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The human protein methyltransferases

Methyltransferases are enzymes that facilitate the transfer of a methyl (CH3) group to specific nucleophilic sites on proteins, nucleic acids or other biomolecules. They share a reaction mechanism in which the nucleophilic acceptor site attacks the electrophilic carbon of S-adenosyl-l-methionine (SAM) in an SN2 displacement reaction that produces a methylated biomolecule and S-adenosyl-l-homocysteine (SAH) as a byproduct. Methylation reactions are essential transformations in small-molecule metabolism, and methylation is a common modification of DNA and RNA. The recent discovery of dynamic and reversible methylation of amino acid side chains of chromatin proteins, particularly within the N-terminal tail of histone proteins, has revealed the importance of methyl marks as regulators of gene expression. Human protein methyltransferases (PMTs) fall into two major familiesprotein lysine methyltransferases (PKMTs) and protein arginine methyltransferases (PRMTs)that are distinguishable by the amino acid that accepts the methyl group and by the conserved sequences of their respective catalytic domains. Given their involvement in many cellular processes, PMTs have attracted attention as potential drug targets, spurring the search for small-molecule PMT inhibitors. Several classes of inhibitors have been identified, but new specific chemical probes that are active in cells will be required to elucidate the biological roles of PMTs and serve as potent leads for PMT-focused drug development.

Protein lysine methyltransferases (PKMTs)

The phylogenetic tree shows 51 genes predicted to encode PKMTs, which are positioned in the tree on the basis of the similarities of their amino acid sequences1. This tree excludes one validated PKMT, DOT1L, which lacks a SET domainthe catalytic domain conserved in this familyand clusters more closely with the PRMTs. The tree has four major branches, and each branch contains enzymes with validated methyltransferase activity (highlighted in red). Some PKMTs add a single methyl group, resulting in a monomethylated product (Kme), whereas others produce di- (Kme2) or trimethylated (Kme3) lysine modifications. Many of the validated PKMTs methylate lysines on histones, though nonhistone substrates have also been identified.
O HN HO O H N O N N H Cl Cl

H H N H PKMT SAM H N H O SAH

N CH3 PKMT SAM SAH

H 3C H N CH3 PKMT SAM SAH

H3C CH3 N CH3

Protein arginine methyltransferases (PRMTs)

Lysine (K)

Kme1

Kme2

Kme3

The human PRMT phylogenetic tree comprises 45 predicted enzymes including the PKMT DOT1L1. There are two major types of PRMT; both catalyze the formation of monomethylarginine (Rme1) but distinct reaction mechanisms yield symmetric (Rme2s) or asymmetric (Rme2a) dimethylarginine. A small number of predicted PRMTs have validated activity (highlighted in blue). In addition to PRMTs, this tree includes validated RNA methyltransferases (highlighted in green) and biosynthetic enzymes (highlighted in violet). It remains uncertain whether these latter enzymes have PRMT activity, despite their shared structural features. Substrates for the enzymes shown include RNA, metabolites, histones and RNA-binding and spliceosomal proteins.

N HN

H N H H PRMT SAM SAH H

CH3 N HN N H H PRMT SAM SAH H 3C

CH3 N HN H N H OR H 3C

N HN

H N

CH3

H N H O

Arginine (R)

Rme1

Rme2a

Rme2s

SUV420H1 SMYD1

SUV420H2

MLL4

MLL SETD1B
N

N NH N N N OMe OMe

METTL11A METTL11B METTL13

O S O O N H S O

AZ505

ref. 5
SETD3 SETD6

SMYD5 SETD4

SMYD3 SMYD2 SMYD4 SETD7 SETD8 EZH1 EZH2

SETD1A

ECE2

COQ3 METTL12 METTL7A

BIX-01294
MLL2 MLL3

ref. 7

H N

ALKBH8 WBSCR22 WBSCR27 COQ5 C20orf7

PRMT7
HO H S N NS O NH O H N O N SN S O

METTL7B AS3MT

ref. 10
OH

PRMT10

METTL20 METTL10 PRMT5

H 2N HO2C

H
N

N O HO N N OH

NH2 N

PRMT2

PRMT6

DOT1L

NH

Chaetocin

ref. 15
PRMT1

PRMT3 CARM1 PRMT8

O H 2N S NH N N CF3 F3C N N O N N H N NH2 S N O

ref. 6
PRDM5 SUV39H1 SUV39H2 EHMT1 EHMT2 SETMAR Q6ZW69 PRDM14 PRDM6 PRDM8 PRDM13 PRDM12 PRDM4 PRDM15 PRDM10 SETD2 ASH1L MLL5 SETD5
N N

MeO HO2C NH HN O HN N HO O N OH N NH2 N H 2N H I N HO O N

N N OH

HN N

PRDM3 PRDM16 PRDM2 PRDM1 PRDM11 PRDM7 PRDM9

IBAO

ref. 13

ref. 11 EPZ004777
ASMT

SETDB1
N NH N N N OMe O N

ref. 4

SETDB2

ref. 12
METTL6 PRMT9 PRMT11 NOP2 NSUN7 NSUN5B NNMT INMT NSUN4 NSUN5 PNMT METTL8 METTL2A METTL2B

UNC-0224

ref. 8

NSD1

NH N N OMe O N HO

NSUN5C
O

WHSC1L1

Br

Br

OH

UNC-0638

ref. 9 ref. 14

NSUN3 NSUN6 NSUN2

WHSC1

Targeting PMTs

inhibitors A selection of small-molecule PMT(minimally with some target selectivity is shown

validated in quantitative in vitro assays) around the trees along with the name of the molecule, citation information and the chemical structure2,3.

a validated therapeutic target for mixed-lineage leukemia . The major DOT1L isleukemias result from chromosomal rearrangements that cause aberity of these
4

rant recruitment of DOT1L to MLL-fusion target genes. Inhibition of DOT1L with EPZ004777 demonstrated that these leukemia cells are addicted to DOT1L activity and established proof of concept for DOT1L inhibition as a therapeutic option.

also Priority therapeutic targetsSETD1B include MLL for leukemias;

and CARM1 for neurodegeneration; as well as EZH2, SMYD3 and EHMTs for multiple cancers.

Additional PMTs have been implicated

in human diseases and may yet emerge as therapeutic targets.

facilitated by Elucidation of the biological function of PMTs would befuture chemicalthe development of selective chemical probes; this is a compelling area for biology studies, given the paucity of available tool compounds, many of which remain to be validated in cells. In particular, the emergence of these enzyme families as therapeutic targets suggests that such chemical probes could yield lead compounds for drug development.

Understanding the mechanisms that govern substrate specificity,

especially for nonhistone targets, merits additional study.

Epizyme is leading the discovery and development of small-molecule protein methyltransferase (PMT) inhibitors, a new class of personally targeted therapeutics for the treatment of genetically defined cancer patients, on the basis of breakthroughs in the field of epigenetics. Epigenetic enzymes are strongly associated with the underlying causes of multiple human diseases. Our patient-driven approach to the creation of personalized therapeutics represents the future of cancer therapy, creating better therapeutics matched to the right patients more quickly and at lower cost than traditional approaches.
www.epizyme.com

Sponsor contacts

Robert A. Copeland and Victoria Richon are at Epizyme, 325 Vassar Street, Suite 2B, Cambridge, MA 02139, USA. Phone: (617) 229-5872 Dr. Robert A. Copeland Executive Vice President & Chief Scientific Officer rcopeland@epizyme.com Dr. Victoria Richon Vice President, Biological Sciences vrichon@epizyme.com

Substrates and products

SAM, S-adenosyl-l-methionine
H 2N HO2C H S Me HO N O N N OH NH2 N

References
SAH, S-adenosyl-l-homocysteine
H 2N HO2C H S HO N O N N OH NH2 N
1. Richon, V.M. et al. Chem. Biol. Drug. Disc. 78, 199210 (2011). 2. Copeland, R.A., Solomon M.E. & Richon, V.M. Nat. Rev. Drug Discov. 8, 724732 (2009). 3. Copeland, R.A. Drug Discov. Today: Therapeutic Strategies, published online 16 September 2011, doi: 10.1016/j.ddstr.2011.08.001. 4. Daigle, S.R. et al. Cancer Cell 20, 5365 (2011). 5. Ferguson, A.D. et al. Structure 19, 12621273 (2011). 6. Mori, S. et al. Bioorg. Med. Chem. 18, 81588166 (2010). 7. Kubicek, S. et al. Mol. Cell 25, 473481 (2007). 8. 9. 10. 11. 12. 13. 14. 15. Liu, F. et al. J. Med. Chem. 52, 79507953 (2009). Vedadi, M. et al. Nat. Chem. Biol. 7, 566574 (2011). Spannhoff, A. et al. Biorg. Med. Chem. Lett. 17, 41504153 (2007). Allan, M. et al. Bioorg. Med. Chem. Lett. 19, 12181223 (2009). Huynh, T. et al. Biorg. Med. Chem. Lett. 19, 29242927 (2009). Yao, Y. et al. J. Am. Chem. Soc. 133, 1674616749 (2011). Cheng, D. et al. J. Med. Chem. 54, 49284932 (2011). Greiner, D. et al. Nat. Chem. Biol. 1, 143145 (2005).

Poster content

Written and edited by Terry L. Sheppard and Amy Donner; copyedited by Yasmin Tayag; art by Katie Vicari; designed by Lewis Long. 2011 Nature Publishing Group Available online at:

http://www.nature.com/nchembio/poster/hpm.pdf