Unveiling epidithiodiketopiperazine as a non-histone arginine methyltransferase inhibitor by chemical protein methylome analyses

Article abstract of DOI:10.1039/c8cc03907k

We present a chemical methylome analysis platform to evaluate the inhibitory activity of small molecules towards poorly characterized protein methyltransferases (PMTs), facsilitating to identify syn-HyPA-ETP-2 as a non-histone arginine methyltransferase inhibitor.

Full text of DOI:10.1039/c8cc03907k

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Unveiling epidithiodiketopiperazine as a
non-histone arginine methyltransferase inhibitor
Cite this:DOI: 10.1039/c8cc03907k
by chemical protein methylome analyses†
Received 16th May 2018,
Accepted 23rd July 2018
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a
a
Yoshihiro Sohtome,
Tadahiro Shimazu, Joaquin Barjau, Shinya Fujishiro,
a
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Mai Akakabe, Naoki Terayama,
Kosuke Dodo,
Akihiro Ito,
DOI: 10.1039/c8cc03907k
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Minoru Yoshida, Yoichi Shinkai and Mikiko Sodeoka
*
rsc.li/chemcomm
11a,13
We present a chemical methylome analysis platform to evaluate the protocol using ProSeAM (1) for enriching the native methylome,
inhibitory activity of small molecules towards poorly characterized which includes the following steps; (i) ProSeAM-mediated pro-
14
protein methyltransferases (PMTs), facsilitating to identify syn- pargylation, (ii) Cu(I)-catalysed alkyne-azide cycloaddition (CuAAC)
HyPA-ETP-2 as a non-histone arginine methyltransferase inhibitor. to incorporate biotin and (iii) enrichment of the biotinylated sub-
8
strates using streptavidin-immobilized beads (Fig. 1). Although
Research on protein methyltransferase (PMT) inhibitors has ProSeAM (1) does not completely correlate to physiologically relevant
1
expanded rapidly over the past decade, in part because selective methylation events, their work opened up opportunities to identify
small-molecular inhibitors of PMTs would be potentially useful the enormous numbers of substrate candidates of endogenous
as probes for investigating the biological functions of PMTs. We have recently established modified protein methylome
2
3
PMTs. High-throughput screening (HTS) using a set of purified
enzymes/proteins (or peptides) is currently the most widely used
1
technology to screen for PMT inhibitors. However, success so far
has been limited, probably due to the unsuitability of single-gene/
single-protein/single-assay methodology for dealing with large
4
numbers of poorly characterized proteins. In particular, there has
5,6
been little work on non-histone methyltransferase inhibitors even
s
though the PhosphoSitePlus database currently contains more
than 15 000 methylation sites (counted redundantly for mono-,
7
di- and trimethylation) in human non-histone proteins. The
development of a robust assay system for efficiently assessing
the inhibitory activity of small-molecules against a wide range of
non-histone PMTs and identifying their substrates would be
extremely useful. Here, we report an approach for evaluating
non-histone methylation inhibitors on the basis of chemical
8
–10
methylome analysis
analogue ProSeAM (1: also called SeAdoYn).
Most strategies for methylome analyses begin by enriching
using the S-adenosylmethionine (SAM)
1
1
1
2
the methylated protein substrates. Luo reported an excellent
a
Synthetic Organic Chemistry Laboratory, RIKEN Cluster for Pioneering Research,
2
-1, Wako, Saitama 351-0198, Japan. E-mail: sodeoka@riken.jp
b
c
d
e
f
RIKEN Center for Sustainable Resource Science, RIKEN, Japan
AMED-CREST, Japan Agency for Medical Research and Development, Japan
Cellular Memory Laboratory, RIKEN Cluster for Pioneering Research, Japan
School of Life Sciences, Tokyo University of Pharmacy and Life Sciences, Japan
Department of Biotechnology, Graduate School of Agricultural Life Sciences, The
University of Tokyo, Japan
Fig. 1 Schematic protocol of chemical methylome profiling using
ProSeAM (1) (A) for identification of unknown PMT-substrate proteins;
(B) for identification of inhibitors and the corresponding PMT-substrate
proteins; (C) control without treatment of lysate.
†
Electronic supplementary information (ESI) available. CCDC 1825180. For ESI and
crystallographic data in CIF or other electronic format see DOI: 10.1039/c8cc03907k
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analyses, which enabled us to correlate recombinant, unchar-
1
5
acterized PMTs with their substrates using ProSeAM (1). A key
feature is the comparison of two sets of samples with or without
exogenous addition of a recombinant PMT (Fig. 1A vs. C). Then,
its substrate proteins are identified based on the increased
intensity of MS signals of the labeled proteins. This chemical
methylome platform using 1 led to identification of various
substrates of both SET-domain-containing- and seven-beta-
1
6
strand PMTs in the nucleus, cytosol and other subcellular
organelles. The success in detecting the differences of labeled
proteins in response to the amount of exogenous recombinant
PMT in both gel- and MS-based analyses prompted us to apply
this chemical methylome profiling platform for the discovery of
8
PMT inhibitors. We envisioned that inhibitory potency and
Scheme 1 Reagents and conditions: (a) L-Ala-OMe, HBTU, DIPEA, MeCN,
selectivity could be evaluated by comparing the enrichment values
in the absence and presence of the inhibitor (Fig. 1B vs. C).
To test the feasibility of this concept, we selected epidithio-
rt, 3 h; (b) H
NaH, DMF, 4 h at 0 1C then 1 h at rt; (e) S
f) (1) NaBH , THF/EtOH = 1/1, 0 1C to rt, 1 h, (2) I
2
2
, Pd/C, MeOH, rt, 8 h; (c) NH
4
OHaq, MeOH, rt, 6 h; (d) MeI,
, NaHMDS, THF, rt, 30 min;
, EtOAc, 0 1C, 5 min;
8
(
4
diketopiperazines (ETPs) as inhibitor candidates (Fig. 2). These (g) 12 M HCl, rt to 80 1C, 2 h.
compounds structurally resemble the fungal metabolite, chaetocin
(
2), which was identified as the first histone lysine methyltransferase
17–19
(HKMT) inhibitor in 2005,
and found to be a potent inhibitor of the corresponding alcohols anti-HyPA-ETP-2 (5a) and syn-HyPA-
20
G9a and SUV39H1. However, ETPs have limited utility as HKMTs ETP-2 (5b).
inhibitors because of their high toxicity, and apparently non-
selective mode of action.
21
To evaluate the potency and protein substrate selectivity of
22–24
We recently demonstrated that the ETPs we compared the ProSeAM (1)-mediated labeling pattern
simple monomeric ETP derivative, (Æ)-PS-ETP-1 (3) having a similar (Fig. 3), wherein the biotin-labeled proteins obtained with
level of G9a-inhibitory activity (2: IC50 = 7.2 mM, 3: IC50 = 5.2 mM) is 1-mediated propargylation/CuAAC were detected by chemi-
2
5
far less toxic than the parent chaetocin (2). This raised the luminescence using streptavidin-horseradish peroxidase. The
question of whether the inhibition pattern could be modulated gel-based chemical methylome assay in the presence [lanes 2–6:
by further structural modification of the ETP core. To answer anti-4a, 8–12: syn-4b, 14–18: anti-5a, 20–24: syn-5b, 26–30:
this question, we focused on the stereochemical and substituent chaetocin (2)] or absence of ETPs (lanes 1, 7, 13, 19 and 25)
effects in the monomeric ETP core and planned to perform provides insights into their inhibitory selectivity against ProSeAM
methylome-based profiling of pilot diastereomer sets of the (1)-mediated protein labeling. The unique histone/non-histone
chiral monomeric anti-HyPA-ETP and syn-HyPA-ETP, which selectivity of ETPs is highlighted in Fig. 3B, which shows the IC50
were synthesized from hydroxyproline and alanine.
values of each ETP against the histone band (17 kDa) and non-
The ETPs were concisely prepared from the protected histone bands (between 20 kDa and 250 kDa). For example, anti-4a
L-hydroxyproline 6 and L-alanine methyl ester (Scheme 1). The more strongly inhibits the histone band than the non-histone
diastereodivergent direct sulfenylation of 8 was achieved with bands. In marked contrast, syn-5b is rather selective for non-
Nicolaou’s procedure using the combination of elemental histone substrates. These gel-based assays using ProSeAM (1)
2
6,27
sulfur S
8
and sodium hexamethyldisilazide (NaHMDS),
suggest that the asymmetric environment of monomeric ETP-type
and the subsequent redox reactions gave the diastereo mixtures inhibitors contributes to their PMT substrate selectivity (propargy-
of anti-HyPA-ETP-1 (4a) and syn-HyPA-ETP-1 (4b). The diastereomers lation inhibition ratio of IC50 values for inhibition of histones/
were separated with usual silica-gel chromatography to isolate anti- non-histones; anti-4a: 0.083 vs. syn-5b: 5.6). These results indicate
4a in 24% yield and syn-4b in 13% yield (three reaction sequences that chemical methylome profiling using 1 could be a powerful
from 8). The relative stereochemistry of anti-4a was confirmed by method to assess the selectivity of PMT inhibitors.
28
X-ray analysis (CCDC 1825180). Finally, the TBS group in anti-4a
Encouraged by the unique non-histone-selective inhibitory
and syn-4b was deprotected under acidic conditions, affording activity of syn-5b, we next characterized the inhibition profiles
Fig. 2 The structures of (A) chaetocin (2), (B) (Æ)-PS-ETP-1 (3), (C) anti-HyPA-ETP and syn-HyPA-ETP.
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Fig. 3 (A) Competitive chemical methylome profiling using 1 in the presence of ETP-based PMT inhibitors at various concentrations: none (lanes 1, 7, 13,
9, and 25), 1 mM (lanes 2, 8, 14, 20, and 26), 10 mM (lanes 3, 9, 15, 21, and 27), 50 mM (lanes 4, 10, 16, 22, and 28), 100 mM (lanes 5, 11, 17, 23, and 29), and
50 mM (lanes 6, 12, 18, 24, and 30); (B) the IC50 values of ETP-based PMT inhibitors. IC50 values for histone were calculated from the intensity at the
7 kDa band, whereas those for non-histone were calculated from the integrated value of bands from 20 to 250 kDa (n = 3).
1
2
1
of syn-5b by means of LC-MS/MS and biochemical assays (Fig. 4). HNRNPK (IC50: 4200 mM). Preliminary investigations using
The quantitative MS comparison analyses led to identification of HeLa cells showed that syn-5b can reduce the formation of
2
8
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a total of 90 proteins as substrate candidates, whose intensities asymmetric dimethyl arginines (ADMA) in living cells. Thus, our
were decreased at least 2-fold by syn-5b compared to the control findings indicate that ETPs, which have been widely recognized as
(
Fig. 3A, lanes 19–24). Among the substrate candidates, 24 PKMT inhibitors, can be converted to PRMT inhibitors by suitably
31
proteins have been reported as substrates of post-translational modifying the ETP core.
methylation. Interestingly, the substrates of protein arginine
7
In summary, this study highlights not only the development
2
9
methyltransferase (PRMT) constitute the majority, while those of a competitive chemical methylome profiling system using
of PKMT are minor (methylation on only R: 17 proteins, both R ProSeAM (1) for assessing PMT inhibitors and identifying the
and K: 4 proteins, only K: 3 proteins). To our knowledge, the corresponding PMT-substrate proteins, but also a characterization
PRMT-inhibitory activity of ETPs has not previously been of the ETP-based pharmacophore of PMT inhibitors. Most
reported. In order to confirm whether syn-5b acts as a non- importantly, the chemical methylome platform allows us to
histone arginine methyltransferase inhibitor, in vitro methylations evaluate the PMT-inhibitory activity of small molecules towards
were examined (Fig. 4B). We focused on HNRNPK (heterogeneous a huge number of poorly characterized PMTs. Our studies using
nuclear ribonucleic protein K) as a non-histone substrate for this a pilot set of simple chiral ETPs revealed that the stereo-
study because several roles of the arginine methylation of chemistry and substituent on the monomeric ETP-type inhibitors
30
HNRNPK by PRMT-1 have been recently reported. As shown in contribute to their substrate protein selectivity (histones vs. non-
Fig. 4B, syn-5b inhibits PRMT1-catalyzed methylation of HNRNPK histones) and residue selectivity (lysine vs. arginine). Specifically,
(
IC50: 71.7 Æ 16.3 mM, n = 4). On the other hand, anti-4a, which syn-HyPA-ETP-2 (syn-5b) is a prototype non-histone methylation
showed histone-selective methylation inhibition activity, was inhibitor. We believe that this chemical methylome strategy will
found to be inactive against PRMT1-catalyzed methylation on accelerate the development of PMT inhibitors with superior target
selectivity and reduced toxicity.
This work was partially supported by a RIKEN’s Strategic Programs
and KAKENHI (JP17H02213, JP18H04277 and JP18K19156) from
JSPS and AMED-CREST (No. JP18gm0710004). J. B. thanks to
JSPS Fellowship Programs for Overseas Researchers and RIKEN
FPR programs. We are grateful to the Support Unit for Bio-
material Analysis, RIKEN BSI, for help with mass spectrometric
analysis. We also thank Dr Daisuke Hashizume for conducting
X-ray single-crystal analysis of anti-4a, and Hiroaki Takagi for
cytotoxicity assay.
Conflicts of interest
There are no conflicts to declare.
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