Studying Histone Deacetylase Inhibition and Apoptosis Induction of Psammaplin A Monomers with Modified Thiol Group

Article abstract of DOI:10.1021/acsmedchemlett.0c00369

Psammaplin A (PsA) is a bromotyrosine disulfide dimer with histone deacetylase (HDAC) inhibition and acts through reduced monomer PsA-SH. We studied the connection of HDAC inhibition, cell growth inhibition, and apoptosis induction of PsA-SH by modifying the -SH group with deletion (6a) or replacement with hydroxamic acid (10b) or benzamide (12g). PsA-SH inhibits HDAC1/2/3 and 6a loses the HDAC inhibition ability. 10b inhibits HDAC1/2/3/6 while 12g shows selective inhibition of HDAC3. PsA-SH and 10b, but neither 6a nor 12g, induce apoptosis in human leukemia HL-60 cells associated with increased acetylation of Histone H3. PsA-SH and 10b inhibit growth of several solid tumor cell lines in vitro and Lewis lung cancer cell growth in vivo. PsA-SH is a simple scaffold for developing selective HDAC inhibitors and induces apoptosis through inhibiting HDAC1/2.

Full text of DOI:10.1021/acsmedchemlett.0c00369

pubs.acs.org/acsmedchemlett
Letter
Studying Histone Deacetylase Inhibition and Apoptosis Induction of
Psammaplin A Monomers with Modified Thiol Group
Yu Bao,# Qihao Xu,# Lin Wang, Yunfei Wei, Baichun Hu, Jian Wang, Dan Liu, Linxiang Zhao,*
and Yongkui Jing*
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ABSTRACT: Psammaplin A (PsA) is a bromotyrosine disulfide dimer with histone deacetylase (HDAC) inhibition and acts
through reduced monomer PsA-SH. We studied the connection of HDAC inhibition, cell growth inhibition, and apoptosis induction
of PsA-SH by modifying the −SH group with deletion (6a) or replacement with hydroxamic acid (10b) or benzamide (12g). PsA-
SH inhibits HDAC1/2/3 and 6a loses the HDAC inhibition ability. 10b inhibits HDAC1/2/3/6 while 12g shows selective
inhibition of HDAC3. PsA-SH and 10b, but neither 6a nor 12g, induce apoptosis in human leukemia HL-60 cells associated with
increased acetylation of Histone H3. PsA-SH and 10b inhibit growth of several solid tumor cell lines in vitro and Lewis lung cancer
cell growth in vivo. PsA-SH is a simple scaffold for developing selective HDAC inhibitors and induces apoptosis through inhibiting
HDAC1/2.
KEYWORDS: Psammaplin A, HDAC, sulfydryl, hydroxamic acid, benzamide
A large group of small-molecules have been developed as
HDAC inhibitors for cancer therapy in the last two
decades.12,13 Vorinostat (SAHA), Romidepsin (FK-228),14
Belinostat (PXD-101), and Panobinostat (LBH-589)15 have
been approved by the USA FDA and Chidamide (CS055/
HBI-8000)16 by the China FDA for T-cell lymphoma and
myeloma therapies (Figure 1). These inhibitors have not been
extended to other types of cancer therapy probably due to the
obvious side effects and/or unconfirmed effectiveness.17−19
More selective inhibitors need to be developed for decreasing
the side effects and/or for increasing the efficacies. HDAC
inhibitors usually contain three portions: zinc binding group
(ZBG), linker region, and surface recognition moiety (SRM).
hromatin is a dense and higher-order structure consisting
Cof DNA and core histone proteins. Histone deacetylases
(HDACs) catalyze deacetylation of the N-terminal lysine
residues of the core histones.1 Deacetylation of histones causes
chromatin aggregation which prevents transcriptional machi-
nery to be close to DNA and represses the gene transcription.2
Aberrant acetylation of histone and/or expression of HDACs
occur in cancer cells and contribute to tumorigenesis.3
Eighteen HDAC subtypes have been found in mammalian
cells and are divided into four classes. Classes I, II, and IV are
Zn2+-dependent HDACs, while class III is NAD+-dependent
sirtuins.4,5 Class I HDACs contain HDAC-1, -2, -3, and -8.
Class II HDACs are further divided into class IIa (HDAC-4,
-5, -7, -9) and class IIb (HDAC-6, -10). Overexpression of
class I HDAC isotypes occurs in aggressive subtypes of tumors
such as stomach, esophagus, colorectal, and breast cancers.6−11
Genetic knockdown of individual HDAC, most notably
HDAC-1, -2, and -3, induced DNA damage, cell cycle arrest,
and apoptosis.6 HDACs, especially class I, have been used as
targets for developing cancer therapeutics.
Received: July 4, 2020
Accepted: November 25, 2020
Published: January 5, 2021
https://dx.doi.org/10.1021/acsmedchemlett.0c00369
© 2021 American Chemical Society
ACS Med. Chem. Lett. 2021, 12, 39−47
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Figure 1. Chemical structures of Psammaplin A (PsA), Psammaplin A monomer (PsA-SH), and approved HDAC inhibitors.
a
Scheme 1. Synthesis of PsA Monomer with a Different ZBG
a
Reagents and conditions: (a) Imidazolidine-2,4-dione, NaHCO3, H2O, 120 °C, 10 h, 83−92%; (b) NaOH, N2, H2O, 95 °C, 12 h, 65−73%; (c)
NaOH, NaHCO3, HONH2·HCl, H2O, r.t., 12 h, 70−84%; (d) HOBt, EDCI, DIEA, DMF, 10 h, 30−47%; (e) DTT, KOH, MeOH, r.t., 30 min; (f)
SOCl2, MeOH, Et2O, r.t., 3 h, 74−83%; (g) LiAlH4, THF, r.t., 1 h; (h) HONH2·H2O, NaOH, MeOH, r.t., 30 min, 73−88%; (i) NaOH, MeOH, 60
°C, 30 min, 90−95%.
The ZBG plays an important role in determining the selectivity
of HDAC inhibitors. Vorinostat, Belinostat, and Panobinostat
containing the hydroxamic acid are pan-HDAC inhibitors.
Chidamide containing the benzamide is a relative Class I
HDAC inhibitor. A great effort of developing HDAC inhibitors
has been focused on compounds with either hydroxamic acid
or benzamide as the ZBG.12,13 Although many new HDAC
inhibitors are put in clinical trials, there is none approved yet.20
HDAC inhibitors with a novel structure need to be considered.
Romidepsin is a natural product derived from Chromobacte-
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Table 1. Activities of PsA Monomers with the Modified −SH to Inhibit HDAC Activity and Cell Growth
a
Compd.
X-Group
HL-60 GI50 [μM]
HDAC1 IC50 [μM]
HDAC2 IC50 [μM]
HDAC3 IC50 [μM]
HDAC6 IC50 [μM]
b
PsA
−S-Dimer
−SH
−CH3
−CH2CH3
−OH
−COOH
0.37 0.04
0.68 0.08
13.5 0.5
0.008 0.006
0.052 0.018
>10
>10
>10
>10
0.033 0.007
0.21 0.16
>10
>10
>10
0.0124 0.0004
0.12 0.02
>10
>10
>10
0.311 0.008
0.96 0.05
>10
>10
>10
PsA-SH
6a
6b
9
36
20
4
2
11b
11.8 1.2
>10
>10
>10
a
b
HL-60 cells were treated for 72 h. Values are means plus SD of three independent experiments. PsA was reduced with TCEP before enzyme
activity inhibition assay.
Figure 2. FACS and Western blot analyses of apoptosis induction by PsA and PsA-SH in HL-60 cells. (A) HL-60 cells were treated with PsA and
PsA-SH at the indicated concentrations for 24 h, and apoptotic cells were determined by FACS after staining with PI/Annexin-V. (B) Western blot
analysis of the protein levels of Ac-H3, H3, Ac-α-tubulin, α-tubulin, PARP, caspase-3, -8, -9, and β-actin of HL-60 cells treated with PsA and PsA-
SH for 24 h.
rium violaceum with a cyclic peptide containing a disulfide
bond (Figure 1). Romidepsin is a class I selective HDAC
inhibitor and needs the reduced thiol group (−SH) to
coordinate the Zn2+ for HDAC inhibition.21 Compounds
with a disulfide bond and/or thiol group would be a great
source of developing Class I HDAC selective inhibitors.
Psammaplin A (PsA) is a symmetrical bromotyrosine-
derived disulfide dimer isolated from the Psammaplysilla
sponge (Figure 1). PsA has various bioactivities such as
antimicrobial activity and cytotoxicity against cancer.22 PsA has
been shown to inhibit several enzymes including DNA gyrase,
DNA topoisomerase, farnesyl protein transferase, and leucine
aminopeptidase.22 More intriguingly, PsA was found to be a
potent inhibitor of HDAC.23,24 Further studies revealed that
PsA, like Romidepsin, is a Class I HDAC inhibitor25 and needs
to be reduced into PsA-SH monomer for HDAC inhib-
ition.24,26 Previous modifications of PsA have been focused on
the bromotyrosine moiety with the dimer structure.24,25,27,28
PsA inhibited cancer cell growth with apoptosis induction, but
the connection with HDAC inhibition was not clearly
understood.25,29−31 We take the advantage of the simple
structure of PsA-SH to dissect connection of HDAC inhibition
and apoptosis induction by modifying the −SH group. We
designed and synthesized PsA-SH monomer analogues with
deleted −SH or replaced by the hydroxamic acid or by the
benzamide, and compared their growth inhibition and
apoptosis induction in tumor cells.
We generated three groups of compounds without changing
the SRM of PsA-SH. The synthetic routes to compounds PsA
(1), PsA-SH (2), 6a−6b, 9, 10a−10g, 11b, and 12a−12g are
depicted in Scheme 1. Coupling of 3-bromo-4-hydroxybenzal-
dehyde with imidazolidine-2,4-dione gave 3, which was then
hydrolyzed in NaOH aqueous to afford 4, which was
subsequently treated with hydroxylamine hydrochloride and
NaHCO3 under basic condition to afford 5. The intermediate
5 was treated with cystamine dihydrochloride in DMF to
afford compound 1, which was subsequently treated with DL-
dithiothreitol under basic condition to afford 2. Coupling of
intermediate 5 with appropriate amine gave the compounds
6a−6b. The appropriate amino acids were treated with SOCl2
in methanol to afford intermediates 7a−7g, then 8a−8g were
prepared by coupling the above methyl ester with intermediate
5. The intermediate 8a was reduced with LiAlH4 to afford
compound 9. The intermediates 8a−8g were treated with 50%
hydroxylamine under basic conditions to afford compounds
10a−10g. The intermediates 8a−8g were hydrolyzed in
aqueous NaOH to afford intermediates 11a−11g. Then
coupling of 11a−11g with benzene-1,2-diamine gave com-
pounds 12a−12g.
The first group of compounds are PsA-SH with deleted
−SH. We kept the scaffold of PsA-SH and converted the −SH
to −Me, −Et, −OH, and −COOH, respectively, and obtained
analogues 6a, 6b, 9, and 11b. The inhibitory effects of these
compounds on HDAC activity and cell growth were tested
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PsA converts to PsA-SH in cells and that −SH is a key
functional group for inhibiting cell growth and HDAC activity.
Apoptosis induction is a key mechanism of HDAC inhibitors
to inhibit tumor cell growth.32 We tested and compared the
apoptosis induction abilities of PsA and PsA-SH by measuring
the content of cells stained positive for Annexin V with flow
cytometry in HL-60 cells. PsA at 4 μM and PsA-SH at 8 μM
induced about 30% of cells undergoing apoptosis (Figure 2A).
Western blot analysis revealed that treatment with PsA and
PsA-SH induced the typical apoptotic signaling changes
including cleavage of PARP, caspase-3, caspase-8, and
caspase-9. Both PsA and PsA-SH increased the levels of
acetylated-Histone 3 (H3), the substrate of HDAC1/2/3.
Although both PsA and PsA-SH exhibited an inhibitory effect
on HDAC6 activity in enzyme activity assay, they did not
increase the levels of acetylated α-tubulin, the specific substrate
of HDAC6 (Figure 2B), suggesting that intracellular HDAC6
activity was not inhibited in cells. Compounds 6a, 9, and 11b
neither induced Annexin-V positive cells nor increased the
levels of acetylated−H3 even at a concentration up to 20 μM
(Figure 3). These data indicate that apoptosis induction by
PsA and PsA-SH associates with inhibition of intracellular
HDAC1, 2, and 3, but not HDAC6.
The second group of compounds are the −SH of PsA-SH
converted to the hydroxamic acid. We generated compounds
10a−g substituted with the hydroxamic acid. These com-
pounds keep the SRM and have different linker lengths (Table
2). 10b is a compound with direct replacement of −SH with
the hydroxamic acid. 10b maintains the abilities of inhibiting
HDAC2 and 3 but with increased abilities of inhibiting
HDAC1 and HDAC6, that is consistent with a previously
reported work.25 The growth inhibitory effect of 10b in HL-60
cells is similar to that of PsA-SH. 10a has one-carbon short
linker compared to 10b and the inhibitory effects on HDAC1,
2, 3, and 6 activity as well as on HL-60 cell growth are
significantly decreased. Compounds 10c−d have one and two
carbons increased linker (Table 2). 10c only exhibits weak
inhibition on HDAC1 with IC50 about 1 μM. 10d exhibits
inhibition on HDAC1, 3, and 6, with an IC50 of 0.436 μM,
0.249 μM, and 1.29 μM, respectively. The IG50 of 10a, 10c,
and 10d of inhibiting HL-60 cell growth are higher than 40
μM. These data indicate that directly converting −SH of PsA-
SH to the hydroxamic acid becomes a pan-HDAC inhibitor.
By comparing the apoptosis induction with PsA and PsA-SH
it was found that 10b was as effective as PsA to induce
apoptosis in HL-60 cells (Figure 4A). Two μM of 10b induced
about 30% of cells undergoing apoptosis. Western blot analysis
showed that 10b induced cleavage of PARP and caspases with
Figure 3. Apoptosis and H3 acetylation levels of HL-60 cells treated
with 6a, 9, and 11b. Apoptotic cells were determined by FACS after
staining with PI/Annexin-V (A), and the acetylated levels of H3 and
α-tubulin were measured with Western blot (B) in HL-60 cells treated
with each compound at 20 μM for 24 h.
using specific recombinant HDAC enzymes and leukemia HL-
60 cells and were compared with that of PsA (1) and PsA-SH
(2). It is to be noted that PsA was reduced with tris(2-
carboxyethyl)phosphine (TCEP) before determining HDAC
activity inhibition as reported.25 PsA inhibited activities of
HDAC1, 2, and 3 with an IC50 of 8 nM, 33 nM, and 12.4 nM,
respectively, and inhibited HDAC6 with an IC50 of 311 nM
(Table 1). PsA-SH has similar inhibitory effects on HDAC1, 2,
and 3, with a little decreased ability of inhibiting HDAC6. PsA
inhibited HL-60 cell growth with a IG50 of 0.37 μM. PsA-SH
has a IG50 of 0.68 μM, 2-fold higher than that of PsA,
suggesting that PsA splits into two molecules of PsA-SH in
cells to act. Compounds 6a, 6b, 9, and 11b did not exhibit
inhibitory effects on cell growth nor on HDAC activity at a
concentration up to 10 μM (Table 1). These data support that
Table 2. Inhibitory Effects of PsA Monomers with the Hydroxamic Acid Substitution on HDAC Activity and Cell Growth
a
Compd.
n
HL-60 IG50 [μM]
HDAC1 IC50 [μM]
HDAC2 IC50 [μM]
HDAC3 IC50 [μM]
HDAC6 IC50 [μM]
10a
10b
10c
10d
1
2
3
4
>40
0.57 0.19
>40
2.93
0.018 0.004
1.01
9.21
0.033 0.001
>10
>10
1.23
0.020 0.002
>10
>10
0.096 0.013
>10
1.29
>40
0.436
0.249
a
HL-60 cells were treated for 72 h. Values are means plus SD of three independent experiments.
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Figure 4. Apoptosis induction and protein regulation by 10b and Tubacin. Apoptotic cells were determined by FACS after staining with PI/
Annexin-V (A), and the protein levels of Ac-H3, Ac-α-tubulin, PARP, caspase-3, -8, -9, and β-actin were determined by Western blotting (B) in
HL-60 cells treated with 10b and Tubacin at the indicated concentrations for 24 h.
Table 3. Inhibitory Effects of PsA Monomers with the Benzamide Substitution on HDAC Activity and Cell Growth
a
Compd.
12a
12b
12c
12d
12e
12f
12g
RGFP966
n
HL-60 IG50 [μM]
HDAC1 IC50 [μM]
HDAC2 IC50 [μM]
HDAC3 IC50 [μM]
HDAC6 IC50 [μM]
1
2
3
4
5
6
7
>40
>40
>40
>40
14.3 3.1
14.0 3.2
5.2 0.4
6.1 1.0
>10
>10
>10
>10
>1
>1
>1
>1
>10
>10
>10
9.76
>1
>1
>1
>1
>10
>10
1.83
1.43
0.18 0.02
0.13 0.04
0.17 0.02
0.19 0.05
>10
>10
>10
>10
>1
>1
>1
>1
a
HL-60 cells were treated for 72 h. Values shown are means plus SD of three independent experiments.
increased levels of both acetylated H3 and acetylated α-tubulin
(Figure 4B). If looking at the dose-dependent effects of 10b
treatment, 10b increased the levels of acetylated α-tubulin at
0.5 μM without inducing PARP cleavage and apoptosis. The
HDAC6 inhibitor Tubacin increased the levels of acetylated α-
tubulin without apoptosis induction. These data indicate that
10b induces apoptosis in a similar way as PsA through
inhibiting class I HDAC1/2/3 but not HDAC6.
The third group of compounds are the −SH of PsA-SH
converted to the benzamide. Benzamide is used as a ZBG to
develop selective Class I HDAC inhibitors.33,34 We replaced
the −SH of PSA-SH with the benzamide and generated several
new analogues 12a−g, which have a linker length of 1 to 7
carbons, respectively (Table 3). 12a and 12b do not show
inhibitory effects on HDAC activity and cell growth. 12c−12d
have weaker inhibitory effects on HDAC3 with IC50 of 1.83
μM and 1.43 μM, respectively. Both compounds do not inhibit
cell growth of HL-60 cells. 12e−g have similar inhibitory
effects on HDAC3 with that of PsA-SH but do not inhibit
HDAC1 and HDAC2 at a concentration of 1 μM. 12e, 12f,
and 12g inhibited growth of HL-60 cells with an IG50 of 14.3
μM, 14.0 μM, and 5.2 μM, respectively, which were much
higher than that of PsA-SH. RGFP966 is a selective HDAC3
inhibitor used as a tool compound. The inhibitory effects on
HDAC activity and cell growth of RGFP966 and 12g were
compared (Table 3). Both compounds exhibited similar
abilities of inhibiting HDAC3 activity and HL-60 cell growth.
12e−g could be considered as novel selective HDAC3
inhibitors with new structures.
The apoptosis induction abilities of 12e, 12f, and RGFP966
in HL-60 cells were measured and compared. These
compounds did not induce apoptosis at 20 μM. When the
concentrations were increased to 40 μM, a small amount of
apoptotic cells were detected (Figure 5A). Western blot
analysis showed that 12e and 12f at 20 μM did not induce
cleavage of PARP and caspases, but with induction of H3
acetylation (Figure 5B). The upregulated H3 acetylation may
come from HDAC3 inhibition, which needs to be further
confirmed. 12e, 12f, and RGFP966 do not increase the levels
of acetylated H3 at 2 μM (data not shown). Comparing to PsA
and 10b, these compounds keep the ability of inhibiting
HDAC3, but not HDAC1/2. Therefore, the apoptosis
induction abilities of PsA, PsA-SH, and 10b should come
from their inhibition of HDAC1/2 but not HDAC3.
PsA-SH inhibits HDAC1 and HDAC3 at the same level
(Table 1), while 12g is more effective to inhibit HDAC3 than
HDAC1 (Table 3). To dissect the difference we did molecular
docking of both PsA-SH and 12g with HDAC1 and HDAC3
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Figure 5. Apoptosis induction and protein regulation by 12e, 12f, and RGFP966 in HL-60 cells. Apoptotic cells were determined by FACS after
staining with PI/Annexin-V (A), and the protein levels of Ac-H3, Ac-α-tubulin, PARP, caspase-3, -8, -9, and β-actin were determined by Western
blotting (B) in HL-60 cells treated with 12e, 12f, and RGFP966 for 24 h.
Figure 6. Putative binding patterns of PsA-SH (A) or 12g (B) within HDAC1 (left panel, green) and HDAC3 (right panel, blue) pockets.
key amino acids within the binding pockets. PsA-SH exhibits
very similar binding patterns within both HDAC1 and HDAC3
pockets, typically forming a coordinate bond with zinc ion
(Figure 6A), suggesting that it could efficiently inhibit both of
them. 12g also exhibits very similar binding patterns within
both HDAC1 and HDAC3 pockets; for example, the carbonyl
and aniline group of 12g forms a coordinate bond with the zinc
ion, and another carbonyl group of 12g is fixed through a
hydrogen bond with Phe204 of HDAC1 and Phe200 of
HDAC3 (Figure 6B). Comparison of the interaction distance
between 12g and the zinc ion reveals that 12g demonstrates
tighter contacts with the zinc of HDAC3 (2.14 Å for the
aniline and 2.39 Å for the carbonyl group, respectively) than
HDAC1 (2.72 Å for the aniline and 2.84 Å for the carbonyl
Table 4. Cell Growth Inhibition Profiles for PsA, PsA-SH,
10b, 12g, and RGFP966 in Four Solid Tumor Cell Lines
MCF-7 IG50 HCT116 IG50
A549 IG
LL/2 IG
a50
a
a
a50
Compd.
[μM]
4.14 0.06
17
1.09 0.02
17.9 0.2
[μM]
[μM]
[μM]
PsA
PsA-SH
10b
12g
RGFP966
2.3 0.3
5.9 0.6
0.90 0.05
11.9 1.7
2.9 0.7
10.7 1.5
1.1 0.4
2.64 0.04
3.9 0.7
5.0 0.9
12.2 1.0
10.8 0.2
3
26
2
30
8
22
4
28.3 1.5
a
Values are means of at least three independent experiments.
protein. By aligning the crystal structures of HDAC1 (PDB
code 5ICN)35 and HDAC3 (PDB code 4A69)36 we find that
they share very similar binding pockets, with almost identical
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Table 5. Antiproliferative Effects of PsA, PsA-SH, 10b, and SAHA on Lewis Lung Cancer LL/2 Cells in Mice
Body weight [g]
Group
Dose[mg/kg]
No. of mice
Initial
End
Tumor weight [g]
Inhibition rate [%]
*p value
Control
PsA
SAHA
PsA-SH
10b
×12
5
5
5
5
5
18.4 0.4
18.2 0.5
17.9 0.5
18.5 0.8
18.9 0.4
18.1 0.2
18.4 0.4
18.1 0.1
18.3 0.9
18.4 0.6
0.98 0.07
0.34 0.09
0.55 0.07
0.48 0.11
0.45 0.04
25 mg/kg ×12
25 mg/kg ×12
25 mg/kg ×12
25 mg/kg ×12
66.04
45.77
55.99
54.68
<0.001
<0.001
<0.001
<0.001
*
p value compared with the control group.
Table 6. Antiproliferative Effects of 12g and PsA-SH on Lewis Lung Cancer LL/2 Cells in Mice
Body weight [g]
Group
Dose
No. of mice
Initial
End
Tumor weight [g]
Inhibitory [%]
*p value
Control
12g
PsA-SH
×12
100 mg/kg ×12
100 mg/kg ×12
5
5
5
18.6 0.8
18.6 0.4
18.6 0.8
17.8 0.7
18.5 0.3
16.9 0.4
1.2 0.3
0.8 0.2
0.5 0.2
36.48
62.00
<0.001
<0.001
*
p value compared with the control group.
group, respectively), which may explain the relative selectivity
of 12g toward HDAC3 over HDAC1.
inhibitor and a simple scaffold to design more selective HDAC
inhibitors by modifying the ZBG.
PsA, PsA-SH, 10b, 12g, and RGFP966 exhibited different
abilities of inhibiting HL-60 cell growth (Tables 1−3). We
extended their antiproliferative studies to several solid tumor
cell lines including human breast cancer MCF-7 cells, colon
cancer HCT116 cells, nonsmall cell lung cancer A549 cells,
and marine Lewis lung cancer LL/2 cells (Table 4). PsA, PsA-
SH, and 10b are more effective than 12g and RGFP966 to
inhibit growth of those cell lines. 10b is the most effective one
among these compounds, and PsA is generally 2-fold more
active than PsA-SH.
We used LL/2 cells to test the in vivo antitumor effects of
PsA, PsA-SH, 10b, and 12g in mice. Since PsA, PsA-SH, and
10b have similar antiproliferative effects in vitro, we gave the
same doses of them to the mice for comparison. Mice
inoculated with LL/2 cells were treated with each agent for 12
days at a dose of 25 mg/kg. The tumor growth inhibition rates
of PsA, PsA-SH, and 10b were 66.0%, 55.9%, and 54.7%,
respectively. SAHA was used as a control with an inhibition
rate of 45.8% (Table5). 12g at 25 mg/kg did not exhibit
antitumor effects. When the dose was increased to 100 mg/kg,
12g only had an inhibition rate of 36.5%, less than that of PsA-
SH at the same dose (Table 6). The body weights of mice did
not decrease after treatment with each agent, suggesting that
they are not toxic at the used doses. PsA, PsA-SH, and 10b
have similar antitumor effects in vitro and in vivo, and 12g has
decreased antitumor effects in vitro and in vivo. PsA, PsA-SH,
10b, and SAHA have similar inhibition rates of tumor growth
in vivo. Increased doses of PsA-SH could not significantly
change the inhibition rate.
ASSOCIATED CONTENT
* Supporting Information
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acsmedchemlett.0c00369.
■
sı
Synthetic methods, characterization of compounds,
biological assays for cell growth, enzyme activity, and
Western blot, and high-solution mass spectra (PDF)
AUTHOR INFORMATION
Corresponding Authors
■
Yongkui Jing − Department of Pharmacology, Liaoning Key
Lab of Targeting Drugs for Hematological Malignancies,
Wuya College of Innovation, Shenyang Pharmaceutical
University, Shenyang 110016, R. P. China; orcid.org/
0000-0002-7915-8593; Phone: 86-24-23986975;
Email: jingyk@syphu.edu.cn
Linxiang Zhao − Key Laboratory of Structure-Based Drugs
Design & Discovery of Ministry of Education, School of
Pharmaceutical Engineering, Shenyang Pharmaceutical
University, Shenyang 110016, China; Phone: 86-024-
43520221; Email: linxiang.zhao@vip.sina.com
Authors
Yu Bao − Department of Pharmacology, Liaoning Key Lab of
Targeting Drugs for Hematological Malignancies, Wuya
College of Innovation, Shenyang Pharmaceutical University,
Shenyang 110016, R. P. China
Qihao Xu − Key Laboratory of Structure-Based Drugs Design
& Discovery of Ministry of Education, School of
Pharmaceutical Engineering, Shenyang Pharmaceutical
University, Shenyang 110016, China
Lin Wang − Department of Pharmacology, Liaoning Key Lab
of Targeting Drugs for Hematological Malignancies, Wuya
College of Innovation, Shenyang Pharmaceutical University,
Shenyang 110016, R. P. China
Yunfei Wei − Key Laboratory of Structure-Based Drugs Design
& Discovery of Ministry of Education, School of
Pharmaceutical Engineering, Shenyang Pharmaceutical
University, Shenyang 110016, China
In summary, PsA-SH is the active molecule of PsA with
abilities of inhibiting HDAC1/2/3 and inducing apoptosis.
The −SH group plays an essential role in HDAC inhibition
and apoptosis induction of PsA-SH. Depletion of the −SH
group loses the abilities of HDAC inhibition and apoptosis
induction. Replacement of the −SH group with the
hydroxamic acid increases the ability of inhibiting HDAC6
which does not contribute to apoptosis induction. Replace-
ment of the −SH group with the benzamide converts PsA-SH
into a selective inhibitor of HDAC3 without apoptosis
induction. Our data indicate that PsA-SH is a Class I HDAC
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Letter
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Baichun Hu − Key Laboratory of Structure-Based Drugs
Design & Discovery of Ministry of Education, School of
Pharmaceutical Engineering, Shenyang Pharmaceutical
University, Shenyang 110016, China
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University, Shenyang 110016, China; orcid.org/0000-
0002-9589-4056
Dan Liu − Key Laboratory of Structure-Based Drugs Design &
Discovery of Ministry of Education, School of Pharmaceutical
Engineering, Shenyang Pharmaceutical University, Shenyang
110016, China
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Complete contact information is available at:
https://pubs.acs.org/10.1021/acsmedchemlett.0c00369
Author Contributions
#Y.B. and Q.X. contributed equally to this work.
Funding
This work was partly supported by Samuel Waxman Cancer
Research foundation.
Notes
The authors declare no competing financial interest.
ABBREVIATIONS
■
Ac-H3, acetyl-Histone H3; COOH, carboxyl; DMSO,
dimethyl sulfoxide; Et, ethyl; FACS, fluorescence-activated
cell sorting; HDAC, histone deacetylase; IC50, half maximal
inhibitory concentration; IG50, half the growth inhibition
concentration; Me, methyl; MTT, methyl thiazolyltetrazolium
bromide; OH, hydroxyl; PARP, poly-(ADP-ribose)-polymer-
ase; PI, propidium iodide; PsA, psammaplin A; PMSF,
phenylmethylsulfonylfluoride; PsA-SH, semipsammaplin A;
SAHA, vorinostat; SDS, sodium dodecyl sulfate; -SH,
sulfhydryl; SRM, surface recognition moiety; ZBG, zinc
binding group
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