Structure-Guided Discovery of a Potent and Selective Cell-Active Inhibitor of SETDB1 Tudor Domain

Article abstract of DOI:10.1002/anie.202017200

SET domain bifurcated protein 1 (SETDB1) is a histone lysine methyltransferase that promotes the silencing of some tumour suppressor genes and is overexpressed in many cancers. SETDB1 contains a unique tandem tudor domain (TTD) that recognizes histone H3 sequences containing both methylated and acetylated lysines. Beginning with the identification of a hit compound (Cpd1), we discovered the first potent and selective small molecule SETDB1-TTD inhibitor (R,R)-59 through stepwise structure-guided optimization. (R,R)-59 showed a K_D value of 0.088±0.045 μM in the ITC assay. The high potency of (R,R)-59 was well explained by the cocrystal structure of the (R,R)-59-TTD complex. (R,R)-59 is an endogenous binder competitive inhibitor. Evidence has also demonstrated its cellular target engagement. Interestingly, the enantiomer (S,S)-59 did not show activity in all the assays, highlighting the potential of (R,R)-59 as a tool compound in exploring the biological functions of SETDB1-TTD.

Full text of DOI:10.1002/anie.202017200

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How to cite: Angew. Chem. Int. Ed. 2021, 60, 8760–8765
International Edition:
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Epigenetics
Hot Paper
Structure-Guided Discovery of a Potent and Selective Cell-Active
Inhibitor of SETDB1 Tudor Domain
Yinping Guo+, Xin Mao+, Liang Xiong+, Anjie Xia, Jing You, Guifeng Lin, Chengyong Wu,
Luyi Huang, Yiwei Wang, and Shengyong Yang*
Abstract: SET domain bifurcated protein 1 (SETDB1) is
a histone lysine methyltransferase that promotes the silencing
of some tumour suppressor genes and is overexpressed in many
cancers. SETDB1 contains a unique tandem tudor domain
(TTD) that recognizes histone H3 sequences containing both
methylated and acetylated lysines. Beginning with the identifi-
cation of a hit compound (Cpd1), we discovered the first potent
and selective small molecule SETDB1-TTD inhibitor (R,R)-
59 through stepwise structure-guided optimization. (R,R)-59
showed a KD value of 0.088 Æ 0.045 mM in the ITC assay. The
high potency of (R,R)-59 was well explained by the cocrystal
structure of the (R,R)-59-TTD complex. (R,R)-59 is an
endogenous binder competitive inhibitor. Evidence has also
demonstrated its cellular target engagement. Interestingly, the
enantiomer (S,S)-59 did not show activity in all the assays,
highlighting the potential of (R,R)-59 as a tool compound in
exploring the biological functions of SETDB1-TTD.
MS31[7b] and VinSpinln,[7c] both of which are inhibitors of
the Tudor domain containing protein, Spindlin1.
Human SETDB1 is a histone lysine methyltransferase
that specifically trimethylates histone H3 lysine
9
(H3K9me3). It has been demonstrated to be an oncogene
and found to be overexpressed in many cancers.[8] SETDB1 is
a multidomain protein containing unique tandem tudor
domains (TTD), a methyl- DNA binding domain (MBD),
and a classical catalytic SET domain.[9] Tudor domains often
recognize methylated lysine.[10] A very recent study by
Jurkowska et al demonstrated that TTD in SETDB1
(SETDB1-TTD) specifically binds to histone H3 tails con-
taining K9 methylation combined with K14 acetylation
(H3K9me/K14ac).[11] However, the exact biological function
of SETDB1-TTD remains unclear. Small molecule inhibitors
that selectively disrupt the binding of SETDB1-TTD to its
endogenous binders could be useful tool compounds for
revealing the biological function of SETDB1-TTD and could
also be potential disease intervention agents. Recently, Mader
et al reported a fragment hit for SETDB1-TTD with a weak
binding affinity (KD: 5 mM).[12] Nevertheless, no potent and
selective small molecule tool compounds targeting SETDB1-
TTD have been reported at present. The work herein
describes the discovery of such a tool compound.
To identify SETDB1-TTD inhibitors, a screening was
performed against an in-house chemical library, which con-
tains about 5000 compounds synthesized by our group, by
using a differential scanning fluorimetry (DSF) assay with
10 mM TTD protein and 100 mM test compounds. One
compound, 3,5-dimethyl-2-{[(3R,5R)-1-methyl-5-phenylpi-
peridin-3-yl]amino}-3,5-dihydro-4H-pyrrolo[3,2-d]pyrimidin-
4-one (Cpd1, Figure 1A), showed a thermal shift (DTm) of
1.588C in the DSF assay. The thermal shift values were also
dose dependent (Figure S1). The bioactivity of this compound
was further validated by an isothermal titration calorimetry
(ITC) assay, which gave a KD value of 4.4 Æ 1.7 mM (Fig-
ure 1B).
H
istone “reader” proteins, which use structurally conserved
domains to recognize and engage histone post-translational
modifications (PTMs), play a critical role in the functional
interpretation of the so-called “histone code” and hence in
regulating gene expression and signal transduction.[1] The
dysregulation of histone reader proteins has been linked to
the development of various human diseases, particularly
cancer.[2] As a consequence, the histone reader proteins have
become promising targets for drug development.[3] Currently,
many histone reader proteins have been identified.[4] Among
them, tudor domains, a type of methyllysine reader proteins,
have recently attracted attention due to their association with
various cancers.[5] Nevertheless, unlike the widely studied
bromodomain (BRD)-containing proteins (acetyllysine read-
ers), which have a large amount of inhibitors reported with
several having already reached clinical trials,[6] a very limited
number of small molecule inhibitors targeting tudor domain-
containing proteins have been reported.[7] And only two
potent and selective inhibitors were disclosed, namely
Further structural optimization of Cpd1 was then carried
out. To this end, we first solved the X-ray crystal structure of
TTD in complex with Cpd1. As shown in Figure 1C and
Figure 1D (PDB entry: 7C9N), Cpd1 binds to the region
between tudor 2 and tudor 3, which is different from the
Spindlin1 inhibitors MS31 and VinSpinln; they bind to tudor 2
(MS31) or the region between tudor 1 and tudor 2 (VinS-
pinln) of Spindlin1 (Figure S2). The 1-methyl-3-phenylpiper-
idine moiety resides in an aromatic cage formed by Y301,
Y268, W275, Y277 and F297. The nitrogen (-NH) linking
pyrrolo[3,2-d]pyrimidin-4-one and piperidine ring forms
a hydrogen bond with the phenol-oxygen of Y268. The
[*] Y. Guo,[+] X. Mao,[+] L. Xiong,[+] A. Xia, J. You, G. Lin, C. Wu, L. Huang,
Y. Wang, Prof. Dr. S. Yang
State Key Laboratory of Biotherapy and Cancer Center, West China
Hospital, Sichuan University
Chengdu, Sichuan 610041 (P. R. China)
E-mail: yangsy@scu.edu.cn
[+] These authors contributed equally to this work.
Supporting information and the ORCID identification number(s) for
the author(s) of this article can be found under:
https://doi.org/10.1002/anie.202017200.
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Figure 1. Bioactivity of Cpd1 and the crystal structure of the Cpd1-TTD complex (PDB entry 7C9N). A) The chemical structure of Cpd1. B) ITC
analysis of Cpd1 (KD =4.4Æ1.7 mM). C) Surface view of the complex of TTD (surface and cartoon representation) and Cpd1 (thick yellow stick
representation) (left) and the electron density of Cpd1 (right). D) The interaction mode of Cpd1 (yellow stick representation) with TTD.
ionized nitrogen in 1-methylpiperidine forms an electrostatic
interaction with D299.
According to the cocrystal structure, we assumed that
three sites of Cpd1 could be modified to improve its binding
affinity. The first site is the methyl group on the pyrrole
nitrogen because replacing the 5-position methyl group with
hydrogen could enhance the binding affinity due to a new
hydrogen bonding interaction between pyrrole nitrogen
(-NH) and E386. The second group is the phenyl group
because there seems to be some incompletely occupied space
in its surroundings. The third group is the 3-methyl group of
pyrrolo[3,2-d]pyrimidin-4-one since there is a large empty
space around the methyl group.
Accordingly, in the first step, we replaced the 5-position
methyl group on the pyrrole nitrogen with hydrogen and
synthesized a new compound (12) (Figure S3A). Scheme 1
shows the synthetic route of compound 12. Commercially
available (R)-1-Boc-3-aminopiperidine reacted with the
bidentate directing group picolinic acid to produce inter-
mediate 2, which underwent C(sp3)-H arylation with iodo-
Scheme 1. Synthesis of compound 12. Reagents and conditions:
a) picolinic acid, HATU, DIEA, DCM, rt, 24 h, 85%; b) aryl iodides,
Pd(OAc)2, Ag2CO3, 2,6- dimethylbenzoic acid, t-BuOH, 1208C, 36 h,
66%, >99% ee; c) TFA, DCM, rt, 4 h; d) 37% formaldehyde, NaBH-
(OAc)3, AcOH, DCM, rt, 6 h, 88%; e) NaOH, i-PrOH, 858C, 18 h,
80%; f) 1 M NaOH (aq), 1008C, 16 h, 92%; g) Boc2O, TEA, DMAP,
DMF, rt, overnight, 82%; h) iodomethane, NaH, anhydrous DMF,
408C, overnight; i) TFA, DCM, rt, 4 h, 46% over two steps; j) DIPEA,
NMP, 1508C, 2 h, 14%.
benzene to afford enantiomerically pure 3. This intermediate
was further converted to key intermediate 6 through Boc
deprotection (4), reductive amination (5), and cleavage of the
bidentate directing group. Commercially available 2,4-
dichloro-pyrrolo[3,2-d]pyrimidine (7) underwent a series of
reactions, including hydrolysation (8), Boc protection (9),
nucleophilic substitution (10) and Boc deprotection, to
generate another key intermediate 11. Finally, 11 underwent
a SNAr reaction with 6 to produce target compound 12.
DSFand ITC assays were adopted to test the bioactivity of
compound 12. This compound indeed showed significantly
improved potency compared with Cpd1; the DTm and KD
values of compound 12 are 5.168C and 1.6 Æ 0.22 mM,
respectively (Table 1, Figure S3B). We then solved the crystal
structure of TTD in complex with compound 12. The crystal
structure (PDB entry: 7CAJ) shows that compound 12
occupies the same binding site of TTD with the same pose
as Cpd1, and key interactions between Cpd1 and TTD remain
(Figure S3C). Replacement of the 5-position methyl with
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Table 1: Bioactivities of the newly synthesized compounds.
We finally modified the 3-methyl group of pyrrolo[3,2-
d]pyrimidin-4-one. Various alkyl groups were used to replace
the methyl group, and five new compounds (56–60) were
synthesized. Scheme 2 shows the reaction routes for these
compounds. Commercially available 3-amino-2-ethoxycarbo-
nylpyrrole reacted with 2-isocyanatopropane to produce 49,
which then underwent a cyclization reaction and successive
chlorination to generate intermediate 51. Nucleophilic sub-
stitution reactions of 9 with bromoethane, 1-bromopropane,
allyl bromide and benzyl bromide gave corresponding
intermediates 52–55. Compounds 51–55 were then reacted
with the previously obtained intermediate 38 to generate the
final products 56–60.
The bioactivities of compounds 56–60 are shown in
Table 1. Compounds 56–58 and 60 bearing isopropyl, ethyl,
n-propyl, and benzyl at the 3-position of pyrrolo[3,2-d]pyr-
imidin-4-one, respectively, exhibited a decreased binding
affinity compared with 45, indicating that a bulkier substitu-
ent is not preferred. However, 59 with a propenyl group
displayed improved potency with a KD value of 0.088 Æ
0.045 mM in the ITC assay (Table 1, Figure 2A, and B). It
also dose-dependently stabilized the TTD protein in the DSF
assay (Figure 2C).
Cpd
R1
R2
R3
DTm [8C][a]
KD [mM]
12
41
42
43
44
45
46
47
48
56
57
58
(R,R)-59
60
(S,S)-59
H
H
H
Br
H
H
BnO
H
H
H
H
H
Me
Me
Me
Me
Me
Me
Me
Me
Me
iPr
Et
Pr
Allyl
Bn
Allyl
5.16
3.26
1.89
4.40
4.03
8.15
2.33
2.79
3.79
3.87
6.71
6.04
5.79
NA[b]
NA
1.6Æ0.22
0.72Æ0.12
2.0Æ1.4
MeO
CF3O
H
PhO
BnO
H
0.54Æ0.05
0.52Æ0.51
0.21Æ0.13
1.9Æ0.25
0.91Æ0.18
1.5Æ0.13
0.47Æ0.12
0.26Æ0.10
0.36Æ0.004
0.088Æ0.045
NB[c]
nBu
OH
BnO
BnO
BnO
BnO
BnO
BnO
H
H
H
H
NB
[a] Protein concentration 10 mM, compound concentration 100 mM;
[b] NA: no activity; [c] NB: no binding.
To explain this unusual phenomenon, we again solved the
crystal structure of TTD in complex with 59 (Figure 2D).
Compound 59 also resides in the same binding site with the
same pose as Cpd1, 12 and 45. All the key interactions
between compound 45 and TTD are maintained. We noticed
hydrogen shortens the distance between E386 and the 5-
position -NH (from 4 ꢀ to 2.6 ꢀ), enabling the formation of
a hydrogen bond between them, as expected.
We then optimized the phenyl group, which resides in
a hydrophobic pocket formed by residues T210, T212, and
W275 in tudor 1 and tudor 2. Various substituents were
installed on the phenyl ring, which generated eight new
compounds (41–48). Compounds 41–47 were synthesized
=
that the phenolic hydroxyl of Y268 faces the C C double
bond of propenyl with C-O distances of 2.995 ꢀ (C1-O) and
2.822 ꢀ (C2-O), implying that a s*-p interaction formed
(Figure 2E). This could explain why 59 has a higher potency
than the others.
following
a similar route to that for compound 12
(Scheme S5). Compound 48 was prepared by the Pd/C
catalysed debenzylation of compound 45.
The bioactivities of the synthesized compounds are shown
in Table 1. Unexpectedly, all the newly synthesized com-
pounds with either small or bulky substituents showed
reduced or at most comparable activity with respect to the
unsubstituted compound 12. The only exception is compound
45 (Figure S3D), which bears a para-benzyloxy group. This
compound showed elevated potency with DTm and KD values
of 8.158C and 0.21 Æ 0.13 mM, respectively (Table 1, Fig-
ure S3E). To understand the intrinsic reason, we solved the
crystal structure of TTD in complex with compound 45. As
shown in Figure S3F, compound 45 resides in the same
binding site with the same pose as Cpd1 and 12. Notably, the
indole ring of W275 undergoes a 238 counter clockwise
rotation, which positions the indole ring parallel to the
benzene ring of benzyloxy, forming an F-type p-p stack
interaction (Figure S3G). Obviously, para-methoxyl (41),
para-trifluoromethoxy (42), meta-bromine (43), para-phe-
noxyl (44), para-n-butyl (47), and para-hydroxyl (48) could
not form such interactions due to either the lack of a benzene
ring (41–43, 47, 48) or insufficient length (44). Although meta-
benzyloxy derivative 46 harbours a benzyloxy group like 45,
the meta-position benzyloxy cannot induce a rotation of the
indole ring of W275 to form p-p interactions and instead
causes unfavourable steric interactions.
Scheme 2. Synthesis of compounds 56–60. Reagents and conditions:
a) 2-isocyanatopropane, Et3N, toluene, rt, 24 h, 95%; b) 1 M KOH
(MeOH), reflux, 2 h, 60%; c) POCl3, 1008C, 24 h, 23%; d) haloalkanes,
NaH, anhydrous DMF, 508C, overnight; e) benzyl bromide, NaH, LiBr,
DME/DMF, 658C, 8 h; f) TFA, DCM, rt; 34–78%, over two steps;
g) DIPEA, NMP, 1508C, 2 h, 12–24%.
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Figure 2. Biophysical activities and the binding mode of (R,R)-59.
A) The chemical structure of (R,R)-59. B) ITC analysis of (R,R)-59
(KD =0.088Æ0.045mM). C) Dose-dependent curve of (R,R)-59 in the
DSF assay. D) Binding modes of (R,R)-59 with SETDB1-TTD (PDB
entry: 7CJT). E) Presumed s*-p interaction between the phenolic
Figure 3. Selectivity and competition assays of (R,R)-59. A) Binding
affinity of (R,R)-59 with 17 tudor domain-containing proteins measured
by ITC. B) A superposition of crystal structures of the TTD-(R,R)-59
complex (PDB entry: 7CJT) and TTD-H3 peptide (PDB ID: 6BHD).
C) ITC analysis of different H3 peptides (H3K9me/K14ac, H3K9me2/
K14ac, H3K9me3/K14ac) and SETDB1-TTD. D) Competition experi-
ment with (R,R)-59 and different H3 peptides using ITC analysis.
SETDB1-TTD protein was pretreated with (R,R)-59 for 20 minutes and
then titrated with different H3 peptides. E) Competition experiment
with (S,S)-59 and different H3 peptides using ITC analysis. SETDB1-
TTD protein was pretreated with (S,S)-59 for 20 minutes and then
titrated with different H3 peptides. F) The HTRF test system consists
of GST-SETDB1-TTD and biotinylated H3 peptides (Bio-H3K9me2/
K14ac, and Bio-H3K9me3/K14ac), which is used to test the inhibitory
activity of compounds against the TTD–endogenous H3 peptide
interaction.
=
hydroxyl of Y268 and the C C double bond of propenyl in (R,R)-59.
F) SPR analysis of (R,R)-59.
Because 59 is a chiral compound with an (R,R) confor-
mation, the other enantiomer (S,S)-59 was also synthesized
(for clarity, 59 will be denoted as (R,R)-59 below). The
synthetic route of (S,S)-59 is very similar to that of (R,R)-59;
the only difference is that the starting material (S)-3-amino-1-
N-boc-piperidine was used instead of (R)-3-amino-1-N-boc-
piperidine in the synthesis of (S,S)-59 (Scheme S6, Fig-
ure S3H). Interestingly, this compound did not show activity
in either the DSF or ITC assay (Table 1, Figure S3I).
To further validate the bioactivity of (R,R)-59, a surface
plasmon resonance (SPR) assay, which is another commonly
used biophysical activity assay method, was performed. In this
assay, (R,R)-59 showed a KD value of 0.106 Æ 0.002 mM
against TTD (Figure 2F). Again, the enantiomer (S,S)-59
did not exhibit activity in this assay.
To examine the selectivity of (R,R)-59, we expressed and
purified 16 other tudor domains from different tudor domain-
containing proteins. The ITC assay was adopted to test the
bioactivity of (R,R)-59 against these proteins (Figure 3A,
Table S2). (R,R)-59 did not show activity against 14 of the 16
tested tudor domains (KD > 100 mM). 53BP1 and JMJD2A
were the only two tudor domain proteins for which (R,R)-59
showed some activity; the KD values were 4.3 mM and 86 mM,
respectively. Furthermore, (R,R)-59 was tested against 32
BRD proteins using the DSF assay. Among the 32 proteins, it
showed no activity or very weak activity (Table S3, Fig-
ure S4A). All these results indicate that (R,R)-59 has
considerable selectivity for SETDB1-TTD.
SETDB1-TTD has been demonstrated to bind to doubly
modified histone H3 with the combination of K9 methylation
and K14 acetylation (H3K9me/K14ac). Further crystal struc-
tures revealed that the H3 peptides bind to the groove
between TD2 and TD3.[11] A superposition of the crystal
structures of the TTD-(R,R)-59 complex (PDB entry: 7CJT)
and TTD-H3 peptide (PDB ID: 6BHD) shows an overlap
between the (R,R)-59 and H3 peptide binding sites (Fig-
ure 3B), implying that (R,R)-59 could prevent or at least
interfere with the binding of H3 peptide. To verify this point,
we performed ITC experiments with differently modified
H3(4–19) peptides and TTD. The dissociation constants (KD)
of TTD and H3K9me/K14ac, H3K9me2/K14ac, and
H3K9me3/K14ac were 3.36 mM, 1.96 mM, and 3.98 mM,
respectively (Figure 3C), which are comparable to the
literature values.[11] After incubating TTD with (R,R)-59 for
20 minutes, the binding of H3 peptides with TTD could not be
detected (Figure 3D), suggesting that (R,R)-59 completely
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prevented the association of H3 peptides and TTD. In the
same assay, the enantiomer (S,S)-59 did not show an impact
on the association of H3 peptides and TTD (Figure 3E).
Further, a homogenous time-resolved fluorescence (HTRF)
assay was used to test the inhibitory activity of (R,R)-59
against the TTD—H3 peptide interaction. The measured IC50
values of (R,R)-59 against the TTD—H3K9me2/K14ac and
TTD—H3K9me3/K14ac interactions were 0.93 mM and
0.75 mM, respectively (Figure 3F). Again, the enantiomer
(S,S)-59 did not exhibit obvious activity in this assay. All the
results suggest that (R,R)-59 is an endogenous binder
competitive inhibitor.
using EGFP tagged SETDB1-TTD (wild-type) and Y268A
mutant plasmids; the Y268A mutation has been demon-
strated to lead a specific loss in the binding of H3 endogenous
binders.[11] Cells were treated with (R,R)-59 (6 mM), (S,S)-59
(6 mM), or DMSO for 18 hours. The normalized half-life
recovery times (t1/2) of (S,S)-59 and DMSO treatment groups
were 1.50 Æ 0.31 s and 1.45 Æ 0.34 s, respectively. Both the
(R,R)-59 treatment group and Y268A mutant group showed
a significantly decreased t1/2 (1.05 Æ 0.33 s, and 1.01 Æ 0.17 s,
respectively) (Figure 4C), meaning that the majority of the
protein is mobile. All the experimental results clearly indicate
(R,R)-59ꢁs cellular target engagement.
To verify whether (R,R)-59 interacts with SETDB1-TTD
in intact cells, we performed a cellular thermal shift assay
(CETSA); CETSA is a method based on the thermal
stabilization of target proteins upon drug binding. In this
assay, HEK293T cells stably transfected with a pLVX-
mCherry-N1-SETDB1-TTD plasmid were used (Figure S4B).
The cells were treated with (R,R)-59 and (S,S)-59 in different
concentrations at 508C and lysed by repeated freeze-thaw
cycles with liquid nitrogen. The protein content of the soluble
fraction in the lysate was determined by western blot analysis.
The results showed that (R,R)-59 at concentrations of >=
5 mM could efficiently and dose-dependently stabilize the
SETDB1-TTD protein in HEK293T cells. In contrast, the
enantiomer (S,S)-59 did not exhibit a stabilizing effect in all
the tested concentrations (Figure 4A, and B). To further
examine the activity of (R,R)-59 in living cells, we performed
a fluorescence recovery after photobleaching (FRAP) assay
Finally a RNA-sequencing (RNA-seq) experiment was
conducted to explore the effect of (R,R)-59 on global gene
expression. In this experiment, human acute monocytic
leukemia THP-1 cells were treated with (R,R)-59 (10 mM),
(S,S)-59 (10 mM), or DMSO for 24 hours, and the cells were
then collected for RNA-seq analysis. (R,R)-59 treatment
significantly affected the expression of 72 genes (> 4-fold).
Among them, 49 genes were uniquely affected by (R,R)-59,
which may be attributed to the effect of SETDB1-TTD
(Figure 4D). Even so, the biological significance of (R,R)-59
causing gene up-regulation or down-regulation still needs to
be further studied.
In conclusion, we report the discovery of a potent and
selective cell-active SETDB1-TTD inhibitor (R,R)-59.
(R,R)-59 and its inactive enantiomer (S,S)-59 could serve as
a pair of early-stage tool compounds for investigating
biological functions and disease associations of SETDB1-
TTD. Moreover, the current study together with recent
discoveries of potent and selective small molecule inhibitors
of Spindlin1[7b,c] clearly demonstrated the feasibility of
targeting tudor domains.
Acknowledgements
This work was supported by the National Natural Science
Foundation of China (Grant No: 81930125, 21772130 and
81773633), and partly by the fast-track research fund of
Sichuan Province (2020YFS0006) and 1.3.5 project for
disciplines of excellence, West China Hospital, Sichuan
University (ZYGD18001). The authors thank the staffs
from BL17U1, BL18U, and BL19U1 beamlines of National
Facility for Protein Science Shanghai (NFPS) at Shanghai
Synchrotron Radiation Facility (SSRF) for assistance during
data collection. The ITC experiments were carried out at the
Core Facilities at College of Life Sciences of Sichuan
University.
Figure 4. Cellular target engagement assays. A) The CETSA assay was
carried out with Flag-SETDB1-TTD transfected HEK293T cells grown in
the presence or absence of (R,R)-59/(S,S)-59 for 6 h. B) Stabilization
effect of Flag-SETDB1-TTD induced by (R,R)-59 or (S,S)-59 normalised
to DMSO. C) The FRAP experiments using 293T cells transfected with
EGFP-tagged SETDB1-TTD wild-type (WT) and Y268A mutant. The
SETDB1-TTD group was treated with 6 mM (R,R)-59 or (S,S)-59 (18 h).
Eighteen cells were imaged for each group and one-way ANOVA with
Dunnett’s correction for multiple comparisons was used to detect
significant differences (P<0.05). D) Heat map of selected genes
showing different regulation by (R,R)-59, (S,S)-59, or DMSO (up- or
down- regulation >4-fold and p<0.05 of (R,R)-59).
Conflict of interest
The authors declare no conflict of interest.
Keywords: epigenetics · SETDB1 ·
structure-based optimization · tool compound · tudor domain
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Manuscript received: December 27, 2020
Accepted manuscript online: January 28, 2021
Version of record online: March 8, 2021
Angew. Chem. Int. Ed. 2021, 60, 8760 –8765
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