Targeting epigenetic modifiers: Inhibitors of histone methyltransferases

Elisabeth-Maria Bissinger a, Ralf Heinke b, Wolfgang Sippl b and Manfred Jung *a
aInstitute of Pharmaceutical Sciences, Albert-Ludwigs-Universität Freiburg, Albertstr. 25, 79104, Freiburg, Germany. E-mail: manfred.jung@pharmazie.uni-freiburg.de
bDepartment of Pharmaceutical Chemistry, Martin-Luther University of Halle-Wittenberg, 06120, Halle/Saale, Germany

Received 29th April 2010 , Accepted 18th June 2010

First published on 9th July 2010


The term epigenetics is defined as inheritable changes that influence the outcome of a phenotype without changes in the genome. Epigenetics is based upon DNA methylation and posttranslational histone modifications. While there is much known about reversible acetylation as a posttranslational modification, research on histone methylation is still emerging, especially with regard to drug discovery. As aberrant epigenetic modifications have been linked to many diseases, inhibitors of histone methylation are very much in demand. This article gives an outline on the different histone methyltransferases, their involvement in disease, the available inhibitors and their potential as drugs.


Elisabeth-Maria Bissinger

Elisabeth-Maria Bissinger

Elisabeth studied Pharmacy at the University of Münster. After graduation, she started in 2007 as a PhD student in the group of Manfred Jung at the Institute of Pharmaceutical Sciences in Freiburg. Her work focuses on the synthesis and in vitro testing of new histone methyltransferase inhibitors.

Ralf Heinke

Ralf Heinke

Ralf studied Pharmacy at the Martin-Luther-University of Halle-Wittenberg and is a PhD student there in the group of Wolfgang Sippl at the Institute of Pharmacy since 2007. His work focuses on the computer-based design of histone methyltransferase inhibitors.

Wolfgang Sippl

Wolfgang Sippl

Wolfgang studied Pharmacy at the University in Berlin. He obtained a PhD in Pharmaceutical Chemistry at the University of Düsseldorf in the group of Hans-Dieter Höltje and was a post-doctoral fellow at the Université Louis-Pasteur in Strasbourg (France) where he worked with Camille G. Wermuth. Wolfgang then took a senior researcher position in Düsseldorf before moving to the University of Halle-Wittenberg as a full professor for Medicinal Chemistry in 2003. Since 2010 he is Director of the Institute of Pharmacy in Halle. His main interests are focused on computational chemistry and structure-based drug design.

Manfred Jung

Manfred Jung

Manfred studied Pharmacy at the University of Marburg where he also did his PhD in the group of Prof. W. Hanefeld working on synthesis of aromatic retinoids. As a postdoctoral fellow he joined Prof. T. Durst (Ottawa, Canada) working on the synthesis of amino acid derivatives. From 1994 to 2003 he was a group leader at the University of Münster. Since 2003 Manfred is a professor of Pharmaceutical Chemistry at the University of Freiburg. His research focus is on Chemical Epigenetics, covering both inhibitor synthesis and in-vitro assay development. He has published over 80 papers, mainly on epigenetics.


1. Introduction

The phenotype of an organism is based on its genetic information stored in the DNA but the final outcome of a certain phenotype is governed by transcriptional regulation. Epigenetics is defined as inheritable changes of the phenotype that take place without any changes in the genetic code. Key biochemical processes that define epigenetics are DNA methylation and histone modifications. In order to be packaged efficiently, DNA is wrapped around basic proteins, the histones. DNA and one histone octamer together form a nucleosome and the assembly of all nucleosomes is called COMPOUND LINKS

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chromatin
. The N-termini of the histones are rich in COMPOUND LINKS

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arginine
and lysine residues and protrude from the nucleosomes. They are subject to many types of post-translational modifications such as acetylation, methylation, phosphorylation, sumoylation and ubiquitinylation.1 These modifications can occur in different combinations at specific sites which is referred to as the “Histone Code” and explains why a great variety of gene expression patterns and thus different phenotypes is possible from one genetic code.2 In this article, we focus on histone methylation because DNA methylation and the role of other histone modifications have recently been reviewed more broadly in other publications.3–6

2. Protein methylation and biological significance

2.1 Histone methylation

Generally, histone methylation is involved in heterochromatin formation and maintenance, X-chromosome inactivation, transcriptional regulation, DNA repair, RNA maturation and genomic imprinting.7 Histones can be methylated either on COMPOUND LINKS

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lysine
or on arginine residues. In contrast to lysines that can be mono-, di- or trimethylated, arginines are only mono- or dimethylated, but the dimethylation can be symmetrical or asymmetrical (see Fig. 1).8 Depending on the site of methylation, COMPOUND LINKS

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lysine
methylation can be linked either to transcriptional activation or repression respectively. Methylation on H3K4, H3K36 and H3K79 for example is considered to be activating whereas methylated H3K9, H3K27 and H4K20 are regarded to be repressive marks.8 Further, loss of trimethylation at H4K20 is a general hallmark of human cancer together with the loss of acetylation at H4K16.9 Just recently, Manuyakorn and co-workers have discovered that cellular histone modification patterns help to predict the prognosis of pancreatic adenocarcinoma. Low cellular levels of H3K4me2, H3K9me2 or H3K81ac were independent predictors of poor survival. If a low cellular level of both H3K4me2 and H3K9me2 was observed, the survival outcome was even worse.10

For a long time, histone methylation was conceived to be a permanent mark11 due to the half-life of methylated lysines that is comparable to the half-life of histones.12 The first discovery of a demethylating activity by Paik and coworkers13 in 1973 describes that an extract obtained from rat kidney was able to convert mono- and dimethylated lysine residues to their unmodified status. Shi et al. were the first to report about a histone demethylase in 2004, namely LSD1, that is able to demethylate H3K4 me1/me2.14 In contrast, methyl groups from trimethylated lysines could not be cleaved off by LSD1. Instead, JmjC-domain-containing enzymes were found to demethylate trimethylated lysines.15–17 Monomethylated arginine residues can be deiminated to COMPOUND LINKS

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citrulline
by PAD4, a peptidylarginine deiminase.18 Dimethylated arginines in contrast, have been reported to be directly converted to COMPOUND LINKS

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arginine
by the demethylase JMJD6.19 The latter has been discussed controversially lately, as Webby and coworkers have shown a lysyl-hydroxylation activity of JMJD6 but they could not observe an argininedemethylation.20 The therapeutic potential of drugs that influence posttranslational modifications is vast as there are many diseases, among them especially cancer, that have been linked to aberrant gene expression patterns or deregulated histone modifying enzymes.21

2.2 Non-histone protein methylation

Besides histoneproteins, many other targets for histonemethyltransferases have been identified so far. To name just a few, COMPOUND LINKS

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arginine
methylation of FOXOtranscription factors by PRMT1 (proteinCOMPOUND LINKS

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arginine
methyltransferase 1) inhibits their phosphorylation by Akt which in turn leads to an increase in oxidative-stress-induced apoptosis mediated by the PI3K-Akt signalling pathway.22 Moreover, PIAS1, a STAT1 inhibitor in the IFN-γsignalling pathway, is methylated by PRMT1 which has an impact on STAT1 target gene expression.23 Furthermore, p53 is subject to methylation. Once methylated by PRMT5 on arginines R333, R335 and R337, which are part of the oligomerization domain, target gene specifity is affected.24 PRMT5 depletion leads to apoptosis because of an altered promoter binding specifity of p53.24 Upon monomethylation of p53 at COMPOUND LINKS

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lysine
382 by Set8 (KMT5A) transcriptional activation by p53 is suppressed. Here, knockdown of Set8 resulted in increased apoptosis.25 When, in contrast, p53 is methylated on COMPOUND LINKS

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lysine
372 by Set9 (KMT7) within the C-terminus, p53 is stabilized and p53-dependent transcription is augmented.26

Interestingly, the HIV transactivator protein Tat is subject both to COMPOUND LINKS

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lysine
methylation by SETDB1 (KMT1E), SETDB2 (KMT1F)27 and Set7/9 (KMT7)28 and COMPOUND LINKS

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arginine
methylation by PRMT6.29 The latter leads to negative regulation of the transactivation activity.29COMPOUND LINKS

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Lysine
methylation of TatK50 and TatK51 by SETDB1 resp. SETDB2 seems to prevent COMPOUND LINKS

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lysine
acetylation of the same residues which in turn prevents viral transactivation.27 On the other hand, data of Pagans et al. indicate that monomethylation of TatK51 by Set7/9 is coactivating Tat transactivation.28 Within the histoneproteins, COMPOUND LINKS

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lysine
methyltransferases are usually highly site-specific as compared to e.g. HDACs.30 But as to their general substrate specificity, it has been shown e.g. for G9a that it methylates the lysines of many protein substrates and not only histones.31

3. Histone methyltransferases and their inhibitors

Histone methyltransferases can be subdivided into three classes: SET-domain containing COMPOUND LINKS

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lysine
methyltransferases, non-SET domain COMPOUND LINKS

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lysine
methyltransferases and COMPOUND LINKS

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arginine
methyltransferases (PRMTs). All of them use COMPOUND LINKS

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S-adenosylmethionine
(SAM) as a co-substrate for the methylation and S-adenosylhomocysteine (SAH) is formed as a byproduct.32 Unlike histone acetylation, histone methylation does not alter the charge of the lysine residue in question. It affects the basicity, hydrophobicity and the size of the amino acid side chain group which affects the affinity of proteins that recognize such side chains. Although these changes are subtle, there are proteins that are able to bind selectively to certain methylated residues. These proteins have so-called chromodomains that recognize the methylated lysines. The structural data available led to an understanding of the motifs involved in COMPOUND LINKS

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methyl lysine
binding.33 Chromodomains recognize mono-, di- or trimethylated lysine residues. Tudor domains bind trimethylated lysines as well but there is also evidence for the recognition of mono- and dimethylated H4K20. PHD (plant homeo domain) fingers preferentially bind di- or trimethylated lysines. There are MBT (malignant brain tumor) domains that specifically bind mono-and dimethylated lysines. The only known proteins that recognize methylated arginines are the so-called WD40 repeats. They consist of about 40 amino acids and often have a COMPOUND LINKS

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tryptophan
- aspartic acid dipeptide (W–D) at the C-terminus. WD40 repeats can bind dimethylated arginine, namely H3R2(me)2 but also dimethylated lysines.33–36

So far, more than 20 COMPOUND LINKS

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lysine
methyltransferases37 and eleven COMPOUND LINKS

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arginine
methyltransferases (PRMT1-11) have been identified, but not for all of the members enzymatic activity could be demonstrated.38–40Lysine methyltransferases have been designated historically with a variety of names. Newly, a common nomenclature for COMPOUND LINKS

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chromatin
modifying enzymes has been proposed instead of these. The human COMPOUND LINKS

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lysine
methyltransferases have been renamed KMTs and are subdivided into 8 classes.41 Many of the methyltransferases have been linked to cancer and therefore the development of inhibitors is desirable.42

3.1 Lysine methyltransferases

Lysine methyltransferases consist of two classes - COMPOUND LINKS

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lysine
methyltransferases with or without a SET-domain.32 As already mentioned, the biological consequences of COMPOUND LINKS

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lysine
methylation depend on the extent of methylation and the target site. Additionally, the methylation may lead to further biochemical changes in a cellular context and this may have varying consequences for different forms of diseases as described below (see Table 1).34

Opposing effects have been discovered concerning the outcome of COMPOUND LINKS

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lysine
methylation: methylation of the tumor suppressor protein p53 on K370 by Smyd2 (KMT3C)43 or on K382 by SET8 (KMT5A)25 leads to inhibition of transcriptional activity whereas methylation on K372 by Set9 (KMT7) results in transcriptional activation.26 The COMPOUND LINKS

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lysine
methyltransferase G9a (KMT1C) has recently been shown to dimethylate p53 on K373. This was correlated with inactivity of p53.44 Its impact on apoptotic processes as well as its overexpression in various cancer types suggests that G9a is a putative oncogene.44 Interestingly, another study revealed a correlation between COMPOUND LINKS

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cocaine
addiction and cellular levels of the lysinemethyltransferase G9a with the conclusion that reduced levels of G9a expression and subsequent hypomethylation on H3K9 correlate with COMPOUND LINKS

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cocaine
addiction. Repeated administration of COMPOUND LINKS

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cocaine
in mice showed a downregulation of G9a. G9a overexpression in mice by direct injections into the Nucleus accumbens with Herpes simplex virus vectors expressing G9a reduced the preference for COMPOUND LINKS

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cocaine
. These insights may help to develop more effective treatments for drug addiction and an improved understanding of the biochemical basis of drug addiction.45

As causes for dysregulated histone methylation, the enzyme in question might be mutated or overexpressed, e.g. in cancer cells. The COMPOUND LINKS

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lysine
methyltransferase EZH2 (KMT6) was found to be increased in breast and prostate cancer46 with a poor prognosis for elevated levels in prostate cancer.47 Mutations of the methyltransferase MLL1 seem to be responsible for various forms of acute leukaemia by blocking hematopoietic differentiation.48 Hypermethylation of H3K79 by the associated complex MLL-AF10 or MLL-hDOT1L respectively is reported to result in leukaemogenesis.49 Additionally, SMYD3 was found to be upregulated in colorectal, hepatocellular50 and breast cancer. For the latter, overexpression led to enhanced cancer cell growth.51 Furthermore, SET7/9 (KMT7) has recently been associated with hyperglycaemia-induced epigenetic changes. Transient high COMPOUND LINKS

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glucose
levels seem to result in SET7/9 methylating histones in the promoter region of nuclear factor κB (NF-κB) causing increased p65 gene expression.52

Table 1 Selected COMPOUND LINKS

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lysine
methyltransferases and links to disease
Epigenetic protein Links to disease Major target sitesa
a If no reference is cited, source is Ref. 41.
SUV39H1 (KMT1A) Elevated mRNA level in colon cancer tissue samples94 H3K9
G9a (KMT1C) Suppressor gene silencing95 H3K9
Eu-HMTase1 (KMT1D) Overexpression in gland tumors96 H3K9
MLL1 (KMT2A) Rearrangement/amplification blocks hematopoietic differntiation48 H3K4
MLL4 (KMT2D) Involved in hepatitis B virus dependent liver carcinogenesis97 H3K4
SMYD2 (KMT3C) Suppression of p53 transcriptional activity43 H3K56, p53K370
SMYD3 Overexpression and enhanced tumor cell growth in breast cancer51 H3K450
Overexpression in colon cancer and hepatocellular carcinoma50
DOT1L (KMT4) Enzymatic activity crucial for leukaemogenesis49 H3K79
EZH2 (KMT6) Amplification and overexpression in multiple cancer types98 H3K27
SET7/9 (KMT7) Hyperglycemia induces SET7/9 causing increased p65 gene expression.52 H3K4, p53K37226


3.2 Inhibitors of COMPOUND LINKS

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lysine
methyltransferases

Generally, methyltransferases can be inhibited by co-substrate analogues. There are three known co-substrate analogues that inhibit a variety of methyltransferases: sinefungin, an antibiotic compound that is structurally similar to SAM, the demethylated co-substrate SAH as a feedback inhibitor, and COMPOUND LINKS

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methylthioadenosine
. This kind of inhibitors is rather unselective and as such not applicable as therapeutic agents.53

There are only a few drug-like inhibitors of COMPOUND LINKS

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lysine
methyltransferases and most of them were discovered by random screening approaches. The first inhibitor was chaetocin (1, see Fig. 2) which is a fungal mycotoxin. It inhibits both the Drosophila melanogasterenzyme Su(var)3–9 with an IC50 of 0.8 μM and G9a with an IC50 of 2.5 μM.54 Recently, chaetocin was also shown to be highly active against myeloma cells by increasing the sensitivity to oxidative stress induced cytotoxicity with hardly any effect on normal human B cells.55



            Lysine
            methyltransferase
            inhibitors (IC50 values and corresponding enzymes in parentheses).
Fig. 2 Lysine methyltransferase inhibitors (IC50 values and corresponding enzymes in parentheses).

The compound Bix-01294 (2, see Fig. 2) was identified in a combined virtual and high-throughput screen approach and shown to be an inhibitor of G9a (KMT1C) with an IC50 of 3 μM. It is selective against SUV39H1 and PRMT1. Cultured cells treated with Bix-01294 showed a reduction of histone H3K9 dimethylation and a decrease in cell number. In the same screening procedure, the compound Bix-01338 (3, see Fig. 2) was discovered as a rather unselective inhibitor of methyltransferases. It did not show any selectivity between COMPOUND LINKS

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lysine
or COMPOUND LINKS

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arginine
methyltransferases with an IC50 of 5 μM for G9a and an IC50 of 6 μM for PRMT1.56

Recently, UNC0224 (4, see Fig. 3) has been presented as a new inhibitor for the lysinemethyltransferase G9a with an IC50 of 15 nM. This 2,4-diamino-7-aminoalkoxyquinazoline has been found by modifying the structure of Bix-01294. It showed a 1000-fold selectivity for G9a compared to SET7/9 (KMT7) and Set8. Additionally, the first crystal structure of G9a with an inhibitor in a complex was introduced with Bix-01294 (see Fig. 4).57Bix-01294 forms two hydrogen bonds to Asp1131 and Asp1140 at the entrance of the substrate binding pocket, whereas the two methoxy groups show van der Waals interactions with the more buried part of the pocket. Interestingly, the inhibitor is not involved in direct interaction with the residues of the catalytic site or the cofactorSAM. Therefore, the dimethylaminopropoxy side chain in UNC0224 was attached in order to obtain binding to the lysine tunnel and hence increased inhibitory activity. The structural information is particularly important because of the known link of G9a to human cancers.44,58



            Lysine
            methyltransferase
            inhibitor obtained by structural variation of BIX-01294 (IC50 value and corresponding enzyme in parentheses).
Fig. 3 Lysine methyltransferase inhibitor obtained by structural variation of BIX-01294 (IC50 value and corresponding enzyme in parentheses).

Crystal structure of the G9a methyltransferase in complex with the inhibitorBix-01294 (coloured orange). The inhibitor is involved in two hydrogen bonds to D1131 and D1140. (The cofactor analogue SAH is coloured green and the protein backbone is shown as purple ribbon. Only interacting residues of G9a are displayed).
Fig. 4 Crystal structure of the G9a methyltransferase in complex with the inhibitorBix-01294 (coloured orange). The inhibitor is involved in two hydrogen bonds to D1131 and D1140. (The cofactor analogue SAH is coloured green and the protein backbone is shown as purple ribbon. Only interacting residues of G9a are displayed).

A new combined epigenetic therapy has been proposed using the EZH2 (KMT6) inhibitor3-deazaneplanocin A (DZNep, 5, see Fig. 2) and the histone deacetylase inhibitorpanobinostat.59 EZH2 is a part of the polycomb repressive complex 2 with COMPOUND LINKS

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lysine
methyltransferase activity and is involved in proliferation and aggressive cell growth. DZNep is a cyclopentenyl analogue of 3-deazaadenosine and was originally synthesized as an inhibitor of S-adenosyl-L-homocysteine hydrolase.60 DZNep treatment of cultured human acute myeloid leukaemia cells induced an increased expression of p16, p21, p27 and FBXO32 as well as apoptosis. These effects were enhanced by cotreatment with the pan-HDAC inhibitorpanobinostat. Also the survival of NOD/SCID mice with leukaemia caused by the AML HL-60 cells was improved upon cotreatment.59

3.3 Arginine methyltransferases

Protein COMPOUND LINKS

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arginine
methylation is mediated by PRMTs that can be subdivided into two classes: type I methyltransferases catalyse the formation of asymmetrically substituted arginine residues and type II methyltransferases mediate the formation of symmetrically methylated arginine residues. PRMTs 1–4, 6 and 8 are type I enzymes and PRMTs 5, 7 and 9 are PRMTs of type II (see Table 2). PRMTs 2 and 9 were shown to be catalytically inactive and for PRMTs 10 and 11 a proof of methyltransferase activity is lacking so far as well.61 PRMTs usually methylate glycine-alanine-COMPOUND LINKS

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arginine
patches (so-called GAR motifs). As an exception, CARM1 shows affinity to COMPOUND LINKS

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proline
-COMPOUND LINKS

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glycine
-COMPOUND LINKS

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methionine
-COMPOUND LINKS

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arginine
sequences (so-called PGM motifs).39 PRMT5 has also been shown to methylate PGM motifs.62

PRMT1 is an essential component of the Mixed Lineage Leukaemia (MLL) oncogenic transcriptional complex. It has transcriptional activation properties when aberrantly expressed and hence is critical for the development of leukemia.63 As quoted above, PRMT1 has also been shown to methylate FOXOtranscription factors that play a role in the homoeostasis of cellapoptosis. By methylationvia PRMT1, the FOXOproteins cannot be further phosphorylated by Akt. This leads to an increase in oxidative-stress-induced apoptosis which is based upon the PI3K-Akt signalling pathway.22 Here, inhibitors of PRMT1 could be beneficial for the therapy of neurodegenerative diseases as one major issue of it is cell damage due to oxidative stress. Methylation of a conserved arginine residue in the Igα subunit of the B cellreceptor by PRMT1 shows a role in immunology as it promotes B cell differentiation.64

Arginine methyltransferases are known to be co-activators of nuclear receptors and therefore an interesting target for the therapy of hormone dependent cancer.65,66E.g., PRMT2 and 5 have been identified as co-activators of the androgen receptor. For PRMT1, 2 and 4 (CARM1) there is evidence of being estrogen receptor associated co-activators. Both PRMT1 and CARM1 seem to stimulate estrogen receptor mediated transcription by interacting with members of the p160 family.67 Furthermore, the histone acetyltransferaseCREB bindingprotein (CBP) is an important regulator for the transcriptional activation by nuclear receptors and other transcription factors. There is evidence that CBP is methylated by CARM1 which enhances its co-activating activity.68

PRMT5 appears to be a regulator of p53 transcription. In the context of DNA damage control reduced levels of PRMT5 lead to apoptosis with an altered p53 promoter binding specifity indicating the regulatory function of PRMT5.24 Additionally, PRMT5 has been connected to proliferation and regulation of cell growth by negatively controlling the expression of tumor suppressor genes ST7 and NM23.69

PRMT7 is also a potential target of cancer therapy because downregulation of PRMT7 seems to sensitize cancer cells to COMPOUND LINKS

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camptothecin
treatment.70 Thus, inhibitors of PRMT7 could be used to potentiate the effects of this and maybe other cytotoxic drugs.

Arginine methylation activity can be regulated viaPRMT-binding proteins such as BTG1 and TIS2/BTG2,71 negative feedback by SAH or posttranslational modifications.39E.g., it has been shown that phosphorylation of CARM1 negatively regulates its COMPOUND LINKS

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arginine
methylating activity. Different serine residues have been identified as potential phosphorylation sites whereas the responsible kinase is still unknown.72,73

Arginine methylation is also involved in RNA maturation62 and DNA repair.74 The checkpoint protein MRE11 plays an important role in DNA damage control and is dependent on COMPOUND LINKS

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arginine
methylation75 implying a possible safety problem for using COMPOUND LINKS

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arginine
methyltransferaseinhibitors as drugs. Nevertheless, there is a great therapeutic potential for inhibitors of COMPOUND LINKS

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arginine
methyltransferases for a variety of diseases.

Table 2 Selected COMPOUND LINKS

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arginine
methyltransferases and links to disease
Epigenetic protein Links to disease Major target sitesa
a If no reference is cited, source is Ref. 61.
PRMT1 Essential component of MLL oncogenic transcriptional complex63 H4R3, RG domains of Npl399
Coactivator of hormonereceptor function76
PRMT2 Androgen receptor coactivator100 No catalytic activity
PRMT4 (CARM1) Overexpression in breast cancer101 H3R2, H3R17, H3R26, CBP/p300R2142103
Overexpression correlates with androgen independence in prostate tumor cells102
Contribution to CBP coactivation due to methylation68
PRMT5 Overexpression in lymphoid cancer cells104 H3R8, H4R3, RG domain of MBD2105,106
PRMT6 Overexpression diminished viral Tat activity29 H2AR3, H3R2, H4R3107
PRMT7 Sensitization of tumor cells to COMPOUND LINKS

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camptothecin
treatment when downregulated70
Not known


3.4 Inhibitors of COMPOUND LINKS

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arginine
methyltransferases

As seen for COMPOUND LINKS

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lysine
methyltransferases, co-substrate analogues like sinefungin can be used as inhibitors of COMPOUND LINKS

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arginine
methyltransferases with the same restrictions as mentioned above.

The first more specific inhibitors of COMPOUND LINKS

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arginine
methyltransferases were discovered in a random screening approach. The screening was performed with the yeast enzyme Hmt1p and with Npl3 as a nonhistone substrate. Nine of the 9000 compounds screened showed a good activity in the low micromolar range and were then tested against human PRMT1. The new inhibitors were called AMIs for COMPOUND LINKS

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arginine
methyltransferaseinhibitors. AMI-1 was the most potent inhibitor with an IC50 of 9 μM against PRMT1 (6, see Fig. 5). It further showed selectivity towards several COMPOUND LINKS

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lysine
methyltransferases and in vivo effects in a cell culture model. The methylation level of Npl3 in HeLa cells that were transiently transfected with GFP-Npl3 was decreased upon AMI-1 treatment and an inhibitory effect on the activation on nuclear androgen and estrogen receptors could be shown.76 As a caveat, AMI-2 contains partial structures well known for promiscuous effects like aggregation and some of the AMIin vitro results have not yet been verified in cell culture.77AMI-1 shows resemblance to the antiparasitic drugsuramin and similarly inhibits sirtuins, yet with decreased activity (IC50 on Sirt1: 300 nM for suramin, 32 μM for AMI-1).78



            Arginine
            methyltransferase
            inhibitors obtained from random screening (IC50 value and corresponding enzyme in parentheses).
Fig. 5 Arginine methyltransferase inhibitors obtained from random screening (IC50 value and corresponding enzyme in parentheses).

Among the AMI compounds, AMI-5, (COMPOUND LINKS

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eosin
, COMPOUND LINKS

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7
, see Fig. 5) has been used as lead structure to synthesize further inhibitors of methyltransferases. One example is compound 6e (15, see Fig. 6).79 It was subsequently modified to give simplified scaffolds that are structurally similar to COMPOUND LINKS

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curcumin
including bromo- or COMPOUND LINKS

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dibromophenol
substructures. Compound 8e (16, see Fig. 6) was selective for CARM1 over PRMT1 at a concentration of 100 μM.80 The newly obtained PRMTinhibitors were also tested against HAT and sirtuins due to the fact that COMPOUND LINKS

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curcumin
is a known HATinhibitor81 and the compound GW5074 with a dibromophenol substructure exhibits sirtuin inhibitory properties.82 Both HAT and sirtuin inhibitory activity was confirmed in vitro for several compounds. Cellular hypomethylation was demonstrated for some inhibitors but data on cellular hypoacetylation is not available.



            Arginine
            methyltransferase
            inhibitors obtained by synthetic variation of screening hits (IC50 value and corresponding enzyme in parentheses).
Fig. 6 Arginine methyltransferase inhibitors obtained by synthetic variation of screening hits (IC50 value and corresponding enzyme in parentheses).

The COMPOUND LINKS

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aminonaphthol sulfonate
core of AMI-1 was used to develop further potent and selective inhibitors for PRMT1 by Bonham and co-workers.83 They combined the structural core with the azo moiety of AMI-976 and the COMPOUND LINKS

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dichlorotriazine
group of AMI-676 on the other hand to give Inhibitor 4 (18, see Fig. 6, IC50 = 4 μM). As PRMT1 activity is involved in T helper cell function84–86 the inhibitory effect of Inhibitor 4 towards T helper cellcytokine production was characterized. Inhibitor 4 enhanced T-helper cell proliferation. Additionally, decreased IFN-γ levels of type 1 T-helper cells and IL-4 levels of type 2 T-helper cells were observed upon inhibitor treatment. Thus, this new compound shows potential in the treatment of autoimmune diseases.

A further compound called inhibitor1 (8, see Fig. 5) which is selective towards CARM1 and has been obtained from high-throughput screening was further modified to give compound 7b (17, see Fig. 6). 7b showed an IC50 of 80 nM and selectivity towards other methyltransferases.87

The first target based virtual screening for COMPOUND LINKS

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arginine
methyltransferases was presented by our group in 2007 using a homology model of hPRMT1.88 The screening procedure was created in a way that potential ligands for the substrate binding pocket were selected but not those that would target the co-substrate binding site. About 40 compounds of the NCI diversity set were selected for in vitro testing by manually inspecting the screening hits. Seven inhibitors with an IC50 below 60 μM were found in the subsequent in vitro assay. The inhibitorsallantodapsone (9, see Fig. 7) and COMPOUND LINKS

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stilbamidine
(COMPOUND LINKS

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10
, see Fig. 7) induced hypomethylation at H4R3 in HepG2 cells. Functional activity was confirmed in a reporter gene assay with MCF7a cells. Both inhibitors showed a reduction of estrogen receptoractivation by COMPOUND LINKS

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estradiol
in a dose-dependent manner.88 Competition studies using histone substrate and cofactor SAM showed that the identified inhibitors are substrate competitive inhibitors, which is in agreement with the obtained docking results. In Fig. 8 the predicted binding mode of allantodapsone and COMPOUND LINKS

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stilbamidine
is presented. Both are making hydrogen bonds to the acidic residue (E152, E161) of the catalytic site.



            Arginine
            methyltransferase
            inhibitors from virtual screening (IC50 value against hPRMT1 in parentheses).
Fig. 7 Arginine methyltransferase inhibitors from virtual screening (IC50 value against hPRMT1 in parentheses).

Docking solutions for the PRMT1 inhibitorsstilbamidine (top, coloured orange) and allantodapsone (bottom, coloured orange). Hydrogen bonds are shown as dashed lines, the protein backbone is shown as pink ribbon and the cofactor analogue SAH is coloured green.
Fig. 8 Docking solutions for the PRMT1 inhibitorsCOMPOUND LINKS

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stilbamidine
(top, coloured orange) and allantodapsone (bottom, coloured orange). COMPOUND LINKS

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Hydrogen
bonds are shown as dashed lines, the protein backbone is shown as pink ribbon and the cofactor analogue SAH is coloured green.

As a second approach, a fragment-based virtual screening for molecules with inhibitory properties and a molecular weight below 200 g mol−1 was performed leading to the discovery of an α–COMPOUND LINKS

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methylthioglycolic amide
as an inhibitor for PRMT1. As this compound exhibited neither good chemical stability nor a drug-like structure, a similarity search was conducted. As a result, the compound RM-65 (11, see Fig. 7) could be identified as inhibitor of PRMT1 in vitro with cellular hypomethylating activity in HepG2 cells at the PRMT1 target H4R3. It does not inhibit the lysinemethyltransferase SET7/9. Although the original search was performed with fragments binding to the substrate binding pocket, RM-65 rather seems to be a bisubstrate mimic as proposed by a docking study on a hPRMT1 homology model.89

In the further course of research, greater databases were screened for new COMPOUND LINKS

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arginine
methyltransferaseinhibitors with a modified screening procedure including a pharmacophore prefilter. This approach conduced to the discovery of inhibitors6, COMPOUND LINKS

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7
and 9 (12, 13, 14 see Fig. 7) from the Chembridge database with inhibitory effects down to 13 μM.90

4. Conclusion

For methyltransferases many links to the pathogenesis of various diseases have been demonstrated. Thus, the further development of inhibitors is extremely important for the future therapy of the various diseases, especially for cancer. The major issue that needs to be addressed is the selectivity among the different histonemethyltransferases in order to better understand the function of the individual subtypes which should eventually lead to optimized therapeutics. Recently obtained structural information should be important information towards obtaining subtype selectivity.91Equally important is a better understanding of the relevance of histonevs.non-histonemethylation for the resulting phenotype.

Even though the field of epigenetic drug discovery is still at its beginning, a dynamic development can be anticipated and clearly further epigenetic drugs based on histonemethyltransferases will be developed in the near future. Of course, also the histone demethylases will be similarly in the focus. Special attention will also be given to the methyl binding proteins, the so-called readers of the histone code. As screening assays have become available for the testing of this recognition,92,93inhibitors of this interaction are to be expected soon which may open up a completely new class of epigenetic therapeutics.

5. Acknowledgements

We would like to thank the Deutsche Krebshilfe for financial support of our work on PRMT inhibitors.

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