Expert Opinion on Therapeutic Patents

ISSN: 1354-3776 (Print) 1744-7674 (Online) Journal homepage: http://www.tandfonline.com/loi/ietp20

A patent review of arginine methyltransferase

inhibitors (2010-2018)

Xiao Li, Chen Wang, Hao Jiang & Cheng Luo

To cite this article: Xiao Li, Chen Wang, Hao Jiang & Cheng Luo (2019): A patent review of
arginine methyltransferase inhibitors (2010-2018), Expert Opinion on Therapeutic Patents, DOI:
10.1080/13543776.2019.1567711

To link to this article: https://doi.org/10.1080/13543776.2019.1567711

Accepted author version posted online: 14

Jan 2019.

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 Publisher: Taylor & Francis

Journal: Expert Opinion on Therapeutic Patents

DOI: 10.1080/13543776.2019.1567711

Review

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A patent review of arginine methyltransferase inhibitors (2010-2018)

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Xiao Li1,2, Chen Wang1,2, Hao Jiang1,2, Cheng Luo1,2*

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1
Drug Discovery and Design Center, CAS Key Laboratory of Receptor Research, State Key
Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese Academy of

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Sciences, 555 Zuchongzhi Road, Shanghai 201203, China
2
University of Chinese Academy of Sciences, 19 Yuquan Road, Beijing 100049, China
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*Corresponding author: Cheng Luo, Drug Discovery and Design Center, Shanghai Institute of
Materia Medica, Chinese Academy of Sciences, 555 Zu Chong Zhi Road, Shanghai 201203,
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China
Email: [email protected]

Abstract
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Introduction: Protein arginine methyltransferases (PRMTs) are fundamental enzymes that

specifically modify the arginine residues of versatile substrates in cells. The aberrant
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expression and abnormal enzymatic activity of PRMTs are associated with many human
diseases, especially cancer. PRMTs are emerging as promising drug targets in both academia
and industry.
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Areas covered: This review summarizes the updated patented inhibitors targeting PRMTs
from 2010 to 2018. The authors illustrate the chemical structures, molecular mechanism of
action, pharmacological activities as well as the potential clinical application including
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combination therapy and biomarker-guided therapy. PRMT inhibitors in clinical trials are also
highlighted. The authors provide a future perspective for further development of potent and
selective PRMT inhibitors.
Expert opinion: Although a number of small molecule inhibitors of PRMTs with sufficient
potency have been developed, the selectivity of most PRMT inhibitors remains to be
improved. Hence novel approaches such as allosteric regulation need to be further studied
to identify PRMT inhibitors. So far, three PRMT inhibitors have entered clinical trials,
including PRMT5 inhibitor GSK3326595 and JNJ-64619178 as well as PRMT1 inhibitor
 GSK3368715. PRMT inhibitors with novel mechanism of action and good drug-like properties
may shed new light on drug research and development progress.

Keywords: Arginine methylation; Protein arginine methyltransferases (PRMTs); Small

molecule inhibitors; Drug research and development.

Article highlights

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 This review summarizes the patented inhibitors targeting PRMTs from 2010 to 2018
and illustrates the chemical structures of PRMT inhibitors as well as their biological

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activities.
 The combination therapies of PRMT inhibitors and immunotherapy antibodies such
as anti-PD-1 or anti-OX40 antibodies show significant efficacy in mouse model.

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 So far, three PRMT inhibitors are being tested in clinical research: PRMT5 inhibitor
GSK3326595 and JNJ-64619178 as well as PRMT1 inhibitor GSK3368715.
 New mechanisms of PRMT inhibition such as allosteric regulation need to be further
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researched for developing inhibitors with high selectivity.
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This box summarizes key points contained in the article.
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Abbreviations:

PRMT: Protein arginine methyltransferases

SAM: S-adenosylmethionine
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SAH: S-adenosyl homocysteine

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MMA: ω NG monomethylarginine

SDMA: ω NG, N’G symmetric dimethylarginine

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ADMA: ω NG, NG asymmetric dimethylarginine

IC50: the concentration of an inhibitor that inhibits 50% of the enzyme.

pIC50: the negative log of the IC50 value when converted to molar

EC50: the concentration for 50% of maximal effect

EC30: the concentration for 30% of maximal effect

 GI50: the concentration for 50% of maximal inhibition of cell proliferation

ICW: In Cell Western

THIQ: tetrahydroisoquinoline

MDS: myelodysplastic syndrome

CMML: chronic myelomonocytic leukemia

AML: acute myeloid leukemia

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MTD: maximum-tolerated dose

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NHL: non-Hodgkin lymphoma

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FTIH: First Time in Humans

DLBCL: diffuse large B-cell lymphoma

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Hsp90: Human Heat Shock Protein 90

HDAC: Histone Deacetylase an

MTA: methylthioadenosine

1.Introduction
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Enzymatic methylation of protein fractions was first found in nuclear thymus extracts over 40
years ago [1]. So far, nine PRMTs (PRMT1-9) have been identified in mammalian cells [2-11].
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When interacting with the cofactor S-adenosylmethionine (SAM) and substrates, PRMTs
catalyze the transfer of methyl groups from SAM to the guanidine of arginine in substrates,
producing methylated arginine and S-adenosyl homocysteine (SAH) [12]. There are three
types of methyl-arginine forms: ω NG monomethylarginine (MMA), ω NG, N’G symmetric
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dimethylarginine (SDMA) and ω NG, NG asymmetric dimethylarginine (ADMA) [13, 14].

According to different methylation patterns, nine PRMTs (PRMT1-9) are divided into three
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groups. Type I enzymes which include PRMT1, 2, 3, 4, 6, 8 catalyze the formation of MMA
and ADMA, while PRMT5 and PRMT9 are categorized as Type II PRMTs that induce MMA and
SDMA [15, 16]. Type III PRMT subfamily, which only contains PRMT7, is responsible for the
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modification of MMA [17]. Among them, PRMT1 accounts for up to 85% of protein arginine
methylation reactions and is expressed in a wide range of normal human tissues which
indicates its widespread importance in mediating cellular physiological processes [18-20].

The methyltransferases that use SAM as methyl groups donor can be categorized into 3
classes based on their structures and different types of substrates [21, 22]. PRMTs belong to
Class I methyltransferases and share several conserved motifs including a common β-sheet
structure as well as the “double E” and “THW” sequence motif [23-25]. The catalytic
methyl-transferring domains of PRMTs are highly conserved among eukaryotes within about
 310 amino acids [25, 26].Type I and type II PRMTs form a head-to-tail homodimer to induce
enzymatic activities in dependence of the central cavity [27, 28], while type III enzyme
PRMT7 maintains the activity in homodimer-like structure without the central cavity [29].
Apart from the common conserved catalytic domains, PRMTs consist of other specific motifs
that contribute to the interaction between PRMTs and other proteins [30], such as the SH3
domain of PRMT2, the F-box of PRMT9 and the zinc finger domain of PRMT3 and PRMT9 [31,
32].

At present, many substrates of PRMTs have been identified and they play indispensable
roles in the cellular physiological processes. It is well known that PRMTs could mediate the

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arginine methylation of histones, including H3R2, H3R8, H3R17, H3R26 and H4R3 [33, 34].

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These histone methylation marks could regulate different transcriptional pathways
depending on whether the chromatin state is transcriptionally active or silent [35, 36].

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Besides, PRMTs could affect other epigenetic modifications by recruiting other epigenetic
modifiers, such as histone acetyltransferases [37]. In addition, PRMTs have a wide range of
non-histone substrates [38, 39]. For example, by methylating the proteins associated with

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DNA-damage response, PRMTs involve in the DNA repair pathways [40, 41]. Several mRNA
splicing factors were identified as the substrates of PRMTs, which implies the role of PRMTs
in pre-mRNA splicing [42]. PRMTs also act as important regulators in immune system through
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regulating the methylation of critical immune modulatory proteins [43-45]. Considering the
widespread substrates of PRMTs and crucial functional pathways modulated by PRMTs, the
dysregulation of PRMTs has been linked to various diseases, notably cancer [46-48].For
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example, the overexpression of PRMT1 has been broadly observed in breast, lung, prostate
and bladder cancer [49].The overexpressed PRMT1 can aberrantly activate oncogenic
transcription [50], which leads to abnormal signal transduction [51].Besides, the
overexpression or enhanced enzymatic activity of PRMT5 may cause transcriptional
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repression, uncontrolled RNA splicing and aberrant signal transduction [52], which may lead
to gastric, colorectal and lung cancer, as well as lymphoma and leukemia [49]. PRMTs
aberration can also result in cardiovascular disease. As ADMA and MMA are endogenously
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nitric oxide synthase (NOS) inhibitors [14], the accumulation of ADMA and MMA reduces
production of nitric oxide (NO), leading to cardiovascular disorders such as elevated blood
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pressure [53, 54]. In addition, many other diseases have been correlated with PRMTs
aberration, such as viral pathogenesis, spinal muscular atrophy, neurodegenerative diseases
and metabolic diseases [55].
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Due to the fundamental roles of PRMTs in various diseases occurrence, these enzymes
especially type I PRMTs and PRMT5 have been regarded as promising drug targets. In recent
years, numerous small molecule inhibitors have been reported and many of them have
applied for patents [56-62]. In this review, we summarize the patents of PRMT small
molecule inhibitors and their corresponding indications from 2010 to 2018.
 2. PRMT inhibitors

2.1 Type I PRMT inhibitors

2.1.1 EPIZYME, INC

The EPIZYME, INC (http://www.epizyme.com/) is a high-producing institute which submitted

ten patents of type I PRMT inhibitors from 2014 to 2016 (Figure 1 and Table 1). As illustrated
in Figure 1, the compounds in WO2014153214A1, WO2014153235A2 and
WO2016044626A1 have similar key structures of ethylenediamine and pyrazole, which
predominantly contribute to the inhibitory activity [63-65]. However, the derivative

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structures among these patents show some differences. For compounds represented by

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formula I in Patent WO2014153214A1, other than the pyrazole moiety, they also have a
substituent Q moiety (a monocyclic or bicyclic heteroaryl with 1-4 heteroatoms selected

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from nitrogen, oxygen, and sulfur), while compounds in Patent WO2014153235A2 have a
moiety of 3aH-indene. In another patent WO2016044626A1, though compounds share the
moiety of pyrazole as the inhibitors in WO2014153214A1, the substitutes are replaced with

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cyclohexenyl, cyclohexyl or tetrahydropyran. In patent WO2018100532A1 submitted by
GLAXOSMITHKLINE IP DEV LTD, the researchers analyzed the co-crystal structure of PRMT1
with one inhibitor produced by the EPIZYME INC and found that the diamine sidechain could
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occupy the putative arginine substrate site of type I PRMTs, suggesting that the diamine
sidechain may mimic the amines of the substrate arginine residue [66].

With similar structure characteristics, the compounds mentioned above show similar
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activities toward type I PRMTs inhibition. For compound 1, the biochemical IC50 value for
PRMT1 and PRMT6 is 71.88 nM and 12.61 nM respectively, while for PRMT8, PRMT3 and
PRMT4, the IC50 values reach to thousands of nanomoles. In the cellular level, the
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compounds were incubated with RKO (colon adenocarcinoma cells) adherent cells and the
degrees of arginine mono-methylation were detected. In Cell Western (ICW) experiment
showed that the EC30 value of 1 and 2 is 6.979 μM and 3.617 μM respectively, suggesting
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these compounds could inhibit protein arginine mono-methylation in cells [57]. In patent
WO2014153235A2, compound 3 and compound 4 show no obvious selectivity among type I
PRMTs. In patent WO2016044626A1, compound 5 and compound 6 show relative strong
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activity in inhibiting arginine methylation with the IC50 and RGG-ICW EC30 values of both less
than 0.1 μM. Apart from the compounds elaborated above, other compounds that share
similar structures from EPIZYME are listed in Table 1 [67-73].
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The compounds summarized in Table 1 have similar structural features of the diamine and
pyrazole moieties that are responsible for type I PRMTs inhibition. Structures of
representative compounds in patent WO2014144659A1, WO2014153090A1,
WO2014153172A1 are shown in Table 1. Compounds in these patents are designed to inhibit
PRMT1 The biochemical IC50 value of compound 7 against PRMT1 is 40.03 nM, and the ICW
EC30 value of compound 7 for detection of arginine mono-methylation in RKO adherent cells
is 6.536 μM, indicating its strong activity inhibiting PRMT1. In patent WO2014153090A1, the
 biochemical IC50 value of compound 8 is 4.45nM, while the ICW EC30 value for arginine
mono-methylation is 305nM. Compounds described in patent WO2014153208A1,
WO2014153226A1, WO2014178954A1 and WO2016044585A1 could inhibit type I PRMTs.
The biochemical IC50 value of compound 10 against PRMT1, PRMT6, PRMT8 and PRMT3 is
38.55 nM, 16.23 nM, 510.41 nM and 8.06923 μM respectively. In RKO adherent cells, the
ICW EC30 value of compound 10 for arginine methylation is 2.607 μM. For compound 11 in
patent WO2014153226A1, the IC50 value for PRMT1, PRMT3, PRMT4, PRMT6 and PRMT8 is
0.01 μM, 8.93 μM, 3.00 μM, 0.01 μM and 0.76 μM respectively. In RKO methylation assay,
the ICW EC30 value of compound 11 for arginine mono-methylation is 5.61 μM. In patent

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WO2014178954A1, the activity of compound 12 is also tested both in molecular and cellular

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level. The biochemical IC50 values against PRMT1, PRMT3, PRMT4, PRMT6 and PRMT8 are all
less than 1 μM. The ICW EC30 value of compound 12 for arginine mono-methylation is 10.198
μM.

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2.1.2 University of South Carolina

In 2010, University of Carolina patented chloro-acetamidine based inhibitors as type I PRMT

inhibitors which comprise an amino acid peptide joined to a chloro-acetamidine warhead
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that mimics the guanidinium of substrate arginine residue (Figure 2). The amino acid peptide
can be replaced with substitutes including aryl and alkyl chain [42]. For compound 14, the 21
amino acids that have an acetylated N-terminus are derived from the first 21 amino acids of
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histone H4 to mimic substrates, so that a high affinity toward PRMTs can be obtained.

To examine whether the inhibitors are able to irreversibly inactivate PRMT1, methylated
substrates formation as a function of time was measured over different inhibitor
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concentrations to get resulting nonlinear progress curves. The Kinact/KI value was
determined to be 4.02×106min-1·M-1, indicating that the inhibitory effect was irreversible. By
covalently modifying the active site of PRMT (confirmed by subsequent rapid dilution assays
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and dialysis experiments), the compounds show great potency to treat heart disease and
breast cancer. Additionally, the compounds can also be fluorescently labeled to screen PRMT
inhibitors from large compound libraries in fluorescence polarization assay [42].
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2.2 PRMT5 inhibitors

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2.2.1 Epizyme, Inc

From 2014 to 2016, the Epizyme, Inc submitted eight patents of PRMT5 inhibitors, which are
listed in Figure 3 and Table 2 [74-81]. For compounds shown in Figure 3, they share the
components of two fused 5- to 6-membered heteroaryl rings (WO2015200677A2) or a
naphthalene, tetrahydronaphthalene (WO2015200680A2) as well as alkyls connected with
an optionally substituted ring (Figure 3). In patent WO2015200680A2, compounds
represented by formula IV have a sulfonyl group between naphthalene and heteroaryl rings
 in addition to the shared components. Structural biology showed that the
tetrahydroisoquinoline (THIQ) moiety of these inhibitors could interact with PRMT5 in a
cation−π binding mode, which contributes to the inhibitory effect toward PRMT5 [82].

The compounds in Figure 3 show similar inhibitory potency in biochemical assays, with IC50
values of less than 100 nM toward PRMT5. Furthermore, Z-138 mantle cell lymphoma cells
were incubated with compounds to conduct methylation assay and proliferation assay.
Results showed the ICW EC50 values of most compounds were less than 100 nM, indicating
their inhibitory effects toward PRMT5 in the cellular level. Besides, these compounds could
inhibit the proliferation of Z-138 cells, demonstrating their potential efficacy in treating

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mantle cell lymphoma. Other PRMT5 inhibitors from patents submitted by the Epizyme, Inc

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are listed in Table 2 (Table 2). Their inhibitory effects are evaluated in both molecular and
cellular level. The biochemical IC50 values of compounds listed in Table 2 (20-24) are all less

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than 0.1μM, while in Z-138 cell lines, the ICW EC50 values of these inhibitors are all less than
1 μM and the EC50 values of the proliferation effect are all less than 10 μM, indicating the
sufficient potency of these compounds.

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2.2.2 Ctxt Pty Ltd
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In patent WO2017153515A1, the compounds from Ctxt Pty Ltd have a structure of C-alkyl
bicyclic amine in which benzene fuses to cyclopentane or heteroaryl rings (Figure 4) [83]. In
biochemical assay, the IC50 values of these compounds are among 19-533 nM, demonstrating
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their inhibitory activity toward PRMT5 in the molecular level. These compounds were also
tested for potency to inhibit symmetrical dimethylation of arginine in biomarker assay. The
TE11 cell line was incubated with compounds to obtain IC50 values in biomarker assay. Except
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for compound 25 in patent WO2017153515A1 whose IC50 value is 430 nM, the other
compounds shown in Figure 4 have IC50 values of less than 4 nM in biomarker assay [84],
suggesting these compounds are effective in arginine methylation inhibition. Other
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compounds provided by Ctxt Pty Ltd are listed in Table 3 (Table 3) [85-88]．The biochemical
IC50 values of compounds 29-32 are all less than 1μM. To test their potency of inhibition of
the arginine symmetric dimethylation, TE11 cells were incubated with the compounds, and
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the ICW IC50 values are all less than 1μM.

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2.2.3 Prelude Therapeutics

To invent compounds that compete with the cofactor SAM as PRMT5 inhibitors, PRELUDE
THERAPEUTICS designed compounds that have similar structures with SAM, which share
structures of the tetrahydrofuran-3,4-diol and a purine [89-93]. In biochemical assay, the IC50
values of compound 33 and compound 34 for PRMT5 are 5.01 μM and 2.38 μM respectively
(Figure 5). Besides, dialysis assays of wildtype and mutant (C449S) PRMT5 showed that the
compounds may inhibit the activity of PRMT5 by covalently modifying C449 in wild type
 PRMT5. These compounds may be applied to treating lymphoid cancer, hemoglobinopathies
such as thalassemia and sickle cell disease (SCD) and other diseases correlated with aberrant
PRMT5 expression.

2.2.4 Other institutes

In addition to the three institutes mentioned above, there are other companies submitting
patents of PRMT5 inhibitors (Table 4) [94-99]. Some of the compound structures are similar
to those from Epizyme, Inc, while some have novel scaffolds. For example, in patent

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WO2014145214A2, Ohio State Innovation Foundation provided compounds that have at

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least one carbazole moiety. The tricyclic or bicyclic heteroaromatic moiety could form
hydrophobic and aromatic interaction with PRMT5. Besides, a linker moiety with at least one

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electron-donating group could form hydrogen bonds with glutamine of PRMT5. As exampled
by compound HLCL-61, the IC50 value of anti-proliferation was 28 μM for 72 h while the IC50
value of H4R3 methylation inhibition is 12 μM. Moreover, in patent WO2018065365A1,

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Janssen Pharmaceutica NV submitted carba nucleoside analogues as PRMT5 inhibitors. They
also show good potential to treat PRMT5-mediated disorders [96]. Take compound 37 for
example, in biochemical assay, the pI50 value is 5.9 μM, while the ICW pI50 value of A549
cell line is 5.3 μM.
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Some of PRMT5 inhibitors are mimics of the cofactor SAM. They have the adenosine group
of SAM that is then connected with the peptide substrate’s guanidine moiety. Notably in the
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structures of these inhibitors, the amino acid moiety of SAM is omitted, which is based on
the findings of Thompson and his colleagues [100]. They mutate the active Arg54 residue
which interacts with the carboxyl group of SAM and find that the catalytic ability of PRMTs is
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less influenced [100]. Thus, in the series of SAM-mimic inhibitors, the carboxyl group is
substituted with other moieties that are more selective to PRMTs other than other kinds of
methyltransferases and the conversion of a PRMT cofactor into an inhibitor is done.
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Besides, many PRMT5 inhibitors mentioned above have the tetrahydroisoquinoline (THIQ)
group, in which the tertiary nitrogen is engaged in the water-mediated interaction with
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residues necessary for enzyme catalysis. The THIQ group can also form a π-π stacking
interaction with the residue of PRMT5 that directs symmetric arginine methylation, further
leading to the residue’s conformational change to accommodate the large THIQ group, thus
the enzymatic activity of PRMT5 is inhibited. Researchers also find that the inhibitory efficacy
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of the compounds is less affected when moieties distal to the THIQ group are changed,
which devotes to the potential improvements in absorption, distribution, metabolism and
excretion (ADME) properties [57].

2.3 PRMT7 inhibitors

PRMT 7 is the only component of type III PRMTs. By modulating the methylation of histones
 (H2A, H4R3, H3R2) and non-histone substrates, PRMT7 can participate in many physiological
processes such as chromatin structure modulation, gene expression, signal transduction and
pluripotency maintenance. In patent CN108503623A submitted by Sichuan University (Figure
6), inhibitors are acrylamide derivatives and the reaction condition is moderate which is
beneficial to industrialization production [101]. The inhibitors activity was both tested in
molecular and cellular level, with a minimum IC50 value of 2.1 μM in enzymatic activity
experiments. They also show relatively good efficacy in inhibition of cancer cell proliferation,
which include prostate cancer cell line (PC-3), lung cancer cell line (H2228, NCI-H1975, PC-9,
NCI-H358, Calu-1), lymphoma cell line (Jeko-1), ovarian cancer cell line (ES-2, H08910,

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A2780S, A2780/T). The pIC50 values of compound 43 for all these cell lines are less than 10

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μM, indicating these inhibitors’ medicinal potential of preventing or treating a substantial
range of cancers.

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3. Indication patents

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PRMT inhibitors could be applied as effective drugs in many indications correlated with the
dysregulation of PRMTs, such as cancer, cocaine addiction, renal fibrosis and hematopoietic
differentiation (Table5). In recent years, many patents about combination therapy and
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biomarker-guided therapy correlated with PRMT inhibitors have been submitted, and we will
introduce them in details as following.

In patent WO2018100532A1, WO2018100534A1 and WO2018100535A1, Glaxo Smith

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Kline pharmaceutical company combined PRMT inhibitors and anti-PD-1 or anti-OX40
antibodies [66, 102, 103]. These combination strategies could be effective in treating
melanoma, breast cancer, lymphoma, triple negative breast cancer (TNBC) and bladder
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cancer. The combination therapy of small molecule inhibitors and antibodies modulating
immune checkpoint showed moderate survival advantage, indicating the synergistic
interaction of the two agents. Due to the widespread use of immune checkpoint inhibitors in
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many tumor types, the combination of small molecular targeted therapy and
immunotherapy will be used more extensively in the future.
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Notably, results show that a profound combination effect on inhibition of tumor growth
can be achieved through the simultaneous inhibition of Type I and Type II PRMTs,
demonstrating the promising treatment strategies of using both of the inhibitors to enhance
the efficacy in killing cancer cells [66]. It is worth mentioning that to date, almost all
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pan-PRMT inhibitors are SAM analogs, which have poor selectivity and can change global
methylation levels. The off-target effects are inescapable for the use of these compounds in
therapy. While on the other hand, the crystal structures of PRMTs and sequences alignment
demonstrate the other binding cavities are different between Type I and Type II enzymes.
Hence it is difficult to develop pan-PRMT inhibitors according to the substrate binding sites.
If type I and type II PRMTs need to be inhibited simultaneously, it is necessary to combine
their respective inhibitors together rather than developing low specificity inhibitors that
 inhibit both type I and type II PRMTs [104].

In addition to the combination therapy of PRMT inhibitors and immune checkpoint

inhibitors as well as the simultaneous inhibition of both type I and type II PRMTs, PRMT
inhibitors could also be combined with inhibitors of other drug targets such as Human heat
shock protein 90 (Hsp90) and histone deacetylase (HDAC). In patent CN105497034A
submitted by Ji’nan University reported the combination of the Hsp90 inhibitor 17-AAG and
PRMT5 inhibitor EPZ015666 to improve the efficacy of treating osteosarcoma [105].
Experiments in cellular level have demonstrated that PRMT5 interacts with ubiquitin ligase
E3 in Hsp90 complex. This interaction would be improved after 17-AGG was used to inhibit

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Hsp90 and the ubiquitination degradation of PRMT5 by E3 ligase would be accelerated. As

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the expression level of PRMT5 is specifically high in osteosarcoma tissues, the combination
therapy shows significant effects in treating osteosarcoma. In patent WO2011079236A1

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submitted by OHIO STATE INNOVATION FOUNDATION, histone deacetylase (HDAC) inhibitor
TSA was combined with PRMT5 inhibitor to treat high grade gliomas [106]. As shown by the
biochemical assay, PRMT5 enzymatic activity on H4R3 and H3R8 arginine residues will be

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improved when the neighboring lysine residues are deacetylated by HDAC enzymes. In the
cellular level, astrocytoma cell lines (U1242 and U251) were treated with either the single
agent or the combination treatment. The combinational treatment showed significant loss of
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S2Me-H4R3 methylation and significant increase of cell apoptosis compared with single
agent treatment. As PRMT5 expression levels are higher in high grade gliomas, the
combination of PRMT5 inhibitor and HDAC inhibitor may have great impact on high grade
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gliomas treatment.

Beside the combination therapy, biomarker-guided therapy correlated with PRMT

inhibitors has also developed in recent years. In patent WO2018100536A1 submitted by
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GlaxoSmithKline and patent WO2016145150A2 submitted by THE BROAD INSTITUTE INC, the
potential patients are those who have a mutation in MTAP or a decreased level of MTAP
polynucleotide or polypeptide, or both [107, 108].In many cancer types, which include 40%
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of glioblastoma, 25% of melanoma and pancreatic adenocarcinoma and 15% of non-small

cell lung carcinoma, the MTAP gene is frequently absent. MTAP loss results in the
accumulation of its metabolite methylthioadenosine (MTA), which is shown to be able to
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inhibit PRMT5 activity, so that type I PRMT inhibitors and PRMT5 inhibitors are more
sensitive to patients who have weak PRMT5 activity due to the loss of MTAP. In patent
WO2018100536A1, both MTAP proficient and deletion lymphoma and melanoma cell lines
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were treated with type I PRMT inhibitors. Results showed that the difference of median GI50
between MTAP proficient and deletion cell lines is more than 5-fold. In patent
WO2016145150A2, PRMT5 inhibitor and MTA were used together and showed significant
effect on growth inhibition of tumor cells.

In addition to the loss of MTAP, the gene fusion of TMPRSS2 and ETS-related gene (EGR) is
another biomarker for tumor therapy. As the gene fusion can lead to N-terminally truncated
or full-length forms of EGR overexpression [109], accumulating EGR can bind and then recruit
 PRMT5 to methylate Androgen Receptor (AR) on arginine 761 (R761). Subsequently, high
methylation level of AR can block its binding to target genes as well as the transcriptional
activity which leads to prostate cancer. As shown in patent WO2016089883A1 that
submitted by NOVARTIS, PRMT5 inhibitor showed selectivity to TMPRSS2: EGR-positive
prostate cancer cells [110].

Apart from cancers, PRMT inhibitors can also be used to treat many other forms of
disorders. In patent CN105125571A submitted by Sichuan University, the PRMT1 inhibitors
AMI-1 and MTA were used together to reduce the level of H4R3me2a, which could inhibit
the transcriptional expression of Cdk5 and CaMK II, and subsequently relieve the cocaine

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addiction [111]. Tongji University also reported that PRMT1 inhibitors like AMI-1 could be

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effective in treating renal fibrosis (patent CN107375257A). As the increased activity of
PRMT1 will enhance the level of H4R3me2a and stimulate the TGF-β signaling pathway and

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then lead to the occurrence of renal fibrosis due to the increased expression of α-SMA,
collagen and fibronectin, the use of PRMT1 inhibitors would relieve syndromes of the renal
fibrosis.

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4.Conclusion
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From 2010 to 2018, type I PRMT inhibitors and PRMT5 inhibitors have been reported in
many patents. Based on the interaction mode of enzymes and substrates, type I PRMT
inhibitors are designed to contain a moiety of ethylene diamino that mimics the amines of
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substrate arginine. Such inhibitors may compete with natural substrates to bind to type I
PRMTs and inhibit their methylation activity. Some of the type I PRMT inhibitors show
sufficient potency against PRMT1, PRMT6 and PRMT8 with IC50 values at the nanomolar level
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and have selectivity against PRMT3 and PRMT4 with IC50 values at the micromolar level. Type
I PRMT inhibitors that are mainly provided by the Epizyme, Inc show relative strong
inhibitory activity and indicate that they have great potential in treating cancers and many
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other disorders. PRMT5 inhibitors are designed to occupy the peptide substrate binding sites
or be mimics of PRMT cofactor, SAM. The biochemical IC50 values of PRMT5 inhibitors are at
the nanomolar or micromolar level, which indicates that PRMT5 inhibitors may have good
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prospects for medicine.

The disease that may be treated by PRMT inhibitors include but are not limited to cancers,
muscular disorder, autoimmune disorder, neurological disorder, vascular disorder, metabolic
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disorder, cardiovascular disease, diabetes, kidney failure, renal disease, pulmonary disease.
Besides, PRMT inhibitors could also be used in combinational therapy or therapy with
biomarker provided. PRMT inhibitors also demonstrate significant efficacy on cocaine
addiction and renal fibrosis in animal models.
 5.Expert opinion

According to the crystal structures, the surface grooves of PRMTs that provide
accommodation for substrate peptide backbones are wide and shallow. The structure
features indicate inhibitors that are designed to mimic the arginine residue may have weak
selectivity among different PRMTs. Such situation is the same with the SAM-mimic inhibitors.
Therefore, other sites such as allosteric cavities that regulate PRMTs activity can be regarded
as novel drug targeting sites. It is important to incorporate the structural information of all
types of PRMTs when developing selective PRMT inhibitors.

t
Based on co-crystal structures of type I PRMTs and their inhibitors, the inhibitors occupy

ip
the substrate binding sites, which indicates that they may compete with peptide substrates,
While noncompetitive pattern of inhibition with respect to substrates is documented in
mechanism of action studies [112]. The contradiction between analysis based on crystal

cr
structure and biological experiments provokes a lot of discussion. One interpretation is that
the combination of inhibitors may change enzymes’ conformation. In crystal structures of

us
type I PRMTs with inhibitors, the N terminus α-helix was totally folded and the binding
pocket for the substrate was locked. Thus in the presence of inhibitors, the peptide would
not bind the cavity even at high concentration [113]. It can also be explained that with a
an
complex of substrate, inhibitor and PRMT may form without prominent penalty of free
energy [114]. Another explanation illustrates that though inhibitors occupy the substrate
binding pocket, the substrate may interact with PRMT additionally outside the catalytic
cavity and be added with methyl group [60].
M

Three inhibitors have been currently reported in clinical trials

(https://www.clinicaltrials.gov/), including PRMT5 inhibitor GSK3326595 and JNJ-64619178
as well as PRMT1 inhibitor GSK3368715 (Figure 7). Currently, PRMT5 inhibitor GSK3326595 is
ed

on phase 1 clinical trial to conduct a dose escalation study assessing its safety,
pharmacokinetics (PK), pharmacodynamics (PD), and preliminary clinical activity in subjects
with advanced or recurrent solid tumors and non-Hodgkin’s lymphoma. On another phase 2
pt

clinical trial, GSK3326595 is tested as an oral treatment for human subjects with relapsed
and refractory myelodysplastic syndrome (MDS), chronic myelomonocytic leukemia (CMML),
ce

and hyperproliferative acute myeloid leukemia (AML). In addition, another PRMT5 inhibitor
JNJ-64619178 is also on phase 1 clinical trial to identify the maximum-tolerated dose (MTD)
in subjects with relapsed/refractory B cell non-Hodgkin lymphoma (NHL) or advanced solid
tumors. The structures and activities of the two PRMT5 inhibitors in clinical trials are shown
Ac

in Figure 7. The inhibitor JNJ-64619178 is designed to occupy the binding sites of SAM
cofactor and guanidino substrate simultaneously. The IC50 value toward PRMT5/MEP50
complex is 0.14 nM and many cell lines are sensitive to JNJ-64619178 treatment [115]. The
inhibitor GSK3326595 is designed to occupy the binding site of substrate peptides and that is
confirmed by the co-crystal of GSK3326595 and PRMT5/MEP50 complex, indicating that the
inhibitor is competitive with substrate peptide. GSK3326595 has a strong inhibitory potency
with the IC50 value of 6.2 ± 0.8 nM. Besides, the inhibitory potency would increase when
 extending the preincubation time, revealing a mode of slow binding inhibition. Apart from
the strong activity, the selectivity of the inhibitor is satisfying too, with > 4000-fold selectivity
over any other methyltransferase (20 methyltransferases tested, including PRMT9) [116].

It is worth noting that PRMT1 inhibitor GSK3368715 is on First Time in Humans (FTIH)
study in subjects with solid tumors and diffuse large B-cell lymphoma (DLBCL). The clinical
trial of GSK3368715 was first posted on September 12, 2018. Unfortunately, we haven’t
found the chemical structure of GSK3368715 with available database. With the prosperous
development in the field of PRMT inhibitors, more and more investigations will pour into
discovery of more potent and selective compound leads, which is such challenging that more

t
accurate, effective designing and evaluating methods need to be developed.

ip
So far, many approaches have been invented to promote the hit identification of PRMTs
including virtual screening techniques and high throughput screening methods, which

cr
account for the discovery of many micromolar and submicromolar small molecule inhibitors.
In recent years, the X-ray crystal structures of most PRMTs have been resolved, which greatly

us
accelerate the modification of PRMT inhibitors based on the structural information.
Nevertheless, though enormous inhibitors have been developed, only three inhibitors are
currently in clinical research. Therefore, we suggest some additional approaches for the
an
development of novel PRMT inhibitors. First, since the limited structure characteristics may
impose restrictions on drug permeability, drug absorption and bioavailability, novel chemical
scaffolds need to be poured into the preliminary hit identification of PRMTs. Second, in
cellular and in vivo validation system of PRMT inhibition needs to be developed. When the
M
inhibitors are exposed to the cellular environment, they may be transformed by the
metabolic systems and present different effects compared with the in vitro system, including
reduced potency or increased toxicity. Therefore, it is important to develop and improve the
ed

in vivo target validation assays of PRMT inhibitors to ensure their safety and effectiveness.
Third, as PRMTs participate in many biological processes by methylating histones and diverse
nonhistone substrates, the network in which PRMTs play regulative roles is very complicated.
pt

It may be more efficient to develop inhibitors targeting one specific pathway in which PRMTs
have aberrant activity. The biological functions of PRMTs need to be further studied and the
design of PRMT inhibitors would be more specific toward certain regulative pathways.
ce

Funding
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This paper received financial support from the National Natural Science Foundation of China
(81625022, 91853205, 81430084, 81821005). K. C. Wong Education Foundation and Chinese
Academy of Sciences (XDA12020353 and XDA12050401), Science and Technology
Commission of Shanghai Municipality （18431907100）.

Declaration of interest
The authors declare that they work for the Drug Discovery and Design Center, CAS Key
Laboratory of Receptor Research, State Key Laboratory of Drug Research, Shanghai Institute
 of Materia Medica, Chinese Academy of Sciences. The authors have no other relevant
affiliations or financial involvement with any organization or entity with a financial interest
in or financial conflict with the subject matter or materials discussed in the manuscript. This
includes employment, consultancies, honoraria, stock ownership or options, expert
testimony, grants or patents received or pending, or royalties

Reviewer disclosures
Peer reviewers on this manuscript have no relevant financial or other relationships to
disclose

t
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 Table 1. Other Type I PRMT inhibitors submitted by Epizyme, Inc

Patent Formula Example

WO2014144659A1

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WO2014153090A1

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WO2014153172A1
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pt
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 Table 1. Continued

WO2014153208A1

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WO2014153226A1

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WO2014178954A1
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ed
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WO2016044585A1
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 Table 2. Other PRMT5 inhibitors from Epizyme, Inc

Patent Formula Examples

WO2014100695A1

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WO2014100716A1

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an
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WO2014100719A2
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pt

WO2014100730A1
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WO2014100734A1
 Table 2. (Continued)

WO2016022605A1

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ed
pt
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 Table 3. Other PRMT5 inhibitors from Ctxt Pty Ltd

Patent Formula Examples

WO2016034675A1

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WO2017153513A1

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WO2017153518A1
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ed
pt

WO2017153521A1
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 Table 4. PRMT5 inhibitors from other institutes

Patent, formula Examples Activity

Institutions
WO20141284 Biochemical Assay
65A1 IC50 =182nM;
Biomarker Assay
CANCER IC50 = 127 nM
THERAPEUTI

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CS CRC PTY

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LTD

WO20141452 Anti-Jeko. cell line

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14A2 proliferation
IC50 = 28 μM (72h);

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OHIO-STATE H4R3 methylation
INNOVATION inhibition
FOUNDATIO IC50 = 12μM
N
an
M
WO2018065 Biochemical Assay
365A1 pIC50 = 5.9 μM
A549 cell line ICW
JANSSEN pIC50 = 5.3 μM
ed

PHARMACEU
TICA NV
pt
ce
Ac
 Table 4. (Continued)

WO201808 PANC1 Cells

1451A1 survival inhibition

INDIANA
UNIVERSIT 38: IC50 = 4.3 μM
Y
RESEARCH
AND

t
TECHNOLO

ip
GY
CORPORATI

cr
ON

us
an 39: IC50 = 8.5 μM
M
ed

40: IC50 = 34 μM
pt
ce

WO201816 Biochemical Assay

1922A1 IC50 = 0.030 μM

SHANGHAI Anti-MCL cell line

Ac

INSTITUTE proliferation
OF IC50 = 0.07 μM (12d)
MEDICAL
MEDICA, Anti-MV4-11 cell
CHINESE line proliferation
ACADAMY GI50 = 0.126 μM
OF
SCIENCES
Table 4. (Continued)

WO201816 Biochemical Assay

7276A1 IC50 = 10nM

ARGONAUT
THERAPEUT
ICS LIMITED

t
ip
cr
us
an
M
ed
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 Table 5. Details on the indication of PRMT inhibitors

Patent, Institutions Indication Details

WO2018100532A1 Anti-tumor Combination of Type I
PRMT inhibitors and
GSK Combination
Therapy Type II PRMT inhibitors

WO2018100534A1 Anti-tumor Combination of Type I

t
PRMT inhibitors and
GSK Combination

ip
anti-PD-1/ OX40
Therapy
antibodies

cr
WO2018100535A1 Anti-tumor Combination of Type II

us
PRMT inhibitors and
GSK Combination
anti-OX40 antibodies
an Therapy

WO2018100536A1 Anti-tumor MTAP destructive

GSK Biomarker Type I PRMT inhibitors
Therapy
M
ed

Table 5. (Continued)

WO2016145150A2 Anti-tumor MTAP loss

THE BROAD INSTITUTE INC Biomarker PRMT5 inhibitors
pt

Therapy
ce

WO2016089883A1 Anti-tumor TMPRSS2:ERG positive

Biomarker
NOVARTIS prostate cancer
Therapy
Ac

PRMT5 inhibitors

WO2011079236A1 Anti-tumor Combination of PRMT5

OHIO STATE INNOVATION FOUNDATION Combination inhibitor (CMP 5) and
Therapy HDAC inhibitor (TSA)
 Table 5. (Continued)

CN105497034A Anti-tumor Combination of PRMT5

Ji'nan University Combination inhibitor (EPZ015666) and

t
Therapy HSP90 inhibitor (17-AAG )

ip
CN105125571A Cocaine addiction PRMT1 inhibitors

cr
Sichuan University (AMI-1, MTA)

us
CN107375257A Renal fibrosis PRMT1 inhibitor
Tongji University an (AMI-1)
M
ed
pt
ce
Ac
 t
ip
cr
us
an
M
ed
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Ac

Figure 1. Representative Type I PRMT inhibitors from Epizyme, Inc

 t
ip
cr
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Figure 2. Inhibitors of type I PRMTs from University of South Carolina
an
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ed
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Ac
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ip
cr
us
an
M
ed
pt
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Ac

Figure 3. Representative PRMT5 inhibitors from Epizyme, Inc

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ip
cr
us
an
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ed
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Ac

Figure 4. PRMT5 inhibitors from Ctxt Pty Ltd

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Figure 5. PRMT5 inhibitors from Prelude Therapeutics

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 Figure 6. PRMT7 inhibitors from Sichuan University

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 t
Figure 7. PRMT5 inhibitors in clinical research

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Figure and table legends:

Table 1: Other Type I PRMT inhibitors submitted by Epizyme, Inc

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Table 2: Other PRMT5 inhibitors from Epizyme, Inc

Table 3: Other PRMT5 inhibitors from Ctxt Pty Ltd

an
Table 4: PRMT5 inhibitors from other institutes

Table 5: Details on the indication of PRMT inhibitors

M
Figure 1. Representative Type I PRMT inhibitors from Epizyme, Inc

Figure 2. Inhibitors of type I PRMTs from University of South Carolina

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Figure 3. Representative PRMT5 inhibitors from Epizyme, Inc

Figure 4. PRMT5 inhibitors from Ctxt Pty Ltd

Figure 5. PRMT5 inhibitors from Prelude Therapeutics

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Figure 6. PRMT7 inhibitors from Sichuan University

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Figure 7. PRMT5 inhibitors in clinical research

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