Lead discovery, chemical optimization, and biological evaluation studies of novel histone methyltransferase SET7 small-molecule inhibitors

Article abstract of DOI:10.1016/j.bmcl.2020.127061

The post-translational modifications of histones, including histone methylation and demethylation, control the expression switch of multiple genes. SET domain-containing lysine methyltransferase 7 (SET7) is the only methyltransferase, which can specifically monomethylate lysine-4 of histone H3 (H3K4me1) and play critical roles in various diseases, including breast cancer, hepatitis C virus (HCV), atherosclerotic vascular disease, diabetes, prostate cancer, hepatocellular carcinoma, and obesity. However, several known SET7 inhibitors exhibit weak activity or poor selectivity. Therefore, the development of novel SET7 inhibitors is highly desirable and of great clinical value. In this study, we identified 2–79 as a new hit compound by structure-based virtual screening and further AlphaLISA-based biochemical evaluation. Via chemical optimization, the synthesized compound DC21 was confirmed as a potent SET7 inhibitor with an IC₅₀ value of 15.93 μM. The interaction between DC21 and SET7 was also validated through SPR experiment. Especially, DC21 retarded proliferation of MCF7 cells with an IC₅₀ value of 25.84 μM in cellular level. In addition, DC21 has good selectivity for several other epigenetic targets, such as SUV39H1, G9a, NSD1, DOT1L and MOF. DC21 can serve as a lead compound to develop more potential SET7 inhibitors and as a chemical probe for SET7 biological function studies.

Full text of DOI:10.1016/j.bmcl.2020.127061

Journal Pre-proofs
Lead Discovery, Chemical Optimization, and Biological Evaluation Studies of
Novel Histone Methyltransferase SET7 Small-molecule Inhibitors
Zeng Hou, Wenjian Min, Rukang Zhang, Ao Niu, Yuanqing Li, Liyuan Cao, Jie
Han, Cheng Luo, Peng Yang, Hong Ding
PII:
S0960-894X(20)30139-6
DOI:
https://doi.org/10.1016/j.bmcl.2020.127061
BMCL 127061
Reference:
Bioorganic & Medicinal Chemistry Letters
To appear in:
Received Date:
Revised Date:
Accepted Date:
25 November 2019
14 February 2020
22 February 2020
Please cite this article as: Hou, Z., Min, W., Zhang, R., Niu, A., Li, Y., Cao, L., Han, J., Luo, C., Yang, P., Ding,
H., Lead Discovery, Chemical Optimization, and Biological Evaluation Studies of Novel Histone Methyltransferase
SET7 Small-molecule Inhibitors, Bioorganic & Medicinal Chemistry Letters (2020), doi: https://doi.org/10.1016/
j.bmcl.2020.127061
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Lead Discovery, Chemical Optimization, and Biological
Evaluation Studies of Novel Histone Methyltransferase
SET7 Small-molecule Inhibitors
Zeng Houa,b#, Wenjian Minc,d#, Rukang Zhange#, Ao Niuc, Yuanqing Lia,b, Liyuan
Caoa, Jie Hana,b, Cheng Luoa,b, Peng Yangc,d*, Hong Dinga,c*
a State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, 555 Zuchongzhi
Road, Shanghai, 201203, China
b University of Chinese Academy of Sciences, 19 Yuquan Road, Beijing 100049, China
c State Key Laboratory of Natural Medicines and Jiangsu Key Laboratory of Drug Design and Optimization, China
Pharmaceutical University, Nanjing 210009, China
d Department of Medicinal Chemistry, School of Pharmacy, China Pharmaceutical University, Nanjing 211198, China
e Department of Hematology and Medical Oncology, Emory University School of Medicine, Atlanta, GA 30322, USA; Winship
Cancer Institute, Emory University School of Medicine, Atlanta, GA 30322, USA.
#These authors contributed equally to this work
Abstract
The post-translational modifications of histones, including histone methylation
and demethylation, control the expression switch of multiple genes. SET domain-
containing lysine methyltransferase 7 (SET7) is the only methyltransferase, which can
specifically monomethylate lysine-4 of histone H3 (H3K4me1) and play critical roles
in various diseases, including breast cancer, hepatitis C virus (HCV), atherosclerotic
vascular disease, diabetes, prostate cancer, hepatocellular carcinoma, and obesity.
However, several known SET7 inhibitors exhibit weak activity or poor selectivity.
Therefore, the development of novel SET7 inhibitors is highly desirable and of great
clinical value. In this study, we identified 2-79 as a new hit compound by structure-
based virtual screening and further AlphaLISA-based biochemical evaluation. Via
chemical optimization, the synthesized compound DC21 was confirmed as a potent
SET7 inhibitor with an IC50 value of 15.93 μM. The interaction between DC21 and
SET7 was also validated through SPR experiment. Especially, DC21 retarded
proliferation of MCF7 cells with an IC50 value of 25.84 μM in cellular level. In addition,
DC21 has good selectivity for several other epigenetic targets, such as SUV39H1, G9a,
NSD1, DOT1L and MOF. DC21 can serve as a lead compound to develop more
potential SET7 inhibitors and as a chemical probe for SET7 biological function studies.
Keywords: Histone methylation, SET7, small-molecule inhibitor, virtual screening,
molecular probe
Histone methylation plays diverse roles in biological process such as gene transcription, gene
silencing, DNA repair and replication, and chromatin structure establishment and maintenance.
Histone methyltransferases and histone demethylases regulate histone methylation in a coordinated

manner. Among histone methyltransferases, SET domain-containing lysine methyltransferase 7
(SET7, also called SETD7, SET9, and KMT7) is the only methyltransferase which can specifically
monomethylate lysine-4 of histone H3 (H3K4me1).1, 2 SET7 belongs to SET domain-containing
protein family that is characterized by an approximately 130 amino-acid-long domain called the
SET domain.3 This protein family can transfer methyl groups from S-adenosyl-L-methionine (SAM,
AdoMet) to lysine on histone H3.4 Wilson et al. obtained crystal structure of SET7 with a resolution
of 2.1 Å. They also found two residues in the C terminus of SET7, Trp-352 and Tyr-353, are
essential for catalytic activity toward Lys-4 of histone H3. Meanwhile, they disclosed that AdoMet
is combined with SET7 through N1 of the adenine with the side chain carbonyl of Asn-296.5
Typically for islet cells, including α- and β-cells, SET7 can regular glucose-stimulated Ca2+
mobilization and insulin secretion by methylation of Lys-4 of histone H3.6
With the development of functional studies of SET7, amount of non-histone substrates were
also detected such as pRb, RelA, TAF10, Tat and ER.7-14 Under DNA damage control, the
methylation of Lys-810 by SET7 blocks pRb binding with CDK, which prevents subsequent
phosphorylation of the associated serine residue.7 NF-κB involves in a variety of biological
processes including inflammation, proliferation and cell survival. However, activation of NF-κB is
closely controlled at multiple levels. RelA will be methylated at Lys-37 by SET7 when treated with
TNFα and IL-1β. The methylation of RelA is important to the expression of NF-κB target genes.8
Meanwhile, SET7 monomethylates RelA lysine residues 314 and 315 in vitro and in vivo in response
to TNF-α stimulation. The methylation state of RelA inhibits NF-κB action by inducing the
proteasome-mediated degradation of promoter-associated protein.9 SET7 can methylate Lys-189 of
TAF10, the TBP-associated factor, which can increase the affinity of TAF10 with RNA polymerase
II. As a result, this modification will promote preinitiation complex formation.10 SET7 associates
with the HIV promoter by monomethylating Lys-51 which is located in the RNA-binding domain
of Tat. The production of Tat leads transcriptional elongation to become highly effective. So the
methylation of Tat by SET7 results in the constant production of full-length HIV transcript.12
Meanwhile, approximately 70% of breast cancers express ERα and their growth depends on
estrogen signaling.13 ER is stabilized by SET7-mediated methylation at Lys-302, but a breast
cancer-associated mutation at Lys-303 alters methylation and blocks the efficient recruitment of
ER.14 SET7 is upregulated in liver tissue under HCV infection and the expression of SET7 is higher
in liver cells of HCV patients than healthy individuals. As the research progressed, it is discovered
that SET7 facilitates HCV replication through the weakening of IFN signaling pathways and IFN-
related factors.15 However, in gastric cancer cells, knocking down SET7 significantly increase cell
proliferation, invasion and migration.16
As the function of SET7 is becoming increasingly clear, researchers are trying to discover its
novel inhibitors as chemical probes or potential clinical agents. S. Mori and K. Iwase et al. designed
and synthesized amine analogues of adenosylmethionine, which were novel bisubstrate-type
inhibitors of SET7.17 In 2014, (R)-PFI-2 was discovered as a substrate-competitive inhibitor of
SET7, which can bind groove of SET7 and also contact directly with adenosylmethionine through
the catalytic lysine-binding channel.18 Via structure-based virtual screening, a substituted N-
phenylbenzamide compound DC-S100 was discovered as a potential inhibitor of SET7 and its
chemical optimization afforded better analog DC-S239 with considerable potency in suppressing
SET7.19 Cyproheptadine, an antiallergy drug, was also identified as a SET7 inhibitor, which can
decrease the expression of ERα in vivo.20

While, most of the known inhibitors of SET7 are analogs of SAM, which exhibit selectivity
issues. To discover more potent and selective SET7 inhibitors, we conducted virtual screening and
biological evaluation to get a new leading compound 2-79. We made further chemical modification
and systematic structure-activity relationship studies, which afforded us compound DC21 with good
inhibitory activity against MCF7 cells.
Discovery of Candidate Compounds by Virtual Screening
Targeting on the SAM-binding site, we identified five inhibitors of SET7 via virtual screening
and biochemical experiments (Fig. 1A). Referring to previous studies, the crystal structure of SET7
in complex with the bioactive cofactor SAM (PDB code 1N6A) was selected for the subsequent
pharmacophore generation and docking studies.19 The including in-house compound library was
first filtered by Lipinski’s rule of five, and “pan-assay interference compounds” were deleted with
Pipeline Pilot, version 7.5 (Pipeline Pilot; Accelrys Software Inc., San Diego, CA) based on SPECS
chemical database.21-24 Then, 20,000 compounds from the preprocessed compound library with all
stereoisomers and different protonation states were used to produce an indexed multi-conformer 3D
database in Accelrys Discovery Studio 3.0. The top-ranked 1,000 candidates were further evaluated
and filtered by Glide SP and XP modes integrated in Maestro 9.2, and clustered with Pipeline Pilot,
version 7.5 (Pipeline Pilot; Accelrys Software Inc., San Diego, CA).25 In the end, 118 compounds
were chosen to be evaluated for their biochemical activities by AlphaLISA assay.
Discovery of the Leading Compound by Biochemical Evaluation and Binding
Assay
The 118 candidate compounds mentioned above were further validated for their inhibitory
activities against SET7 by AlphaLISA assay, taking Sinefungin as a positive control. In the catalysis
of SET7, the methyl group on the SAM is transferred to the biotinylated histone polypeptide
substrate, which can be combined with the streptavidin alpha donor beads. When the AlphaLISA
acceptor beads conjugated to anti-methyl-Histone H3 Lysine-4 (H3K4me1-2) is combined with the
special epigenetic modification site, short-lived singlet oxygen molecules produced by the donor
beads upon laser irradiation of the beads-target complexes at 680 nm can reach the acceptor beads
in proximity to generate an amplified chemiluminescent signal at 615 nm.26 The intensity of light
emission is proportional to the level of biotinylated substrate modification. Depending on the result
of inhibition assay, we chose 5 compounds with ability to inhibit SET7 activity more than 90% at
the concentration of 100 μM to determine their IC50 values (Fig. 1B). Among these 5 compounds,
compound 2-79 exhibited the highest potency with an IC50 value of 46.06 μM (Fig. 1C). The
remaining four compounds could not fit appropriate curves, so that the appropriate IC50 values were
not obtained.
Then, we evaluated the binding ability of compound 2-79 to SET7 by surface plasmon
resonance (SPR) assays. The equilibrium dissociation constant (KD) value of compound 2-79 was
29.00 μM, which was consistent with the detected inhibitory activity in AlphaLISA assays (Fig.
S1). All in all, these biophysical and biochemical evidence demonstrated that compound 2-79 can
bind to SET7 and inhibit it in vitro.

Fig. 1. Discovery and confirmation of hit compounds. (A) Workflow of the discovery of hit
compounds. (B) Inhibitory activities of 118 compounds against SET7 at 100 μM. (C) IC50 curve of
compound 2-79 against SET7. (D) Chemical structures of the hit compounds inhibiting SET7
enzyme activity greater than 90% at 100 μM.
Design and Synthesis of New Compounds for Further Structural Optimization
For further structural optimization, we designed and synthesized a series of derivatives based
on compound 2-79. The synthetic routes of p-benzoquinone derivatives are outlined in Scheme 1,
2, and 4. Firstly, the commercially available methyl 4-bromobenzoate was reacted with 2,5-
dimethoxyphenyl boronic acid to give the intermediate DC1, which was used in the following
coupling reaction, hydrolysis reaction and condensation reaction to yield 2,5-dimethoxyphenyl
substituted compounds (Scheme 1: DC3a-e, and Scheme 4: DC23). And then, those 2,5-
dimethoxyphenyl substituted compounds were treated with Oxone and 4-iodophenoxyacetic acid to
yield p-benzoquinone derivatives (DC4h-m and DC24). The protective Boc group in compound
DC24 was removed using HCl gas to give compound DC25. Compounds DC4g and DC21 were
obtained via oxidation from intermediates DC1 and DC20, respectively.
In Scheme 2, intermediates DC5, DC10a and DC10b were obtained from 4-bromo-2-
nitrobenzaldehyde through a reductive amination reaction, which was used for the subsequent
coupling reaction, reductive reaction and condensation reaction to get DC8a-f and DC13a-f. And
then, compounds DC8a-f and DC13a-f were oxidized by Oxone and 4-iodophenoxyacetic acid to
yield the final compounds DC9a-f and DC14a-f.

The synthetic route to obtain the cyanobiphenyl derivatives are outlined in Scheme 3. The
coupling reaction between cyanophenyl boronic acid and methyl p-bromobenzoate yielded the
corresponding compounds DC15a and DC15b. Then, DC15a and DC15b were hydrolyzed to get
carboxylic acids in THF/H2O. Finally, the condensation reaction between intermediate DC16a (or
DC16b) and N, N-dimethylethane-1, 2-diamine or 1-boc-piperazine yielded the corresponding
compounds DC17a-b and DC18a-b. The protective Boc group in compound DC18b was removed
using HCl gas to give compound DC19a.
All the target compounds were purified by flash column chromatography and characterized
with 1H NMR and liquid chromatography mass spectra (LCMS).
O
O
O
O
OH
B
O
O
O
O
O
O
O
ⅰ
ⅳ
OH
O
+
Br
ⅱ
DC4g
DC1
O
R3
O
R3
O
R2
R1
R2
O
O
OH
O
O
N
O
O
N
ⅳ
ⅲ
N
N
R2
O
O
DC2
DC4h
R1 =
R1 =
R2 = H
R3 = H
R3 = H
DC3a R1 =
DC3b R1 =
R2 = H
R3 = H
O
O
O
O
O
O
R2 = H
R2 = H
R3 = H
S
S
DC4i
DC4j
CH3
CH3
R1 = -Boc
R1 = -Boc
R1 =
R2 = H
R3 = -CH3
R3 = H
DC3c R1 = -Boc
DC3d R1 = -Boc
DC3e R1 =
R2 = H
R3 = -CH3
R3 = H
DC4k
R2 = -CH3
R2 = -CH3
R2 = -CH3
R2 = -CH3
R3 = H
R3 = H
DC4m
N
N
Scheme 1. Reagents and conditions: (i) Toluene, CH3OH, 2 M Na2CO3, Pd(PPh3)4, reflux, 6 h; (ii) THF/H2O = 2:1,
LiOH·H2O, rt, 1 h; (iii) Piperazine derivatives, EDCI, HOBt, DMF, TEA, rt, 3 h; (iv) CF3CH2OH/H2O = 2:1, Oxone,
4-iodophenoxyacetic acid, rt, 1 h.

R1
R1
R1
O
O
O
ⅲ
ⅰ
ⅱ
O
NO2
NH2
DC7
R1 = pyrrolidinyl
Br
NO2
Br
NO2
DC12a R1 = morpholinyl
O
DC12b R1 = 1-Boc-piperazinyl
DC5
R1 = pyrrolidinyl
DC6
R1 = pyrrolidinyl
DC10a R1 = morpholinyl
DC11a R1 = morpholinyl
ⅳ
DC10b R1 = 1-Boc-piperazinyl
DC11b R1 = 1-Boc-piperazinyl
R1
R1
O
O
O
O
O
ⅴ
O
S
N
S
R2
N
R2
H
H
O
O
DC8a R1 = pyrrolidinyl; R2 = phenyl
DC9a R1 = pyrrolidinyl; R2 = phenyl
DC8b R1 = pyrrolidinyl; R2 = 4-methoxyphenyl
DC8c R1 = pyrrolidinyl; R2 = 4-methylphenyl
DC8d R1 = pyrrolidinyl; R2 = 4-fluorophenyl
DC8e R1 = pyrrolidinyl; R2 = 4-trifluoromethylphenyl
DC8f R1 = pyrrolidinyl; R2 = n-butyl
DC9b R1 = pyrrolidinyl; R2 = 4-methoxyphenyl
DC9c R1 = pyrrolidinyl; R2 = 4-methylphenyl
DC9d R1 = pyrrolidinyl; R2 = 4-fluorophenyl
DC9e R1 = pyrrolidinyl; R2 = 4-trifluoromethylphenyl
DC9f R1 = pyrrolidinyl; R2 = n-butyl
DC13a R1 = morpholinyl; R2 = phenyl
DC14a R1 = morpholinyl; R2 = phenyl
DC13b R1 = morpholinyl; R2 = 4-methylphenyl
DC13c R1 = morpholinyl; R2 = 4-chlorophenyl
DC13d R1 = morpholinyl; R2 = 4-nitrophenyl
DC13e R1 = morpholinyl; R2 = 4-trifluoromethylphenyl
DC13f R1 = 1-Boc-piperazinyl; R2 = 4-chlorophenyl
DC14b R1 = morpholinyl; R2 = 4-methylphenyl
DC14c R1 = morpholinyl; R2 = 4-chlorophenyl
DC14d R1 = morpholinyl; R2 = 4-nitrophenyl
DC14e R1 = morpholinyl; R2 = 4-trifluoromethylphenyl
DC14f R1 = 1-Boc-piperazinyl; R2 = 4-chlorophenyl
Scheme 2. Reagents and conditions: (i) nitrogen-containing heterocyclic derivatives, ClCH2CH2Cl, NaBH(OAc)3, rt,
3 h; (ii) toluene, CH3OH, 2 M Na2CO3, Pd(PPh3)4, reflux, 6 h; (iii) NH4Cl, Fe, 70% ethanol (20 mL), 78 oC, 3 h; (iv)
sulfonyl chloride derivative, CH2Cl2, pyridine, rt, 1 h; (v) CF3CH2OH/H2O = 2:1, Oxone, 4-iodophenoxyacetic acid, rt,
1 h.
O
O
O
N
OH
N
O
OH
B
H
ⅲ
ⅱ
ⅰ
OH
O
R1
R1
R1
R1
N
H2N
R2
R2
DC17a
O
R2
R2
Br
DC15a
DC15b
R1 = 4-CN
R1 = H
R2 = H
DC16a
DC16b
R1 = 4-CN
R1 = H
R2 = H
R1 = 4-CN
R1 = H
R2 = H
R2 = 3-CN
R2 = 3-CN
DC17b
R2 = 3-CN
H
N
ⅳ
N
Boc
O
N
O
N
ⅴ
N
NH
Cl
Boc
H
R1
R1
R2
R2
R1 = H
R2 = 3-CN
DC18a
DC18b
R1 = 4-CN
R1 = H
R2 = H
DC19a
R2 = 3-CN
Scheme 3. Reagents and conditions: (i) Toluene, CH3OH, 2 M Na2CO3, Pd(PPh3)4, reflux, 6 h; (ii) THF/H2O = 2:1,
LiOH·H2O, rt, 1 h; (iii) N,N-dimethylethane-1,2-diamine, HATU, DIPEA, TEA, rt, 3 h; (iv) 1-boc-piperazine, EDCI, HOBt,
DMF, TEA, rt, 3 h; (v) HCl gas, rt, 1 h.

O
O
O
O
O
O
OH
B
O
O
O
O
O
O
O
ⅰ
ⅳ
OH
O
O
Br
+
ⅱ
DC20
DC21
O
O
O
O
O
Boc
Boc
N
N
NH
N
OH
N
N
O
O
O
O
O
O
O
O
O
O
ⅴ
ⅳ
ⅲ
Cl
H
O
DC23
DC25
DC24
DC22
Scheme 4. Reagents and conditions: (i) toluene, CH3OH, 2 M Na2CO3, Pd(PPh3)4, reflux, 6 h; (ii)
THF/H2O = 2:1, LiOH·H2O, rt, 1 h; (iii) 1-Boc-piperazine, EDCI, HOBt, DMF, TEA, rt, 3 h; (iv)
CF3CH2OH/H2O = 2:1, Oxone, 4-iodophenoxyacetic acid, rt, 1 h; (ⅴ) HCl gas, rt, 1 h.
Structure−Activity Relationship (SAR) Analysis of Compounds
Compared the binding modes of compound 2-79 with SET7 (Fig. S2), there are still suitable
cavities in the 3' and 4' positions of 2-phenyl-p-benzoquinone. In order to find more effective
compounds, we modified the structure of compound 2-79 and synthesized several series of
compounds. Compounds DC4a-f and DC19b-c were purchased from Specs
(https://www.specs.net/), and other compounds were synthesized in our laboratory. Their inhibitory
activity against SET7 were shown in Table 1.
We first modified the 3' and 4' positions of 2-phenyl-p-benzoquinone side chain to investigate
their effect on the activity. If the R1 group was substituted with a small alkyl side chain, compounds
DC4a-f exhibited almost no inhibitory activity against SET7. Replaced small alkyl side chain with
an ester (compound DC4g), it exhibited moderate inhibitory activity against SET7 with an IC50
value of 85.00 μM. The binding model of compound 2-79 in the protein (Fig. S2) indicated that the
space nearby R1 and R2 groups may accommodate larger substitutes. Thus, the piperazine-amide
and sulfonamide groups were introduced into the compounds (DC4h-m, DC9a-f and DC14a-f). R2
substituted by a sulfonamide group and R1 with pyrrolidinyl (DC9a-f) manifested a better inhibitory
activity against SET7, while R1 replaced by morpholino-methyl or piperazine-methyl (DC14a-f)
decreased the inhibitory activity. Compared with the lead compound 2-79, compounds DC4h, DC4i
and DC4j showed better activity and their IC50 values were 24.00 μM, 23.00 μM and 42.00 μM,
respectively. So, we concluded that a special size of the R1 substitute is essential to keep good
inhibitory activity against SET7, and the piperazine-methyl is the best substitute.
The binding mode of compound 2-79 (Fig. S2B) suggested that the p-benzoquinone group is
capable of forming hydrogen bond with His-297. To further investigate whether the p-benzoquinone
ring is an essential group for activity, we changed p-benzoquinone into a benzene ring bearing 2,5-
dimethoxy or cyano group. Unfortunately, those compounds exhibited no inhibitory activity on
SET7 (Table 2). We also used a naphthalene-1,4-dione ring to replace the p-benzoquinone ring
(DC19b and DC19c). Similarly, these compounds showed no inhibitory activity. Based on these
results, we inferred that the p-benzoquinone group plays an indispensable role in this series of

compounds to keep good inhibitory activity against SET7, but the naphthalene-1,4-dione is too big
for the binding pocket.
Finally, we replaced the benzene ring of compound 2-79 with a furan ring and synthesized
several compounds. Compounds DC21 and DC24 displayed increased inhibitory activity with an
IC50 value of 15.93 μM and 20.40 μM, respectively (Table 2). Replacing the middle benzene ring
with a furan ring, we got three compounds DC21, DC24 and DC25. Two of them (DC21and DC24)
showed greatly improved inhibitory activity. Especially, compound DC21 showed the best
inhibitory activity against SET7 with an IC50 value of 15.93 μM (Fig. 2B), which was chosen for
further biological assays.
Table 1. Chemical structures and biological activities of compounds DC4a-m, DC9a-f and DC14a-
f.
5'
R1
4'
6'
O
3'
R2
1'
2'
O
IC50
IC50
No.
R1
R2
No.
R1
R2
(μM)
(μM)
NH
S
CH3
O
O
DC4a
DC4b
—H
>200
>200
DC9a
DC9b
62.00
72.00
NH
S
F
F
O
O
F
—H
O
NH
S
O
O
CH3
CH3
DC4c
DC4d
DC4e
—H
>200
> 200
>200
DC9c
DC9d
DC9e
83.00
88.00
>200
N
CH3
NH
S
O
O
CH3
CH3
F
NH
O
S
O
—H
Br
CF3

NH
S
O
O
CH3
DC4f
DC4g
>200
85.00
DC9f
85.00
80.00
NH
S
O
O
O
DC14a
O CH3
O
NH
S
O
O
N
DC4h
DC4i
DC4j
DC4k
24.00
23.00
42.00
55.00
DC14b
DC14c
DC14d
DC14e
97.00
>200
>200
>200
N
N
O
H3C
O
CH3
O
O
N
NH
S
O
O
O
O
O
N
O
S
O
Cl
O
N
NH
S
O
Me
N
NO2
Boc
N
O
NH
S
O
O
N
N
Me
Boc
CF3
O
NH
S
O
N
N
DC4m
55.00
DC14f
90.00
N
N
Cl
Boc
N
The IC50 values were calculated from AlphaLISA assay.

Table 2. Chemical structures and biological activities of other new compounds.
IC50
IC50
No.
DC6
Scaffold
R1
R2
No.
Scaffold
R1
R2
(μM)
(μM)
O
NC
HN
CH3
CH3
NO2
NH2
>200
DC17a
DC17b
DC18a
DC18b
>200
>200
>200
>200
N
O
HN
CH3
CH3
CN
N
N
DC7
>200
>200
>200
O
N
R2
NC
DC8a
DC13g
N
H3C
R1
R2
Boc
R1
O
O
O
N
NH
O S O
CH3
N
CN
N
Boc
N
Boc
O
N
CN
N
DC13a
DC12b
>200
>200
DC19a
DC19b
>200
>200
NH.HCl
O
N
O
O
NH2
OH
R1
R2
N
Boc
H
O
CH3
DC19c
DC21
>200
15.93
NC
DC16a
DC16b
>200
>200
O
O
CH3
O
OH
R2
O
N
R1
DC24
DC25
20.40
66.80
R1
N
Boc
H
O
O
O
R2
O
CN
O
OH
N
NH.HCl

The IC50 values were calculated from AlphaLISA assay.
Biophysical Binding Affinity and Selectivity over Histone Methyltransferases
To confirm the binding affinity between compound DC21 and SET7, SPR assay was
implemented. The KD value of compound DC21 was 18.00 μM, which is basically the same as the
result of the in vitro enzyme activity experiment (Fig. 2C).
The SET domains, which include SAM pocket, are conserved in these known SET domain-
containing histone methyltransferases (HMTases).27 Therefore, we first chose SUV39H1, G9a and
NSD1, which are HMTases containing SET domains, to evaluate the selectivity of compound
DC21.28-30 Besides, we selected DOT1L that is HMTases without SET domains31 and MOF that is
histone acetyltransferase32 to measure the selectivity of compound DC21. The results showed that
compound DC21 exhibited significant selectivity over these epi-targets at 100 μM (Fig. 2D) and
could act as a selective inhibitor against SET7.
Fig. 2. Biophysical and biochemical validation of compound DC21. (A) Chemical structure of
compound DC21. (B) IC50 curve of compound DC21 against SET7 by AlphaLISA. (C) Binding
ability of compound DC21 to SET7 by SPR assay. (D) Inhibitory activities of compound DC21 to
other enzymes at 100 μM.
Structure-Activity Relationship (SAR) Analysis of the Modified Compound
In order to figure out the molecular mechanism of inhibition of SET7 by compound DC21, we
conducted molecular docking studies to investigate possible binding patterns between SET7 and
compound DC21. The results indicated that compound DC21 occupies the SAM-binding site, which
accounts for its in vitro activity (Fig. 3A, B). As the binding modes showing, the carbonyl oxygen
on the ester group of compound DC21 forms a hydrogen bond with the amide of Asn-282 and the

amino group of Lys-294, respectively. And there is an interaction between the p-benzoquinone
group of the molecule with the indole ring of Trp-352 through π−π stacking. Additionally, the other
hydrogen bond was observed between the carbonyl oxygen on quinone group away from the furan
ring of compound DC21 and the amino group of His-297 (Fig. 3C, D). Previous mutational analysis
showed that His-293 and His-297 are essential for HMTase activity of SET domain-containing
protein family.1, 2 The nitrogen N6 and N7 of SAM have interaction with the backbone carbonyl
group of His-297.33 SET7, SUV39H1, G9a and NSD1, which are histone methyltransferases
(HMTases) containing SET domains, have many conserved polar residues in the SET domain. As
shown in the sequence alignment (Fig. S3), only a conserved residue, His-297, forms a hydrogen
bond with compound DC21. Compound DC21 forms hydrogen bonds with nonconserved residues
Asn-282 and Lys-294. Meanwhile, there is an interaction between the p-benzoquinone group of the
molecule with the indole ring of nonconserved residues Trp-352 through π−π stacking. As a result,
compound DC21 exhibited significant selectivity just to SET7 not to SUV39H1, G9a and NSD1
(Fig. 2D). As a contrast, the hydrogen bonds link compound 2-79 with the His-297 and Asn-265 of
SET7. Obviously, the binding modes illustrated that the benzoquinone group of the molecule
interacts with the indole ring of Trp-352 through π−π stacking (Fig. S2B). The replacement of furan
ring with larger groups, for example, benzoquinone group, displays significant steric clashes, which
provides the structural explanation for depressed activity of compound 2-79 and its derivative. The
hydrophobic pocket is not able to provide sufficient structural adjustment for the larger group to
provide efficient binding, which is consistent with biochemical activity in vitro (Table 1).
Fig. 3. Putative binding mode between compound DC21 and SET7. (A) Comparison of the status
of compound DC21 and SAM in the pocket. Compound DC21 is shown as light blue sticks and
SAM is shown as light green sticks. SET7 is shown in dark gray surface. (B) The binding pocket of
SET7 for compound DC21. Compound DC21 is depicted as light blue sticks. SET7 is shown in

electrostatic potential surface. (C) Close-up view of the key interactions stabilizing compound
DC21 in SAM-binding pocket. Compound DC21 is depicted as light blue sticks, the surrounding
key residues are shown in green sticks and labelled. Hydrogen bonds are shown as yellow dot lines.
The amino acid that produces the π-π interaction is shown as red stick. SET7 is shown in pink
cartoon. (D) Schematic diagram showing detailed interactions between SET7 and compound DC21.
Hydrogen-bonding interactions are shown as purple line with arrow.
Cell-based Assays of the Modified Compound
Set7-mediated Lys-302 methylation of ERα is associated with the stability of ERα.14 Therefore,
to further assess the inhibitory ability of compound DC21 at the cellular level, the anti-proliferation
activities of compound DC21 against the MCF7 cell line, which expresses endogenous ER, were
determined by Cell Titer-Glo luminescent assay (Promega, Madison, WI, USA). Compound DC21
inhibited the proliferation of MCF7 cells in a dose-dependent manner with an IC50 values of 25.84
μM. Moreover, compound DC21 had little effect on the proliferation of normal cells, including
normal embryonic lung fibroblast cell line MRC-5 and umbilical vascular endothelial cell line
HUV-EC-C (Fig. 4A). Furthermore, we found that compound DC21 could decrease the stability of
ERα which leads to the decrease of ERα protein level in MCF7 cell line in a dose-dependent manner
by western blot (Fig. 4B). Taken together, these results proved the high target participation of the
identified SET7 inhibitor in MCF7 cell line.
Fig. 4. Biological activity of compound DC21 at cellular level. (A) Cell viability curves of
compound DC21 toward the MCF7 cell line (red), HUV-EC-C cell line (brown) and MRC-5 cell
line (black). (B) Quantification and Western blot of ERα protein expression after treatment with
compound DC21 in MCF7 cell line.
The post-translational modifications of histones, including histone methylation and
demethylation, control the gene expression in different space and different time.34, 35 If the balance
is broken, there will be a lot of proliferative diseases, particularly cancer.36, 37 As a member of SET
domain-containing protein family, SET7 obtains methyl from SAM in the natural state. A series of
amine analogues of adenosylmethionine were synthesized to act as competitive inhibitor with little

selectivity over other SET domain-containing proteins.38 In addition to these analogues, (R)-PFI-2
was found to be an effective and selective SET7 inhibitor, while other inhibitors had little potency
to SET7. Based on these conditions, we intend to explore new small molecule inhibitors targeting
SET7. First, a hit compound 2-79 was identified by structure-based virtual screening and its
biochemical activity was confirmed by AlphaLISA. Then, based on the hit compound, series of
compounds were purchased and synthesized, and the most potent compound was evaluated by
biochemical and biophysical experiments. Next, the predicted binding modes and interaction
schematics of the best compound with SET7 were illustrated by molecular docking studies. Finally,
the cellular level studies further indicated that the best compound DC21 could efficiently damage
the stability of ERα by against SET7 to impact the proliferation of breast cancer cells. In sum, we
discovered a novel inhibitor targeting SET7 with an IC50 value of 15.93 μM. Meanwhile, it
performed satisfactory target selectivity, and exhibited cell selectivity between breast cancer cells
and normal cells. This provides a novel scaffold that lays a root for novel drug development and
mechanism study of SET7.
Declaration of Competing Interest
The authors declare that they have no known competing financial interests or personal
relationships that could have appeared to influence the work reported in this paper.
Acknowledgments
This study was supported by the Ministry of Science and Technology of China (National Key
R&D Program of China, 2017YFB0202600), the “Personalized Medicines Molecular Signature-
based Drug Discovery and Development” (Strategic Priority Research Program of the Chinese
Academy of Sciences, XDA12020368) to Hong Ding.
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Declaration of interests
☒ The authors declare that they have no known competing financial interests or personal
relationships that could have appeared to influence the work reported in this paper.
☐The authors declare the following financial interests/personal relationships which may be
considered as potential competing interests: