± 0.51
2.01 ± 0.99
10.17 ± 1.22
11.65 ± 1.09
1.71 ± 0.79
2.03 ± 0.92
0.98 ± 0.07
3.24 ± 0.75
9.64 ± 1.21
9.30 ± 0.66
1.18 ± 0.42
9.83 ± 1.96
9.07 ± 0.92
4.83 ± 0.97
7.76 ± 1.09
3.00 ± 0.38
5.45 ± 0.82
7.91 ± 1.06
9.36 ± 0.59
9.87 ± 1.08
4.92 ± 0.63
6.79 ± 1.01
19.76 ± 2.11
3.97 ± 0.87
3.46 ± 0.95
2.68 ± 0.47
7.22 ± 0.99
10.07 ± 1.02
4.83 ± 0.69
13.16 ± 3.14
8.50 ± 0.93
1
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
1
17
1
2
3
a
IC values are the mean of at least three independent assays, presented as mean ±
5
0
SD.
HDACs Isoform Selectivity of Compound 14d. In order to test the HDAC isoform
selectivity profile, compound 14d was selected to assay the enzyme inhibitory
activities against HDAC1ꢀ3, HDAC6 and HDAC8 (Table 4). The results revealed that
compound 14d exhibited remarkable selectivity for HDAC6 (IC50 = 17.5 nM) over
other isoforms. In contrast, it inhibited HDAC1 (IC50 = 820 nM), HDAC2 (IC50 = 420
nM) and HDAC3 only in the subꢀmicromolar range. Similar to positive compound 1,
compound 14d was less active activity against HDAC8 (IC50 = 1224 nM).
α
Table 4. HDACs Isoform Selectivity of Compounds 1 and 14d (IC50, nM)
1
14d
HDAC1
45.0 ± 3.1
821 ± 12
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1
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1
1
2
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3
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5
6
HDAC2
HDAC3
HDAC6
HDAC8
153.1 ± 41.0
29.0 ± 3.1
16.3 ± 1.6
3970 ± 379
420.5 ± 13.0
178.3 ± 6.2
17.5 ± 1.3
1224 ± 172
0
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0
a
IC50 values are the mean of at least three independent assays, presented as mean ±
SD.
Effects of Compound 14d on p53 and MDM2 Expression. Next, we investigated
the effects of compound 14d on the levels of p53 and MDM2 expression in A549
cells by Western blotting analysis using compound 3 as the positive control (Figure
4
). Treatment with compound 14d or 3 in A549 cells both doseꢀdependently increased
the protein level of p53, as well as MDM2 at the 24 h time point. The results indicated
that compound 14d had similar effects to compound 3 in inducing the p53 and MDM2
expression within A549 cells.
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Figure 4. Western blotting analysis of p53 and MDM2 expression by compound 14d.
(A) Effects of compounds 14d and 3 on the level of p53 and MDM2 in human lung
cancer A549 cells using Western blotting analysis after the treatment for 24 h.
GAPDH was used as a loading control. (B) Quantitative analysis of Western blotting
using ImageQuant (Molecular Dynamics, U.S.). Protein p53 and MDM2 were
analyzed in A549 cells, respectively. Data were presented as means ± SEM, n = 3. * P
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
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2
2
3
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3
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0
1
2
3
4
5
6
7
8
9
0
<
0.05 and ** P < 0.01 vs control group.
Effects of Compound 14d on Histone and αꢀTubulin Acetylation. In order to probe
the contribution of HDACs inhibition to the cytotoxic activity of compound 14d, the
intracellular acetylation level of histone 3 and histone 4 (Figure 5), two common
substrates for HDAC (1, 2 and 3), were determined in A549 cells by the Western
blotting. Similar to HDAC inhibitor 1, compound 14d doseꢀdependently increased the
acetylation level of histone 3 and of histone 4 in A549 cells. Compound 14d was
further subjected for evaluation of intracellular HDAC6 inhibition. The effects of
compound 14d on the acetylation level of αꢀtubulin, a known substrate for HDAC6,
are shown in Figure 5. Exposure to compound 14d for 24 h led to the increase of
αꢀtubulin acetylation in A549 cells. These results demonstrated that the inhibition of
intracellular HDAC1 and HDAC6 was one of the mechanisms of bioactivity of
compound 14d.
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0
1
2
3
4
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8
9
0
1
2
3
4
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6
7
8
9
0
Figure 5. Western blotting analysis of histone and αꢀtubulin acetylation by compound
14d. (A) Effects of compounds 14d and 1 on the levels of histone 3 and histone 4
acetylation, αꢀtubulin acetylation in lung cancer A549 cells using Western blotting
analysis after the treatment for 24 h. (B) Quantitative analysis of Western blotting
with ImageQuant (Molecular Dynamics, U.S.). Histone 3 and 4 acetylation and
αꢀtubulin acetylation were analyzed in A549 cells, respectively. Data were presented
as means ± SEM, n = 3. * P < 0.05 and ** P < 0.01 vs control group.
Flow Cytometry Analysis. It was reported that p53ꢀMDM2 inhibitor 3 arrested
wildꢀtype p53 cancer cell lines at the G0/G1 phase and depleted the Sꢀphase
47
compartment. HDAC inhibitor 1 induced cell cycle arrest at the G0/G1 phase
through transcriptional activation of genes such as cell cycleꢀregulated genes in a
3
7
p53ꢀindependent manner. Compound 1 could also arrest the G2/M phase in some
58, 59
human cancer cell lines.
To investigate the cellular mechanisms of compound 14d,
its effects on the cell cycle progression were tested in A549 cells by the flow
cytometry analysis (Figure 6). As compared to the vehicle, treatment with 1 ꢁM of
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compounds 14d, 1, and 3, respectively, led to the increase of G0/G1 fraction.
Compounds 14d and 3 were also able to arrest A549 cellꢀcycle progression in the
G0/G1 segment at higher concentration (5 ꢁM). Meanwhile, compound 1 also
resulted in the increase of the G2/M fraction at the same concentration. The results
indicated that compound 14d could significantly arrest the A549 cell cycle at the
G0/G1 phase.
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
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3
3
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0
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0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Figure 6. Cell cycle analysis of compound 14d by flow cytometry. Human nonꢀsmall
lung cancer cells (A549) were treated with increasing concentrations of compound
14d or reference compounds for 24 h. The data are representative of two independent
experiments.
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6
Previous studies revealed that inactivation of HDAC and/or MDM2 could increase the
47, 60, 61
percentage of apoptosis cells.
Flow cytometry analysis was performed to
0
1
2
3
4
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0
1
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determine the cell apoptosis effects of compound 14d towards A549 cells. As shown
in Figure 7, compound 14d, as well as 1 and 3, could significantly induce apoptosis
in a doseꢀdependent manner. At the concentration of 5 ꢁM, compound 14d induced
33.57% of cell apoptosis, which was higher than that of compounds 1 (31.99%) and 3
(15.32%), indicating that inactivation of HDAC and/or MDM2 could increase the
percentage of apoptosis cells.
Figure 7. Effects of compounds 1, 3 and 14d on the induction of apoptosis in A549
cells. The cells were exposed to increasing concentrations of each compound for 48 h
and analyzed by annexin V/propidium iodide double staining. The data are
representative of two independent experiments.
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Biological Activity of Enantiomers of Compound 14d. The enantiomers of
compound 14d were separated by chiral column chromatography and tested the
optical rotation by polarimeter. Furthermore, the configuration of enantiomers of 14d
could be identified by the comparison of optical rotation to that of enantiomers of 3
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
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3
3
3
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0
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(
Figure 8A and Supporting Information Figure S2). Fluorescence polarization
binding assay revealed that their MDM2 inhibitory activities were significantly
different (Figure 8B). Enantiomers 14dꢀ2 (K = 0.06 ꢁM) was about 180ꢀfold more
potent than enantiomer 14dꢀ1 (K = 10.89 ꢁM). Furthermore, Western blotting was
i
i
used to verify the MDM2 inhibitory activity of enantiomers 14dꢀ1 and 14dꢀ2 in A549
cells. Consistent with the fluorescence polarization binding assay, enantiomer 14dꢀ2
significantly increased the level of p53 and upꢀregulated the expression of MDM2 via
negative feedback loop after 12 h, whereas enantiomer 14dꢀ1 caused the increase of
p53 only at the high concentration (10 ꢁM) (Figure 8C).
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Figure 8. MDM2 inhibitory activities of compound 14d enantiomers. (A) Chemical
structures of compound 14d enantiomers, which were separated by chiral column
chromatography. (B) The MDM2 binding affinity of compound 14d enantiomers. (C)
Effects of compounds on the level of p53 and MDM2 in A549 cells using the Western
blotting analysis. GAPDH was used as the loading control. Quantitative analysis of
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Western blotting was performed with ImageQuant. Data were presented as means ±
SEM, n = 3. * P < 0.05 and ** P < 0.01 vs control group.
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Subsequently, HDAC1 inhibitory activity of compounds 14dꢀ1 and 14dꢀ2 were
evaluated (Table 5). Interestingly, enantiomer 14dꢀ1 (IC50 = 0.91
activity to enantiomer 14dꢀ2 (IC50 = 0.89 M). The results indicated that the chiral
centers on the imidazole scaffold had significant impact on the binding of the two
ꢀchloropnenyl groups with MDM2 hot spots. In contrast, as the cap group, the
ꢀM) showed similar
ꢀ
4
conformation of 4ꢀchloropnenyl groups had little effect on HDAC1 binding.
Consistently, enantiomer 14dꢀ2 showed better antiproliferative activity than 14dꢀ1.
The different antiproliferative activity of two enantiomers also indirectly revealed that
simultaneously inhibited HDAC and MDM2 by a singleꢀmolecule were capable of
generating synergistic antitumor activities.
α
Table 5. Biological Activities of Enantiomers of 14d
p53ꢀMDM2
, ꢁM)
IC50 (ꢁM)
HCT116
Compound
(
K
i
HDAC1
A549
14dꢀ1
10.89 ± 1.12 0.91 ± 0.08 10.03 ± 0.93 9.02 ± 0.47
0.06 ± 0.01 0.89 ± 0.12 1.11 ± 0.24 0.99 ± 0.12
14dꢀ2
3
0.14 ± 0.03
＞100
9.97 ± 0.86 9.58 ± 0.63
a
K and IC values are the mean of at least three independent assays, presented as
i
50
mean ± SD.
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Binding Mode of Enantiomers of Compound 14d with MDM2 and HDAC1. To
investigate the binding mode of enantiomers of compound 14d with MDM2 and
HDAC1, computational docking studies were performed. As shown in Figure 9A,
14dꢀ2 mimicked the interactions of p53 binding site on MDM2. Three aromatic
substituents of the scaffolds projected into the pockets normally occupied by the three
key residues (Phe19, Trp23, and Leu26) of p53. Ethyl ether side chain in 14dꢀ2
directed toward the Phe19 pocket, the two 4ꢀchlorophenyl groups of 14dꢀ2 projected
into the Leu26 and the Trp23 pocket, respectively. Moreover, the 4ꢀchlorophenyl
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moiety formed πꢀπ stacking interaction with His92 (Figure 9A and Figure S3A in
Supporting Information). In contrast, the ethyl ether side chain of 14dꢀ1 was located
at the outside of the Phe19 pocket (Figure 9C) and the hydrophobic interactions were
lost (Figure 9C and Figure S3C in Supporting Information). It might be the main
reason why the MDM2 binding affinity of 14dꢀ1 was inferior to that of 14dꢀ2.
The binding mode of enantiomers of compound 14d with HDAC1 was shown in
Figure 9B and 9D. Compound 14dꢀ1 and 14dꢀ2 bound with HDAC1 mainly through
the linker and ZBG. The aliphatic linker was projected into the hydrophobic cavity of
2
+
HDAC1. Besides chelating with Zn , the terminal hydroxamic acid group of 14dꢀ2
formed two hydrogen bonds with Tyr308 and His145, respectively. Moreover, the
4ꢀchloropnenyl group in the cap formed πꢀπ stacking interactions with Arg275
(Figure 9B and Figure S3B in Supporting Information). Similarly, the hydroxamic
2+
acid group of 14dꢀ1 chelated with Zn and formed two hydrogen bonds with Gly143
and His145, respectively. In addition, hydrogen bonding interaction was observed
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between the carbonyl group of 14dꢀ1 and Phe210 (Figure 9D and Figure S3D in
Supporting Information). The computational docking result was consistent with the
fact that the two enantiomers had similar HDAC1 inhibitory activity.
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Figure 9. The binding modes of compound 14d enantiomers with MDM2 and
HDAC1. (A) Compound 14dꢀ2 (red) projected into the three hot spots of MDM2
(PDB ID: 4IPF). (B) Proposed binding mode of 14dꢀ2 (red) in the active site of
HDAC1 (PDB ID: 4BKX). (C) Binding mode of compound 14dꢀ1 (green) with
MDM2 (PDB ID: 4IPF). The ethyl ether was buried out the Phe19 pocket. (D)
Proposed binding mode of 14dꢀ1 (green) in the active site of HDAC1 (PDB ID:
4BKX). The figure was generated using PyMol. (http://www.pymol.org/).
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In vivo Antitumor Efficacy of Compound 14d. Compound 14d was evaluated for in
vivo antitumor effcacy in the A549 xenograft nude mouse model. When tumors had
3
reached an average volume of 100 mm in the 11th day after implantation, compound
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14d was administered orally (po) at 100 mg/kg/day and 150 mg/kg/day for 21
consecutive days, respectively. Compounds 1 (100 mg/kg/day, po) and 3 (100
mg/kg/day, po) were used as the positive controls. As shown in Figure 10, compound
14d effectively inhibited the tumor growth in a doseꢀdependent manner compared to
the vehicle control. Oral administration of 100 mg/kg/day of compound 14d achieved
the tumor growth inhibition (TGI) of 65.4%. At 150 mg/kg/day, the TGI of compound
14d was increased to 74.5%, whose in vivo potency was better than compound 1 (100
mg/kg/day, TGI = 57.3%) (P < 0.01) and 3 (100 mg/kg/day, TGI = 44.0%) (P < 0.01).
Notably, no significant body weight loss and no adverse effects were observed for
compound 14d during the in vivo studies. The results highlighted the advantages of
dual inhibition of MDM2 and HDAC for the treatment of A549 lung cancer.
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Figure 10. In vivo antitumor potency of compound 14d. (A) Antitumor activity of
compound 14d, in comparison to compounds 1 and 3, in the A549 lung cancer
xenograft model in nude mice. Compound 14d was treated with oral gavage at 100 or
150 mg/kg daily for 21 days and reference compounds was treated with oral gavage at
100 mg/kg once per day for 21 days. The antitumor potency was expressed by TGI.
TGI was calculated according to the formula, TGI = (1ꢀ tumor volume of treatment
group/ tumor volume of control group) × 100%, at the end of treatment. Data are
expressed as the mean ± standard deviation. * P < 0.05 and *** P < 0.001,
determined with 1ꢀway ANOVA test for significance. (B) Changes in body weight of
mice during treatment period. (C) The picture of dissected A549 tumor tissues.
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