A potent, selective and cell-active allosteric inhibitor of protein arginine methyltransferase 3 (PRMT3)

Article abstract of DOI:10.1002/anie.201412154

PRMT3 catalyzes the asymmetric dimethylation of arginine residues of various proteins. It is essential for maturation of ribosomes, may have a role in lipogenesis, and is implicated in several diseases. A potent, selective, and cell-active PRMT3 inhibitor would be a valuable tool for further investigating PRMT3 biology. Here we report the discovery of the first PRMT3 chemical probe, SGC707, by structure-based optimization of the allosteric PRMT3 inhibitors we reported previously, and thorough characterization of this probe in biochemical, biophysical, and cellular assays. SGC707 is a potent PRMT3 inhibitor (IC₅₀=31±2 nM, K_D=53±2 nM) with outstanding selectivity (selective against 31 other methyltransferases and more than 250 non-epigenetic targets). The mechanism of action studies and crystal structure of the PRMT3-SGC707 complex confirm the allosteric inhibition mode. Importantly, SGC707 engages PRMT3 and potently inhibits its methyltransferase activity in cells. It is also bioavailable and suitable for animal studies. This well-characterized chemical probe is an excellent tool to further study the role of PRMT3 in health and disease. High selectivity: The first PRMT3 chemical probe, SGC707, was discovered by structure-based optimization. SGC707 is a potent PRMT3 inhibitor with outstanding selectivity. The mechanism of action studies and crystal structure of the PRMT3-SGC707 complex confirm the allosteric inhibition mode. SGC707 engages PRMT3 and potently inhibits its methyltransferase activity in cells. It is also bioavailable and suitable for animal studies.

Full text of DOI:10.1002/anie.201412154

Angewandte
Chemie
DOI: 10.1002/anie.201412154
Chemical Probes
A Potent, Selective and Cell-Active Allosteric Inhibitor of Protein
Arginine Methyltransferase 3 (PRMT3)**
H. ꢀmit Kaniskan, Magdalena M. Szewczyk, Zhengtian Yu, Mohammad S. Eram,
Xiaobao Yang, Keith Schmidt, Xiao Luo, Miao Dai, Feng He, Irene Zang, Ying Lin,
Steven Kennedy, Fengling Li, Elena Dobrovetsky, Aiping Dong, David Smil, Sun-Joon Min,
Melissa Landon, Jennifer Lin-Jones, Xi-Ping Huang, Bryan L. Roth, Matthieu Schapira,
Peter Atadja, Dalia Barsyte-Lovejoy, Cheryl H. Arrowsmith, Peter J. Brown, Kehao Zhao,*
Jian Jin,* and Masoud Vedadi*
Abstract: PRMT3 catalyzes the asymmetric dimethylation of mode. Importantly, SGC707 engages PRMT3 and potently
arginine residues of various proteins. It is essential for inhibits its methyltransferase activity in cells. It is also
maturation of ribosomes, may have a role in lipogenesis, and bioavailable and suitable for animal studies. This well-
is implicated in several diseases. A potent, selective, and cell- characterized chemical probe is an excellent tool to further
active PRMT3 inhibitor would be a valuable tool for further study the role of PRMT3 in health and disease.
investigating PRMT3 biology. Here we report the discovery of
the first PRMT3 chemical probe, SGC707, by structure-based
P
rotein arginine methyltransferase (PRMT) 3 is a type I
optimization of the allosteric PRMT3 inhibitors we reported PRMT that catalyzes mono- and asymmetric dimethylation of
previously, and thorough characterization of this probe in arginine residues. It was shown to be a cytosolic protein
biochemical, biophysical, and cellular assays. SGC707 is methylating 40S ribosomal protein S2 (rpS2) which is critical
a potent PRMT3 inhibitor (IC50 = 31 Æ 2 nm, KD = 53 Æ 2 nm) for proper maturation of 80S ribosome.[1] Recently it was
with outstanding selectivity (selective against 31 other methyl- shown that in cells treated with palmitic acid or T0901317 (a
transferases and more than 250 non-epigenetic targets). The liver X receptor a (LXRa) agonist) PRMT3 co-localizes with
mechanism of action studies and crystal structure of the LXRa in the cell nucleus, regulating hepatic lipogenesis.[2]
PRMT3-SGC707 complex confirm the allosteric inhibition However, this effect appears to be independent of PRMT3
[*] Dr. H. ꢀ. Kaniskan,[+] Dr. X. Yang, K. Schmidt, Dr. S.-J. Min,
Prof. Dr. J. Jin
Prof. Dr. M. Schapira, Prof. Dr. M. Vedadi
Department of Pharmacology and Toxicology, University of Toronto
Toronto, ON M5S 1A8 (Canada)
Departments of Structural and Chemical Biology, Oncological
Sciences, and Pharmacology and System Therapeutics
Icahn School of Medicine at Mount Sinai
New York, NY 10029 (USA)
Prof. Dr. C. H. Arrowsmith
Department of Medical Biophysics
University of Toronto and Princess Margaret Cancer Centre
101 College St., MaRS South Tower, Suite 707
Toronto, ON M5G 1L7 (Canada)
E-mail: jian.jin@mssm.edu
Dr. M. M. Szewczyk,[+] Dr. M. S. Eram,[+] Dr. S. Kennedy, Dr. F. Li,
E. Dobrovetsky, A. Dong, Dr. D. Smil, Dr. M. Landon,
Prof. Dr. M. Schapira, Dr. D. Barsyte-Lovejoy,
Prof. Dr. C. H. Arrowsmith, Dr. P. J. Brown, Prof. Dr. M. Vedadi
Structural Genomics Consortium, University of Toronto
Toronto, ON M5G 1L7 (Canada)
E-mail: m.vedadi@utoronto.ca
Dr. Z. Yu,[+] Dr. X. Luo, M. Dai, Dr. F. He, I. Zang, Dr. Y. Lin,
Dr. P. Atadja, Dr. K. Zhao
Novartis Institutes for Biomedical Research (China)
Zhangjiang Hi-Tech Park
[+] These authors contributed equally to this work.
[**] This research was supported by the grant R01GM103893 (to J.J.)
from the U.S. National Institutes of Health. The S.G.C. is
a registered charity (no. 1097737) that receives funds from AbbVie,
Bayer, Boehringer Ingelheim, Genome Canada through Ontario
Genomics Institute Grant OGI- 055, GlaxoSmithKline, Janssen, Lilly
Canada, the Novartis Research Foundation, the Ontario Ministry of
Economic Development and Innovation, Pfizer, Takeda, and Well-
come Trust Grant 092809/Z/10/Z. H.ꢀ.K. was supported by
a postdoctoral fellowship from the SGC. We thank Jacob Stuckey
and Dr. Ashutosh Tripathy for assisting with ITC studies, Dr. Anqi
Ma for checking NMR spectra, and Drs. Yuan Mi, Bin Xiang, and
Justin Gu for valuable discussion.
Pudong New Area, Shanghai 201203 (China)
E-mail: kehao.zhao@novartis.com
Dr. J. Lin-Jones
DiscoveRx Corporation
Fremont, CA 94538 (USA)
Supporting information for this article (including detailed synthetic
procedures and compound characterization as well as methods for
scaffold hopping, crystallization, structure determination, bio-
chemical assays, SPR, ITC, PRMT3 InCELL Hunter assay, cellular
PRMT3 assay, cell viability assay, and mouse PK studies) is available
on the WWW under http://dx.doi.org/10.1002/anie.201412154.
Dr. X. P. Huang, Prof. Dr. B. L. Roth
Department of Pharmacology, School of Medicine
University of North Carolina at Chapel Hill
Chapel Hill, NC27599 (USA)
Dr. S.-J. Min
Center for Neuro-Medicine, Brain Science Institute
Korea Institute of Science and Technology
Seoul 136-791 (South Korea)
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methyltransferase activity. PRMT3 as well as PRMT1 meth-
ylate the recombinant mammalian nuclear poly(A)-binding
protein (PABPN1)[3] and have been implicated in oculophar-
yngeal muscular dystrophy, which is caused by polyalanine
expansion in PABPN1.[3c,4] PRMT3 has been shown to be
overexpressed in human myocardial tissues from patients
with coronary heart disease.[5] It also methylates a histone
peptide (H4 1–24) in vitro.[6] Histone H4R3 is a modification
site associated with increased transcription of a number of
genes, including those under control of estrogen and andro-
gen receptors.[7] Interaction of PRMT3 with the tumor
suppressor DAL-1/4.1B and consequent inhibition of
its methyltransferase activity suggest a possible role of
PRMT3 in tumor growth and possible epigenetic
regulation of gene expression.[8]
Figure 1. Discovery of SGC707 and its inactive control XY1.
Well-characterized chemical probes[9] that display
high potency and selectivity, and robust on-target
activities for protein(s) of interest in cells would be
extremely valuable for elucidating biological functions
of the protein(s) and testing therapeutic hypotheses.
However, selective and cell-active PRMT inhibitors
have not been reported to date.[10] We recently discov-
ered a novel allosteric site for PRMT3 and the first
allosteric inhibitor of PRMT3 (Compound 1;
Figure 1).[11] We later reported structure–activity rela-
tionship studies of this inhibitor, leading to the iden-
tification of a sub-micromolar PRMT3 inhibitor.[12]
To discover the first chemical probe of PRMT3, we
further optimized both the left-hand side (LHS) and
right-hand side (RHS) moieties of this scaffold through
structure-guided design and synthesis. In particular, we
conducted a scaffold hopping exercise (see the Sup-
porting Information (SI) for details) for replacing the
LHS benzothiadiazole moiety, which resulted in the
selection of several replacement candidates, including
an isoquinoline moiety. We also explored various
substituents at the isoquinoline ring. For the RHS
moiety, we extensively investigated various amides,
ketones, and bicyclic heterocycles. In total, more than
100 analogues were designed, synthesized, and tested.
From this study, we discovered SGC707 (Figure 1),
a potent, low molecular weight (MW= 298) PRMT3
inhibitor with an IC50 value of 31 Æ 2 nm (n = 3) by
scintillation proximity assay (SPA) and 66 nm (n = 2) by
LC-MS detection assay at balanced conditions (sub-
strate and cofactor concentrations equal to their Km
values; Figures 2A and S1, SI). We also designed and
synthesized an inactive control, XY1 (Figure 1), for
chemical biology studies based on the crystal structure
of the PRMT3-SGC707 complex. XY1, a close ana-
logue of SGC707, was completely inactive against
PRMT3 at concentrations as high as 100 mm (Fig-
ure 2A). Binding of SGC707 was confirmed by iso-
thermal titration calorimetry (ITC) with a KD value of
53 Æ 2 nm (n = 3) (Figure S2, SI) and surface plasmon
resonance (SPR) with a KD value of 85 Æ 1 nm (n = 3)
(kon of 1.17 Æ 0.05 ꢀ 105 mÀ1 sÀ1 and koff of 0.99 Æ 0.03 ꢀ
10À2 sÀ1; Figure 2B). SGC707 exhibited a reasonably
Figure 2. SGC707 is a potent, selective, and non-competitive inhibitor of
*
*
PRMT3. A) IC50 determination for SGC707 ( ) and XY1 ( ) was performed at
balanced condition (Km of both substrates). B) SPR studies confirmed that
SGC707 had a high binding affinity to PRMT3 (KD =85Æ1 nm (n=3)).
C) Effect of SGC707 on the activity of 27 protein methyltransferases as well as
DNMT1, DNMT3A-3L, DNMT3B-3L, and BCDIN3D (an RNA-methyltransfer-
ase) were assessed at 1 (red), 5 (green), and 20 mm (purple) of compound and
no inhibition was observed. No change in IC50 values at varying D) SAM or
E) peptide concentrations was consistent with a noncompetitive pattern,
long residence time, a significant improvement over confirming the allosteric mode of inhibition.
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compound 1 (kon = 5.1 Æ 0.57 ꢀ 104 mÀ1 sÀ1 and koff = 0.76 Æ
0.09 sÀ1).[11] We confirmed that SGC707 had a longer resi-
dence time (9.7 min) than compound 1 (6.4 min) using jump
dilution experiments (Figure S3, SI). Remarkably, SGC707
was selective for PRMT3 over 31 protein-, DNA-, and RNA-
methyltransferases (Figure 2C) as well as a broad range of
non-epigenetic targets including more than 250 kinases,
G protein-coupled receptors (GPCRs), ion channels, and
transporters (Table S1, SI). It showed modest inhibition at
10000 nm for only six (5HT2B 69%, BRSK1 56%, DLK1
60%, MSK2 55%, PKG2 58%, and PRKX 61%) out of more
than 250 targets. Its binding affinity (Ki) for 5HT2B was
determined to be higher than 15000 nm with a maximum
inhibition of about 50% at 32000 nm (Figure S4, SI). In
mechanism of action studies (Figure 2D and E), SGC707
displayed a noncompetitive inhibition pattern with respect to
both the cofactor S-(5’-adenosyl)-l-methionine (SAM) and
peptide substrate, suggesting it is an allosteric inhibitor of
PRMT3.
prevents formation of a catalytically competent state. We
attribute the increased potency of SGC707 over parent
compounds to the presence of the isoquinoline ring that
optimally occupies the PRMT3 cavity, and the pyrrolidine
amide that could form direct or water-mediated contacts with
the side chain of K392.[12] Our inactive control XY1, which
contains a naphthyl group replacing the isoquinoline group,
lacks the key hydrogen bond with T466. This hydrogen bond
is absolutely desolvated, thus contributing strongly to the
PRMT3–SGC707 interaction. There is no other hydrogen-
bond acceptor in the vicinity of the T466 side chain, which, in
the absence of an inhibitor, forms a hydrogen bond with
a water molecule (PDB code: 2FYT). The naphthyl ring of
XY1 could act as a weak hydrogen-bond acceptor, but this
should come with a substantial enthalpic penalty. The more
than 1000-fold potency loss of XY1 compared with SGC707
supports this analysis. It is unclear whether other factors such
as electronic effects also contributed to the potency loss of
XY1 compared with SGC707.
To further confirm that SGC707 binds to the allosteric site
of PRMT3, we solved the crystal structure of the PRMT3-
SGC707 complex (PDB code: 4RYL; Table S2, SI). As
observed with parent compounds,[11] SGC707 occupies
a cavity located in the b-barrel of PRMT3, at the base of
the dimerization arm, more than 15 ꢁ away from the site of
methyl transfer (Figure 3). The isoquinoline group forms
a hydrogen bond at the bottom of the cavity with T466, the
urea group forms hydrogen bonds with E422 and R396 side
chains, and the pyrrolidine amide is buttressed against the a-
helix of the other PRMT3 subunit. This a-helix is a dynamic
secondary element that is conserved in class I PRMTs and is
critical for catalytic activity.[13] The N-terminal section of this
helix, that wraps around the cofactor in the active conforma-
tion of the enzyme (PDB code: 2FYT), is unresolved in
inhibitor-bound structures, and we believe that SGC707
induces conformational constraints on the a-helix that
To demonstrate the target engagement of SGC707 in cells,
we used an InCELL Hunter Assay, which measures intra-
cellular binding of SGC707 to the methyltransferase domain
of PRMT3 in cell lines expressing the methyltransferase
domain of PRMT3 tagged with a short fragment of b-
galactosidase (enhanced ProLabel or ePL). Binding of
a compound to PRMT3(211-531)-ePL increases the fusion
protein half-life. SGC707 stabilized PRMT3 in both HEK293
and A549 cells with EC50 values of 1.3 mm and 1.6 mm,
respectively (Figure 4A). We have previously shown that
PRMT3 can methylate histone peptides in vitro.[11] To deter-
mine if SGC707 can inhibit the PRMT3 catalytic activity in
cells we investigated its effect on H4R3 asymmetric dimethy-
lation. Methylated arginine residues have relatively slow
turnover[14] thus we overexpressed human Flag-tagged
PRMT3 and looked at the methylation of both endogenous
H4 and exogenously introduced GFP-tagged H4. Overex-
pressed PRMT3 increased the endog-
enous H4R3me2a from the baseline
levels and SGC707 was able to reduce
this increase (Figure 4B and C) with
an IC50 of 225 nm. The asymmetric
dimethylation of exogenous H4R3
was also inhibited (IC50 = 91 nm) indi-
cating potent cellular effect of
SGC707 (Figure 4B and D). The
dependency on the transfected
PRMT3 catalytic activity was deter-
mined by using the catalytically dead
PRMT3 mutant (E335Q) that did not
affect endogenous or exogenous
H4R3me2a levels and therefore was
used to establish the baseline levels of
the mark. SGC707 at 1 mm was almost
as effective at reducing the
H4R3me2a mark as the catalytically
dead PRMT3 mutant E335Q (Fig-
ure 4B) indicating full PRMT3 inhib-
Figure 3. Structure of the PRMT3–SGC707 complex. SGC707 (magenta) binds an allosteric site at
the interface of the two PRMT3 subunits that is distant from the site of methyl transfer (red star).
The allosteric site comprises a PRMT3-specific pocket in one subunit (yellow/orange) and a portion ition at this concentration. Taken
of the “activation a-helix” (light blue) of the other subunit.
together, these results clearly indicate
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dose was well tolerated by the test animals. These results
suggest that SGC707 is suitable for animal studies in addition
to cell-based studies.
In summary, we discovered SGC707, a first-in-class
PRMT3 chemical probe which is a potent and cell-active
allosteric inhibitor of PRMT3. SGC707 is remarkably selec-
tive for PRMT3 over 31 other methyltransferases and more
than 250 kinases, GPCRs, ion channels, and transporters. In
multiple cell-based assays, SGC707 engages PRMT3 and
potently inhibits its methyltransferase activity. SGC707 is
bioavailable and suitable for animal studies. In addition, we
discovered XY1, which is a very close analogue of SGC707,
but is completely inactive. Thus, SGC707 and XY1 are a pair
of excellent tools for the biomedical community to further
elucidate biological functions and disease associations of
PRMT3.
Received: December 18, 2014
Revised: January 31, 2015
Published online: && &&, &&&&
Keywords: allosteric inhibition · chemical probes ·
.
enzyme inhibitors · histone methylation · X-ray diffraction
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Figure 4. SGC707 binds to PRMT3 in cells and reduces PRMT3-
dependent H4R3me2a. A) SGC707 stabilized PRMT3 in both HEK293
and A549 cells with EC50 values of 1.3 mm and 1.6 mm in PRMT3
InCELL Hunter Assays. RLU, relative light units. B) Western blot
analysis of H4R3me2a levels. HEK293 cells were co-transfected with
FLAG-tagged PRMT3 (WT) or its catalytically dead mutant E335Q
(Mut) and treated with different concentrations of SGC707, as
indicated. The color panels below endogenous and exogenous
H4R3me2a and H4 panels are overlays of the above panels in which
the methylated H4R3 signal is in green and total H4 is in red thus
both signals generating yellow. C) Quantitation of SGC707 effect on
the endogenous H4R3me2a from the top panels in (B). D) Quantita-
tion of SGC707 effect on the exogenous H4R3me2a from the bottom
panels in (B). The graphs represent nonlinear fits of H4R3me2a signal
intensities normalized to intensities of GFP or H4 for exogenous and
endogenous H4, respectively, and subtracted for the baseline signal
from mutant PRMT3. The results are mean ÆSEM of three replicates.
that SGC707 can engage PRMT3 and effectively inhibit its
catalytic activity in cells and that overexpressed PRMT3 can
methylate histone H4 in cells. No toxicity was observed under
the conditions of the 24 h assays. Compound treatment for
72 h resulted in some toxicity, but only at very high concen-
trations, 50 and 100 mm (Figure S5, SI).
Lastly, we assessed in vivo pharmacokinetic (PK) proper-
ties of SGC707. Intraperitoneal injection of SGC707 at
30 mgkgÀ1 gave good plasma exposure in CD-1 male mice
over 6 h with the peak plasma level of 38000 nm (Figure S6,
SI). The plasma level of SGC707 at 6 h post injection was
208 nm, more than 2-fold higher than its IC50 value in the
cellular assay and the half-life of SGC707 was about 1 h. This
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Communications
Chemical Probes
High selectivity: The first PRMT3 chem-
ical probe, SGC707, was discovered by
structure-based optimization. SGC707 is
a potent PRMT3 inhibitor with outstand-
ing selectivity. The mechanism of action
studies and crystal structure of the
PRMT3–SGC707 complex confirm the
allosteric inhibition mode. SGC707
engages PRMT3 and potently inhibits its
methyltransferase activity in cells. It is
also bioavailable and suitable for animal
studies.
H. ꢁ. Kaniskan, M. M. Szewczyk, Z. Yu,
M. S. Eram, X. Yang, K. Schmidt, X. Luo,
M. Dai, F. He, I. Zang, Y. Lin, S. Kennedy,
F. Li, E. Dobrovetsky, A. Dong, D. Smil,
S.-J. Min, M. Landon, J. Lin-Jones,
X. P. Huang, B. L. Roth, M. Schapira,
P. Atadja, D. Barsyte-Lovejoy,
C. H. Arrowsmith, P. J. Brown, K. Zhao,*
J. Jin,* M. Vedadi*
&&& — &&&
A Potent, Selective and Cell-Active
Allosteric Inhibitor of Protein Arginine
Methyltransferase 3 (PRMT3)
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