Identification of a novel selective small-molecule inhibitor of protein arginine methyltransferase 5 (PRMT5) by virtual screening, resynthesis and biological evaluations

Article abstract of DOI:10.1016/j.bmcl.2018.03.087

As one of the most promising anticancer target in protein arginine methyltransferase (PRMT) family, PRMT5 has been drawing more and more attentions, and many efforts have been devoted to develop its inhibitors. In this study, three PRMT5 inhibitors (9, 16

Full text of DOI:10.1016/j.bmcl.2018.03.087

Bioorganic & Medicinal Chemistry Letters xxx (2018) xxx–xxx
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Identification of a novel selective small-molecule inhibitor of protein
arginine methyltransferase 5 (PRMT5) by virtual screening, resynthesis
and biological evaluations
Kongkai Zhu a,c, Chengshi Jiang a,c, Hongrui Tao , Jingqiu Liu , Hua Zhang , Cheng Luo b,
a
b
a,
⇑
⇑
a
School of Biological Science and Technology, University of Jinan, Jinan 250022, PR China
Drug Discovery and Design Center, State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203, PR China
b
a r t i c l e i n f o
a b s t r a c t
Article history:
As one of the most promising anticancer target in protein arginine methyltransferase (PRMT) family,
PRMT5 has been drawing more and more attentions, and many efforts have been devoted to develop
its inhibitors. In this study, three PRMT5 inhibitors (9, 16, and 23) with novel scaffolds were identified
by performing pharmacophore- and docking-based virtual screening combined with in vitro radiomet-
ric-based scintillation proximity assay (SPA). Substructure search based on the scaffold of the most active
Received 23 February 2018
Accepted 29 March 2018
Available online xxxx
Keywords:
PRMT5 inhibitor
Resynthesis
Virtual screening
Molecular docking
Molecular dynamics simulation
9
afforded 26 additional analogues, and SPA results indicated that two analogues (9–1 and 9–2) showed
increased PRMT5 inhibitory activity compared with the parental compound. Resynthesis of 9, 9–1, and 9–
confirmed their PRMT5 enzymatic inhibition activity. In addition, compound 9–1 displayed selectivity
2
against PRMT5 over other key homological members (PRMT1 and CARM1 (PRMT4)). While the structure–
activity relationship (SAR) of this series of compounds was discussed to provide clues for further struc-
ture optimization, the probable binding modes of active compounds were also probed by molecular dock-
ing and molecular dynamics simulations. Finally, the antiproliferative effect of 9–1 on MV4-11 leukemia
cell line was confirmed and its impact on regulating the target gene of PRMT5 was also validated. The hit
compounds identified in this work have provided more novel scaffolds for future hit-to-lead optimization
of small-molecule PRMT5 inhibitors.
Ó 2018 Elsevier Ltd. All rights reserved.
Protein arginine methyltransferases (PRMTs) play important
roles in diverse and essential biological processes including cell
cancer; both PRMT1 and PRMT5 are overexpressed in lung cancer
1
2
and leukaemia
; PRMT6 accumulates in bladder and lung
1
,2
12
growth, cell proliferation, cell cycle regulation, cell death, gene
cancer ; PRMT9 is involved in lymphoma, melanoma, testicular,
and pancreatic cancers. Owing to the pivotal roles of PRMTs in
transcription, RNA splicing, ribosome biogenesis,6 kinase sig-
3
4,5
15
7
nalling, etc. Nine members (PRMT1–9) of mammalian PRMTs have
the occurrence and progression of tumor, they have recently
received more and more attention and become an important and
been identified and demonstrated to fulfil their functions by
methylating arginine residues of their cytoplasm and nuclear sub-
9
promising class of anticancer targets. Up to date, quite a few
8
strate proteins. Modification of histone is the most important
micromolar and submicromolar small-molecule inhibitors have
been obtained for nearly all PRMT members, especially for those
key ones such as PRMT1, CARM1 (PRMT4), and PRMT5.9 Among
the reported PRMT1, PRMT3, CARM1, PRMT5, and PRMT6-specific
inhibitors, a small molecule (GSK-3326595) against PRMT5 has
function of PRMTs, which represents a significant component
9
accounting for the complexity of the overall epigenetic system.
By catalysing the methylation of nucleosomal histone tails, PRMTs
participate in the regulation of the adaptive switching between
transcriptionally active and silent chromatin states.1
0,11
Dysregula-
been put into clinical trial indicative of the promising potential
16
tion or aberrant expression of PRMTs are associated with a variety
of PRMT5 as a good drug development target.
1
2
13
of human diseases especially cancer. For example, PRMT1, -2,
-
PRMT5 belongs to the symmetric dimethylation enzyme
group and can perform the methylation of an arginine residue
up to two methyls. This process is assisted by its cofactor methy-
losome protein 50 (MEP50) which has also been known as p44.
1
2
12
14
3
,
-4, and -7, are found overexpressed or aberrant in breast
⇑
Corresponding authors.
E-mail addresses: bio_zhangh@ujn.edu.cn (H. Zhang), cluo@simm.ac.cn (C. Luo).
These authors contributed equally.
Histone H4 arginine
3 (H4R3) and histone H3 arginine 8
(
H3R8) are two general methylating sites of PRMT5 and can be
c
https://doi.org/10.1016/j.bmcl.2018.03.087
960-894X/Ó 2018 Elsevier Ltd. All rights reserved.
0
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2
K. Zhu et al. / Bioorganic & Medicinal Chemistry Letters xxx (2018) xxx–xxx
symmetrically dimethylated resulting in repression of gene
of active compounds were proposed and their preliminary struc-
ture activity relationship (SAR) was also discussed. Cell level assay
showed that 9–1 displayed moderate antiproliferative activity
1
7,18
expression.
It is shown that PRMT5 promotes cell survival
and growth by directly methylating epidermal growth factor
receptor (EGFR), E2F1, and p53.1
9–22
Studies have indicated that
against MV4-11 cells with an EC50 value of 22.5 lM and upregu-
PRMT5 could promote tumorigenesis in multiple tissues by reg-
lated the expression of the target gene (Gas1) of PRMT5. The pre-
sent search for new PRMT5 inhibitors has provided new chemical
templates for future hit-to-lead optimization and will contribute
to further development of new therapeutic candidates for cancer
treatment.
23
24
ulating various partners such as p53, cyclin D1, and Janus-
2
5
activated kinase-2. In addition, in mouse embryonic fibroblast
MEF) cells, PRMT5 can be recruited by Menin to the promoter
of growth arrest specific 1 (Gas1) gene and suppresses Gas1
(
2
6
expression. In agreement with its promising anticancer target
role, PRMT5 has been found overexpressed in lymphoma, leuke-
mia, and glioblastoma, as well as colorectal, ovarian, lung, and
52 Small-molecule PRMT5 candidate inhibitors were obtained
by pharmacophore- and docking-based virtual screening
2
7
prostate cancers.
In addition to SAM analogues (Chart 1) that are Pan-MTase
methyltransferase) inhibitors, only four classes of PRMT5 inhibi-
(
Pharmacophore- and docking-based virtual screening was used
to screen small-molecule PRMT5 candidate inhibitors. Crystal
structure of human PRMT5 had been determined by X-ray diffrac-
tion and deposited in RCSB protein data bank (PDB), which laid the
foundation for structure-based virtual screening targeting PRMT5.
At present, there are ten human PRMT5 crystal structures available
27–30
tors (Chart 1) have been reported so far.
Considering the sig-
nificance of PRMT5 as an anticancer target, the discovery and
development of more PRMT5-specific inhibitors remains a pressing
task for medicinal chemists.
In this study, six PRMT5 inhibitors of three novel scaffolds were
3
1
31
31
31
identified with IC50 values ranging from 14 to 56
lM by employing
in PDB with codes of 5C9Z, 5EMJ, 5EMK, 5EML, 5EMM,
3
2
28
28
28
33
structure-based virtual screening and radiometric-based scintilla-
tion proximity assay (SPA). Resynthesis of top three active com-
pounds 9, 9–1, and 9–2 confirmed their PRMT5 enzymatic
inhibition activity. Moreover, the most potent compound 9–1 dis-
played well selectivity with no inhibition against other key PRMT
members (PRMT1 and CRAM1). Through molecular docking and
molecular dynamics simulations, the binding modes of this series
5FA5, 4X60, 4X61, 4X63, and 4GQB, respectively, and all
structures are in the form of PRMT5:MEP50 complex with different
ligands in SAM and substrate binding sites. Among these crystal
structures, only 5EML and 4X61 contain the cofactor SAM, while
4GQB is the sole one incorporating a histone H4 peptide at the sub-
strate binding site. 5EML with higher resolution (2.39 Å) than 4X61
(2.85 Å) and 4GQB (2.06 Å) was eventually chosen in the virtual
Chart 1. Reported PRMT5 inhibitors.
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K. Zhu et al. / Bioorganic & Medicinal Chemistry Letters xxx (2018) xxx–xxx
3
screening for new inhibitors targeting SAM binding sites. In the
constructed structure, the coordinates of PRMT5:MPE50 and SAM
were derived from 5EML while the H4 peptide was derived from
on the constructed structure of PRMT5, a pharmacophore model
(Fig. 1B) was mimicked with nine features consisting of two hydro-
gen bond donors (HB_Donor1 and HB_Donor5), four hydrogen
bond acceptors (HB_Acceptor2, HB_Acceptor3, HB_Acceptor4, and
4
GQB. The screening workflow (Fig. 1A) was as follows. First, based
Fig. 1. (A) Workflow of the virtual screening strategy adopted in the present study; (B) The diagram of pharmacophore model aligned with SAM.
Fig. 2. (A) Inhibitory activities of the 52 PRMT5 candidate inhibitors at 100 and 50 lM, respectively. (B) The structures and IC50 values of the hit compounds. Data shown are
mean ± SD of three replicates.
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K. Zhu et al. / Bioorganic & Medicinal Chemistry Letters xxx (2018) xxx–xxx
HB_Acceptor9), and two positive (POS_Ionizable6 and POS_Ioniz-
able7) and one negative ionizables (NEG_Ionizable8). This model
was then used to screen SPECS database (http://www.specs.net)
containing 212,255 compounds, and a total of 2166 candidates that
matched at least four above-mentioned pharmacophore features
were obtained. Next, the 2166 candidates were further docked into
SAM binding sites of PRMT5 by molecular docking to remove com-
pounds with low binding scores. The top 600 small molecules
ranked by the XP Gscore were acquired for the subsequent cluster
analysis and manual selection. Finally, according to cluster results
and the selection criteria that we previously reported, 5 2 small-
molecule PRMT5 inhibitor candidates were selected and purchased
for further biological evaluation against the methylation activity of
PRMT5.
2
7
Table 1
Inhibitory activity of 9 and its three analogues (9–1 to 9–3) against PRMT5.a
Compounds
Structure
Inhibition rate (%)
80.0
IC50 (lM)
9
35 ± 2.83
14 ± 1.41
22 ± 0.19
44 ± 2.26
9–1
9–2
9–3
95.0
71.0
61.0
a
Initial inhibition rate was tested at 50
three replicates.
lM for all compounds, and only those with <50% inhibition rate were selected for IC50 measurements. Data shown are mean ± SD of
Fig. 3. (A) The structures and IC50 values of the three active analogues of compound 9 and the positive control SAH. (B) IC50 determination of 9–1 against PRMT1 and CARM1
using SPA method. Data shown are mean ± SD of three replicates.
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K. Zhu et al. / Bioorganic & Medicinal Chemistry Letters xxx (2018) xxx–xxx
5
Radiometric-based SPA results revealed three candidates with
PRMT5 inhibitory activity at enzymatic level
ranging from 35 to 56
inhibitory activity.
lM, of which 9 exhibited the most potent
Radiometric-based assay has become a highly accepted protocol
for quantitating in vitro activity of PRMTs owing to its high sensi-
tivity. In this work, radiometric-based SPA was used to detect the
inhibitory activity of the 52 candidates against PRMT5 at enzy-
matic level. The inhibition rate of each candidate was tested at
Two of 26 analogues displayed better PRMT5 inhibitory activity
than 9 and their SAR was discussed
As 9 showed the best activity among the aforementioned three
candidates, similarity search was performed to retrieve its ana-
logues for further evaluation. Then 26 analogues in SPECS database
were collected and their PRMT5 inhibitory activities were tested.
The assay results were summarized in Tables 1 and S1. As shown
the concentrations of 100 and 50
showed that three compounds displayed inhibition rate above
0% at 100 M (Fig. 2A), and the IC50 values of them were then
lM, respectively. SPA results
5
l
determined and shown in Fig. 2B. As we can see, the three active
compounds represented three different scaffolds with IC50 values
(Fig. 3A), two analogues, i.e. 9–1 and 9–2, showed better activity
than 9 with IC50 values of 14 and 22 M, respectively, while the
l
3 3
Scheme 1. Synthesis of 9, 9–1, and 9–2. Reagents and conditions: (a) i) Benzoyl isothiocyanate, acetone, rt; ii) 1 N NaOH(aq), THF, reflux; (b) PhMe NBr , THF; (c) EtOH,
reflux.
Fig. 4. Binding modes of compounds 9, 9–1, and 9–2 with PRMT5. PRMT5 and MEP50 are shown in cartoon diagram in purplish and yellow, respectively. Compounds 9, 9–1,
and 9–2 occupy the SAM binding site represented as yellow, magenta, and cyan sticks, respectively, while SAM is shown as green sticks. Arginine residue 3 in H4 peptide is
also shown as green sticks.
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K. Zhu et al. / Bioorganic & Medicinal Chemistry Letters xxx (2018) xxx–xxx
third one (9–3) exhibited decreased activity with an IC50 value of
M. S-adenosyl homocysteine (SAH) was used as the positive
The synthetic strategy for target compounds 9, 9–1, and 9–2 is
depicted in Scheme 1. Briefly, thiourea 9b was prepared by starting
from aniline 9a and benzoyl isothiocyanate in two steps according
44 l
control when testing the IC50 values of the targeted compounds.
Based on the structures and activities of this series of compounds,
their SAR was discussed as follows: 1) The carboxylic acid group
appeared to be an essential factor for their activity and was spec-
ulated to interact with the key residues K333 and Y334 of the
SAM binding sites of PRMT5, which was further validated by later
analysis of their proposed binding modes. The only exception was
the thiazolium salt 9–4. 2) Replacement of the hydroxybenzoic
acid moiety by alkoxy- and halogen-substituted benzene rings
3
4
to a previously reported protocol.
a-Bromoacetophenones 9d
were generally obtained from corresponding aceto compounds 9c
and phenyltrimethylammonium tribromide. Finally, aminothiazole
3
4
formations of 9b and 9c generated the target compounds 9, 9–1,
and 9–2.
Compound 9–1 showed selective inhibitory activity against
PRMT5
(
9–5 to 9–13) or benzo[b]thiophene-dioxide ring (9–14) dramati-
cally reduced the inhibition rates to the range of 20–33% at 50
M, presumably due to the loss of H-bond or salt bridge interaction
with the key residues Y334, K333, and E435. 3) Substitution of the
-benzene fragment of thiazole moiety by more bulky aromatic
rings, such as benzofuran (9–15), naphthalene (9–16), coumarin
9–17 to 9–19), and benzyloxyphenyl (9–20 and 9–21) led to the
The most active 9–1 was chosen for further test on its inhibition
against two other key homological members of PRMT5, PRMT1 and
CARM1. As shown in Fig. 3B, the assay results revealed that 9–1
was inactive toward the latter two enzymes with IC50 values larger
than 100 lM, which suggested that this class of compounds were
selective PRMT5 inhibitors.
l
4
(
loss of activity (inhibition rates <10%). In addition, to expand the
structural diversity of tested compounds, five more analogues with
benzamido (9–22 to 9–24) or benzoylthioureido (9–25 and 9–26)
were also screened, but none of them showed inhibitory activity
against PRMT5.
Table 2
The binding free energy of 9, 9–1 and 9–2 to PRMT5 calculated by the MM/PBSA
a
method.
D
G
gas
D
G
solv
DG
9
9–1
9–2
À11.50
À14.74
À21.08
À8.21
À22.55
À8.24
À19.71
À37.29
À29.32
Resynthesis of 9, 9–1, and 9–2 confirmed their PRMT5
inhibitory activity
a
All calculated values were given in kcal/mol.
D
G =
gas solv
DG + DG . DGgas rep-
The top three active compounds 9, 9–1, and 9–2 were synthe-
sized to further confirm their PRMT5 enzymatic inhibition activity.
resents the binding free energy in vacuum while
free energy change.
DGsolv represents the solvation
Fig. 5. The detailed interactions of compounds 9, 9–1, and 9–2 with PRMT5.
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K. Zhu et al. / Bioorganic & Medicinal Chemistry Letters xxx (2018) xxx–xxx
7
Fig. 6. (A) Effect of compound 9–1 on the proliferation of MV4-11 cells; (B) RT-qPCR analysis of the gene expression of Gas1 when treated with 9–1 at 30
l
M. Results shown
are mean ± SD of three replicates. *P < 0.05.
Binding modes of the active compounds to PRMT5 were
obtained by molecular docking and verified by molecular
dynamics simulation
and could be used for further optimization to improve their activ-
ities. 26 analogues of compound 9 in SPECS database were
retrieved and purchased, and two of them displayed more potent
inhibitory activity against PRMT5 indicated by SPA results. Resyn-
thesis of 9, 9–1, and 9–2 confirmed their PRMT5 enzymatic inhibi-
tion activity. From the proposed binding modes, the top three
active hits (9, 9–1, and 9–2) had hydrophobic, hydrogen bond, salt
As shown in Figs. 4 and S1, molecular docking was used to
acquire the binding modes of 9, 9–1, 9–2, and 9–3, with reasonable
binding poses similar to that of SAM. It was interesting to note that
first three compounds showed exactly the same binding modes,
whereas the N-phenyl ring in 9–3 displayed opposite spatial orien-
tation leading to reduced interactions (Fig. S2) with PRMT5, which
could be responsible for its lower activity. The detailed interactions
of 9, 9–1, and 9–2 with PRMT5 were shown in Fig. 5. As can be
bridge and cation-p interactions with PRMT5. MD simulation
results indicated that the order of the binding free energy was in
well agreement with the activity of the three compounds. Finally,
the antiproliferative effect of the most active 9–1 was confirmed
in MV4-11 cells, and the qPCR results validated the target on
PRMT5 of 9–1. Besides, 9–1 displayed well selectivity against
PRMT5 among the key PRMT members (PRMT1, and CARM1).
Therefore, the hits discovered in this study will provide novel scaf-
folds for further hit-to-lead optimization and lay the foundation for
further development of therapeutic candidates for cancer
treatments.
observed, hydrophobic, hydrogen bond, salt bridge, and cation-
p
interactions were the main forces in stabilizing the binding confor-
mation. Not surprisingly, 9–1 showed an extra hydrogen bond with
Met420 residue of PRMT5, which might be a significant factor
accounted for its best activity among these analogues. To further
analyse the reason of 9 and 9–2 showing different activities,
molecular dynamics (MD) simulation was used to obtain more
information on their SAR. 100 ns MD simulation was thus per-
formed on the complexes of 9, 9–1, and 9–2 with PRMT5:MEP50.
By calculating the binding free energy using MM/PBSA method
based on their MD trajectories, 9, 9–1, and 9–2 were demonstrated
to have different binding affinity (Table 2) to PRMT5 with values of
À19.71, À37.29, and À29.32 kcal/mol, respectively. The binding
free energy order of these three compounds was in well agreement
with their experimental activities.
Acknowledgements
This work was supported by Natural Science Foundation of
Shandong Province [Nos. ZR2017BH038, JQ201721], the Natural
Science Foundation of China [21672082], Shandong Key Develop-
ment Project [No. 2016GSF201209], the Young Taishan Scholars
Program [No. tsqn20161037], and Shandong Talents Team Cultiva-
tion Plan of University Preponderant Discipline [No. 10027].
Compound 9–1 showed antiproliferative activity on MV4-11
cells and exhibited impact on the target gene of PRMT5
A. Supplementary data
Supplementary data associated with this article can be found, in
the online version, at https://doi.org/10.1016/j.bmcl.2018.03.087.
PRMT5 was validated as anticancer target and found overex-
pressed in leukemia, so MV4-11 leukemia cell was used to test
the antiproliferation effect of the most active 9–1. As shown in
Fig. 6A, 9–1 displayed concentration-dependent antiproliferative
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Please cite this article in press as: Zhu K., et al. Bioorg. Med. Chem. Lett. (2018), https://doi.org/10.1016/j.bmcl.2018.03.087