Cellular differentiation is one of the most important components of embryonic development and postnatal tissue maintenance and repair. Almost every nucleated cell of the human body contains the same, complete complement of genomic DNA. However, the ability of pluripotent cells to differentiate into distinct lineages and ultimate cell types is conferred by specific patterns of transcription of subsets of genes in the genome. A large and growing body of data support the idea that epigenetic regulation of gene transcription is a key biological deter-minant of cellular differentiation1.

The chromosomes within eukaryotic cell nuclei are packaged together with structural proteins (histones) to form the complex known as chromatin. Four major histones (H2A, H2B, H3 and H4) form an octameric, disc-shaped aggregate — composed of two copies of each histone type — around which the DNA is wound to form regular, repeating units known as nucleosomes (FIG. 1). Chromatin exists in two main conformational states: a condensed state (heterochromatin) in which the nucleo-somes are tightly packed together and gene transcription is largely repressed; and a more relaxed state (euchro-matin) in which gene transcription is activated. Epigenetic regulation of gene transcription is mediated by selective, enzyme-catalysed, covalent modification of specific nucleo tides within the genes and also by post-translational modifications of the histone proteins (FIG. 1). Modification of DNA can silence gene transcription directly, whereas the post-translational modifications of histones control the conformational transition between the heterochroma-tin and euchromatin states2. The enzymes that covalently modify DNA and histones are therefore the key mediators of epigenetic regulation of gene transcription.

Several putative epigenetic enzymes have recently been identified and, in some cases, their catalytic mechanism and three-dimensional structures have been determined2,3.

Epigenetic enzymes that are encoded in the human genome catalyse group transfer reactions and can be categorized according to the nature of the covalent modifications that they catalyse and by the substrates upon which they act. In humans, these enzymes include DNA methyltransferases (DNMTs), which methylate the carbon atom at the 5-position of cytosine in the CpG dinucleotide sites of the genome; protein methyl-transferases (PMTs), which methylate lysine or arginine residues on histones and other proteins; protein demethyl-ases, which remove methyl groups from the lysine or arginine residues of proteins; histone acetyltransferases, which acetylate lysine residues on histones and other proteins; histone deacetylases (HDACs), which remove acetyl groups from lysine residues on histones and other proteins; ubiquitin ligases, which add ubiquitin to lysine residues on histones and other proteins; and specific kinases that phosphorylate serine residues on histones4,5.

Given that small-molecule inhibitors have been suc-cess fully designed for HDACs and DNMTs (discussed below), it is likely that additional families of histone-modifying enzymes will also be amenable to small-mol-ecule modulation. The opportunity for chemical-probe development and pharmacological control of epigenetic gene transcription is therefore of great interest in the fields of basic biology and drug discovery4,5. Indeed, the role of these enzymes in human diseases is high-lighted by the recent approval of three drugs by the US Food and Drug Administration6 that act as selective, small-molecule inhibitors of HDACs and DNMTs for the treatment of specific human cancers (TABLE 1).

In recent years, there have been numerous reviews in the literature that highlight different aspects of the biology, disease association and/or structural biology of various histone-modifying enzymes. In this Review, we

Epizyme, Inc., 840 Memorial Drive, Cambridge, Massachussets 02139, USA.Correspondence to R.A.C. e-mail: RCopeland@epizyme.comdoi:10.1038/nrd2974

EpigeneticsA stably heritable change in phenotype or gene expression in an organism or cell, resulting from changes in a chromosome that are not caused by a change in DNA sequence. The process of eukaryotic cell differentiation is one of the most well-known examples of epigenetic changes.

Protein methyltransferases as a target class for drug discoveryRobert A. Copeland, Michael E. Solomon and Victoria M. Richon

Abstract | The protein methyltransferases (PMTs) — which methylate protein lysine and arginine residues and have crucial roles in gene transcription — are emerging as an important group of enzymes that play key parts in normal physiology and human diseases. The collection of human PMTs is a large and diverse group of enzymes that have a common mechanism of catalysis. Here, we review the biological, biochemical and structural data that together present PMTs as a novel, chemically tractable target class for drug discovery.

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Nature Reviews | Drug Discovery

Me

Ac MeK

K

UbK

SP R

PKMTs52 family members

PRMTs≥10 family members

Demethylases~30 family members

Deacetylases~18 family members

Acetyltransferases

Kinases

Ligases

Target classA group of proteins that are related by a common type of drug-binding pocket, but sufficiently diverse that selective inhibition of specific proteins can be achieved, using medicinal chemical elaboration of the basic chemotype structures.

SAMS-adenosyl-l-methionine, the universal methyl group donor of all enzymatic methyltransferase reactions.

focus on the PMTs, and in particular on those aspects that make PMTs attractive targets for drug discovery efforts. We summarize the data that contribute to the validation of PMTs as targets for specific human diseases, as well as the structural and mechanistic data that suggest PMTs are a tractable (that is, druggable) target class.

PKMTs and PRMTs in human diseaseIn surveying the histone-modifying enzymes of the human genome, the enzymes that catalyse methylation of lysine residues (protein lysine methyltransferases (PKMTs)) and arginine residues (protein arginine methyltransferases (PRMTs)) are of substantial interest from the perspective of drug discovery and medicinal chemistry. The action of these enzymes is crucial in controlling gene regulation, and there is an increasing amount of biochemical and biological data to suggest that the enzymatic activities of several of these proteins have pathogenic roles in cancer, inflammatory diseases, neurodegenerative diseases and other conditions of importance7–15.

For example, with the exception of DoT1-like, histone H3 methyltransferase (DoT1l; also known as KMT4), all human PKMTs contain a ~130 amino-acid domain, referred to as the SET domain, which constitutes the catalytic domain of these enzymes14–16. Enhancer of zeste homologue 2 (EZH2; also known as KMT6) is a SET domain protein that forms the catalytic subunit of the 4–5- protein core of polycomb repressive complex 2 (PRC2). PRC2 is a PKMT that catalyses the methyla-tion of lysine 27 of histone H3 (in the nomenclature of histone modification, this site is referred to as H3K27). Although EZH2 contains the catalytic active site, all of the proteins of the PRC2 complex are required for full PKMT activity. overexpression of EZH2 or another PRC2 subunit, suppressor of zeste 12 homologue (SUZ12), has been associated with numerous human cancer types, including prostate, breast, bladder, colon, skin, liver, endometrial, lung and gastric cancers, as well as lymphomas and myelomas15. In breast carcinomas, increased levels of EZH2 have been shown to correlate with increased invasiveness and proliferation rate; it has been suggested that EZH2 could be a prognostic indica-tor of patient outcome for breast cancer10. In cell culture, overexpression of EZH2 in breast epithelial cells causes anchorage-independent cell growth and increased invasiveness. Additionally, when EZH2-overexpressing cells were injected into the mammary fat pads of nude mice, the animals developed tumours, demonstrating the tumorigenicity of EZH2 overexpression. Importantly, the phenotypic effects of EZH2 overexpression are correlated with increased H3K27 methylation and are dependent on the presence of an intact SET domain, both of which imply a role for EZH2 enzymatic activity in pathogenesis10,15. Several other human PKMTs and PRMTs are strongly associated with human cancers, as summarized in TABLE 2. Similarly, there is compelling evidence that other PKMTs and PRMTs have a pathogenic role in serious human diseases other than cancer7–9. For example, SET domain, bifurcated 1 (SETDB1)17 and coactivator-associated

arginine methyltransferase 1 (CARM1; also known as PRMT4)18 have been implicated in the neurodegenera-tive diseases Huntington’s disease and spinal muscular atrophy, respectively. SET domain-containing lysine methyltransferase 7 (SETD7; also known as KMT7)19, CARM1 (REF. 20) and PRMT1 (REF. 21) have been associated with nuclear factor-κB-related inflamma-tory diseases, and SET domain-containing protein 1A (SETD1A)22 and CARM1 (REF. 23) have been associated with viral infections involving Herpes simplex virus and human T lymphotrophic virus, respectively. PKMTs and PRMTs are therefore emerging as compelling targets for drug discovery efforts4,5.

PMTs as a drug target classFrom a chemical biology and medicinal chemistry perspective, the PKMTs and PRMTs are of interest because they have a common mechanism of catalysis (discussed below), involving a small, organic cofactor. As other druggable classes of enzymes, such as the protein kinases, share this mechanistic feature, it is likely that the PMTs will be similarly amenable to inhibition by small, organic molecules.

The PKMTs and PRMTs catalyse methyl transfer from their universal methyl donor, S-adenosyl-l-methionine (SAM; also known as AdoMet) (FIG. 2), to a nitrogen atom of lysine or arginine side chains3. Protein substrate spe-cificity can be stringent in these enzymes; some PKMTs seem to selectively methylate a particular lysine residue on a specific histone, and the extent of methylation on a single lysine residue (that is, mono-, di- or tri-methyl-ation) that is catalysed by a particular enzyme can also

Figure 1 | A nucleosome and the post-translational histone protein modifications that can influence epigenetic regulation of gene transcription. Modifications of the histone protein tail are shown: changes in acetylation (Ac) by acetyltransferases and deacetylases, phosphorylation (P) by kinases, ubiquitylation (Ub) by ligases and changes in methylation (Me) by methyltransferases and demethylases. The enzyme families that are responsible for the various post-translational modifications are shown. PKMT, protein lysine methyltransferase; PRMT, protein arginine methyltransferase.

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SAHS-adenosyl-l-homocysteine, the universal product of all enzymatic methyltransferase reactions, formed by methyl group transfer from S-adenosyl-l-methionine.

be stringent (discussed below). Clearly, the structure of each enzyme is unique16,24, as is the resulting biology and pathobiology associated with each enzyme. Yet, the shared chemical mechanisms of the PKMTs and PRMTs allows for certain efficiencies and economies in the dis-covery of selective drugs for these enzymes, by treating them as a target class25–27. Several of these enzymes have been found to catalyse methyl transfer to lysine or arginine residues on a number of cellular proteins; this is especially the case for the PRMTs, for which sev-eral cytosolic substrates have been identified24,28. With respect to gene regulation, however, the most important targets for both PKMTs and PRMTs are likely to be the histones, as post-translational modification of these pro-teins is clearly a determinant of chromatin remodelling and therefore regulation of gene transcription.

Representation of PMTs in the human genomeThe PMT target class is represented in many species, and the human genome encodes several PKMTs and PRMTs. Attempts to quantify the representation of PKMTs in a particular organism, and to understand the related-ness of these proteins to one another, have focused on the sequence alignment of the SET domain because, as discussed above, this domain is common to all PKMTs (except DoT1l).

Several attempts have been made to systematically group the PKMTs on the basis of sequence homology and substrate14,29. For example, in 2007, a nomenclature con-vention was proposed for the PKMTs, along with other types of chromatin-modifying enzymes29. In this study, 24 human PKMTs were identified. These SET domain PKMTs have been divided into related families on the basis of sequence alignment; initially four, and later seven, major families were defined in this manner14,16: the

suppressor of variegation 3–9 (SUV39) family; the SET1 (also known as Mll) family; the SET2 (also known as NSD) family; the retinoblastoma protein-interacting zinc finger protein (RIZ) (also known as PRDM) family; the SET and MYND domain-containing (SMYD) family; the enhancer of zeste (EZ) family; and the SUV4–20 family. An eighth family, known as ‘others’, included the enzymes SETD7 and SETD8 (also known as PRSET7). Finally, DoT1l, the human, non-SET domain PKMT can be considered a ninth family of PKMTs.

our group has recently extended this work to system-atically identify all of the SET domains that are encoded by the human genome. This study has more than doubled the number of putative human PKMTs to 52 (51 SET domain proteins plus DoT1l) (l.F. Jerva, K.o. Elliston, V.M.R., M.E.S. and R.A.C., unpublished observations). From the perspective of drug discovery, the salient point is that these enzymes are numerous in humans.

The PRMTs are similarly well represented in humans. There are at least eight human PRMTs for which some level of methyltransferase activity has been shown. These proteins have a canonical sequence domain that is associ-ated with the binding sites for the cofactor and substrate (arginine), although the sequence conservation among these proteins is low. Estimates of the total number of PRMTs that are encoded by the human genome vary, depending on the method of sequence alignment and the level of alignment stringency that is applied. Nevertheless, it is clear that 10–50 of these enzymes are represented in humans.

The human PMTs are thus a large class of enzymes, and several of them already have well established dis-ease association (discussed above). Furthermore, owing to the common features of their chemical mechanism of catalysis (discussed below), the PMTs are likely to be inherently tractable as targets for small-molecule drug intervention. The PMT target class, as defined here, therefore provides an important pool of potential targets for drug discovery efforts.

The PMT active siteThe pursuit of the PMTs as a drug target class is facilitated by a rich literature base of crystallographic and enzyme kinetic studies of these enzymes that have helped to define their mechanisms of catalysis. All of these enzymes probably use a common bimolecular nucleophilic sub-stitution (SN2) methyl transfer mechanism3,24,30. The lone pair electrons of a nitrogen atom (from lysine or arginine) ‘attacks’ the electrophilic methylsulphonium cation of SAM at a 180° angle to the leaving group, to form a penta-coordinate carbon transition state. The transition state structure then collapses, with methyl group relo-cation to the nitrogen atom of the lysine or arginine side chain and formation of S-adenosyl-l-homocysteine (SAH; also known as AdoHcy) (FIG. 2) as a product.

The use of a naturally occurring adenosyl analogue as the universal group transfer donor by PMTs is remi-niscent of protein kinases — another large family of druggable enzyme targets, the ATP-binding pockets of which have proved to be highly tractable targets for drug discovery31,32. Furthermore, despite binding a

Table 1 | Epigenetic-enzyme inhibitors for cancer therapy

generic name Alernative names

clinical status*

DNMT inhibitors

5-azacitidine Vidaza Approved in the United States for myelodysplastic syndrome

Decitabine Dacogen Approved in United States for myelodysplastic syndrome

HDAC inhibitors

Vorinostat Zolinza Approved in United States for cutaneous manifestation in cutaneous T cell lymphoma

Romidepsin FK228 New drug application filing

Panobinostat LBH-589 Phase II

Belinostat PXD-101 Phase II

Entinostat MS-275 SNDX-275

Phase II

MGCD-0103 MG-0103 Phase II

JNJ-26481585 None Phase I

Givinostat ITF2357 Phase II

*See REFS 51,52 for comprehensive reviews of novel cancer therapies, including those described in the table. DNMT, DNA methyltransferase; HDAC, histone deacetylase.

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common natural ligand, the ATP-binding pockets of protein kinases have afforded medicinal chemists a rich diversity of chemical scaffolds, which have resulted in a range of drug molecules of varying degrees of target selectivity32. Similarly, the commonality of SAM use by the PMTs belies the structural, biological and pathologi-cal diversity of these enzymes. From the perspective of drug discovery and medicinal chemistry, the diversity of SAM-binding modes and catalytic mechanisms of these enzymes is of key importance.

A common structural feature of PKMTs and PRMTs that distinguishes these enzymes from other pro-teins that use SAM is the overall architecture of their extended catalytic active sites. This generally consists of a SAM-binding pocket that is accessed from one face of the protein, and a narrow, hydrophobic, acceptor (that is, lysine or arginine) channel that extends to the opposite face of the protein surface, such that the two substrates enter the active site from opposite sides of the enzyme surface.

Table 2 | Selected PKMTs and PRMTs that have shown an association with human cancers

Protein methyltransferase

Methylation substrates

cancers cancer association refs

SUV39H1 H3K9 Colon cancer Increased expression in colorectal tumours; associated with transcriptional repression

53

EHMT2 H3K9 Lung, prostate and hepatocellular carcinoma

Increased expression in lung cancer cell lines; regulates centrosome duplication, presumably through chromatin structure

54,55

MLL H3K4 Leukaemia Chromosomal aberrations involving MLL are a cause of acute leukaemias; the SET domain is lost in translocation

56–58

NSD1 H3K36 Acute myeloid leukaemia

Translocation fuses NSD1 to nucleoporin 98 in human acute myeloid leukaemia

59

WHSC1 H3K36 and H4K20

Myeloma Translocated and increased expression in myeloma; associated with transcriptional regulation

60–62

WHSC1L1 H3K4 Lung and breast cancers, and childhood acute myeloid leukaemia

Amplified in lung cancer and breast cancer; translocation with nucleoporin 98; mediates transcriptional activation

63–64

DOT1L H3K79 MLL-rearranged leukaemias

Recruited by MLL fusion partners MLLT1, MLLT2, MLLT3 and MLLT10 to homeobox genes; associated with transcriptional activation and elongation

11, 66,67

SMYD3 H3K4 Breast, liver, colon and gastric cancers

Overexpressed in multiple tumour types; associated with transcriptional activation

68,69

EZH2 H3K27 Breast, prostate, colon, gastric, bladder and liver cancers, melanoma and lymphoma

Amplified and increased expression in several tumour types; a member of the polycomb repressive complex 2; associated with transcriptional repression

10,15,70,71

SETD7 H3K4 Breast cancers SET7-mediated methylation stabilizes the oestrogen receptor and is necessary for the recruitment of the oestrogen receptor to its target genes and target gene transactivation

72

PRDM14 No known substrate

Breast cancers Amplified and overexpressed in cancers; associated with transcriptional repression

73

CARM1 H3R17, EP300–CBP and NCOA3

Breast and prostate cancers

Increased expression correlates with androgen independence in human prostate carcinoma; overexpressed in breast tumours and associated with transcriptional activation

74,75

PRMT5 H3R8, p53, SNRPD1, SNRPD3 and SUPT5H

Lymphoma PRMT5 expression and H3R8 methylation levels are increased in lymphoid cancer cells; PRMT5 mediates p53 methylation, which promotes cell arrest rather than cell death; H4R3 methylation promotes recruitment of DNMT3A, subsequent promoter CpG methylation and gene silencing

12, 76

CARM1, coactivator-associated arginine methyltransferase 1 (also known as PRMT4); CBP, CREB-binding protein; DNMT3A, DNA (cytosine-5-)-methyltransferase 3α; EHMT2, euchromatic histone-lysine N-methyltransferase 2 (also known as G9A and KMT1C); EP300, E1A-binding protein p300; EZH2, enhancer of zeste homologue 2 (also known as KMT6); DOT1L, DOT1-like, histone H3 methyltransferase (also known as KMT4); MLL, myeloid, lymphoid or mixed-lineage leukaemia (also known as KMT2A); MLLT1, myeloid, lymphoid or mixed-lineage leukemia, translocated to 1; NCOA3, nuclear receptor coactivator 3; NSD1, nuclear receptor-binding SET domain protein 1; PKMT, protein lysine methyltransferase; PRDM14, PR domain-containing protein 14; PRMT, protein arginine methyltransferase; SETD7, SET domain-containing lysine methyltransferase 7 (also known as KMT7); SMYD3, SET and MYND domain-containing protein 3; SNRPD1, small nuclear ribonucleoprotein D1 polypeptide 16kDa (also known as SMD1); SNRPD3, small nuclear ribonucleoprotein D3 polypeptide 18kDa (also known as SMD3); SUPT5H, suppressor of Ty 5 homologue; SUV39H1, suppressor of variegation 3–9 homologue 1 (also known as KMT1A); WHSC1, Wolf–Hirschhorn syndrome candidate 1 (also known as MMSET and NSD2); WHSC1L1, Wolf–Hirschhorn syndrome candidate 1-like protein 1 (also known as NSD3).

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PMTs

SAM SAH

N

O

H

NH

O

NHH

CH3

NH3+

–O2C

S+

OHH H

NO

N

N

N

NH2

H

OH

H

N

O

H

NH

O

NCH3

HH

NH3+

–O2C

S+

OHH H

NO

N

N

N

NH2

H

OH

H

Nu– LG Nu LG–+LGNuδ– δ+ ‡

a

b

Crystallographic studies have revealed two distinct binding modes for SAM or SAH in the cofactor-binding pockets of PMTs24. For the SET domain PKMTs that have been co-crystallized with SAM or SAH, it is known that the cofactor adopts a ‘U-shaped’ configuration within the active site (FIG. 3) that aligns the methylsulphonium cation of SAM at the base of the narrow lysine channel, in perfect juxtaposition to the ε-amino group of the acceptor lysine residue, which facilitates group transfer. This U-shaped configuration is induced by a conserved aspartate or glutamate residue that binds to the ribose hydroxyl groups, and a positively charged lysine or arginine residue that forms a salt bridge with the car-boxylate of SAM. In striking contrast to the U-shaped configuration that is adopted by the cofactor when bound to PKMTs, SAM bound within the active site of PRMTs adopts an extended configuration that resem-bles the extended SAM configuration seen in the DNA methyltransferases; again, the binding motif results in alignment of the SAM methylsulphonium cation with the base of the acceptor-binding channel. Another dis-tinction between cofactor binding within the PKMTs and the PRMTs is that, in PRMTs, dimer formation seems to be a crucial component of SAM binding and catalysis, whereas this is not the case for PKMTs3,24. The mechanistic consequences of obligate dimer formation in the PRMTs is not yet clear, but it may be involved in multiple methylations of the arginine residue.

From the above discussion, it could be concluded that the configuration of the bound SAM is structurally related to the identity of the methyl acceptor nitrogen species upon which the enzymes act; that is, U-shaped for PKMTs and extended for PRMTs. However, data on the non-SET domain PKMT, DoT1l, do not support this conclusion. In the co-crystal structures of human DoT1l bound to SAM33, and the yeast homologue DoT1P bound to SAH34, the cofactor is bound in the extended configu-ration, similar to that seen in the PRMTs. Additionally, the solvent-exposed surface area of the bound cofactor in DoT1l is more similar to that seen in the PRMTs than the PKMTs, as is the overall amino-acid sequence around the cofactor-binding pocket24,33. Therefore, from a struc-tural perspective, DoT1l seems to link the PKMT and PRMT groups of PMTs.

The discovery and optimization of selective drugs for the PMTs will depend not only on the static structure of the active site of the enzyme, as revealed through crys-tallographic studies, but also on the structural dynamics of the active site that accompany catalytic turnover27,35. Studies on the kinetic mechanisms of the PMTs may provide some information in this area.

Some of the SET domain PKMTs, such as SETD7, perform a single round of catalysis on a lysine residue, resulting in a mono-methylated product, whereas other SET domain PKMTs catalyse multiple rounds of methyl-ation on a specific lysine residue. Crystallographic studies suggest that the difference between single-turnover and multiple-turnover SET domain enzymes results from the degree of steric crowding and hydrogen-bonding patterns in the lysine-binding channel of these enzymes3,24,36,37. In particular, the identity of an aromatic residue within the lysine-binding pocket seems to be the key determinant of the multiplicity of lysine methyl-ation. In the PKMT DIM5, this residue is a phenylalanine (F281), and the enzyme can tri-methylate the acceptor lysine residue of its protein substrate. The correspond-ing residue in SETD7 is a tyrosine (Y305), and this enzyme can only mono-methylate its protein substrate. Remarkably, the mutant F281Y transforms DIM5 into a mono-methylating PKMT, and the corresponding mutant Y305F in SETD7 results in an enzyme that is capable of multiple rounds of lysine methylation38. These mutagenesis results have been extended to the PKMT euchromatic histone lysine N-methyltransferase 2 (EHMT2; also known as G9A)39, and the ‘tyrosine–phenylalanine switch’ seems to be a general determinant of product specificity among the SET domain PKMTs24. Molecular dynamics and hybrid quantum mechanical–molecular mechanical studies also suggest a key role for bound water molecules (a water channel) in the extent of lysine methylation by PKMTs30.

An outstanding question that has yet to be reconciled with the mechanistic hypothesis described above is how the quaternary nitrogen atom is deprotonated to gener-ate a neutral amine methyl acceptor. At physiological pH, the lysine amine is protonated (the negative log-arithm of the acid dissociation constant (pKa) of the side chain amine is ~10.8 (REF. 35)), and so there are no lone pair electrons to act as the attacking nucleophile in the

Figure 2 | PMT-catalysed methylation of proteins by an sN2 reaction with sAM as the methyl donor. The protein methyltransferases (PMTs) catalyse methyl transfer from their universal methyl donor, S-adenosyl-l-methionine (SAM; also known as AdoMet) to a nitrogen atom of lysine or arginine side chains to form S-adenosyl-l-homocysteine (SAH; also known as AdoHcy). a | The methyl group (shown in red) of the SAM sulphonium cation is ‘attacked’ by the lone pair electrons of a lysine (shown here) or arginine (not shown) side-chain nitrogen atom. The reaction results in transfer of the methyl group to the attacking nitrogen atom and the production of SAH from the reaction cofactor. b | A more generalized chemical scheme of a bimolecular nucleophilic substitution (S

N2)

group transfer reaction, illustrating the attacking nucleophile (Nu–; lysine or arginine in the case of PMTs), the leaving group (LG; the methyl group in the case of PMTs), and the transient but essential formation of a penta-coordinate carbon transition state (‡).

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b DOT1L

a PRMT

c SET domain

Nature Reviews | Drug Discovery

General base catalysisA mechanism that can occur in enzyme catalysis, in which a basic group accepts protons from a substrate molecule, usually to stabilize a charged transition-state species.

SN2-mediated methyl transfer reaction. A potential mech-anism of deprotonation is through general base catalysis. However, inspection of the amino acids in the active sites of PKMTs reveals no obvious basic side chains that could act in this capacity. Another hypothesis is that the solvent acts as a proton ‘sponge’; however, this seems inconsistent with the fact that the lysine side chain is buried deeply in the protein, with no clear access to bulk solvent. An alternative hypothesis has recently been proposed, based on molecular dynamics simulations30. According to this model, binding of SAM and protein substrates creates a ‘water shuttle’ that can remove a proton from the buried lysine side chain and ferry this proton along a contiguous chain of water molecules to be deposited into the bulk solvent. Additionally, the electrostatic repulsion created by the quarternary nitrogen atom and the positively charged SAM cofactor lowers the pKa of the lysine side-chain amine to ~8.2, thereby facilitating this deprotonation

process. Furthermore, the water shuttle hypothesis pro-vides an alternative mechanism to explain the differences in extent of lysine methylation by the PKMTs. The molec-ular dynamics studies suggest that the ability to form a water shuttle will determine the extent of methylation that is catalysed by a given enzyme. For example, simulations of SETD7-mediated catalysis suggest that mono-methyl-ation of lysine prevents re-formation of a new water shuttle, and so this enzyme terminates catalysis after one round of methylation. The same simulations suggest that other PKMTs, such as the ribulose bisphosphate carboxylase–oxygenase large subunit methyltransferase, can readily re-form the water shuttle, leading to multiple rounds of methylation.

Enzymes that perform multiple rounds of catalysis on a macromolecular substrate can do so by one of two mechanisms: a distributive enzyme mechanism, in which each round of catalysis results in macromolecular product dissociation and rebinding, or a processive mechanism, in which multiple rounds of catalysis proceed before dis-sociation of the macromolecular product. PMTs use both of these mechanisms: some SET domain PKMTs that perform multiple rounds of lysine methylation have been found to use a processive mechanism3,24, whereas DoT1l has been shown to perform multiple rounds of H3K79 methylation through a non-processive (distributive) mechanism40.

The PRMTs are also capable of performing multiple rounds of arginine methylation to produce either mono- or di-methylated arginine products. The PRMTs that have been studied so far follow an ordered, sequential mechanism in which SAM binds before the arginine-containing substrate, and di-methyl arginine production occurs through a processive mechanism3. on the basis of product specificity, PRMTs can be subdivided into two types: type I PRMTs, which produce an asymmetrical N,N ′-dimethyl arginine; and type II PRMTs, which produce a symmetrical N,N-dimethyl arginine3.

The variations in active-site structure and chemical mechanism that are summarized above reflect a target class with the potential for substantial chemical diversity among small-molecule modulators of individual enzymes in the class. Therefore, the opportunity for the develop-ment of different chemotypes that compete with the com-mon, natural ligands of these enzymes (for example, SAM, lysine and arginine), and can be modified to produce enzyme-selective inhibitors, seems promising.

Known inhibitors of PMTsDespite the convergence of data concerning PMTs, the search for potent, selective inhibitors of these enzymes has only recently begun in earnest. Some indirect approaches to inhibiting or depleting PMTs have been reported. For example, the antiviral compound 3-deazaneplanocin (DZNep) inhibits the enzyme SAH hydrolase and thereby increases intracellular levels of the universal product of PMTs, SAH41. Product inhibition by SAH would therefore be expected for all PMTs and other SAM-dependent enzymes, with the degree of inhibition for specific enzymes being related to their relative inhibi-tion constant (Ki) and Michaelis constant (Km) values for

Figure 3 | variations in the configuration of sAM or sAH bound within the active sites of different PMTs. a | The representative conformation shown for the protein arginine methyltransferases (PRMTs) was taken from the crystal structure of S-adenosyl-l-homocysteine (SAH; also known as AdoHcy) bound to coactivator- associated arginine methyltransferase 1 (CARM1)49. b | The conformation shown for DOT1-like, histone H3 methyltransferase (DOT1L) was taken from the crystal structure of S-adenosyl-l-methionine (SAM; also known as AdoMet) bound to this protein33. c | The representative conformation shown for the protein lysine methyl-transferases (PKMTs) was taken from the crystal structure of SAH bound to SET domain-containing lysine methyl transferase 8 (REF. 50). Carbon atoms are represented by grey circles; nitrogen atoms are represented by blue circles; oxygen atoms are represented by red circles; and sulphur atoms are represented by yellow circles.

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SAH and SAM, respectively27. Similarly, the activity of all SAM-dependent enzymes in a cell could be reduced by blocking SAM biosynthesis — for example, by inhibit-ing dihydrofolate reductase or SAM synthase, which are

two enzymes involved in SAM biosynthesis42. Also, the pan-HDAC inhibitor panobinostat has recently been shown to cause depletion of cellular levels of the PMT EZH2 (REF. 43). Although the mechanism by which this

Table 3 | Chemical structures and biochemical data for small-molecule inhibitors of PMTs

compound structure Mechanism and potency selectivity* refs

SAH

H2N S

CO H2O

OHOH

N

N

N

N

NH2 Product of the reactions catalysed by PMTs IC50 values range from 0.1 to 20 µM

Non-selective 77,78

Sinefungin

CO H2O

H2N

OHOH

N

N

N

N

NH2

NH2

Natural product analogue of SAM and SAH IC50 values range from 0.1 to 20 µM

Non-selective 36

ChaetocinHN N

HN

O OH

O

S S

SSNH

NH

N

O

O OH

SAM-competitive inhibitor of SUV39 IC

50 = 0.6 µM

> 4-fold 79

BIX-01294

N

NH

N

NMeO

MeO

NN

SAM-non-competitive inhibitor of EHMT2 IC

50 = 2.7 µM

> 4-fold 80

Methylgene compound 7a of REF. 45

CH3O

HN

O

NN

F3C

S

NH

O NH2

CARM1 inhibitor IC

50 = 60 nM

> 100-fold for PRMT1 and SETD7

45

Bristol–Myers Squibb compound 7f of REF. 47

N N

O

NS

NN

F3C

HN

O

NH2

CARM1 inhibitor IC

50 = 40 nM

>100-fold for PRMT1 and PRMT3

46,47

CARM1, coactivator-associated arginine methyltransferase 1 (also known as PRMT4); EHMT2, euchromatic histone lysine N-methyltransferase 2 (also known as G9A and KMT1C); IC

50, half-maximal inhibitory concentration; PMT, protein methyltransferase;

PRMT, protein arginine methyltransferase; SAH, S-adenosyl-l-homocysteine (also known as AdoHcy); SAM, S-adenosyl-l-methionine (also known as AdoMet); SETD7, SET domain-containing lysine methyltransferase 7 (also known as KMT7); SUV39, suppressor of variegation 3–9; *Selectivity is given as the ratio of the IC

50 value for the most potent inhibition at a non-target PMT over the IC

50 value

for the primary target. See REF. 27.

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Structure–activity relationshipThe relationship between the chemical structure of a compound and its pharmacological activity.

occurs is not yet fully understood, an approach of this type would nevertheless deplete the protein levels of EZH2 and so abolish the PMT catalytic activity of the enzyme along with any other non-enzymatic functions of EZH2.

Direct inhibitors of PMTs have recently been reviewed4, along with other probes of histone-modifying enzymes. Some natural ligands for these enzymes have been known for some time, including the reaction product, SAH, and a natural inhibitor isolated from Streptomyces spp. cultures, sinefungin (TABLE 3). More selective inhibitors have been identified for SUV39 (chaetocin; reported half-maximal inhibitory concentration (IC50) = 0.6 μM) and for EHMT2 (BIX-01294; reported IC50 = 1.6 μM), but no further opti-mization of these compounds has been reported to date4. A co-crystal structure of BIX-01294 bound to EHMT2 has recently been published44. Surprisingly, the compound was found to bind to the enzyme non-competitively with respect to SAM, in a groove that is normally occupied by a portion of the protein substrate.

More recently, two groups have reported potent, selec-tive, pyrazole-based inhibitors of the PRMT CARM1 (REFS 45–47) (TABLE 3). These compounds are the first examples of inhibitors of a specific PMT that are effective at nanomolar concentrations and display >100-fold selec-tivity for the primary target over related enzymes. The compound series reported by Methylgene45 was found to be inactive in cellular assays; no cellular data have been reported for the compound series from Bristol–Myers Squibb46,47. Therefore, although an exciting first step has been made towards developing selective inhibitors of PMTs, substantial work remains to be done before these findings can be translated into pharmacologically tractable species.

The paucity of potent, selective, pharmacologically tractable inhibitors of the PMTs creates a crucial thera-peutic gap which medicinal chemists should strive to fill. As described here, the pathobiological relevance of these enzymes, together with the structural and mecha-nistic information that suggests their druggability as a target class, converge to make the PMTs an attractive and important class of novel enzymes for contemporary drug discovery.

ConclusionsThere is a growing body of evidence that enzymes in this target class have important pathogenic roles in human diseases. The structures and enzymatic mechanisms of the PMTs support the view that pharmacological modu-lation of these enzymes by small-molecule inhibitors will be an effective means of therapeutic intervention in cancer and numerous other unmet medical needs. The discovery of small-molecule inhibitors of PMTs as starting points for drug development should clearly be a key focus of new research efforts. Beyond this goal, there are many opportunities to use chemical probes of PMT function to define the underlying biology and pathobiology that are associated with protein modifi-cation by these enzymes. The nature of PMT catalysis, and the available structural information about these enzymes, should facilitate the discovery of PMT ligands through mechanism- and structure-guided discovery methods48, as well as methods that do not rely on mech-anistic knowledge, such as high-throughput screening of diverse chemical libraries.

A key remaining question when considering the PMTs as a drug discovery target class is whether or not selective inhibition of particular enzymes can be achieved through targeting the SAM-binding pocket. This is analogous to the question that hindered the early acceptance of protein kinases as drug targets: whether it was possible to achieve selectivity among the ATP-binding pockets of the kinases. In retrospect, it is clear that the diversity of binding-site architecture and the binding-site dynamics associated with enzyme catalysis provide ample opportunities for selective inhibition of kinases through medicinal chemistry efforts. Will the same be true for the SAM-binding pockets of PMTs? Ultimately, structure–activity relationship profiles, selec-tivity and collateral inhibition of off-target enzymes by PMT inhibitors will need to be determined empirically. Despite these limitations, it is our hope that the data pre-sented here will help to stimulate systematic exploration of the human PMT target class towards the goal of devel-oping selective inhibitors of PMTs as therapeutic agents for human diseases.

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AcknowledgementsWe are grateful to K. Shiosaki, C. T. Walsh, H. R. Horvitz, Y. Zhang, and R. Gould for their insights, constant support and encouragement. We also thank K. Boater, E. Olhava, L. Jin and T. Luly for expert help in preparation of this manuscript.

Competing interests statementThe authors declare competing financial interests: see web version for details.

DATABASESUniProtKB: http://www.uniprot.orgCARM1 | DOT1L | EHMT2 | EZH2 | PRMT1 | SETD7 | SETD8 | SETD1A | SETDB1 | SUZ12

FURTHER INFORMATIONAuthor’s homepage: http://www.epizyme.com

All liNks Are AcTive iN THe oNliNe PDf

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