Rational Design, synthesis and biological evaluation of novel triazole derivatives as potent and selective PRMT5 inhibitors with antitumor activity

Article abstract of DOI:10.1007/s10822-019-00214-y

Protein arginine methyltransferase 5 (PRMT5) is responsible for the mono-methylation and symmetric dimethylation of arginine, and its expression level and methyl transferring activity have been demonstrated to have a close relationship with tumorigenesis, development and poor clinical outcomes of human cancers. Two PRMT5 small molecule inhibitors (GSK3326595 and JNJ-64619178) have been put forward into clinical trials. Here, we describe the design, synthesis and biological evaluation of a series of novel, potent and selective PRMT5 inhibitors with antiproliferative activity against Z-138 mantle cell lymphoma cell line. Among them, compound C_4 exhibited the highest potency with enzymatic and cellular level IC₅₀ values of 0.72 and 2.6 μM, respectively, and displayed more than 270-fold selectivity toward PRMT5 over several other isoenzymes (PRMT1, PRMT4 and PRMT6). Besides, C_4 demonstrated obvious cell apoptotic effect while reduced the cellular symmetric arginine dimethylation levels of SmD3 protein. The potency, small size, and synthetic accessibility of this compound class provide promising hit scaffold for medicinal chemists to further explore this series of PRMT5 inhibitors.

Full text of DOI:10.1007/s10822-019-00214-y

Journal of Computer-Aided Molecular Design
https://doi.org/10.1007/s10822-019-00214-y
Rational Design, synthesis and biological evaluation of novel triazole
derivatives as potent and selective PRMT5 inhibitors with antitumor
activity
Kongkai Zhu1 · Jingwei Shao2 · Hongrui Tao1 · Xue Yan1 · Cheng Luo3 · Hua Zhang1 · Wenhu Duan2
Received: 24 April 2019 / Accepted: 11 July 2019
© Springer Nature Switzerland AG 2019
Abstract
Protein arginine methyltransferase 5 (PRMT5) is responsible for the mono-methylation and symmetric dimethylation of
arginine, and its expression level and methyl transferring activity have been demonstrated to have a close relationship
with tumorigenesis, development and poor clinical outcomes of human cancers. Two PRMT5 small molecule inhibitors
(GSK3326595 and JNJ-64619178) have been put forward into clinical trials. Here, we describe the design, synthesis and
biological evaluation of a series of novel, potent and selective PRMT5 inhibitors with antiproliferative activity against Z-138
mantle cell lymphoma cell line. Among them, compound C_4 exhibited the highest potency with enzymatic and cellular
level IC50 values of 0.72 and 2.6 μM, respectively, and displayed more than 270-fold selectivity toward PRMT5 over several
other isoenzymes (PRMT1, PRMT4 and PRMT6). Besides, C_4 demonstrated obvious cell apoptotic efect while reduced
the cellular symmetric arginine dimethylation levels of SmD3 protein. The potency, small size, and synthetic accessibility of
this compound class provide promising hit scafold for medicinal chemists to further explore this series of PRMT5 inhibitors.
Keywords PRMT5 inhibitor · Anti-proliferative · Cellular target validation · Design and synthesis
Introduction
Kongkai Zhu and Jingwei Shao have contributed equally to this
work.
Protein arginine methylation catalyzed by protein argi-
nine methyltransferases (PRMTs) is an important type of
post-translational modifcation that afects crucial cellular
processes including cell growth and proliferation, apop-
tosis, metastasis and angiogenesis via modulation of gene
transcription and/or protein function [1–3]. The identifed
nine human PRMTs (PRMT1–9) are further subdivided
into type I, type II, and type III enzymes according to the
states of the methylated arginine. PRMT5 belongs to the
predominant type II PRMTs which transfer methyl groups
from S-adenosylmethionine (SAM) to a guanidine nitrogen
of protein arginine residues, resulting in the reaction prod-
ucts ω-NG-monomethylarginine (MMA), symmetric ω-NG,
N′G-dimethylarginine (sDMA) and S-adenosylhomocysteine
(SAH). Proteomic analysis of human tissues revealed that
PRMT5, -1 and -4 exhibited more universal expression
compared to PRMT2, -3, -6, -7, and -8 [4]. Through the
symmetric dimethylation of diverse substrate proteins in
the nucleus and cytoplasm, PRMT5 mediated various cel-
lular processes, such as transcription [5], RNA metabolism
[6], signal transduction [7], and cellular diferentiation [8],
Electronic supplementary material The online version of this
article (https://doi.org/10.1007/s10822-019-00214-y) contains
supplementary material, which is available to authorized users.
* Kongkai Zhu
hkhhh.k@163.com
* Hua Zhang
bio_zhangh@ujn.edu.cn
* Wenhu Duan
whduan@simm.ac.cn
1
School of Biological Science and Technology, University
of Jinan, Jinan 250022, People’s Republic of China
2
Department of Medicinal Chemistry, Shanghai Institute
of Materia Medica, Chinese Academy of Sciences (CAS),
Shanghai 201203, People’s Republic of China
3
Drug Discovery and Design Center, CAS Key Laboratory
of Receptor Research, State Key Laboratory of Drug
Research, Shanghai Institute of Materia Medica,
Chinese Academy of Sciences, Shanghai 201203,
People’s Republic of China
Vol.:(0123456789)
1 3

Journal of Computer-Aided Molecular Design
etc. The symmetric dimethylation of histones (H4, H3, and
H2A), Sm proteins (B/B′, D1, and D3) are the hallmarks
associated with PRMT5 activity [9, 10].
Tremendous efforts from a number of groups have
been made to discover PRMT5 inhibitors [20, 23, 25–33],
and all reported PRMT5 inhibitors are summarized in
Fig. 1. Among them, LLY-283 [31], Sinefungin [34],
and A9145C [10] are SAM binding site inhibitors, while
EPZ015666 [23] together with its analogue GSK3326595
[35] are substrate binding site inhibitors. EPZ015666 is
the frst PRMT5 inhibitor to be described with potent
activities in both in vitro and in vivo models of Mantle
cell lymphoma. Besides, the phase I of clinical trial of
GSK3326595 for the treatment of solid tumors and non-
Hodgkin’s lymphoma is going on. And compound JNJ-
64619178 [36] was reported to bind into the SAM bind-
ing pocket and meanwhile to reach the substrate binding
pocket. Although more than 10 classes of PRMT5 inhibi-
tors have been reported, most of them exhibit low potency
or lack in vivo activity. Therefore, the search for more
potent PRMT5 inhibitors remains a pressing task.
Interestingly, accumulating studies implicated that
PRMT5 was an attractive therapeutic target in cancer. It
is overexpressed or upregulated in several human cancers,
including lymphomas [11–13], breast cancer [14], lung can-
cer [15], colorectal cancer [16], hepatocellular carcinoma
[17], multiple myeloma [18], and glioblastoma [19]. It was
reported that selective inhibition of PRMT5 could block ini-
tiation and maintenance of B cell transformation [20]. The
importance of PRMT5 in breast cancer stem cell (BCSC)
maintenance was also highlighted [21]. Besides, PRMT5
was documented to control melanoma growth through the
SKI/SOX10 regulatory axis [22]. PRMT5 has been proposed
as an efective target in mantle cell lymphoma (MCL) [23],
glioblastoma [19], multiple myeloma [18], and mixed line-
age leukemias (MLL)-rearranged acute myeloid leukemia
[24].
Fig. 1 Previously reported
O
O
O
HN
N
N
N
N
HO
H2N
HO
PRMT5 inhibitors
NH2
N
N
N
HN
NH2
NH2
N
NH2
HO
NH2
H2N
S
O
N
N
O
N
O
N
OH
OH
OH
HO
HO
Sinefungin (IC50 = 8.6 µM)
DS-437 (IC50 = 5.9 µM)
A9145C (IC50 = 0.035 µM)
Br
N
N
N
N
N
OH
NH2
NH2
OH
N
NH2
H2N
F
O
O
N
N
N
N
Cl
HO
HO
OH
OH
HO
OH
Inhibitor 1 (IC50 = 0.001 µM)
LLY-283 (IC50 = 0.022 µM)
JNJ-64619178 (IC50 = 0.14 nM)
O
Br
O
N
O
N
N
N
OH
S
OH
H
N
N
Cl
NH2
N
O
N
H
H
S
O
GSK3326595 (IC50 = 0.0062 µM)
Inhibitor 19 (IC50 = 6.6 µM)
P5i-6 (IC50 = 0.57 µM)
O
O
O
H
N
N
N
O
O
O
N
H
S
N
H
N
O
O
NH OH
P1608K04 (IC50 = 1.5 µM)
Inhibitor 17 (IC50 = 0.33 µM)
DC-C01 (IC50 = 2.8 µM)
O
N
H
H
N
N
H
N
N
O
O
N
HO
N
Cl
NH2
O
O
S
N
N
HO
C9a (IC50 = 0.7 µM)
C2 (IC50 = 0.1 µM)
Inhibitor 9-1 (IC50 = 14 µM)
O
N
N
OH
O
H
N
N
N
F
N
N
N
NH
H
H
H
O
O
NC
DC_Y134 (IC50 = 1.7 µM)
EPZ015666 (IC50 = 0.022 µM)
4b14 (IC50 = 2.7 µM)
1 3

Journal of Computer-Aided Molecular Design
In the present study, we report the identifcation of a
series of PRMT5 inhibitors with potent antiproliferative
activity against Z-138 mantle cell lymphoma cell line. The
most potent one (C_4) was employed to characterize its
cell apoptotic efect and cellular target validation.
Western blot analysis
Exponentially growing Z-138 cells were incubated with
compound C_4 with diferent concentrations for 4 days.
Then cells were harvested and lysed in 100 μL of total lysis
bufer, and total cell lysates were separated on 4–12% SDS-
PAGE and transferred to PVDF membrane (Millipore).
Afterward, blocking bufer (5% nonfat milk in 0.1% Tween
20 PBS bufer) was used to block the Blots. Subsequently,
primary antibodies [SDMA (CST, 13222s), GAPDH (CST,
5174) and SmD3 (Sigma, HPA001170-100UL), all prepared
in a 1:3 dilution of blocking bufer] were added to Blots
and incubated overnight at 4 °C. The next day, after being
washed three times with PBST, the Blots were incubated
with 1:10,000 dilution of donkey anti-rabbit secondary
antibody (HRP conjugated) for 2 h. Finally, the signal was
detected using ChemiScope3400 imaging system (Clinx Sci-
ence Instruments).
Materials and methods
PRMT5, PRMT1, PRMT4 and PRMT6 enzymatic
activity inhibition assays
Using a radioactivity-based assay to monitor the transfer
3
of the methyl group from H-SAM to peptide substrate,
we tested the inhibitory activity of our compounds against
PRMT5:MEP50. The detailed methods to test the inhibi-
tion activity of compound C_4 against PRMT5, PRMT1,
PRMT4 and PRMT6 were as we have previously described
[29].
Flow cytometric analysis
Exponentially growing Z-138 cells were plated in 6-well
plates at a density of 5 × 105 and treated with compound
C_4, EPZ015666 (positive control) or DMSO (control).
Cells were harvested at 48 h and were then measured using
AnnexinV-FITC Apoptosis Detection Kit (Vazyme Biotech).
FlowJo V7.6.1 was used to detect samples and to analyze
data.
Molecular docking
Molecular docking was performed using Glide pro-
gram inserted in Maestro 7.5. The coordinates of
PRMT5:EPZ015666 (PDB code: 4X61) were used to
construct docking model. First, the protein part was mini-
mized by Protein Preparation Wizard Workfow. After that,
docking grids were created by defning residues within 15
Å around EPZ015666. Then the prepared (using LigPrep
panel) GSK3326595 was docked to the pre-defned dock-
ing grids with standard precision mode.
Chemistry
All purchased chemicals and solvents were used without
further purifcation unless otherwise noted. Flash chroma-
tography was performed using silica gel (300–400 mesh).
All reactions were monitored by thin-layer chromatography
(TLC), using silica gel plates with fuorescence F254 and
visualized under UV light. 1H NMR data were recorded on
Varian Mercury 300 or 400 NMR spectrometers. Chemical
shifts (δ) were reported in parts per million (ppm), coupling
constant (J) values were in hertz, and the splitting patterns
were abbreviated as follows: s for singlet; d for doublet; t
for triplet; q for quartet and m for multiplet. High resolution
ESI–MS spectra were recorded on a Finnigan/MAT95 spec-
trometer. All the fnal compounds are racemates. Compound
5 was synthesized according to the reported procedures [37].
All the compounds were synthesized as illustrated in
Schemes 1, 2, and 3. Compound 1 was reacted with 2-(chlo-
romethyl)oxirane in the presence of base to aford the inter-
mediate epoxide 2. Benzenesulfonyl chloride derivatives
were treated with ammonium hydroxide to aford benze-
nesulfonamides. Compounds A_1–A_10 were prepared
through ring-opening reaction of epoxide (compound 2)
under solid–liquid phase-transfer catalytic conditions.
In vitro anti‑proliferation assay
Long-term proliferation assays were performed on Z-138
cells using the method we previously reported. In brief,
exponentially growing cells were seeded in 24-well cell
plates in growth medium (RPMI1640 medium (Invitrogen)
supplemented with 10% fetal bovine serum and 1% peni-
cillin/streptomycin) at a density of 1 × 105 cells per well.
Cells were incubated in the presence of increasing con-
centrations of the tested compounds up to 200 μM. Viable
cell number was determined every 4 days for up to 8 days
using the Cell Titer-Glo Luminescent Cell Viability kit
(Promega). On days of cell counting, growth media and
compounds were replaced and cells split back to a density
of 1×105 cells per well. IC50 values were determined from
at least three independent assays and calculated from the
concentration-dependence curves.
1 3

Journal of Computer-Aided Molecular Design
a
Scheme 1 Synthesis of com-
pounds A_1–A_10a. aReagents
and conditions: a 2-(chlorome-
thyl)oxirane, K2CO3, MeOH,
rt; b NH2OH, MeCN, 0 °C; c
K2CO3, TEBAC, 1,4-dioxane,
90 °C
O
NH
N
R1
1
3
2
H
O
c
H
N
N
S
O
O
O
O
−
A 1 A 10
b
_
_
S
O
S
O
l
C
NH2
R1
R1
4
O
Scheme 2 Synthesis of
O
compounds B_1 and B_2a. aRe-
agents and conditions: a tetrahy-
dro-2H-pyran-4-amine or 11b,
K2CO3, DMF, 115 °C; b i Pd/C,
H2, MeOH; ii 2, EtOH, 120 °C,
sealed; c i Pd/C, H2, MeOH; ii
NaBH3CN, dihydro-2H-pyran-
4(3H)-one, MeOH
O2N
S
N
N
S
a
b
O2N
Cl
S
NH
NH
N
N
H
N
OH
5
6
B 1
_
a
NO2
NH
N
S
S
N
N
c
N
N
N
H
H
O
OH
OH
7
B
_
2
Si
r
B
a
b
c
R1
R1
R1
1
1
1
1
1
1
a:
8
=
=
-
=
=
-
N
O2
a:
10
=
=
-
3
N
O2
a:
R
R
3
N
O2
9
R
R
3
R
R
d
:
-
:
-
:
-
8b
4 N
O2
9b
4 N
10b
4 N
O2
O2
N
N
N
R1
O
H
N
N
R2
N
O
1
:
R
= -
3
N
O2
4 NO2
C
_
1
R
OH
1
:
12
=
-
2
a:
=
=
11
R
R
N3
NH2
2
2
:
11b
e
N
N
N
N
N
N
f
R1
R1
O
H
O
H
N
N
3
1
:
2
:
5
=
=
-
NH
C
_
C
_
R
R
2
C
_
C C
3 6
4
, ,
_ _
1
-
4 NH2
a
Scheme 3 Synthesis of compounds C_1–C_6a. Reagents and con-
ditions: a trimethylsilylacetylene, CuI, Pd(PPh3)2Cl2, Et3N, DMF,
microwave irradiation, 120 °C; b K2CO3, MeOH, rt; c for 11a:
NaN3, EtOH, refux; for 11b: NH3 in MeOH, 80 °C in sealed tube;
d CuSO4(H2O)5, sodium ascorbate, THF/H2O; e Pd/C, H2, MeOH; f
NaBH3CN, ketone, MeOH
The synthesis of compounds B_1 and B_2 was shown
in Scheme 2. Compound 5 was treated with tetrahydro-
2H-pyran-4-amine or 11b, afording 6 or 7. Reduction of
6 yielded the amine intermediate which was converted to
compound B_1 by reacting with 2. Compound B_2 was
obtained by a two-step procedure: reduction of 7 and reduc-
tive amination reaction.
Sonogashira cross-coupling with TMS-acetylene and then
deprotected with K2CO3 to yield 10. Compound 2 was treated
with NaN3 or NH3 in MeOH to furnish 11. Compounds C_1
and 12 were prepared via click chemistry from intermediates
10 and 11a. Reduction of C_1 and 12 with hydrogen aforded
C_2 or C_5 that were reacted with ketones to aford the target
compounds.
The synthesis of compounds C_1–C_6 was started from
3- or 4-bromonitrobenzene, which was frst subjected to a
1 3

Journal of Computer-Aided Molecular Design
Fig. 2 Binding mode analysis.
a
The detailed interactions of
respectively. b The comparison of binding modes of GSK3326595
and EPZ015666. The protein and the ligands were shown as surface
and sticks, respectively. c Only the ligands were shown for the pur-
pose of clarity
EPZ015666 with PRMT5. Residues involved in H-bond and hydro-
phobic interactions with EPZ015666 and SAM which established
cation–π interaction with EPZ015666 were shown as sticks. Yel-
low and pink dashed lines indicate H-bond and cation–π interaction,
N-(3-(3,4-dihydroisoquinolin-2(1H)-yl)-2-hydroxypro-
pyl) amino fragment of EPZ015666 forms crucial inter-
action with many residues (L319, T323, F327, E435,
L437, E444 and W579) contributing to its high inhibi-
tory potency against PRMT5. It is worth to note that the
cation-π interaction between tetrahydroisoquinoline ring
and the cofactor SAM was frst proposed and believed to
contribute to the good selectivity of EPZ015666. Most
importantly, the hydroxyl group in EPZ015666 forms
stable H-bond interactions with L437, E444 and W579,
which indicates that it is the key pharmacophore. So we
retained this hydroxyl group in the subsequent molecular
design. Giving that the chirality of the hydroxyl-bonding
carbon was diferent in EPZ015666 and GSK3326595,
Results
Rational design, synthesis, and structure–activity
relationship analysis
As the best two previously reported substrate binding
site inhibitors are GSK3326595 and EPZ015666, we
thus choose them as template compounds for further
structure optimization. Fortunately, the complex crystal
structure of PRMT5 with EPZ015666 was previously
determined, which provided many clues for structure-
based molecular design. Therefore, the detailed inter-
action between EPZ015666 and PRMT5 was analyzed
and shown in Fig. 2a. As can be seen from Fig. 2a, the
Fig. 3 Design of new PRMT5
O
R
R
inhibitors
S
N
N
O
H
H
O
-
A 1 A 10
_
_
N
O
H
N
Structure optimization
S
N
H
N
N
H
N
N
O
N
H
O
N
H
O
B_1 B_2
,
N
R
H
N
O
-
N
C 1 C 6
_
_
1 3

Journal of Computer-Aided Molecular Design
Table 1 PRMT5 inhibitory
Compound
A_1
Structure
IC50
SD b (µΜ)
a
activity of compounds
3.40 0.62
6.09 0.73
A_2
4.27 0.45
A_3
5.72 0.56
A_4
A_5
14.80 2.38
24.74 2.95
59.88 6.34
A_6
A_7
42.66 5.63
A_8
21.69 4.14
21.72 3.67
A_9
A_10
1 3

Journal of Computer-Aided Molecular Design
Table 1 (continued)
Compound
Structure
IC50
SD b (µΜ)
a
4.1 0.35
B_1
6.6 0.64
B_2
5.5 0.64
34 3.54
C_1
C_2
2.4 0.85
C_3
0.72 0.20
31 3.54
C_4
C_5
5.1 1.27
C_6
0.047 0.015
EPZ015666
IC50 half maximal inhibitory concentration, SD standard deviation
and the co-crystal structure of PRMT5 complexed with
GSK3326595 was not determined, molecular docking was
thus used to probe the binding mode of GSK3326595. As
can be seen from Fig. 2b, c, the N-(3-(3,4-dihydroisoquin-
olin-yl)-2-hydroxypropyl) group in GSK3326595 could be
well overlapped with that in EPZ015666, which suggested
that it was the privileged fragment and the chirality of the
hydroxyl-bonding carbon had little efect on the PRMT5
inhibitory activity. As shown in Fig. 2a, apart from the
N-(3-(3,4-dihydroisoquinolin-2(1H)-yl)-2-hydroxypro-
pyl) amino fragment, the remaining fragment (left part)
of EPZ015666 established well-defined hydrophobic
1 3

Journal of Computer-Aided Molecular Design
Table 2 Inhibitory activity of C_4 and EPZ015666 against PRMT1,
the free amino group with extra rings signifcantly improved
the activity by 14- to 17-fold (C_3, C_4 vs. C_2) or sixfold
(C_6 vs. C_5), and the best inhibition against PRMT5 was
found for compound C_4 with a cyclobutyl substitution on
the amino moiety, showing a IC50 value of 0.72 0.20 µM.
-4 and -6 (IC50 in μM) at enzymatic level
Compd No.
PRMT1
PRMT4
PRMT6
C_4
EPZ015666
>200
>200
>200
>200
>200
>200
Selectivity testing indicated that C_4 is a selective
PRMT5 inhibitor
Table 3 The in vitro efects of
B and C series of compounds
on cell proliferation
Compound
IC50 (μM)
As C_4 displayed the most potency, it was selected to evalu-
ate the selectivity over other PRMTs (PRMT1, PRMT4, and
PRMT6). As shown in Table 2, the IC50 values of compound
C_4 against PRMT1, PRMT4, and PRMT6 were all above
200 μM, which indicated that it was a selective PRMT5
inhibitor.
B_1
6.25 1.07
12.85 2.35
17.35 1.65
40.5 3.26
3.23 0.36
2.60 0.81
57.57 5.21
7.81 2.24
0.11 0.02
B_2
C_1
C_2
C_3
C_4
C_5
Anti‑proliferative activity on Z‑138 cells
C_6
EPZ015666
Z-138 cells were used to assess the anti-proliferative efects
upon treatment with our synthesized compounds. A long-
term proliferation assay was developed to measure the cell
growth over 8 days. B and C series of compounds dem-
onstrated potent concentration-dependent anti-proliferative
efects, with IC50 values ranging from 2.60 to 57.57 μM
(Table 3). As indicated by the assay results shown in Table 3,
the cell level activities of these compounds were consist-
ent with those of the enzyme level, and compound C_4
also proved to be the most potent one with an IC50 value of
2.60 0.81 μM. The dose response curve for C_4 against
Z-138 cell lines was shown in Fig. 4a.
interactions with PRMT5, which indicated that the rela-
tive pocket was a hydrophobic pocket. Thus, the design
strategy was to keep the N-(3-(3,4-dihydroisoquinolin-
yl)-2-hydroxypropyl) amino moiety and to replace the
left side part of EPZ015666 with groups that could form
hydrophobic interactions with PRMT5. A total of 18 com-
pounds (Fig. 3) were synthesized according to our design
strategy by utilizing the traditional structure optimization
methods, such as scafold hopping, bioisosteric replace-
ment and aromatic ring replacement (see Supplementary
Material for synthetic details).
Cellular symmetric dimethylation efects
and inducement of cell apoptosis of C_4
The inhibitory activities against PRMT5 at enzymatic
level (Table 1) of the 18 compounds and EPZ015666
were evaluated using the method we previously reported
[29, 38]. From Table 1, it could be seen that the activity of
EPZ015666 (47 nM) remained the same level as reported
(22 nM) in the literature, demonstrating the reliability of
our assay, whereas replacing the amide moiety with the sul-
fonamide group (A_1–A_10), benzo[d]thiazole ring (B_1
and B_2) or triazole-derived fragment (C_1–C_6) caused a
distinct loss of activity. Compounds A_2–A_4 with an elec-
tron-donating substituent on the benzene ring showed much
better inhibitory activity than compounds (A_5–A_10) with
corresponding electron-withdrawing groups, which was dif-
ferent from the C series of compounds. Compound C_1 with
an electron-withdrawing nitro substituent on the benzene
ring showed much better inhibitory activity than C_2 with a
corresponding electron-giving amino group, while the sub-
stituting position of the amino group did not seem to impact
the result (C_2 vs. C_5). Further structural modifcation of
The efect of C_4 on cellular symmetric arginine dimeth-
ylation in Z-138 cells was tested by immunoblot using the
pan-dimethyl symmetric arginine antibody symmetric dime-
thyl arginine (SDMA). As shown in Fig. 4b, concentration-
dependent decrease in the intensity of SmD3me2s was
observed. SmD3 is one of the PRMT5 substrate proteins
and can be used to track the cellular biochemical activity of
PRMT5, so from the western blot result, we can conclude
that compound C_4 could target PRMT5 in cellular context.
Since PRMT5 inhibitors usually induce cancer cell apop-
tosis [19, 33], the efect of C_4 on cell apoptosis was stud-
ied in Z-138 cells for 48 h by fow cytometry. As shown in
Fig. 5, concentration-dependent increase of apoptosis upon
treatment with compound C_4 was observed. This result
further confrmed the cellular target engagement of C_4 on
PRMT5.
1 3

Journal of Computer-Aided Molecular Design
Fig. 4 Cellular anti-proliferative activity and target validation of com-
pound C_4. a Dose response curve for compound C_4 against Z-138
cell line. b Efect of C_4 on cellular symmetric dimethyl arginine
substrate SmD3 in Z-138 cells. Concentration-dependent inhibition of
SmD3me2S after 4 days of treatment with a dose-titration from 5 to
20 μM of compound C_4 in Z-138 cells
Fig. 5 Cell apoptosis induction after treatment with C_4. Treatment with C_4 induced apoptosis of Z-138 cells measured after 48 h
1 3

Journal of Computer-Aided Molecular Design
alternative splicing by PRMT5 reveals a role for Mdm4 pre-
mRNA in sensing defects in the spliceosomal machinery. Genes
Dev 27(17):1903–1916. https://doi.org/10.1101/gad.219899.113
7. Jansson M, Durant ST, Cho EC, Sheahan S, Edelmann M, Kes-
sler B, La Thangue NB (2008) Arginine methylation regulates
the p53 response. Nat Cell Biol 10(12):1431–1439. https://doi.
org/10.1038/ncb1802
Discussion
In the present study, new PRMT5 inhibitors were suc-
cessfully discovered by combination of structure-based
drug design, synthesis and biological evaluation. Among
our synthesized 18 compounds, compound C_4 showed
the best PRMT5 inhibitory activity and profound cellular
anti-proliferative activity with IC50 values of 0.72 0.21
and 2.60 0.81 μM, respectively. Besides, fow cytomet-
ric analysis demonstrated that C_4 exhibited an obvious
apoptotic efect. Compound C_4 also reduced the cellu-
lar symmetric arginine dimethylation level of SmD3, a
protein that can be used to track the biochemical activ-
ity of PRMT5 in cells. The compounds identifed in this
work provide a new scafold for future PRMT5 inhibitor
development.
8. Dacwag CS, Ohkawa Y, Pal S, Sif S, Imbalzano AN (2007) The
protein arginine methyltransferase Prmt5 is required for myo-
genesis because it facilitates ATP-dependent chromatin remod-
eling. Mol Cell Biol 27(1):384–394. https://doi.org/10.1128/
MCB.01528-06
9. Karkhanis V, Hu YJ, Baiocchi RA, Imbalzano AN, Sif S (2011)
Versatility of PRMT5-induced methylation in growth control and
development. Trends Biochem Sci 36(12):633–641. https://doi.
org/10.1016/j.tibs.2011.09.001
10. Brahms H, Meheus L, de Brabandere V, Fischer U, Luhrmann R
(2001) Symmetrical dimethylation of arginine residues in spliceo-
somal Sm protein B/B’ and the Sm-like protein LSm4, and their
interaction with the SMN protein. RNA 7(11):1531–1542
11. Chung J, Karkhanis V, Tae S, Yan F, Smith P, Ayers LW, Ago-
stinelli C, Pileri S, Denis GV, Baiocchi RA, Sif S (2013) Pro-
tein arginine methyltransferase 5 (PRMT5) inhibition induces
lymphoma cell death through reactivation of the retinoblastoma
tumor suppressor pathway and polycomb repressor complex 2
(PRC2) silencing. J Biol Chem 288(49):35534–35547. https://
doi.org/10.1074/jbc.M113.510669
Acknowledgements This research work was fnancially supported by
the National Natural Science Foundation of China (No. 81803438),
National Science and Technology Major Project (2018ZX09711002-
004-013), Shandong Provincial Natural Science Foundation (Nos.
JQ201721, ZR2017BH038), the Young Taishan Scholars Program
(No. tsqn20161037), and Shandong Talents Team Cultivation Plan of
University Preponderant Discipline (No. 10027).
12. Wang L, Pal S, Sif S (2008) Protein arginine methyltransferase 5
suppresses the transcription of the RB family of tumor suppressors
in leukemia and lymphoma cells. Mol Cell Biol 28(20):6262–
6277. https://doi.org/10.1128/MCB.00923-08
13. Pal S, Baiocchi RA, Byrd JC, Grever MR, Jacob ST, Sif S
(2007) Low levels of miR-92b/96 induce PRMT5 translation
and H3R8/H4R3 methylation in mantle cell lymphoma. EMBO J
26(15):3558–3569. https://doi.org/10.1038/sj.emboj.7601794
14. Powers MA, Fay MM, Factor RE, Welm AL, Ullman KS (2011)
Protein arginine methyltransferase 5 accelerates tumor growth
by arginine methylation of the tumor suppressor programmed
cell death 4. Cancer Res 71(16):5579–5587. https://doi.
org/10.1158/0008-5472.CAN-11-0458
References
1. Blanc RS, Richard S (2017) Arginine methylation: the coming
of age. Mol Cell 65(1):8–24. https://doi.org/10.1016/j.molce
l.2016.11.003
2. Cheung N, Chan LC, Thompson A, Cleary ML, So CW (2007)
Protein arginine-methyltransferase-dependent oncogenesis. Nat
Cell Biol 9(10):1208–1215. https://doi.org/10.1038/ncb1642
3. Copeland RA (2013) Molecular pathways: protein methyltrans-
ferases in cancer. Clin Cancer Res 19(23):6344–6350. https://doi.
org/10.1158/1078-0432.CCR-13-0223
15. Wei TY, Juan CC, Hisa JY, Su LJ, Lee YC, Chou HY, Chen JM,
Wu YC, Chiu SC, Hsu CP, Liu KL, Yu CT (2012) Protein arginine
methyltransferase 5 is a potential oncoprotein that upregulates
G1 cyclins/cyclin-dependent kinases and the phosphoinositide
3-kinase/AKT signaling cascade. Cancer Sci 103(9):1640–1650.
https://doi.org/10.1111/j.1349-7006.2012.02367.x
4. Kim MS, Pinto SM, Getnet D, Nirujogi RS, Manda SS, Chaerkady
R, Madugundu AK, Kelkar DS, Isserlin R, Jain S, Thomas JK,
Muthusamy B, Leal-Rojas P, Kumar P, Sahasrabuddhe NA, Bal-
akrishnan L, Advani J, George B, Renuse S, Selvan LD, Patil AH,
Nanjappa V, Radhakrishnan A, Prasad S, Subbannayya T, Raju
R, Kumar M, Sreenivasamurthy SK, Marimuthu A, Sathe GJ,
Chavan S, Datta KK, Subbannayya Y, Sahu A, Yelamanchi SD,
Jayaram S, Rajagopalan P, Sharma J, Murthy KR, Syed N, Goel
R, Khan AA, Ahmad S, Dey G, Mudgal K, Chatterjee A, Huang
TC, Zhong J, Wu X, Shaw PG, Freed D, Zahari MS, Mukherjee
KK, Shankar S, Mahadevan A, Lam H, Mitchell CJ, Shankar SK,
Satishchandra P, Schroeder JT, Sirdeshmukh R, Maitra A, Leach
SD, Drake CG, Halushka MK, Prasad TS, Hruban RH, Kerr CL,
Bader GD, Iacobuzio-Donahue CA, Gowda H, Pandey A (2014)
A draft map of the human proteome. Nature 509(7502):575–581.
https://doi.org/10.1038/nature13302
16. Cho EC, Zheng S, Munro S, Liu G, Carr SM, Moehlenbrink J, Lu
YC, Stimson L, Khan O, Konietzny R, McGouran J, Coutts AS,
Kessler B, Kerr DJ, Thangue NB (2012) Arginine methylation
controls growth regulation by E2F-1. EMBO J 31(7):1785–1797.
https://doi.org/10.1038/emboj.2012.17
17. Jiang H, Zhu Y, Zhou Z, Xu J, Jin S, Xu K, Zhang H, Sun Q, Wang
J, Xu J (2018) PRMT5 promotes cell proliferation by inhibiting
BTG2 expression via the ERK signaling pathway in hepatocellular
carcinoma. Cancer Med 7(3):869–882. https://doi.org/10.1002/
cam4.1360
18. Gulla A, Hideshima T, Bianchi G, Fulciniti M, Kemal Samur M,
Qi J, Tai YT, Harada T, Morelli E, Amodio N, Carrasco R, Tag-
liaferri P, Munshi NC, Tassone P, Anderson KC (2018) Protein
arginine methyltransferase 5 has prognostic relevance and is a
druggable target in multiple myeloma. Leukemia 32(4):996–1002.
https://doi.org/10.1038/leu.2017.334
5. Chen H, Lorton B, Gupta V, Shechter D (2017) A TGFbeta-
PRMT5-MEP50 axis regulates cancer cell invasion through
histone H3 and H4 arginine methylation coupled transcriptional
activation and repression. Oncogene 36(3):373–386. https://doi.
org/10.1038/onc.2016.205
19. Yan F, Alinari L, Lustberg ME, Martin LK, Cordero-Nieves
HM, Banasavadi-Siddegowda Y, Virk S, Barnholtz-Sloan J, Bell
EH, Wojton J, Jacob NK, Chakravarti A, Nowicki MO, Wu X,
Lapalombella R, Datta J, Yu B, Gordon K, Haseley A, Patton
6. Bezzi M, Teo SX, Muller J, Mok WC, Sahu SK, Vardy LA,
Bonday ZQ, Guccione E (2013) Regulation of constitutive and
1 3

Journal of Computer-Aided Molecular Design
by structure-based virtual screening and hit optimization. J Med
Chem 60(14):6289–6304. https://doi.org/10.1021/acs.jmedc
hem.7b00587
JT, Smith PL, Ryu J, Zhang X, Mo X, Marcucci G, Nuovo G,
Kwon CH, Byrd JC, Chiocca EA, Li C, Sif S, Jacob S, Lawler
S, Kaur B, Baiocchi RA (2014) Genetic validation of the protein
arginine methyltransferase PRMT5 as a candidate therapeutic
target in glioblastoma. Cancer Res 74(6):1752–1765. https://doi.
org/10.1158/0008-5472.CAN-13-0884
30. Prabhu L, Chen L, Wei H, Demir O, Safa A, Zeng L, Amaro
RE, O’Neil BH, Zhang ZY, Lu T (2017) Development of an
AlphaLISA high throughput technique to screen for small mol-
ecule inhibitors targeting protein arginine methyltransferases. Mol
BioSyst 13(12):2509–2520. https://doi.org/10.1039/c7mb00391a
31. Bonday ZQ, Cortez GS, Grogan MJ, Antonysamy S, Weichert K,
Bocchinfuso WP, Li F, Kennedy S, Li B, Mader MM, Arrowsmith
CH, Brown PJ, Eram MS, Szewczyk MM, Barsyte-Lovejoy D,
Vedadi M, Guccione E, Campbell RM (2018) LLY-283, a potent
and selective inhibitor of arginine methyltransferase 5, PRMT5,
with antitumor activity. ACS Med Chem Lett 9(7):612–617. https
://doi.org/10.1021/acsmedchemlett.8b00014
20. Alinari L, Mahasenan KV, Yan F, Karkhanis V, Chung JH, Smith
EM, Quinion C, Smith PL, Kim L, Patton JT, Lapalombella R,
Yu B, Wu Y, Roy S, De Leo A, Pileri S, Agostinelli C, Ayers L,
Bradner JE, Chen-Kiang S, Elemento O, Motiwala T, Majumder
S, Byrd JC, Jacob S, Sif S, Li C, Baiocchi RA (2015) Selective
inhibition of protein arginine methyltransferase 5 blocks initiation
and maintenance of B-cell transformation. Blood 125(16):2530–
2543. https://doi.org/10.1182/blood-2014-12-619783
21. Chiang K, Zielinska AE, Shaaban AM, Sanchez-Bailon MP, Jar-
rold J, Clarke TL, Zhang J, Francis A, Jones LJ, Smith S, Barbash
O, Guccione E, Farnie G, Smalley MJ, Davies CC (2017) PRMT5
is a critical regulator of breast cancer stem cell function via his-
tone methylation and FOXP1 expression. Cell Rep 21(12):3498–
3513. https://doi.org/10.1016/j.celrep.2017.11.096
32. Ye F, Zhang W, Ye X, Jin J, Lv Z, Luo C (2018) Identifcation
of selective, cell active inhibitors of protein arginine methyl-
transferase 5 through structure-based virtual screening and bio-
logical assays. J Chem Inf Model 58(5):1066–1073. https://doi.
org/10.1021/acs.jcim.8b00050
33. Zhu K, Tao H, Song JL, Jin L, Zhang Y, Liu J, Chen Z, Jiang CS,
Luo C, Zhang H (2018) Identifcation of 5-benzylidene-2-phe-
nylthiazolones as potent PRMT5 inhibitors by virtual screening,
structural optimization and biological evaluations. Bioorg Chem
81:289–298. https://doi.org/10.1016/j.bioorg.2018.08.021
34. Antonysamy S, Bonday Z, Campbell RM, Doyle B, Druzina Z,
Gheyi T, Han B, Jungheim LN, Qian Y, Rauch C, Russell M,
Sauder JM, Wasserman SR, Weichert K, Willard FS, Zhang A,
Emtage S (2012) Crystal structure of the human PRMT5:MEP50
complex. Proc Natl Acad Sci USA 109(44):17960–17965. https
://doi.org/10.1073/pnas.1209814109
22. Tamiya H, Kim H, Klymenko O, Kim H, Feng Y, Zhang T, Han
JY, Murao A, Snipas SJ, Jilaveanu L, Brown K, Kluger H, Zhang
H, Iwai K, Ronai ZA (2018) SHARPIN-mediated regulation of
protein arginine methyltransferase 5 controls melanoma growth.
J Clin Investig 128(1):517–530. https://doi.org/10.1172/JCI95410
23. Chan-Penebre E, Kuplast KG, Majer CR, Boriack-Sjodin PA,
Wigle TJ, Johnston LD, Rioux N, Munchhof MJ, Jin L, Jacques
SL, West KA, Lingaraj T, Stickland K, Ribich SA, Raimondi A,
Scott MP, Waters NJ, Pollock RM, Smith JJ, Barbash O, Pap-
palardi M, Ho TF, Nurse K, Oza KP, Gallagher KT, Kruger R,
Moyer MP, Copeland RA, Chesworth R, Duncan KW (2015) A
selective inhibitor of PRMT5 with in vivo and in vitro potency
in MCL models. Nat Chem Biol 11(6):432–437. https://doi.
org/10.1038/nchembio.1810
35. Gerhart SV, Kellner WA, Thompson C, Pappalardi MB, Zhang
XP, Montes de Oca R, Penebre E, Duncan K, Boriack-Sjodin A,
Le B, Majer C, McCabe MT, Carpenter C, Johnson N, Kruger RG,
Barbash O (2018) Activation of the p53-MDM4 regulatory axis
defnes the anti-tumour response to PRMT5 inhibition through its
role in regulating cellular splicing. Sci Rep 8(1):9711. https://doi.
org/10.1038/s41598-018-28002-y
24. Kaushik S, Liu F, Veazey KJ, Gao G, Das P, Neves LF, Lin K,
Zhong Y, Lu Y, Giuliani V, Bedford MT, Nimer SD, Santos MA
(2018) Genetic deletion or small-molecule inhibition of the argi-
nine methyltransferase PRMT5 exhibit anti-tumoral activity in
mouse models of MLL-rearranged AML. Leukemia 32(2):499–
509. https://doi.org/10.1038/leu.2017.206
36. Brehmer D, Wu T, Mannens G, Beke L, Vinken P, Gafney D, Sun
W, Pande V, Thuring JW, Millar H, Poggesi I, Somers I, Boeckx
A, Parade M, van Heerde E, Nys T, Yanovich C, Herkert B, Ver-
hulst T, Du Jardin M, Meerpoel L, Moy C, Diels G, Viellevoye
M, Schepens W, Poncelet A, Linders JT, Lawson EC, Edwards JP,
Chetty D, Laquerre S, Lorenzi MV (2017) A novel PRMT5 inhibi-
tor with potent in vitro and in vivo activity in preclinical lung
cancer models [abstract]. In: Proceedings of the American Asso-
ciation for Cancer Research Annual Meeting 2017, Washington,
DC Philadelphia (PA): AACR; Cancer Res 77(13 Suppl):Abstract
nr DDT02-04 2017
25. Smil D, Eram MS, Li F, Kennedy S, Szewczyk MM, Brown PJ,
Barsyte-Lovejoy D, Arrowsmith CH, Vedadi M, Schapira M
(2015) Discovery of a dual PRMT5-PRMT7 inhibitor. ACS Med
Chem Lett 6(4):408–412. https://doi.org/10.1021/ml500467h
26. Duncan KW, Rioux N, Boriack-Sjodin PA, Munchhof MJ, Reiter
LA, Majer CR, Jin L, Johnston LD, Chan-Penebre E, Kuplast
KG, Porter Scott M, Pollock RM, Waters NJ, Smith JJ, Moyer
MP, Copeland RA, Chesworth R (2016) Structure and property
guided design in the identifcation of PRMT5 tool compound
EPZ015666. ACS Med Chem Lett 7(2):162–166. https://doi.
org/10.1021/acsmedchemlett.5b00380
37. Reddy GR, Thompson WC, Miller SC (2010) Robust light
emission from cyclic alkylaminoluciferin substrates for frefy
luciferase. J Am Chem Soc 132(39):13586–13587. https://doi.
org/10.1021/ja104525m
27. Ji S, Ma S, Wang WJ, Huang SZ, Wang TQ, Xiang R, Hu YG,
Chen Q, Li LL, Yang SY (2017) Discovery of selective protein
arginine methyltransferase 5 inhibitors and biological evaluations.
Chem Biol Drug Des 89(4):585–598. https://doi.org/10.1111/
cbdd.12881
38. Zhu K, Jiang C, Tao H, Liu J, Zhang H, Luo C (2018) Iden-
tifcation of a novel selective small-molecule inhibitor of pro-
tein arginine methyltransferase 5 (PRMT5) by virtual screening,
resynthesis and biological evaluations. Bioorg Med Chem Lett
28(9):1476–1483. https://doi.org/10.1016/j.bmcl.2018.03.087
28. Kong GM, Yu M, Gu Z, Chen Z, Xu RM, O’Bryant D, Wang
Z (2017) Selective small-chemical inhibitors of protein arginine
methyltransferase 5 with anti-lung cancer activity. PLoS ONE
12(8):e0181601. https://doi.org/10.1371/journal.pone.0181601
29. Mao R, Shao J, Zhu K, Zhang Y, Ding H, Zhang C, Shi Z, Jiang
H, Sun D, Duan W, Luo C (2017) Potent, selective, and cell active
protein arginine methyltransferase 5 (PRMT5) inhibitor developed
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