even though all nucleated 
 cells contain the same genomic dna , multicellular 
 organisms have developed a machinery of differentiation that maintains 
 unique biological functions of specific cell types , tissues , and organs . growing evidence suggests that gene expression is a key component 
 of cellular differentiation and is not only controlled by dna sequence 
 and transcription factors but also by epigenetic regulation . epigenetics is typically referred to as heritable 
 changes in gene expression or phenotype without changes in dna sequence . the human genome is encoded in dna and tightly packed into 
 23 pairs 
 of chromosomes that contain repeating nucleosome units . each nucleosome 
 consists of eight histone proteins ( two copies of each of four core 
 histones h2a , h2b , h3 , and h4 ) and the dna double helix that wraps 
 around the histone octomer . nucleosomes are further 
 condensed to form chromatin , which can reside in two main conformational 
 states . in the heterochromatin state , nucleosomes are tightly packed together , and gene transcription is 
 mainly repressed . on the other hand , in the euchromatin state , nucleosomes 
 are more loosely packed and accessible , leading to gene expression 
 and transcription activation . thus , epigenetic regulation of gene 
 expression depends on the state of chromatin , which is mainly controlled 
 by dna methylation , noncoding rnas , nucleosome remodeling histone 
 variants , and post - translational modifications ( ptms ) of histones . histones are small basic proteins with a flexible 
 and charged n - terminus called histone tails , which are rich in arginine 
 and lysine residues . ptms of histones include , but are not limited 
 to , methylation , acetylation , phosphorylation , sumoylation , ubiquitination , 
 and glycosylation . the histone code hypothesis suggests that various ptms of histones ( often referred as histone 
 marks ) would promote interaction affinities for chromatin - associated 
 proteins and may act in numerous combinations or successively on the 
 same or different histone tails , affecting specific cellular outcomes . the proteins that are directly involved in ptms of histones are divided 
 into three categories : the enzymes that create these modifications 
 ( the writers ) , the proteins that recognize the modifications ( the 
 readers ) , and the enzymes that remove the modifications ( the erasers ) . as shown by mounting evidence , dysregulation of gene expression 
 contributes to many human diseases including inflammation , brain disorders , 
 metabolic diseases , and cancer , which can be caused not only by genetic 
 mutations but also by epigenetic alterations . given the importance of epigenetic regulation 
 in cell differentiation , proliferation , development , and maintaining 
 cell identity , the epigenetic regulatory enzymes have been increasingly 
 recognized as potential therapeutic targets . hence , there is growing 
 interest in the scientific community to discover and develop selective 
 small - molecule inhibitors of these enzymes . such inhibitors would 
 be valuable chemical tools for investigating biological functions 
 and disease associations of the target enzymes and for assessing the 
 potential of these enzymes as therapeutic targets . recently , small - molecule 
 inhibitors of dna methyltransferases ( dnmts ) for the treatment of 
 myelodysplastic syndrome and histone 
 deacetylases ( hdacs ) for the treatment of t - cell lymphoma have been approved by the u.s . food and drug administration ( fda ) 
 as the first epigenetic drugs , validating these epigenetic regulatory 
 enzymes as drug targets . in this perspective , we focus on selective 
 small - molecule inhibitors of protein methyltransferases ( pmts ) , also 
 commonly referred as histone methyltransferases ( hmts ) . we describe 
 past and present advances on discovering these inhibitors and discuss 
 future directions for pmt inhibitor discovery . histone methylation by pmts 
 is one of the most studied post - translational 
 modifications since it is implicated in heterochromatin formation 
 and maintenance , transcriptional regulation , dna repair , x - chromosome 
 inactivation , and rna maturation . in 
 addition to histones , pmts ( the methyl writers ) catalyze the transfer 
 of the methyl group from the cofactor s-5-adenosyl - l - methionine ( sam ) to either lysine or arginine residues of 
 proteins ( figure 1a ) . therefore , pmts are further 
 divided in two categories : protein lysine methyltransferases ( pkmts ) 
 and protein arginine methyltransferases ( prmts ) . the known methylation 
 sites for histone 3 ( h3 ) and histone 4 ( h4 ) tails are shown in figure 1b . lysine residues can be mono- , di- , and/or trimethylated 
 by pkmts , whereas arginine guanidinium groups can be mono and/or dimethylated . dimethylation of terminal guanidino nitrogens 
 followed by monomethylation of arginine ( mma ) could be either asymmetrical 
 on the same nitrogen ( adma ) or symmetrical on two different guanidino 
 nitrogens ( sdma ) ( scheme 1 ) . methylation of 
 lysine or arginine residues does not change the charge of these residues , 
 but it alters the bulkiness and hydrophobicity of the protein , in 
 turn affecting protein  protein interactions . in the case of 
 histone lysine and arginine methylation , each methylation mark on 
 different residues or on the same residue establishes a specific signal 
 that is recognized by reader proteins . ( a ) 
 methylation of lysine ( k ) or arginine ( r ) residues of proteins 
 by protein methyltransferases ( pmts ) . ( b ) location of known methylation 
 sites of histone 3 ( h3 ) and histone 4 ( h4 ) . sam , s-5-adenosyl - l - methionine ; sah , s-5-adenosyl - l - homocysteine . both pkmts and prmts have a 
 cofactor binding site and a substrate 
 binding site . upon activation , these enzymes recruit lysine or arginine 
 residues of substrate proteins to the substrate binding pocket and 
 sam to the cofactor binding site . these 
 two binding sites are linked by a narrow hydrophobic channel to allow 
 the substrate and cofactor to come into close proximity to transfer 
 the methyl group from the cofactor sam via a sn2 transition 
 state to a lysine or arginine residue , releasing the cofactor product s-5-adenosyl - l - homocysteine ( sah ) . this 
 process can be repeated to achieve higher level of methylations of 
 lysine or arginine residues . all of the known pkmts , with the exception of dot1l , contain a 
 conserved , approximately 130 amino acid long set domain . the set domain was originally identified in three drosophila genes : su(var.)3 - 9 ( the suppressor of position - effect variegation 
 3 - 9 ) , en(zeste ) ( an enhancer 
 of the eye color mutant zeste ) , and trithorax ( the homeotic gene regulator ) . therefore , pkmts can be divided into two classes : set domain - containing 
 pkmts and non - set domain pkmts ( dot1l is the only member of this class ) . the set domain folds into several small -sheets that surround 
 a knot - like structure , which brings together the two highly conserved 
 motifs of the set domain and forms an active site next to the sam 
 binding pocket . set domain - containing 
 pkmts are categorized according to similarities in the sequence around 
 the set domain and collected under five major families : suv , set1 , 
 set2 , ez , and riz . more recently , an alternative 
 classification and nomenclature has been suggested to give more generic names to histone modifying enzymes 
 according to the type of their enzymatic activity and the type of 
 residue(s ) they modify , as these enzymes have also been shown to target 
 nonhistone proteins . therefore , pkmts were divided into eight major 
 groups : kmt1 ( lysine methyltransferases 1 ) to kmt8 , including set 
 domain - containing and non - set enzymes ( e.g. , dot1l is the only member 
 of the kmt4 subfamily ) . we summarize all of the official gene names 
 and alternative names that have been used for the enzymes discussed 
 in this perspective in table 1 . it is also found 
 in a large number of other eukaryotic proteins and in several bacterial 
 proteins . based on hgnc ( hugo gene nomenclature 
 committee ) histone lysine methylation catalyzed 
 by pkmts has been recognized 
 as a major mechanism in regulating gene expression and transcription . depending on the methylation site and methylation state ( e.g. , mono- , 
 di- , or trimethylation ) , histone lysine methylation can lead to either 
 transcription activation or repression . for example , h3k4 ( histone 
 h3 lysine 4 ) , h3k36 , and h3k79 methylation are generally associated 
 with transcription activation . on the other hand , h3k9 di- and trimethylation 
 ( h3k9me2 and h3k9me3 ) and h3k27 trimethylation ( h3k27me3 ) are typically 
 associated with repression . in the following section , we organize inhibitors of pkmts according 
 to their histone methylation site(s ) ( figure 2 and table 2 ) . only selected nonhistone targets 
 and function(s ) and link(s ) to diseases were included , not a comprehensive 
 list . methylation of 
 h3k9 in humans is controlled by pkmts : suv39h1 ( suppressor of variegation 
 3 - 9 homologue 1 ) , suv39h2 , g9a ( euchromatic histone - lysine n - methyltransferase 2 ( ehmt2 ) ) , glp ( g9a - like protein 1 , 
 also known as ehmt1 ) , setdb1 ( set domain , bifurcated 1 ) , setdb2 , prdm2 
 ( pr domain containing 2 , with znf domain also referred as riz1 ) , prdm3 , 
 and prdm16 . h3k9 dimethylation ( h3k9me2 ) 
 and trimethylation ( h3k9me3 ) are repressive marks recognized by heterochromatin 
 protein 1 ( hp1 ) , which is directly involved in the formation of transcriptionally 
 silent chromatin . mounting evidence suggests 
 that h3k9 methyltransferases are implicated in various human diseases . to date , selective inhibitors of suv39h1 , g9a , and glp have been 
 reported . in the following section , we review the discovery and biological 
 characterization of these inhibitors . suv39h1 is the first identified 
 histone lysine methyltransferase and is the human orthologue of drosophila su(var.)3 - 9 and schizosaccharomyces 
 pombe clr4 . in this enzyme , 
 the -sheets of the set domain are packed together with pre - set 
 and post - set domains . the latter contains 
 three conserved cysteine residues , which are essential for the enzymatic 
 activity . it has been clearly shown in mouse models that genome stability 
 during mammalian development is directly related to suv39h1/2-dependent 
 h3k9 methylation at pericentric heterochromatin . it has also been suggested that suv39h1 and suv39h2 play 
 a role as tumor suppressors by maintaining h3k9 methylation at pericentric 
 heterochromatin . furthermore , it has been reported that increased 
 metastatic potential of tumor cells is linked to reduced heterochromatic 
 accumulation of hp1. besides heterochromatin 
 silencing , the suv39h1hp1 complex plays a part in repression 
 of euchromatic genes by retinoblastoma protein ( rb ) . it has also been shown that the active transcriptional 
 state of nfb - p65 gene is associated with reduced 
 h3k9 methylation , in part mediated by suv39h1 . furthermore , in a recent study , suv39h1 and setdb1 expression 
 was upregulated in glioma cell lines . the field of selective pkmt inhibitors was commenced by the 
 report 
 of the fungal mycotoxin chaetocin ( 1 ) ( figure 3 ) as the first selective inhibitor of drosophila su(var.)3 - 9 histone methyltransferase 
 in 2005 . however , it is still a controversial 
 subject as to whether chaetocin is a truly selective inhibitor ( see 
 below ) . greiner et al . screened a library of 2976 compounds in a standard 
 radioactive filter - binding assay and found chaetocin , which is a member 
 in an epidithiodiketopiperazine ( etp ) family of alkaloids , as the 
 most potent inhibitor with an ic50 of 0.6 m . furthermore , inhibition assays were performed in the presence 
 of increasing concentrations of chemical reductant dithiothreitol 
 ( dtt ) to reduce the disulfide bond of chaetocin . under these assay 
 conditions , it was found that the inhibition was maintained and the 
 inhibitory activity was not dependent on the disulfide functionality . it terms of selectivity , chaetocin inhibited the human suv39h1 ( ic50 = 0.8 m ) , mouse g9a ( ic50 = 2.5 m ) , 
 and neurospora crassa dim5 ( ic50 = 3.0 m ) , but it was less potent for drosophila e(z)-complex ( recombinant protein , ic50 = > 90 m ) , setd8 , and setd7 ( bacterially expressed 
 proteins , ic50 = > 180 m ) . like other members 
 of 
 etps , chaetocin shows cytotoxicity depending upon initial cell density . the toxicity of other known etps was due to formation of mixed thiols 
 with cellular protein . greiner et al . 
 suggested that the toxicity of chaetocin was not caused by the inhibition 
 of su(var.)3 - 9 since the disulfide bond in chaetocin was not involved 
 in inhibition . in addition , chaetocin reduced h3k9me2 and h3k9me3 
 levels in sl-2 drosophila tissue culture 
 cells at a low concentration ( 0.5 m ) , but it did not reduce 
 h3k4 , h3k27 , h3k36 , and h3k79 methylation levels in these cells . however , 
 the possibility of the drosophila orthologue 
 of mammalian g9a being responsible for the reduced h3k9me2 levels 
 was not ruled out . published the first 
 total synthesis of natural ( + ) -chaetocin 
 ( 1 ) ( figure 3 ) and its enantiomer 
 and reported that both enantiomers inhibited g9a ( ic50 = 
 2.5 and 1.7 m , respectively ) in 2010 . interestingly , the sulfur - deficient analogue of chaetocin , 2 ( figure 3 ) , and its enantiomer were 
 inactive against g9a ( ic50 > 50 m ) . thus , it 
 was 
 concluded that the disulfide bridge of chaetocin was crucial for the 
 inhibitory activity and that g9a was not sensitive to absolute stereochemistry 
 of chaetocin . activity 
 relationship ( sar ) studies of chaetocin and reported that simple derivatives such as ps - etp-1 ( 3 ) ( figure 3 ) were significantly less toxic 
 but effectively inhibited g9a ( ic50 = 5.2 m ) . these 
 results suggest that the dimeric etp structure is not necessary for 
 g9a inhibition . in 2013 , cherblanc et al . reported 
 that chaetocin inhibits su(var.)3 - 9 in a time - dependent and nonspecific 
 manner via chemical modification of the enzyme by the disulfide group 
 of chaetocin , differing from the published findings and conclusions 
 by greiner et al . thus , it has been suggested that chaetocin or related 
 natural products could not be used as selective chemical probes of 
 pkmt function . , 
 cherblanc et al . found that compound 4 ( figure 3 ) , which does not have the bisulfide bridge , was 
 inactive and that structurally simple etp compound 5 ( figure 3 ) had an inhibitory activity with an ic50 of 3.2 m . interestingly , in their mechanism of action ( moa ) 
 studies , chaetocin was not competitive with sam . therefore , it was 
 concluded that any specific interaction of chaetocin with su(var.)3 - 9 
 was due to the increased sensitivity of this enzyme to thiol - reactive 
 compounds . these conclusions and suggestions were disputed by greiner 
 et al . , emphasizing the 30-fold lower 
 potency of simple etp compound 5 as well as the differences 
 between the assay conditions ( e.g. , preincubation time ) of the two 
 studies . in their following publication , cherblanc et al . the structurally simple etp compound 5 ( ic50 = 4.9 m ) showed comparable potency as that 
 of chaetocin ( ic50 = 2.6 m ) . , demonstrated that the disulfide 
 bridge is crucial for the activity of the inhibitor while the rest 
 of the complex structure is not essential . furthermore , in moa studies , 
 cherblanc et al . found that inhibition of g9a by chaetocin is reversible 
 and that activity of g9a was recovered in the presence of dtt . on 
 the other hand , in the absence of dtt , inhibition of g9a by chaetocin 
 was maintained . therefore , it has been concluded that chaetocin and 
 related etp compounds inhibit g9a in vitro via mixed disulfide linkages 
 formed between cysteine residues of enzyme and inhibitor . the mechanism 
 of inhibition is dependent on the assay conditions and incubation 
 time . inhibition of the g9a enzymatic activity by structurally 
 simple 
 etp compounds , as shown by both iwasa et al . and cherblanc et al . , 
 demonstrated that the etp core , and in turn the disulfide bridge , is 
 essential for inhibition . these results , together with the findings 
 from reversibility studies and denaturing mass spectrometry ( ms ) studies 
 showing the involvement of different pre - set domain cysteines , support the view that chaetocin and related 
 compounds with the thiol - reactive functionality are unlikely to be 
 suitable for investigating biological functions of the pkmt(s ) of 
 interest as selective inhibitors . therefore , results from cellular 
 studies using these inhibitors should be interpreted with caution . g9a and glp are the main methyltransferases that catalyze mono- 
 and dimethylation of h3k9 . in addition 
 to h3k9 , these enzymes dimethylate many nonhistone targets , including the tumor suppressor p53 at lysine 
 373 . g9a is overexpressed in various 
 cancers , and knockdown of g9a inhibits prostate cancer , leukemia and lung cancer cell 
 growth . in mouse models , significant 
 delays in acute myeloid leukemia ( aml ) progression and reduction of 
 leukemia stem cell frequency were observed with loss of g9a . in addition , a recent report suggested that 
 g9a functions as a coactivator for p21 transcription , 
 leading cells to undergo apoptosis . involvement 
 of g9a in maintenance of hiv-1 latency , cocaine addiction , and mental retardation was also documented . in addition , g9a has been 
 implicated in stem cell function , maintenance , differentiation , and 
 reprogramming . for example , g9a is critical for early mouse embryonic development 
 and esc ( embryonic stem cell ) differentiation and mediates h3k9me2 
 patterning during hematopoietic stem and progenitor cells ( hspcs ) 
 lineage specification . furthermore , it 
 was recently reported that t cell - intrinsic expression of g9a was 
 required for development of pathogenic t cells and intestinal inflammation 
 in a colitis model . glp has been implicated 
 in kleefstra syndrome , a disorder affecting intellectual 
 ability . more recently , glp was reported to be an essential lysine 
 methyltransferase in the prdm16 ( pr domain containing protein 16 ) 
 transcriptional complex and controls brown adipose cell fate and energy 
 homeostasis . in 2007 , the discovery 
 of this selective inhibitor was a major advancement in the pkmt inhibitor 
 field . 125  000 preselected 
 compounds resulted in two confirmed hits : compound 6 ( bix-01338 ) , which contains a 2-(n - acyl)-aminobenzimidazole 
 core , and compound 7 ( bix-01294 ) , which is a 2,4-diamino-6,7-dimethoxyquinazoline 
 derivative ( figure 4 ) . both compounds were tested against a panel of methyltransferases 
 including g9a and glp along with prmt1 ( protein arginine methyltransferase 
 1 ) , setd7 , setdb1 , wild - type ( wt ) suv39h1 , and a hyperactive suv39h1 
 ( h320r ) mutant . compound 6 inhibited all of them in a 
 concentration range of 515 m ; thus , it was not selective . 
 on the other hand , 7 selectively inhibited g9a ( ic50 = 1.7 m ) and glp ( ic50 = 38 m ) 
 ( other enzymes were not inhibited even at 45 m ) . it is worth 
 mentioning that the inhibition of glp by 7 was measured 
 under oversaturated reaction conditions where almost all of the substrate 
 transformed to h3k9me3 , whereas g9a inhibiton was assayed under linear 
 reaction conditions . in another study , 7 was reported to be slightly more potent for glp ( ic50 = 0.7 m ) than g9a ( ic50 = 1.9 m ) 
 when using the same linear assay conditions for both enzymes . initial mechanistic studies showed that 7 did not inhibited g9a in a sam - competitive manner . chang et al . obtained a crystal structure of 
 the glp set domain in complex with 7 and sah , which reveals 
 that inhibitor 7 binds to the substrate binding groove 
 of glp ( figure 5 ) . inhibitor 7 was also characterized in multiple 
 cell - based 
 assays . in mouse escs , 7 at 4.1 m reduced global 
 levels of h3k9me2 , increased unmodified h3k9 , and did not change the 
 h3k9me1 and h3k9me3 marks . similar 
 effects on the reduction of h3k9me2 global levels were observed in 
 mouse embryonic fibroblast ( mef ) and human hela cells . furthermore , 
 effects of 7 on the reduction of h3k9me2 at promoters 
 of g9a target genes were investigated using chromatin immunoprecipitation 
 ( chip ) . treatment of wt mouse es cells ( embryonic stem cells ) with 7 ( at 4.1 m for 2 days ) reduced the h3k9me2 mark at 
 promoters of g9a target genes mage - a2 , bmi1 , and serac1 . on the other hand , compound 7 did not affect the h3k9me2 mark at promoters of g9a nonresponsive 
 genes such as mage - b4 and tubulin . it is worth noting that 7 was toxic at concentrations 
 above 4.1 m in cellular assays . taken together , these results 
 demonstrated that compound 7 is the first selective small - molecule 
 inhibitor of g9a and glp and that it is competitive with the peptide 
 substrate and selectively reduces the h3k9me2 mark in cells . x - ray co - crystal structure of the glp7 complex 
 ( pdb code : 3fpd ) . protein residues , gray colored stick models ; inhibitor , green 
 colored stick model ; and hydrogen bonds , red dashed line . in 2009 , liu et al . discovered compound 8 ( unc0224 ) 
 as a potent and selective inhibitor of g9a and glp by studying sar 
 of the 2,4-diaminoquinazoline scaffold represented by 7 ( figures 6 and 7 ) . after establishing initial sar for the 2- and 
 4-amino regions , compound 8 was designed and synthesized 
 on the basis of the crystal structure of the glp7 complex . in particular , the 7-dimethylaminopropoxy 
 group of 8 was designed to occupy the lysine binding 
 channel , which was not occupied by 7 . as expected , 8 was more potent than 7 in multiple biochemical 
 and biophysical assays including isothermal titration calorimetry 
 ( itc ) with a kd ( dissociation constant ) 
 of 23  8 nm . it was more than 1000-fold selective for g9a and 
 glp over setd7 and setd8 and also selective against a broad range 
 of g - protein coupled receptors ( gpcrs ) , ion channels , and transporters . a high0resolution ( 1.7  ) x - ray crystal structure of the g9a8 complex confirmed the occupation of the g9a lysine binding 
 channel by the 7-dimethylaminopropoxy group ( figure 6 ) . the co - crystal structure also reveals that ( 1 ) the secondary 
 amine at the 4-position forms a hydrogen bond with asp1083 , ( 2 ) the 
 distal n - methyl group off the piperidine group is 
 solvent - exposed , and ( 3 ) the 7-dimethylaminopropoxy group does not 
 fully occupy the lysine channel ( figure 6 ) . further explored 
 the 7-aminoalkoxy group and discovered 9 ( unc0321 ) , which 
 has a longer ethoxyethyl chain instead of the 3-carbon chain of 8 ( figure 7 ) . compound 9 has been the most potent g9a inhibitor to 
 date with a morrison ki of 63 pm and was 
 40- and 250-fold more potent than 8 and 7 , respectively . additionally , it had high potency for glp and showed 
 a similar selectivity profile as that of 8 . x - ray co - crystal 
 structure of g9a8 complex 
 ( pdb code : 3k5k ) . inhibitor is shown as green colored stick model , and a fragment 
 of the histone peptide ( magenta ) is transposed into this crystal structure 
 to illustrate the lysine binding channel . structures of g9a and glp inhibitors 8 and 9 . in 2010 , chang et al . published 
 their g9a and glp inhibitor 10 ( e72 ) ( figure 8) by adding a lysine 
 mimic to the quinazoline scaffold based on the crystal structure of 
 the glp7 complex . compound 10 reactivated 
 k - ras - mediated epigenetic silencing of the proapototic fas gene in 
 nih 3t3 cells with modest potency and was less cytotoxic compared 
 to that of 7 , which could be due to its high polarity 
 and thus low cell membrane permeability . similarly , the relatively 
 high polarity and poor cell membrane 
 permeability were likely key contributors to the poor cellular potency 
 of 9 even though 9 was significantly more 
 potent than 7 in biochemical assays . therefore , liu et al . further optimized the quinazoline 
 scaffold to simultaneously improve physicochemical properties and 
 maintain in vitro potency . from these 
 studies , the g9a and glp cellular chemical probe 11 ( unc0638 ) ( figure 8) , along with 
 several back - up probes including unc0646 and unc0631 , was discovered ( figure 8) . chemical 
 probe 11 had high in vitro potency for g9a ( ic50 < 15 nm ) and glp ( ic50 = 19 nm ) in multiple biochemical 
 assays . in moa studies , 11 was competitive with the peptide substrate ( ki = 3.0  0.05 nm ) and noncompetitive with sam . the high 
 binding affinity was confirmed in biophysical assays such as differential 
 scanning fluorimetry ( dsf ) and surface plasmon resonance ( spr ) . the 
 x - ray crystal structure of g9a in complex with 11 and 
 sah clearly shows that this inhibitor occupies the substrate binding 
 groove and does not interact with the sam binding pocket , thus confirming 
 its mechanism of action . major interactions of 11 in 
 the g9a co - crystal structure are in good agreement with the ones of 8 ( figure 9 ) overlay of the x - ray co - crystal structure of 
 g9a8 complex ( pdb code : 3k5k ) with the one of 
 the g9a11 complex ( pdb code : 3rjw ) . inhibitor 8 , green colored stick model ; inhibitor 11 , red 
 colored stick model . a fragment of the histone peptide ( magenta ) was 
 transposed into the crystal structures to illustrate the lysine binding 
 channel . a high - quality chemical probe 
 should have ( 1 ) an excellent and 
 well - characterized selectivity profile and ( 2 ) robust on - target activities 
 in cells . therefore , 11 was characterized in a 
 broad range of epigenetic and nonepigenetic targets . it was more than 200-fold selective for g9a and glp over 
 16 other methyltransferases and epigenetic targets including suv39h1 , 
 suv39h2 , ezh2 , setd7 , mll , smyd3 , setd8 , dot1l , prdm1 , prdm10 , prdm12 , 
 prmt1 , prmt3 , histone acetyltransferase htatip , jimonji demethylase 
 jmjd2e , and dna methyltransferase dnmt1 . it was also at least 100-fold 
 selective over more than 80 gpcrs , kinases , ion channels , and transporters . 
 using an in - cell western assay , vedadi et al . showed that 11 ( ic50 = 81  9 nm ) was more potent and efficacious 
 than 7 ( ic50 = 500  43 nm ) at reducing 
 global levels of h3k9me2 in mda - mb-231 cells , a human breast carcinoma 
 cell line . g9a protein and mrna levels 
 were not affected during compound 11 treatment , suggesting 
 that the reduction of h3k9me2 results from inhibition of enzymatic 
 function , not from changes in protein abundance . in a standard mtt 
 ( 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide ) assay , 11 ( ec50 = 11  000  710 nm ) was found 
 to be significantly less toxic than 7 ( ec50 = 2700  76 nm ) in mda - mb-231 cells . thus , 11 has 
 an excellent separation of functional potency and cell toxicity with 
 a function / toxicity ratio of 138 , whereas 7 has a relatively 
 poor separation of functional potency and cell toxicity with a function / toxicity 
 ratio of 5.6 . compound 11 also exhibits a good separation 
 of functional potency and cell toxicity in seven other tumor and normal 
 cell lines . the effect of 11 on cellular levels of h3k9me2 
 was confirmed using quantitative ms - based proteomics . in the same 
 study , the effect of 11 on cellular levels of other 20 
 common histone modifications was also assessed . with the exception 
 of h3k14ac , 11 did not significantly change the other 
 histone marks , suggesting that cellular actions of 11 are specific and that there is a possible cross - talk between h3k9me2 
 and h3k14ac . in addition , 11 reduced the h3k9me2 mark 
 at promoters of g9a target genes magea1 , tbc1d5 , and magea2 and had no effect on 
 h3k9me2 at the promoter of the g9a nonresponsive gene mageb4 in chip  chip ( chromatin immunoprecipitation furthermore , 11 reactivated a silent retroviral 
 vector and g9a target genes in mes cells . it also reduced the h3k9me2 
 mark at promoters of those genes and the retroviral long terminal 
 repeat ( ltr ) region and indirectly induced dna hypomethylation in 
 mes cells . taken together , these results demonstrated that 11 is a highly selective inhibitor of g9a and glp and has robust on - target 
 activities in cells . in terms of phenotypic effects , 11 reduced clonogenecity 
 in mcf7 cells , but it had no effect on clonogenicity in mda - mb-231 
 cells , suggesting different phenotypic effects depending upon cell 
 types and/or epigenetic states . in addition , 
 it was recently reported that 11 significantly suppressed 
 the growth of primary human acute myeloid leukemia ( aml ) cells by 
 inducing leukemia stem cell differentiation . mechanistically , it was found that the methyltransferase 
 activity of g9a and its interaction with the leukemogenic transcription 
 factor hoxa9 regulate fast proliferating myeloid progenitors . these 
 results highlight a clinical potential of g9a inhibition as a means 
 to block the proliferation and self - renewal of aml cells by attenuating 
 hoxa9-dependent transcription . furthermore , it was recently reported 
 that 11 induced differentiation of wild - type t cells 
 into regulatory t cells and th17 cells , and adult hematopoietic stem cells continuously treated with 11 retained stem cell - like phenotypes and function better 
 than the those that are untreated during in vitro expansion . while compound 11 is an excellent 
 chemical probe of g9a and glp for cell - based 
 studies as discussed , it displayed 
 poor in vivo pharmacokinetic ( pk ) properties and consequently is not 
 suitable for animal studies . to achieve 
 an in vivo chemical probe that is suitable for animal studies , liu 
 et al . further optimized compound 11 and discovered 12 ( unc0642 ) as the first in vivo chemical probe of g9a and 
 glp ( figure 8) . compound 12 has high in vitro potencies for both g9a 
 and glp ( ic50 < 2.5 nm ) and is a substrate competitive 
 inhibitor with a ki of 3.7  1 nm . it is 2000-fold selective for g9a and glp over pcr2-ezh2 and > 20  000-fold 
 selective over 13 other methyltransferases ( suv39h2 , mll1 , setdb1 , 
 setd7 , setd8 , prmt3 , prmt5 , smyd2 , smyd3 , suv420h1 , suv420h2 , dot1l , 
 and dnmt1 ) . it was tested against a broad panel of 50 kinases and 
 44 gpcrs , transporters , and ion channel . with the exception of the 
 histamine h3 receptor , 12 is more than 300-fold 
 selective for g9a and glp over these nonepigenetic targets . similar 
 to 11 , it displayed high potency at reducing the h3k9me2 
 mark and low cell toxicity in a number of tumor and normal cell lines . it also reduced clonogenicity in panc-1 cells ( a human pancreatic 
 epithelioid carcinoma cell line ) . importantly , 12 displayed 
 much improved exposure in plasma compared to that of 11 in mouse pk studies , making it suitable for animal studies as an 
 in vivo chemical probe of g9a and glp . more recently , a biotinylated 
 derivative of probe 11 , compound 13 ( unc0965 ) 
 was also designed and synthesized ( figure 10 ) . this new derivative retained high 
 in vitro potency for g9a and was active in cellular assays . have shown that 13 can selectively precipitate 
 g9a from whole - cell lysates ( chemiprecipitation ) and is an effective 
 chemical tool for exploring the localization of g9a on chromatin both 
 in vitro and in vivo in chem  chip studies . reported 
 the discovery of 14 ( brd4770 , 
 figure 11 ) by 
 synthesizing a focused library of 2-substituted benzimidazoles to 
 mimic sam based on compound 6 ( figure 4 ) , a sam - competitive but nonselective inhibitor of pkmts . compound 14 is the methyl ester 
 of its carboxylic acid derivative 15 ( brd9539 ) for cell - based 
 studies ( figure 11 ) . compound 15 inhibited g9a with an ic50 of 
 6.3 m and was selective for g9a over suv39h1 , suv39h2 , mll1 , 
 setd7 , setd8 , prmt1 , prmt3 , prmt5 , dnmt1 , and hdac19 . however , 
 it also inhibited prc2ezh2 with a similar potency and nsd1 
 only at 40 m . the potency of carboxylic acid 15 against glp was not reported ; thus , it is not clear whether 15 is a pan g9a and glp inhibitor . methyl ester 14 at 10 m significantly reduced 
 cellular levels of h3k9me2 and h3k9me3 and increased cellular levels 
 of h3k9me1 . interestingly , cellular levels 
 of h3k27me3 were unaltered during compound 14 treatment , 
 suggesting that 14 did not inhibit prc2ezh2 in 
 cells . the caspase3/7 activity , as a measure of cell apoptosis , was 
 also assessed . methyl ester 14 did not induce caspase 
 activity in panc-1 cells even after 72 h treatment , whereas inhibitor 7 increased caspase activity after only 24 h , suggesting that 14 has low cell toxicity . compound 14 treated 
 panc-1 cells showed enlarged and flattened cell morphology with increased 
 senescence - associated -galactosidase staining . 
 the total number of cells as well as clonogenicity was reduced after 
 72 h of treatment with the inhibitor . mechanistically , increase in 
 phosphorylation of atm ( ataxia telangiectasia mutated ) and nuclear 
 translocation of phosphorylated atm after the treatment with compound 14 were observed , whereas atr ( ataxia telangiectasia and rad3-related ) 
 was not activated . it was therefore suggested that 14 causes 
 cellular senescence similar to that resulting from activation of phosphorylation 
 of atm by hdac inhibitors . in 2014 , sweis et al . reported the 
 discovery of a potent g9a ( ic50 = 3.3 nm ) and glp ( ic50 = 38 nm ) inhibitor 16 ( a-366 , figure 11 ) . this inhibitor 
 contains a new spiro(cyclobutane-1,3-indol)-2-amine 
 core and is selective for g9a and glp over other methyltransferases 
 including suv39h2 , mll1 , setdb1 , setd7 , setd8 , prmts ( 1 , 3 , 5 , 6 and 
 8) , smyd2 , smyd3 , ezh1 , ezh2 , suv420h1 , suv420h2 , and dnmt1 . moa studies 
 of 16 showed noncompetitive inhibition with respect to 
 sam but competitive inhibition with the peptide substrate . this finding 
 is confirmed by the x - ray crystal structure of g9a in complex with 16 . this co - crystal structure reveals hydrogen - bonding interactions 
 with asp1074 and asp1078 , and the 7-aminopropoxy group interacts the 
 lysine binding channel , similar to that of 8 and 11 interacting with g9a ( figure 12 ) . 
 in a human prostate cancer cell line , pc3 , after treatment for 72 
 h with compound 16 at 3 m , cellular levels of 
 h3k9me2 were reduced by about 50% , whereas other histone marks , such 
 as h3k27me3 and h3k36me2 , were not affected . x - ray co - crystal structure of the g9a16 complex 
 ( pdb code : 4nvq ) . polycomb repressive 
 complex 2 ( prc2 ) is the multisubunit protein complex that catalyzes 
 methylation of h3k27 . the main biological function of prc2 is transcriptional silencing 
 of genes involved in differentiation and development via trimethylation 
 of h3k27 . prc2 is a crucial chromatin - modifying 
 complex that is conserved from drosophila to humans , and the core prc2 complex 
 includes four subunits : ezh1 ( enhancer of zeste homologue 1 , also 
 known as kmt6b ) or ezh2 ( enhancer of zeste 2 polycomb repressive complex 
 2 subunit , also known as kmt6a ) , suz12 ( suppressor of zeste 12 ) , eed 
 ( embryonic ectoderm development ) , and histone binding proteins rbap46/48 . ezh1 or ezh2 is the catalytic subunit of the complex , and its c - terminal 
 set domain is essential for the methyltransferase activity . ezh1 and ezh2 are highly homologous , 
 with 76% overall sequence identity and 96% sequence identity in their 
 set domains . however , ezh1 and ezh2 
 have different expression patterns . while the former is present in 
 both dividing and differentiated cells , the latter is found only in 
 actively dividing cells . furthermore , prc2 
 complex - containing ezh1 ( prc2ezh1 ) has lower methyltransferase 
 activity as compared to that of prc2 complex - containing ezh2 ( prc2ezh2 ) . nevertheless , both of these complexes are involved 
 in the maintenance of h3k27 methylation in cells . methylation of h3k27 occurs 
 successively to give the h3k27me3 mark , which is a well - known repressive 
 mark . even though ezh2 is the catalytic 
 subunit of prc2ezh2 , it does not have enzymatic activity on 
 its own and needs at least two other subunits ( eed and suz12 ) to gain 
 the methyltransferase activity . it has recently been 
 shown that prc2 can contain several other protein subunits such as 
 aebp2 , pcls , and jarid2 . point mutations at tyrosine 641 ( y641 ) in 
 the c - terminal set domain of ezh2 have been identified . these mutants including y641f , y641n , y641s , 
 y641h , and y641c were observed in 7% of follicular lymphomas and 22% 
 of germinal center b - cell ( gcb ) and diffuse large b - cell lymphomas 
 ( dlbcls ) . these gain - of - function y641 mutations prefer 
 h3k27me2 as the substrate , resulting in enhanced enzymatic activity 
 for trimethylation of h3k27 . in contrast , wild - type 
 ezh2 has a substrate preference for unmethylated h3k27 . thus , wild - type ezh2 and the y641 mutant work cooperatively , leading 
 to increased levels of h3k27me3 in tumor tissues . recently , a new 
 ezh2 mutation , a677 g , was identified in lymphoma cell lines and primary 
 tumor specimens . interestingly , the 
 a677 g mutant has no substrate preference among unmethylated h3k27 , 
 h3k27me1 , and h3k27me2 . overexpression of ezh2 and/or hypertrimethylation 
 of h3k27 have been associated with a number of human cancers , such as breast , prostate , lymphoma , and 
 leukemia . in 2012 , knutson et 
 al . hts of a 175  000-compound 
 diversity library led to the identification of compound 17 ( ic50 = 620 nm ) as a tractable hit ( figure 13 ) . compound 18 ( epz005687 , figure 13 ) ( ic50 = 54  5 nm ) was discovered 
 by optimization of this hit including ( 1 ) addition of an amine functionality 
 at the 4-position of the phenyl ring , ( 2 ) change of the 5,6-fused 
 pyrrazolapyridine to the indozole , and ( 3 ) introduction of a larger 
 lipophilic group at the indazole nitrogen ( figure 13 ) . menten kinetic studies indicated that this 
 inhibitor 
 was competitive with sam with a ki of 
 24  7 nm and noncompetitive with the peptide substrate . it was previously reported that the cofactor 
 product sah inhibited ezh2 in a sam - competitive manner . therefore , with this indirect 
 evidence , binding of 18 in the ezh2 sam pocket has been 
 suggested . compound 18 was more than 500-fold selective 
 for prc2ezh2 over 14 other methyltransferases , including g9a , 
 glp , setd7 , smyd2 , smyd3 , whsc1l1 , mmset , prmts ( 1 , 3 , 4 , 5 , 6 , and 
 8) , and dot1l . it also displayed similar 
 potencies for ezh2 y641 mutants ( y641n , f , s , h , and c ) compared to 
 that for wild - type ezh2 . on the other hand , 18 was approximately 
 5-fold more potent for the a677 g mutant than that for wild - type ezh2 . in addition , 18 showed no significant inhibition at 10 
 m against most of the 77 gpcrs and ion channels tested ( hit 
 only 4 targets with the lowest extrapolated ic50 of 1.5 
 m , corresponding to more than 60-fold selectivity ) . in 
 oci - ly19 cells , a wild - type ezh2 lymphoma cell line , 18 reduced h3k27me3 levels with an ic50 of 80  30 
 nm . h3k27me3 and h3k27me2 were the only 
 major ptms that were significantly changed ( a slight increase in h3k27ac 
 was also observed ) . similarly , this inhibitor significantly reduced 
 the h3k27me3 mark in wsu - dlcl2 cells , a lymphoma cell line harboring 
 the y641f mutant . although 18 did not affect the growth 
 of oci - ly19 cells ( with wild - type ezh2 ) , it significantly inhibited 
 the growth of wsu - dlcl2 ( with ezh2 mutant ) and pfeiffer 
 ( with ezh2 mutant ) cells with a delayed onset of activity . these results suggest that hypertrimethylation of h3k27 is essential 
 for proliferation and survival of lymphoma cells harboring ezh2 mutants . in addition , gene set enrichment analysis ( gsea ) studies showed that 
 inhibition of ezh2 by 18 in wsu - dlcl2 cells resulted 
 in derepression of known ezh2 target genes . taken together , these 
 results have demonstrated that 18 is a potent and selective 
 inhibitor of ezh2 and that it is competitive with sam and engages 
 in the target in cells . shortly after the publication of inhibitor 18 , mccabe 
 et al . reported another selective ezh2 inhibitor , 19 ( gsk126 , 
 figure 14 ) , which 
 shares the core scaffold with 18 . hts of the corporate 
 compound collection resulted in the identification of a small - molecule 
 inhibitor ( ki = 700 nm ) 
 as a hit . optimization of this hit led 
 to the discovery of 19(133 ) along 
 with several other potent ezh2 inhibitors including gsk343 and gsk503 ( figure 14 ) with the same core template . compound 19 contains the same pyridone group as 18 but has an indole 
 group instead of the indazole group of 18 ( figure 13 ) . compound 19 potently inhibited both wild - type 
 and 
 mutant ezh2 with ki of 0.53 
 nm . it was more than 1000-fold 
 selective for ezh2 over 20 other methyltransferases including g9a , 
 suv39h1 , suv39h2 , mll14 , setd8 , setd7 , smyd2 , mmset , setmar , 
 prmts ( 1 , 3 , 4 , 5 , and 6 ) , dot1l , dnmt3a , and dnmt3b . additionally , 19 showed no significant inhibition against a broad panel 
 of kinases , gpcrs , ion channels , and transporters as well as other 
 chromatin - modifying enzymes such as hdac111 , jmjd2d , jmjd3 , 
 and lsd1 . mccabe et al . also investigated the effect of 19 on 
 cell proliferation in a panel of b - cell lymphoma cell lines and found 
 that six of the seven most sensitive dlbcl cell lines harbored y641n , 
 y641f , or a677 g mutations , whereas most of the insensitive dlbcl cells 
 in the panel had no mutations . these 
 results suggest that the growth of dlbcl cells harboring the gain - of - function 
 mutations is dependent on prc2ezh2 methyltransferase activity . the timing of 19-induced effects on the proliferation 
 and cell death was also studied in two of the most sensitive dlbcl 
 cell lines : pfeiffer ( with a677 g mutation ) and karpas-422 ( with y641n 
 mutation ) . in pfeiffer cells , inhibition of cell proliferation by 19 was started after 2 days , and a clear decrease in cell 
 number was observed after 3 days . the observed cell death was attributed 
 to caspase - mediated apoptosis . on the other hand six to seven days 
 were required to reach the maximal potency in inhibiting the growth 
 of these cells . mechanistically , a mainly cytostatic effect was observed 
 in karpas-422 cells with minimal caspase activity . in addition , the 
 effect of 19 on gene expression in dlbcl cell lines was 
 examined . the treatment with 19 led to clear transcriptional 
 activation in the most sensitive dlbcl cell lines . the gene expression 
 changes caused by ezh2 inhibition via 19 versus ezh2 
 knockdown via shrna were very similar in both pfeiffer and karpas-422 
 cell lines , suggesting that the observed effects were due to on - target 
 activity of the inhibitor . the chip - seq ( chromatin immunoprecipitation 
 followed by sequencing ) analysis for the three most sensitive cell 
 lines , pfeiffer , wsu - dlcl2 ( harboring y641f ) , and karpas-422 , showed 
 that the genes upregulated in response to compound 19 treatment displayed enrichment in h3k27me3 before treatment , suggesting 
 that the ezh2 target genes are transcriptionally repressed by the 
 h3k27me3 mark . furthermore , 19 was efficacious 
 in karpas-422 and 
 pfeiffer tumor xenograft mouse models . in particular , intraperitoneal ( ip ) administration of 19 at 50 mg / kg once daily , 150 mg / kg once daily , or 300 mg / kg twice 
 per week for 35 days resulted in a drastic reduction in tumor volume 
 and a marked improvement in survival in the more aggressive karpas-422 
 xenograft model . after the treatment with 19 was stopped , 
 tumor stasis was seen in the 50 mg / kg once daily group , whereas complete 
 tumor eradication had been observed in the 150 mg / kg once daily and 
 300 mg / kg twice per week groups for 25 days . a dose - dependent decrease 
 in the h3k27me3 mark and an increase in expression of ezh2 target 
 genes compound 19 was well - tolerated at the doses and schedules used in these 
 mouse models . in summary , 19 is a highly potent and selective 
 ezh2 inhibitor , has robust on - target activity in cells , and is efficacious 
 in multiple cell - based and in vivo models . importantly , 19 has been advanced into phase 1 clinical trials for the treatment 
 of gcb - dlbcl . reported 20 ( ei1 , figure 14 ) , a selective ezh2 
 inhibitor , which shares the same pyridone and indole / indazole core 
 with inhibitors 18 and 19 . compound 20 potently inhibited the wild - type ezh2 and y641f mutant 
 with ic50 s of 15  2 and 13  3 nm , respectively . it was competitive with the cofactor sam with a ki of 13  3 nm calculated by using cheng compound 20 was more than 10  000-fold 
 selective for ezh2 over 10 other pmts including g9a , suv39h2 , mll , 
 setd2 , setd7 , setd8 , smyd2 , whsc1l1 , carm1 , and dot1l and was also 
 about 90-fold selective for ezh2 over ezh1 . the effect of 20 on dlbcl cell lines harboring ezh2 
 gain - of - function mutations ( y641f and y641n ) and a rhabdoid tumor 
 cell line g4001 ( with wild - type ezh2 ) was investigated.20 concentration - dependently decreased 
 cellular levels of h3k27me3 and h3k27me2 , but it did not affect h3k27me1 
 and other di- and trimethylation marks on h3k4 , h3k9 , h3k36 , and h3k79 , 
 suggesting that the cellular action of 20 is specific . the expression levels of p16 were increased 20-fold after 5 days of treatment with 20 . furthermore , chip experiments revealed that both h3k27me3 and ezh2 
 were enriched at the p16 promoter , but upon the treatment 
 with 20 , the h3k27me3 mark was drastically reduced at 
 the p16 promoter , whereas ezh2 levels were not changed , 
 suggesting that p16 expression was activated by the 
 suppression of the h3k27me3 mark but not by ezh2 occupancy at the 
 promoter . moreover , similar to 18 and 19 , compound 20 strongly inhibited the proliferation of 
 dlbcl cell lines ( wsu - dlcl2 , su - dhl6 , karpas-422 , db , su - dhl4 ) harboring 
 ezh2 gain - of - function mutants . on the other hand , the proliferation 
 of dlbcl cell lines ( oci - ly19 , ga10 , and toledo ) with wild - type ezh2 
 was not or was only weakly inhibited by 20 . in addition , 
 qi et al . found that inhibition of ezh2 by 20 significantly 
 blocked cell cycle progression and induced apoptosis in dlbcl cell 
 lines harboring ezh2 mutants . the time - dependent gene expression changes 
 in karpas-422 ( ezh2 ) cells suggest that the reduction 
 of h3k27me3 at the gene promoters is associated with 20-dependent transcriptional upregulation . in 2013 , konze et 
 al . reported an orally bioavailable chemical 
 probe of ezh2 and ezh1 , 21 ( unc1999 , figure 15 ) . docking of 18 into an ezh2 homology model built on the basis of the x - ray 
 crystal structure of glp suggested that the morpholinomethyl group 
 was solvent - exposed . therefore explored this region to 
 improve physicochemical properties of this chemical series . from this 
 study , 21 was discovered as a chemical probe of ezh2 
 and ezh1 . it displayed high in vitro potency for ezh2 ( ic50 < 10 nm ) and possessed more desirable lipophilicity ( clogp = 
 3.1 ) compared with that of 18 and 19 . in 
 moa studies , 21 was competitive with sam with a ki of 4.6  0.8 nm and noncompetitive with 
 the peptide substrate . importantly , 21 was more than 10  000-fold 
 selective for ezh2 over 15 other methyltransferases including g9a , 
 glp , suv39h2 , suv420h1 , suv420h2 , mll1 , setdb1 , setd7 , setd8 , smyd2 , 
 prmts ( 1 , 3 , and 5 ) , dot1l , and dnmt1 . interestingly , unlike the ezh2 
 inhibitors discussed earlier , 21 was only about 1015-fold 
 selective for ezh2 over ezh1 . thus , it would be a useful tool in cellular 
 and disease settings where the h3k27 methylation by prc2ezh2 
 is compensated by prc2ezh1 . in addition , 21 was 
 highly selective over a broad panel of more than 90 kinases , gpcrs , 
 transporters , and ion channels , with the exception of sigma2 . inhibitor 21 reduced h3k27me3 levels with an ic50 of 124 
  11 nm in mcf10a cells and exhibited low cell toxicity ( ec50 = 19  200  1200 nm ) . thus , 21 had an excellent separation of cellular potency 
 and toxicity , with a function - to - toxicity ratio of more than 150 . in addition , 21 at 5000 nm for 72 h did not significantly 
 change ezh2 protein levels in mcf7 cells even though the h3k27me3 
 mark was completely removed . furthermore , 21 concentration - dependently 
 inhibited the proliferation of db cells , a dlbcl cell line harboring 
 the ezh2 mutant . consistent with the results 
 generated in mcf7 cells , 21 at 3000 nm for 72 h significantly 
 reduced h3k27me3 levels in db cells and did not affect ezh2 protein 
 levels . importantly , 21 was orally bioavailable , likely 
 due to its improved physicochemical properties . a single 50 mg / kg 
 oral dose of 21 attained good plasma exposure levels 
 and high cmax of 4700 nm in swiss albino 
 mice . in summary , compound 21 is a potent and selective 
 inhibitor of ezh2 and ezh1 , is competitive with sam , has robust on - target 
 activity in cells , and is orally bioavailable , making it a valuable 
 tool for assessing in vivo efficacy and potential toxicity of dual 
 inhibition of ezh2 and ezh1 in chronic animal studies . konze 
 et al . also developed a number of inhibitor 21-based 
 tool compounds . first , unc2400 
 ( figure 15 ) , which is a dimethylated analogue , 
 was designed and synthesized as a negative control for cell - based 
 studies . even though this dimethylated derivative is structurally very similar to 21 , it was 
 more than 1000-fold less potent for ezh2 than 21 in biochemical 
 assays , did not affect h3k27me3 levels in cells , and displayed similar 
 ( low ) cell toxicity as that of 21 . second , unc2399 ( figure 15 ) , a biotinylated derivative of 21 , was designed and synthesized for chem  chip and chemoproteomics 
 studies . this tool compound displayed 
 high in vitro potency for ezh2 ( ic50 = 17  2 nm ) 
 and can selectively pull down ezh2 from whole - cell lysates . third , 
 unc2239 ( figure 15 ) , a cell - penetrant dye conjugate 
 of 21 , was developed for live cell imaging studies . this conjugate also exhibited high in vitro 
 potency for ezh2 ( ic50 = 21  1 nm ) and has been used 
 for nuclear colocalization studies in live cells . structures of ezh2 and 
 ezh1 inhibitor 21 and 21-based tool compounds . shortly after the publication 
 of inhibitor 21 , knutson 
 et al . reported an orally active ezh2 inhibitor , 22 ( epz-6438 , 
 figure 16 ) , which has better potency and pharmacokinetic 
 properties than that of their early compound 18 . inhibitor 22 contains the same 
 pyridone core as other ezh2 inhibitors discussed earlier , but it does 
 not have the indole / indazole motif . it displayed high in vitro potency 
 for wild - type ezh2 ( ki = 2.5  0.5 
 nm ) as well as ezh2 y641f , c , h , n , and s and a677 g mutants . in moa 
 studies , 22 was competitive with sam and noncompetitive 
 with the peptide substrate . it was around 35-fold selective for ezh2 
 over ezh1 and more than 4500-fold selective for ezh2 over 14 other 
 ptms including g9a , glp , setd7 , smyd2 , smyd3 , mmset , whsc1l1 , prmts 
 ( 1 , 3 , 4 , 5 , 6 , and 8) , and dot1l . specific inactivating mutations 
 in subunits of the chromatin remodeling 
 complex swi / snf ( switch / sucrose nonfermentable ) have been found in 
 human cancers . one of such example is 
 the smarcb1 subunit that is inactivated in nearly all malignant rhabdoid 
 tumors ( mrts ) , one of the most common malignancies in pediatric oncology . inhibitor 22 showed a concentration - dependent 
 reduction of global levels of h3k27me3 as well as h3k27me2 and h3k27me1 , 
 but it did not affect other h3 ptms ( k4me3 , k9me3 , k36me2 , k79me2 , 
 and k27ac ) in smarcb1-deficient mrt cells ( g401 ) . the treatment of g401 cells with 22 caused 
 antiproliferative effects with nanomolar potency and reached the maximal 
 activity at day 14 . on the other hand , 22 did not suppress 
 proliferation of rd cells , which contain wild - type smarcb1 . furthermore , 
 the treatment of g401 cells with 22 induced g1 arrest 
 at day 7 and apoptosis at day 11 , whereas this inhibitor did not affect 
 cell cycle or induce apoptosis in rd cells . these results suggest 
 that the proliferation of smarcb1-deficient mrt cells such as g401 
 cells is dependent on overexpression of ezh2 and hypertrimethylation 
 of h3k27 and that ezh2 inhibitors are effective at blocking the proliferation 
 of these aggressive tumor cells . importantly , 22 ( twice 
 daily oral dosing at 250 or 500 mg / kg for 28 days ) completely eliminated 
 g401 xenografts in scid ( severe combined immunodeficiency ) mice . the 
 complete tumor regression was sustained for 32 days after the cessation 
 of compound 22 treatment . in the tumors that were harvested 
 from a subset of the animals treated with 22 for 21 days , 
 a strong correlation was observed between inhibition of h3k27me3 levels 
 and antitumor activity . in addition , 22 was well - tolerated 
 in all treatment groups . in june 2013 , compound 22 became 
 the first ezh2 inhibitor that entered human clinical trials . it is 
 being evaluated for the treatment of advanced solid tumors or b - cell 
 lymphomas . in 2013 , garapaty - rao 
 et al . reported an ezh2 small - molecule inhibitor 
 containing a new scaffold that differs from that of previously reported 
 pyridone indole / indazole - based ezh2 inhibitors . hts of a 150  000-compound corporate collection led 
 to the identification of a hit containing a tetramethylpiperidinyl 
 benzamide scaffold . optimization of this hit resulted in the discovery 
 of compound 23 ( figure 16 ) , which 
 displayed good in vitro potency for wild - type ezh2 ( ic50 = 21  4 nm ) and ezh2 mutant ( ic50 = 197  14 nm ) . this inhibitor 
 was also competitive with sam , similar to other known ezh2 inhibitors . it was selective for ezh2 over five other pkmts including g9a , setd7 , 
 setd8 , whsc1 , and dot1l and was about 10-fold selective for ezh2 over 
 ezh1 ( ic50 = 213  70 nm ) . compound 23 reduced global levels of h3k27me3 and 
 h3k27me2 levels with a modest potency ( ec50 = 7 m ) 
 and did not affect h3k27me1 , h3k4me3 , h3k9me3 , and h3k36me3 levels 
 in hela cells . this inhibitor did not 
 reduce protein levels of ezh2 , ezh1 , suz12 , and eed . the effect of 
 compound 23 on 46 common histone modifications was studied 
 using mass spectrometry in two germinal center b cell - like ( gcb ) dlbcl 
 cell lines : ht ( with wild - type ezh2 ) and sudhl6 ( with mutant ezh2 ) . this study confirmed the effect of compound 23 on reducing 
 h3k27me3 and h3k27me2 marks . in addition , compound 23 inhibited the growth of pfeiffer ( with ezh2 ) cells 
 with a delayed onset of activity but did not affect the growth of 
 oci - ly19 ( with wild - type ezh2 ) cells , even though h3k27me3 levels 
 were reduced in both cell lines . transcription levels of previously 
 known ezh2-regulated genes including abat , epb41l1 , apol1 , ceacam1 , pigz , sesn3 , and sox9 were increased in a concentration - dependent manner in pfeiffer cells . 
 on the other hand , genes that are important for cell cycle progression 
 were either not affected ( cdkn2a , cdkn1a , and cdkn2b ) or were downregulated ( cdkn1c and cdkn2d ) . it was also found that compound 23 did not inhibit the growth of prostate cancer cell lines 
 pc3 and du145 even though it significantly reduced h3k27me3 levels 
 in both cell lines , suggesting that the proliferation of these prostate 
 cancer cell lines is independent of ezh2 activity and h3k27 hypertrimethylation . methylation 
 of h3k4 in humans is controlled by pkmts : setd1a , setd1b , and setd7 
 as well as the mll family proteins mll15 , setmar , and smdy1 , 
 2 , and 3 . mll ( also known as lysine ( k)-specific 
 methyltransferase 2a ( kmt2a ) , trx1 , and mll1 ) is a large multidomain 
 ( several n - terminal dna domains and a c - terminal set domain with an 
 essential post - set region ) protein that 
 is specific for h3k4 mono- , di- , and trimethylation . chromosomal rearrangements associated with mll have been shown to 
 cause acute myeloid leukemia ( aml ) , acute lymphoblastic leukemia ( all ) , 
 or mixed lineage leukemia ( mll ) . more 
 than 50 functionally diverse mll fusion proteins have been identified 
 in human leukemias . af4 , af9 , af10 , af6 , and enl 
 are the most commonly seen mll fusion partners in mll - rearranged leukemias . mll has also been shown to be essential for 
 homeotic gene regulation and embryonic development via regulation 
 of hox gene expression in mice . although peptide- or peptidomimetic - based inhibitors of 
 mll are known , selective small - molecule 
 inhibitors of mlls have not been reported . it is worth noting that 
 small - molecule inhibitors of wdr5 ( wd repeat - containing protein 5 ) , 
 as an indirect way of disrupting mll activity , have been reported . setd7 ( set domain containing ( lysine 
 methyltransferase ) 7 , also 
 known as kmt7 , set7 , set9 , and set7/9 ) monomethylates h3k4 and many 
 nonhistone proteins including p53 and dnmt1 . it has recently been linked with hyperglycemia via its contribution 
 to upregulation of the gene encoding p65 subunit of nf-b in 
 response to glucose . it has been suggested 
 that setd7 is a potential target for the treatment of diabetes . compound 24 ( ( r)-pfi-2 ) , the first 
 chemical probe of setd7 , was very recently reported by barsyte - lovejoy 
 et al . ( figure 17 ) . an initial hit ( ic50 = 2.1 m ) was discovered by 
 hts of a 150  000-compound collection . optimization of this 
 initial hit resulted in the discovery of 24 , which was 
 highly potent for setd7 ( ic50 = 2.0  0.2 nm , ki = 0.33  0.04 nm ) . interestingly , 
 its enantiomer was about 500-fold less potent and is an excellent 
 negative control for cell - based studies . chemical probe 24 was more than 1000-fold selective for setd7 over 18 other methyltransferases 
 including g9a , glp , ezh2 , ezh1 , suv39h2 , suv420h1 , suv420h2 , setd2 , 
 setd8 , smyd2 , mll , whsc1 , prmt1 , prmt3 , prmt5 , prmt8 , and dot1l as 
 well as dnmt1 . it was also selective for setd7 over 134 gpcrs , ion 
 channels , and other enzyme targets ( less than 35% inhibition at 10 
 m ) . it occupied the substrate binding groove 
 in the x - ray crystal structure of the setd724 complex ( pdb code : 4jlg ) . however , 24 bound to setd7 only in the presence of 
 sam in spr experiments . therefore , inhibition of setd7 by 24 was not purely substrate competitive , and sam had a significant 
 role in the binding of 24 to setd7 . the direct binding 
 of 24 to setd7 in cells was demonstrated by the pull - down 
 studies using a biotinylated derivative of 24 as well 
 as by compound 24 increasing the stability of setd7 in 
 a cellular thermal shift assay ( cetsa ) . furthermore , inhibitor 24 increased nuclear localization of the transcriptional coactivator 
 yes - associated protein ( yap ) in a concentration - dependent manner and 
 induced expression of yap - silenced genes in cells . therefore , 24 is a well - characterized chemical probe and will be a valuable tool 
 for elucidating the role of setd7 in various human diseases . the smyd ( set and mynd domain containing ) family of proteins is 
 uniquely defined by a set domain that is split into two fragments 
 by a zinc ion binding domain mynd ( myeloid translocation protein-8 , 
 nervy , and deaf-1 ) and followed by a cysteine - rich post - set domain . the smyd family of proteins might be important 
 in developmental regulation , as the disruption of the smyd1 gene results in impaired cardiomyocyte maturation , flawed cardiac 
 morphogenesis , and embryonic lethality in mice . smyd1 ( also known as kmt3d ) and smyd3 ( also known as kmt3e ) 
 modify chromatin structure through their h3k4-specific enzymatic methylation 
 activity . smyd3 has been shown to be involved in cancer cell proliferation 
 and is overexpressed in most hepatocellular and colorectal carcinomas 
 as well as breast cancer . a recent report showed 
 that the methylation of map3k2 by smyd3 increases map kinase signaling 
 and promotes the formation of ras - driven carcinomas . smyd2 ( also known as kmt3c ) was reported to methylate h3k4 
 as well as h3k36 . methylation is another 
 hallmark that is associated with transcriptional 
 activation and in humans is controlled by pkmts : nsd1 , mmset , whsc1l1 , 
 setd2 , ash1l , and smyd2 . smyd2 has been shown to methylate the tumor 
 suppressors p53 and rb in addition to histone h3 . very recently , smyd2 has been 
 demonstrated to be highly expressed in pediatric acute lymphoblastic 
 leukemia and consitutes a poor prognostic indicator . overexpression of smyd2 was also connected to tumor cell 
 proliferation and resulted in malignant esophageal squamous cell carcinoma . in 2011 , ferguson et al . reported the 
 discovery of 25 ( az-505 ) , a potent and selective inhibitor 
 of smyd2 ( figure 17 ) . hts of a 1  230  000-compound 
 collection resulted in the discovery of 25 with an ic50 of 0.12 m and a kd of 
 0.5 m ( by itc ) . this inhibitor was around 700-fold selective 
 for smyd2 over six other pkmts including closely related smyd3 as 
 well as g9a , glp , setd7 , ezh2 , and dot1l . menten 
 kinetics studies revealed that 25 was competitive with 
 a peptide substrate ( 361380 of the c - terminal regulatory domain 
 of p53 ) and noncompetitive with sam . as mentioned previously , smyd 
 proteins comprise a unique split set domain . the s - sequence and the 
 core set domain combine to form the catalytically active set domain 
 of smyd2 , and the mynd , i - set , and post - set domains surround the core 
 set domain . to understand the structural 
 basis of p53 recognition and inhibitor binding , a series of x - ray 
 crystal structures of smyd2 in complex with the cofactor sam , a peptide 
 substrate ( 366378 of the c - terminal regulatory domain of p53 ) , 
 and/or 25 were obtained . there were no significant conformational 
 changes observed on binding with the p53 peptide substrate or the 
 inhibitor . the two residues that are highly conserved in smyd proteins , 
 y240 and y258 , are very important for activity . while y258 positions 
 the amino group of the substrate lysine in the lysine binding channel , 
 the hydroxyl group of y240 is essential for the catalytic activity , 
 as the mutation of this residue eradicates the catalytic activity 
 of smyd2 , as shown by brown et al . inhibitor 25 features three different functional groups : benzooxazinone , 
 cyclohexyl , and dichlorophenethyl moieties ( figure 17 ) . the benzooxazinone moiety lies deep in the lysine binding 
 channel , interacting with y258 as well as sam . the cyclohexyl group 
 is placed in the interface of the core set and i - set domains . the 
 dichlorophenethyl moiety extends across the peptide binding groove 
 and interacts with a secondary hydrophobic pocket . the x - ray co - crystal 
 structures confirm that compound 25 is a substrate - competitive 
 inhibitor ( figure 18 ) . x - ray co - crystal structure of the smyd225 complex 
 ( pdb code : 3s7b ) . more recently , 26 ( lly-507 , figure 17 ) , a chemical probe of 
 smyd2 , was discovered . compound 26 , which does not share 
 a common scaffold with 25 , inhibited smyd2 with an ic50 of less than 15 nm and was more than 100-fold selective 
 over other methyltransferases and nonepigenetic targets . it inhibited monomethylation 
 of p53 k370 in cells with an ic50 of about 600 nm . human setd2 
 ( set domain containing 2 , also known as kmt3a and set2 ) 
 has been shown to be a tumor suppressor associated with p53-dependent 
 gene regulation , transcription elongation , and intron a recent study suggests that the disruption 
 of setd2 h3k36 trimethylation pathway is a distinct mechanism for 
 leukemia development . evidence for the 
 tumor suppressor role of setd2 in human breast cancer was also provided . the disruption of h3k36 trimethylation by loss - of - function 
 setd2 mutations has been suggested to be central to the genesis of 
 hemispheric high - grade gliomas ( hggs ) in older children and young 
 adults . zheng et al . designed 
 and synthesized n - alkyl 
 derivatives of sinefugin ( 27 ) ( figure 19 ) , which is a close analogue of the cofactor sam and nonselective 
 inhibitor of pmts , and tested them against 
 various methyltransferases . from this 
 study , n - propyl sinefungin ( 28 ) ( pr - snf ) 
 ( figures 17 and 19 ) 
 was discovered as a selective inhibitor of setd2 ( ic50 = 
 0.8  0.02 m ) . 28 was highly selective for 
 setd2 over g9a , glp , setd8 , ezh2 , mll , suv39h2 , suv420h1 , suv420h2 , 
 prmt3 , dot1l , and dnmt1 . it displayed 
 modest potency for setd7 ( ic50 = 2.2  0.4 m ) , 
 carm1 ( ic50 = 3.0  0.3 m ) , and prmt1 ( ic50 = 9.5  0.4 m ) . the x - ray crystal structure 
 of setd2 in complex with 28 suggests that this inhibitor 
 forms hydrogen bonds with the two backbone carbonyl groups in the 
 setd2 active open conformer , resulting in the observed selectivity 
 for setd2 over other methyltransferases . findings from sar studies 
 also suggest that the lysine binding pocket of setd2 is flexible enough 
 to accommodate a large group such as the n - propyl 
 group . the kinetic data obtained from these studies together 
 with structural information revealed by the co - crystals suggest that 
 the setd2-catalyzed methylation goes through a random sequential mechanism 
 and that inhibition occurs via either a 28setd2 
 binary complex or a 28setd2substrate 
 ternary complex . methylation 
 of h4k20 , which is considered to be a transcriptionally repressive 
 mark , is catalyzed by pkmts : suv420h1 , suv420h2 , and setd8 ( set domain 
 containing ( lysine methyltransferase ) 8) in humans . the latter , also known as set8 , pr - set7 , or kmt5a , is the 
 sole methyltransferase that catalyzes monomethylation of h4k20 . setd8 and h4k20me1 ( h4k20 monomethylation ) have been implicated 
 in regulating a diverse set of biological processes including the 
 dna damage response , dna replication , and mitotic condensation . in 
 addition to h4k20 , setd8 methylates many nonhistone substrates including 
 the tumor suppressor p53 and proliferating 
 cell nuclear antigen ( pcna ) . the monomethylation 
 of p53 lysine 382 by setd8 represses p53 target genes . the monomethylation of pcna by setd8 at lysine 
 248 stabilizes pcna protein and increases the interaction between 
 pcna and the flap endonuclease fen1 , which promotes the proliferation 
 of cancer cells . the first selective 
 inhibitor of setd8 is a marine natural product nahuoic acid a ( 29 ) ( figure 20).29 inhibited setd8 with an ic50 of 6.5  0.5 m and was competitive with the cofactor 
 sam ( ki = 2.0  0.3 m ) and 
 noncompetitive with the peptide substrate . it was selective for setd8 
 over 10 other methyltransferases including g9a , glp , setd7 , suv39h2 , 
 suv420h1 , suv420h2 , prmt3 , prmt5 , carm1 , dot1l , prc2-ezh2 , dnmt1 , 
 and mll . very recently , ma et al . reported the first substrate - competitive 
 inhibitor of setd8 , 30 ( unc0379 , figure 20 ) . this small - molecule inhibitor 
 was discovered by cross - screening of about 150 quinazoline - based compounds 
 against setd8 . compound 30 displayed inhibitory activity 
 against setd8 with micromolar potency in multiple biochemical assays . binding of this inhibitor to setd8 was confirmed by biophysical studies 
 such as itc and spr . importantly , 30 was selective for 
 setd8 over 15 other methyltransferases including g9a , glp , setdb1 , 
 setd7 , suv39h2 , suv420h1 , suv420h2 , prc2ezh2 , mll1 , smyd2 , 
 prmt1 , prmt3 , prmt5 , dot1l , and dnmt1 . in moa studies , inhibitor 30 was competitive with the peptide substrate and noncompetitive 
 with sam . however , in vitro potencies of 29 and 30 are modest , and cellular activities of these inhibitors 
 have not 
 been reported . in addition , selective inhibitors of suv420h1 and suv420h2 , 
 the other two h4k20 pkmts , have not been published . most recently , 
 another report on small - molecule inhibitors of setd8 
 was published by blum et al . screening 
 of more than 5000 commercial compounds resulted in the discovery of 
 three setd8 inhibitors : 31 ( sps8i1 , also known as nsc663284 , 
 ic50 = 0.21  0.03 m ) , 32 ( sps8i2 , 
 also as known as bvt948 , ic50 = 0.50  0.20 m ) , 
 and 33 ( sps8i3 , as known as ryuvidine , ic50 = 0.70  0.20 m ) ( figure 20 ) . the selectivity of these inhibitors was evaluated 
 against other pmts including setd2 , glp , g9a , smyd2 , and setd7 as 
 well as prmt1 , prmt3 , and carm1 . inhibitor 31 was only 
 2.5-fold selective over smyd2 and > 6-fold selective over other 
 pmts 
 tested . similarly , 32 showed modest selectivity over 
 setd2 , g9a , smyd2 , carm1 , and prmt3 , whereas 33 was less 
 selective . further mechanistic studies suggested that 31 ( substrate dependent ) , 32 ( no substrate or sam dependence ) , 
 and 33 ( both substrate and sam dependent ) inhibited setd8 
 via distinct modes of action . since structures of these three inhibitors 
 shared a common quinonic motif , which could react with active cysteine 
 residues , further mechanistic studies were performed . from these studies , it was concluded that 3133 inhibited 
 setd8 via an irreversible slow - onset process . it is worth noting that 
 other commercially available compounds containing a simple and related 
 quinonic motif did not inhibit setd8 , suggesting that the full structures 
 of these inhibitors are necessary for the inhibition . cys270 of setd8 
 was identified as the reactive residue for 31 and 32 , whereas 33 targeted cysteine residues in 
 a nonspecific manner . in hek293 t cells , the h4k20me1 mark was reduced 
 within 24 h of treatment with the inhibitors , whereas other histone 
 marks ( e.g. , h4k20me2/3 , h3k9me ) were not affected . in addition , these 
 inhibitors at 15 m produced a cell cycle arrest phenotype , 
 similar to that of setd8 knockdown . however , off - target effects on 
 other pmts ( 31 for smyd2 and 33 for prmt3 
 and setd2 ) and other cellular targets ( 31 , inhibition 
 of cdc25 ; 32 , inhibition of cyclin - dependent kinase 4 
 and 2 ( cdk4/2 ) ; and 33 , inhibition of protein tyrosine 
 phosphatase ptb1b ) were observed and documented . dot1l ( disruptor 
 of telomeric silencing 1-like ) is the only pkmt identified in humans 
 that does not contain the set domain . dot1l , also known as kmt4 , has a non - set catalytic domain , which 
 adopts a folding topology that is also observed in prmts and dnmts . it has been shown that dot1l is responsible for mono- , di- , and 
 trimethylation of h3k79 . methylation of h3k79 , 
 which is generally associated with transcriptional activation , has 
 been linked with transcriptional regulation , dna repair , embryonic 
 development , cell cycle regulation , hematopoiesis , and cardiac function . importantly , dot1l has been shown to interact with af4 , af9 , af10 , 
 af6 , and enl , the most commonly seen mll fusion proteins in mll - rearranged 
 leukemias . dot1l interacts with these mll fusion proteins and is recruited 
 to their target genes including leukemogenic genes such as hoxa9 and meis1 . therefore , dot1l has been considered as a potential therapeutic target 
 for the treatment of mll - rearranged leukemia . in 2011 , daigle et al . reported the first selective dot1l 
 inhibitor , 34 ( epz004777 ) , which was designed and synthesized 
 based on the cofactor sam and the crystal structure of the enzyme 
 active site ( figure 21 ) . inhibitor 34 displayed very high in vitro 
 potency ( ic50 = 400  100 pm ) and was remarkably selective 
 ( more than 1000-fold ) for dot1l over nine other pmts including g9a , 
 setd7 , whsc1 , ezh1 , ezh2 , prmt1 , prmt5 , prmt8 , and carm1 , despite 
 being structurally close to sam , the universal methyl donor for all 
 methyltransferases . as expected , 34 was competitive with 
 sam and noncompetitive with the peptide substrate in moa studies . the very high binding affinity ( ki = 300 pm ) of 34 was mainly driven by its 
 slow off rate , leading to a very long residence time the x - ray crystal 
 structure of dot1l34 published in 2012 ( figure 22 ) not only confirmed that 34 was a 
 cofactor - competitive inhibitor but also 
 revealed that the high affinity and long residence time of 34 was driven by a ligand - induced conformational adaptation of dot1l . a global reduction in h3k79me2 levels 
 was observed in cell lines 
 molm-13 , mll - af9 ( derived from human mll - rearranged aml cell line ) , 
 mv4 - 11 , mll - af4 ( mll - rearranged biphenotypic leukemia ) , and jurkat 
 ( non - mll - rearranged t - cell acute leukemia ) after the treatment with 34 . the full effect on the reduction 
 of h3k79me2 levels was observed in 45 days . levels 
 were also reduced by the inhibitor , whereas trimethylation levels 
 could not be measured . in addition , no significant reduction of other 
 histone methylation marks ( e.g. , h3k4me3 , h3k9me3 , h3k27me2 , h3k27me3 , 
 h3k36me2 , h4k20me2 , h3r17me2a , h4r3me2s ) was observed , suggesting 
 that the cellular action of 34 is specific . compound 34 concentration - dependently inhibited the 
 expression of hoxa9 and meis1 , the 
 overexpression of which is the hallmark of mll - rearranged leukemia . the maximum reduction of hoxa9 and meis1 mrna levels in molm-13 and mv4 - 11 cells 
 was observed after 68 days of inhibitor treatment . in these 
 cell lines , it is worth 
 mentioning that a significant delay ( 68 days ) was observed 
 for the antiproliferative effect , consistent with the time course 
 of this inhibitor s effect on hoxa9 and meis1 . furthermore , 34 displayed an antiproliferative 
 effect against six other mll - rearranged leukemia cell lines with low 
 micromolar potencies , but it was largely ineffective in six non - mll - rearranged 
 leukemia cell lines ( ic50 > 10 m ) . in addition , 
 gsea of the genes up- and downregulated in the treated mv4 - 11 and 
 molm-13 cells suggests that dot1l inhibition by 34 reverses 
 the mll - rearranged leukemia gene signature . taken together , these 
 results suggest that the dot1l methyltransferase activity is essential 
 for the proliferation of mll - rearranged leukemia cells and mll fusion 
 mediated transformation but is nonessential for the proliferation 
 of non - mll - rearranged leukemia cells . importantly , continuous 
 infusion of 34 via an implanted 
 osmotic minipump for 14 days dose - dependently increased survival in 
 nsg ( nod scid gamma ) mice after intravenous injection of mv4 - 11 cells . the h3k79me2 levels in subcutaneous mv4 - 11 tumors derived from 34-treated animals were significantly reduced . thus , it was 
 demonstrated for the first time that selective inhibition of dot1l s 
 methyltransferase activity had antitumor activity in animal models 
 of mll - rearranged leukemia . these results highlight 
 a clinical potential of selective dot1l inhibition as a means for 
 treating mll - rearranged leukemia . shortly after the publication 
 of inhibitor 34 , yao 
 et al . reported the discovery of compound 35 ( figure 21 ) , a selective , mechanism - based inhibitor of dot1l . this inhibitor displayed high in vitro potency 
 for dot1l ( ic50 = 38 nm ) and was more than 29-fold selective 
 for dot1l over other methyltransferases tested : carm1 , prmt1 , g9a , 
 and suv39h1 . it was suggested that compound 35 would 
 undergo an intramolecular cyclization to form a reactive aziridinium 
 intermediate , which would further react with the -nh2 group of the lysine 79 to covalently link to h3k79 . in addition , 
 yao et al . noticed that the 6-nh2 group of sam ( adenosine 
 moiety nh2 group ) forms only one hydrogen bond with the 
 enzyme and that there is a relatively large hydrophobic pocket available 
 in the co - crystal structure of the dot1l  sam complex . on the 
 other hand , set domain - containing pkmts such as g9a form two hydrogen 
 bonds with the 6-nh2 group . therefore , yao et al . designed 
 and synthesized 6-n - methyl sah and found that it 
 was indeed highly selective for dot1l ( ki = 290 nm ) over carm1 , prmt1 , g9a , and suv39h1 ( ki > 20  000 nm ) . the x - ray crystal structure 
 of 
 dot1l in complex with 6-n - methyl sah confirms that 
 the n - methyl group sits in the hydrophobic pocket . activities of these dot1l inhibitors in cell - based assays were not 
 reported . in a continuation of this work , anglin et al . reported 
 extensive 
 sar studies , which led to the discovery of compound 36 ( figure 21 ) . compound 36 had high in vitro potency ( ki = 0.46 nm ) and was more than 4500-fold selective for 
 dot1l over carm1 , prmt1 , and suv39h1 . an alkyl group such as methyl , 
 ally , and benzyl on the 6-amino group was well - tolerated ( dotl1l ki of 0.76 , 12 , and 22 nm , respectively ) and 
 led to high selectivity for dot1l . compound 36 inhibited 
 the proliferation of mv4 - 11 cells with a slow onset of activity , but 
 it did not affect the proliferation of nb4 cells , which harbor wild - type 
 mll . yu et al . , in december 2012 , reported a chemical probe 
 of dot1l 
 with improved in vitro and cellular potencies . the co - crystal structure of dot1l34 that they obtained revealed remodeling of the catalytic site ( figure 22 ) , consistent with 
 the ligand - induced conformational adaptation reported by basavapathruni 
 et al . it was noticed that a hydrophobic 
 cleft near the 7-position of the deazaadenosine moiety was not exploited . therefore , a focused set of analogues aimed at exploiting this hydrophobic 
 pocket was synthesized , resulting in the discovery of 37 ( sgc0946 ) , which has a bromo substitution at the 7-position of the 
 deazaadenosine ring ( figure 21 ) . this inhibitor was more potent than 34 in biochemical and biophysical assays ( e.g. , kd = 0.06 nm versus 0.25 nm in spr ) . similar to 34 , compound 37 was highly 
 selective for dot1l over 13 other methyltransferases including g9a , 
 glp , suv39h2 , suv420h1 , suv420h2 , mll , setdb1 , setd7 , setd8 , prc2ezh2 , 
 prmt3 , prmt5 , and dnmt1 . 37 was also almost 10-fold more 
 potent at reducing h3k79 methylation levels in mcf10a cells , with 
 an ic50 of 8.8  1.6 nm , than that of 34 ( ic50 = 84  20 nm ) . furthermore , 37 ( at 1 m ) was more effective than 34 at killing 
 human cord blood cells transformed with an mll af9 fusion oncogene , 
 whereas it ( at 1 and 5 m ) did not affect viability of cord 
 blood cells transformed with an unrelated oncogene , tls - erg . structures 
 of dot1l inhibitors . in 2013 , daigle et al . reported a new dot1l inhibitor , 38 ( epz-5676 ) , as a result of their structure - guided design 
 and optimization 
 of the compound 34 series ( figure 21 ) . the x - ray co - crystal structure of 38 in complex with dot1l clearly showed that the inhibitor 
 occupied the sam binding pocket and induced conformational changes 
 in dot1l ( pdb code : 4hra ) . compound 38 inhibited dot1l with a morrison ki of 0.08  0.03 nm , which is more potent 
 than that of 34 ( morrison ki = 0.3  0.02 nm ) . it was more than 37  000-fold selective 
 for dot1l over 16 other pmts including g9a , glp , setd7 , smyd2 , smyd3 , 
 mmset , whsc1l1 , prmts ( 1 , 3 , 4 , 5 , 6 , and 8) , ezh1 , and ezh2 . co - crystal 
 structure of dot1l34 complex ( pdb 
 code : 4er3 ) 
 ( left ) . overlay of dot1l34 and dot1l37 complexes ( pdb code : 4er6 ) ( right ) . inhibitor 38 reduced h3k79me2 levels in mv4 - 11 
 cells 
 ( a mll - af4 expressing acute leukemia cell line ) with an ic50 of 3 nm and in hl-60 cells ( a non - mll - rearranged cell line ) with 
 a similar potency . no significant reduction of other 
 histone methylation marks ( h3k4me3 , h3k9me3 , h3k27me2 , h3k27me3 , h3k36me2 , 
 h4k20me2 , h3r17me2a , and h4r3me2s ) was observed . additionally , 38 concentration - dependently inhibited hoxa9 and meis1 mrna levels in mv4 - 11 cells . the proliferation 
 of mv4 - 11 cells treated with 38 for 
 14 days was inhibited with an ic50 of 3.5 nm . the antiproliferative 
 activity was observed as early as 4 days , but it reached a maximum 
 at day 7 . this delayed onset of activity is likely due to a cascade 
 of epigenetic events including the depletion of the h3k79me2 mark , 
 inhibition of mll fusion target gene expression , and a reversal in 
 leukemogenic gene expression . in addition , 38 exhibited nanomolar antiproliferative 
 activity against most of other mll - rearranged cell lines tested but 
 weaker potencies against non - mll - rearranged cell lines . importantly , 
 continuous intravenous ( iv ) infusion of 38 at 70 mg / kg 
 per day for 21 days resulted in complete elimination of the established 
 subcutaneous ( sc ) mv4 - 11 tumors in immunocompromised rats . the tumor 
 regression was sustained for more than 30 days after the cessation 
 of compound 38 treatment . all doses were well - tolerated 
 by the test animals , and no significant body weight loss was observed . furthermore , h3k79me2 levels and hoxa9 and meis1 mrna levels were significantly reduced in mv4 - 11 sc 
 xenograft tissue harvested from rats dosed by continuous iv infusion 
 for 14 days . inhibition of h3k79 methylation was also observed in 
 bone marrow cells and pbmcs isolated from the same rats . taken together , 
 these results suggest that 38 displays on - target activity 
 in vivo and has a potential to be an effective therapeutics for the 
 treatment of mll - rearranged leukemia . in 2013 , compound 38 became the first pmt inhibitor advanced to the clinic , a watershed 
 event in the pmt inhibitor field . it is currently being evaluated 
 in phase 1 clinical trials for the treatment of aml and all . protein arginine 
 methylation catalyzed by prmts is another important 
 and common type of ptm in eukaryotic cells . arginine is unique among amino acids with its ability to form interactions 
 via its five potential hydrogen - bond donors with surrounding hydrogen - bond 
 acceptors . every methylation of arginine would take away a potential 
 hydrogen bond as well as create steric bulkiness and increased hydrophobicity . importantly , methylation does not neutralize the cationic charge of 
 arginine residues , and it was suggested that methylation could enhance 
 their interactions toward aromatic rings via cation 
 interactions . thus , methylation of arginine 
 residues in proteins can change their recognition and in turn affect 
 their physiological functions . nine prmts have been identified to date , and they are responsible 
 for mono- and/or dimethylation of the guanidino group of arginine . as we mentioned earlier , there are two possible 
 ways for dimethylation to occur after monomethylation of arginine 
 ( mma ) : either by methylating the same nitrogen , yielding asymmetrical 
 dimethyl arginine ( adma ) , or by methylating another nitrogen to give 
 symmetrical dimethyl arginine ( sdma ) ( scheme 1 ) . on the basis of their methylation functions , prmts are divided 
 into three categories : type i , type ii , and type iii . type i prmts , which include prmt1 ( protein 
 arginine methyltransferase 1 ) , prmt2 , prmt3 , carm1 ( coactivator - associated 
 arginine methyltransferase 1 , also known as prmt4 ) , prmt6 , and prmt8 , 
 catalyze monomethylation and asymmetric dimethylation of arginine . on the other hand , prmt5 is a type ii prmt that catalyzes monomethylation 
 and symmetrical dimethylation of arginine . prmt7 is categorized as 
 a type iii prmt , as it catalyzes monomethylation of arginine only . prmt9 ( also known as f - box only protein 11 ( fbxo11 ) ) has not been 
 classified yet because its activity has not been fully characterized . they typically have additions to the n - terminus 
 with the exception of carm1 , which also has c - terminal additions . the monomeric structure of the prmt core comprises 
 a mtase domain , a -barrel that 
 is unique to prmts , and a dimerization arm . a homodimeric structure 
 was observed in prmt1 and prmt3 , and it has been suggested that dimer 
 formation is a conserved feature for prmts . prmts generally methylate glycine and arginine - rich ( gar ) motifs 
 in their substrates with the exception 
 of carm1 , which specifically methylates proline , glycine , and methionine - rich 
 ( pgm ) motifs . prmt5 , on the other hand , can 
 symmetrically dimethylate both of these motifs . dysregulation of prmts and arginine 
 methylation have been implicated in cancer and other diseases . it has been shown that prmt1 is responsible for most of 
 the type i arginine methyltransferase activity in mammalian cells . prmt1 catalyzes asymmetrical dimethylation 
 of h4r3 ( h4r3me2a ) , which is associated with transcriptional activation . overexpression as well as aberrant splicing of prmt1 has been implicated 
 in diseases such as breast , prostate , lung , colon , and bladder cancers 
 and leukemia . additionally , prmt1 has been associated with human telomeres and shown to directly regulate the akt signaling 
 pathway . large numbers of nonhistone 
 substrates such as dna repair proteins mre11 , p53 binding protein 1 ( 53bp1 ) , ash2l , and the tumor suppressor brca1 have been identified for prmt1 . even 
 though there has been continuous interest in the discovery of selective 
 prmt1 inhibitors over the past decade , most of the reported prmt1 
 inhibitors ( figure 2 and table 2 ) lack sufficient potency and selectivity against a broad 
 panel of pmts , thus limiting their potential use in functional studies . in 2004 , cheng et al . reported the discovery of the first small - molecule 
 inhibitors of prmts , named amis ( arginine methyltransferase inhibitors ) , 
 by hts of a 9000-compound library . among 
 the nine hits identified , only 39 ( ami-1 ) , a symmetrical 
 sulfonated urea salt , and ami-6 ( figure 23 ) 
 showed specificity for prmts over pkmts . therefore , it was suggested that this inhibitor binds in 
 the substrate binding pocket . it was reported that 39 inhibited the methylation of npl3p in hela cells in a concentration - dependent 
 manner . in 2007 , ragno et al . published their structure- and ligand - based 
 modeling studies on amis and their close analogues , and , although 
 ami-5 was confirmed to be a prmt1 inhibitor ( ic50 = 1.4 
 m ) , selectivity of these inhibitors was not reported ( figure 23 ) . in the same year , 
 a target - based approach to discover 
 inhibitors of prmts by spannhoff et al . resulted in prmt inhibitors 
 stilbamidine and allantodapsone with ic50 s of 57 
  6.2 and 1.7  3 m for prmt1 , respectively . spannhoff et al . also reported the prmt1 inhibitor 
 rm65 ( ic50 = 55  3.4 m ) ( figure 23 ) , which was identified via virtual screening . structures of small - molecule 
 inhibitors of prmt1/prmts . in an effort to develop more potent inhibitors of prmts , 
 bonham 
 et al . discovered compound 40 , which inhibited prmt1 
 ( ic50 = 4.2  1.6 m ) and carm1 ( ic50 = 2.6  0.6 m ) . however , 40 ( figure 23 ) also inhibited prmt5 , 
 prmt6 , and prmt8 even though it was selective against setd7 . in the 
 same year ( 2010 ) , feng et al . reported the discovery of compound 41 , named ns1 ( naphthalene - sulfo derivative 1 ) , via virtual 
 screening of 400  000 compounds ( figure 23 ) . compound 41 inhibited 
 prmt1 with an ic50 of 13  0.1 m . moa studies 
 suggested that this inhibitor was competitive with the substrate with 
 a ki of 1.7  0.54 m . however , 
 even though 41 did not inhibit carm1 , it inhibited prmt3 
 and prmt6 with similar potencies . in 2011 , dowden et al . reported 
 a sam derivative as a prmt1 inhibitor . compound 42 inhibited prmt1 with an ic50 of 
 3.9  1.8 m and was inactive against carm1 and setd7 ( figure 23 ) . in 2012 , dillon 
 et al . reported the discovery of two mechanism - based inhibitors , cid 
 5380390 and cid 2818500 ( figure 23 ) , which 
 inhibited prmt1 and prmt8 , the only two prmts that contain a reactive 
 cysteine in their active sites . in the same year , 
 wang et al . reported the discovery of prmt1 inhibitor 43 ( a36 ) via pharmacophore - based virtual screening ( figure 23 ) . it was about 7-fold more potent for prmt1 over carm1 , but 
 it was only 2-fold more potent over prmt5 . yan et 
 al . reported compound 44 , a diamidine containing prmt1 
 inhibitor ( figure 23 ) . it inhibited prmt1 with an ic50 of 9.4  
 1.1 m and was selective for prmt1 over carm1 ( more than 42-fold ) , 
 prmt5 ( around 18-fold ) , and prmt6 ( around 30-fold ) . it was also found that cell lines derived from down syndrome patients 
 and mll - af9 patients ( cmy , chrf-288 - 1 , and molm-13 cells ) were more 
 sensitive to compound 44 treatment than other cell lines 
 tested ( hel , jurkat , and hl-60 ) . this 
 type i prmt is located mainly in cytosol and has a zinc finger domain 
 at its n - terminus . asymmetric dimethylation of 
 rps2 by prmt3 results in stabilization of rps2 and influences ribosomal 
 biosynthesis . prmt3 has also been reported to methylate the recombinant mammalian 
 nuclear poly(a)-binding protein ( pabpn1 ) and a histone peptide ( h4 124 ) in vitro . the protein complex consisting of prmt3 , the von hippel - lindau 
 ( vhl ) tumor suppressor , and arf ( alternative reading frame ) has been 
 shown to methylate the tumor suppressor p53 . in addition , the tumor suppressor dal-1 ( differentially expressed 
 in adenocarcinoma of the lung-1 ) inhibits the methyltransferase activity 
 of prmt3 by interacting with it , suggesting that dal-1 may affect 
 tumor growth by regulating prmt3 function . furthermore , prmt3 expression levels are 
 elevated in myocardial tissues from patients with atherosclerosis , potentially implicating the involvement of 
 prmt3 . additionally , prmt3 function has been reported to be essential 
 for dendritic spine maturation in rats . in 2012 , siarheyeva et al . reported the discovery of the first 
 selective prmt3 inhibitor , compound 45 ( figure 24 ) , via screening a library of 16  000 diversity 
 compounds . compound 45 inhibited 
 prmt3 with an ic50 of 1.6  0.3 m and was selective 
 for prmt3 over other methyltransferases including g9a , glp , suv39h2 , 
 setd7 , setd8 , prmt1 , carm1 , prmt5 , and prmt8 . this inhibitor displayed 
 rapid on and off rates with kd of 9.5 
  0.5 m . interestingly , in moa studies , this inhibitor 
 was noncompetitive with both sam and the peptide substrate . the x - ray 
 crystal structure of the prmt3inhibitor 45 complex 
 reveals that it occupies a novel allosteric binding site located at 
 the interface of the two subunits of the prmt3 homodimer ( figure 25 ) . the cyclohexenyl moiety interacts with the alpha - y 
 segment of the activation helix of the opposite subunit . it has 
 been shown that the proper folding of the alpha - x segment is crucial 
 for both cofactor and substrate binding . thus , it is most likely that 
 the binding of 45 to the allosteric site prevents the 
 proper positioning / folding of the alpha - x segment , which in turn inhibits 
 the enzymatic activity of prmt3 . protein interactions 
 revealed by the co - crystal structure include ( 1 ) a hydrogen bond between 
 the middle nitrogen of the tightly fit benzothiadiazole moiety with 
 the hydroxyl group of t466 , ( 2 ) two hydrogen bonds between the two 
 nitrogens of the central urea moiety and the carboxylate group of 
 e422 , ( 3 ) and a hydrogen bond between the oxygen of the urea moiety 
 with the guanidinium group of r396 ( figure 25 ) . the key hydrogen - bond interactions were confirmed by sar studies 
 in addition to site - directed mutagenesis studies . subsequent 
 sar studies by liu et al . resulted in the discovery 
 of more potent inhibitors compounds 46 ( ic50 = 0.48 m ) and 47 ( ic50 = 0.23 m ) 
 ( figure 24 ) . these 
 inhibitors possess the same benzothiodiazole and urea moieties but 
 differ in the right - hand side functional group . compound 46 contains a benzoyl group , whereas compound 47 has a 
 piperidinyl amide . similar to 45 , compound 46 showed excellent selectivity for prmt3 over other methyltransferases 
 including g9a , glp , suv39h2 , prmt5 , setd7 , prc2 , setd8 , setdb1 , suv420h1 , 
 suv420h2 , mll1 , smyd3 , smyd2 , dot1l , and dnmt1 . the crystal structure 
 of the prmt346 complex confirmed that this inhibitor 
 occupies the same allosteric binding site . taken 
 together , these results suggest that the allosteric binding site of 
 prmt3 can be exploited to yield potent and selective inhibitors co - crystal structure of the dimeric prmt345 complex 
 ( pdb code : 3smq ) ( left ) . co - activator - associated 
 arginine methyltransferase 1 ( carm1 , also known as prmt4 ) activates 
 the transcription by asymmetric dimethylation of h3r17 . it was first identified as a steroid receptor coactivator and was 
 the first member of the prmts to be associated with transcriptional 
 regulation . the loss of carm1 results 
 in neonatal lethality , evidenced by the death of newborn knockout 
 mice shortly after birth . carm1 has 
 been shown to be involved in mrna splicing , rna processing and stability , cell 
 cycle progression , and dna damage response . in addition to histones , carm1 methylates a 
 variety of proteins such as cbp / p300 , pabp , hur , hud , ca150 , sap49 , 
 smb , and u1c . carm1 levels have shown to be elevated in castration - resistant prostate 
 cancer and aggressive breast tumors . because of the involvement of carm1 in a wide 
 variety of biological processes and diseases , it has been pursued as a potential therapeutic target . reported the discovery of the carm1 inhibitor compound 48 ( figure 26 ) via optimization of 
 a pyrrazole containing hit , which was identified by a hts campaign . this inhibitor was potent with an ic50 of 40 nm and more 
 than 600-fold selective for carm1 over prmt1 and prmt3 . however , additional 
 selectivity , moa , and cellular activity data were not reported . therrien 
 et al . also reported the discovery of potent carm1 inhibitors , which 
 were based on the reported pyrrazole core . structures 
 of selected inhibitors of carm1 ( prmt4 ) . in 2010 , selvi et al . identified a carm1 inhibitor , tbbd 
 ( ellagic 
 acid , 49 ) , which was isolated from pomegranate crude 
 extract ( figure 26 ) . compound 49 concentration - dependently inhibited carm1 , 
 but it did not inhibit g9a and histone acetyltransferase cbp / p300 . burk plots that 49 was noncompetitive with both h3 and sam . however , itc experiments 
 showed minimal interaction between 49 and carm1 alone . it was suggested that the partial inhibition of carm1 by 49 could be mediated via its interaction with the enzyme in addition , 49 significantly 
 reduced expression levels of p21 in h1299 and hek293 t 
 cells but not in hela cells . as we discussed throughout this perspective , a 
 growing body of 
 evidence suggests that protein methyltransferases play a key role 
 in the regulation of transcriptional activity and are implicated in 
 cancer and many other human diseases . due to these key functions , there has been a steadily growing interest in pursuing these enzymes 
 as potential therapeutic targets . therefore , discovery of selective , 
 small - molecule inhibitors of these methyltransferases has become a 
 very active area of research over the past decade . there has been 
 tremendous progress in the pmt inhibitor field as a result of collective 
 advances made in assay development , high - throughput screening , structural 
 biology , and medicinal chemistry . this research area was kicked off 
 by the reports of the first pkmt inhibitor chaetocin in 2005 and the 
 first prmt inhibitor 39 in 2004 and culminated by the 
 initiations of human clinical trials for the dot1l inhibitor 38 in 2012 and the ezh2 inhibitors 22 in 2013 
 and 19 in 2014 . highly potent , selective , well - characterized 
 chemical probes with robust on - target activities in cells have been 
 developed . these chemical probes including 11 , 12 , 18 , 19 , 20 , 21 , 22 , 34 , 37 , and 38 are valuable tools for further investigating the biological 
 functions of the targeted enzymes and assessing the potential of these 
 proteins as therapeutic targets . the discoveries of substrate - competitive 
 inhibitors of g9a / glp ( e.g. , 6 , 11 , 12 , 16 ) , smyd2 ( 25 and 26 ) , setd8 ( 30 ) , and setd7 ( 24 ) suggest that 
 the substrate binding groove of pkmts can be targeted to yield potent 
 and selective inhibitors . similarly , the discoveries of highly potent , 
 selective , and sam - competitive inhibitors of dot1l ( e.g. , 34 , 37 , 38 ) and ezh2 ( e.g. , 18 , 19 , 20 , 21 , 22 ) provided experimental evidence that extremely high selectivity 
 can be achieved by targeting the sam binding site of pkmts , which 
 is analogous to targeting the atp binding site of protein kinases . in addition , the discovery of the prmt3 allosteric inhibitors ( e.g. , 46 and 47 ) suggests that the allosteric binding 
 site of prmt3 and potentially other prmts can be exploited to create 
 potent and selective inhibitors . despite the significant progress 
 that has been made over last 10 
 years first and foremost , 
 a systematical coverage of pmts as a protein family with potent and 
 selective inhibitors is needed . currently , many individual targets 
 and subgroups of targets on the pmt phylogenetic tree lack selective 
 inhibitors ( figure 2 ) . for example , there are 
 no selective inhibitors reported for smyd3 , mmset ( nsd2 ) , and prmt5 , 
 which are potentially important therapeutic targets . potent , selective , 
 and cell - penetrant inhibitors of these pmts would be invaluable tools 
 for therapeutic hypothesis testing and target validation . on the other 
 hand , there is limited understanding of biological functions and potential 
 disease implications for many pmts on the phylogenetic tree , and no 
 selective inhibitors have been reported for many of these targets 
 ( e.g. , prdms ) . chemical probes of these proteins that have sufficient 
 potency , selectivity , and cell permeability would be extremely valuable 
 for investigating and understanding their biological functions . second , 
 thorough characterization in biochemical , biophysical , and cellular 
 assays is needed for some of the existing inhibitors and future inhibitors . 
 while the chemical probes discussed earlier were well - characterized , 
 a number of other inhibitors need to be thoroughly characterized . 
 for the inhibitors to be used in in vitro studies , in addition to 
 activities in biochemical assays , a direct interaction between the 
 target and the small - molecule inhibitor should be demonstrated by 
 a biophysical method ( e.g. , itc , spr ) or by a nmr solution or x - ray 
 crystal structure of the protein  ligand complex . selectivity 
 of these inhibitors for the target pmt(s ) over a broad panel of other 
 methyltransferases should also be assessed and achieved . for the inhibitors 
 to be used in cellular studies , sufficient cell permeability and target 
 engagement in cells should be demonstrated in addition to the in vitro 
 target engagement and selectivity described above . these characterizations 
 are necessary for associating the observed biological effects with 
 the inhibition of the target pmt(s ) by the ligands . third , it has 
 been challenging to discover potent , selective , and cell - penetrant 
 inhibitors of prmts . a breakthrough in this area will be truly exciting 
 and is keenly awaited . fourth , understanding of the molecular basis 
 for high subtype selectivity is needed . while the ligand - induced dot1l 
 conformation adaptation provides an excellent explanation for the 
 extremely high selectivity observed for dot1l inhibitors 34 and 37 , there is no satisfactory 
 explanation for the high selectivity ( > 150-fold ) of 19 for ezh2 over ezh1 , as ezh2 and ezh1 share 96% sequence identity 
 in their set domains . high - resolution structures of the ezh2inhibitor 
 and ezh1inhibitor complexes would likely shed light on how 
 such high subtype selectivity is achieved . it would also be valuable 
 to demonstrate that this level of high selectivity can be achieved 
 for other closely related pkmts . for example , g9a and glp share 80% 
 sequence identity in their set domains , and all reported inhibitors 
 have similar potencies for both g9a and glp . a highly selective g9a 
 or glp inhibitor would be a useful tool for dissecting the role of 
 g9a or glp in biological systems . fifth , improvement on pk properties 
 of several in vivo probes would be beneficial . as described earlier , the development of inhibitor 38 , the first pkmt clinical 
 candidate , was a watershed event in the field . however , continuous 
 intravenous infusion of this dot1l inhibitor via an osmotic minipump 
 was required to achieve sustained tumor regression . the next generation 
 of dot1l inhibitors that are orally bioavailable would be beneficial 
 for patients . it would be interesting to see whether good oral bioavailability 
 can be achieved for the inhibitors derived from the sam scaffold . similarly , improvement on plasma exposure and therapeutic window of 12 , the first in vivo chemical probe of g9a and glp , would 
 make it more suitable for chronic animal studies . sixth , there are 
 opportunities to generate chemical tools such as biotinylated ligands 
 of pmts by exploiting recently developed high - affinity inhibitors . 
 these tools would be very useful in chemical biology studies such 
 as chemoproteomics , chem  chip , and chem  seq . in 
 this perspective , we highlight the key progress on the discovery , 
 characterization , and application of selective pmt inhibitors for 
 investigating physiological functions and disease implications of 
 the target pmts . we also discuss challenges and future directions 
 and opportunities in the pmt inhibitor field . it is our hope that 
 this perspective will inspire new and original discoveries . over the 
 last 10 years , we have witnessed amazing progress in this emerging 
 research field , culminated by three pkmt inhibitors entering clinical 
 trials in 20122014 . it is anticipated that the next 10 years 
 will be even more exciting for this now very active area . the biomedical 
 community eagerly awaits the clinical successes of selective pmt inhibitors 
 and discoveries of many potent , selective , cell - penetrant , first - in - class 
 pmt inhibitors .
mounting 
 evidence suggests that protein methyltransferases ( pmts ) , 
 which catalyze methylation of histone and nonhistone proteins , play 
 a crucial role in diverse biological processes and human diseases . 
 in particular , 
 pmts have been recognized as major players in regulating 
 gene expression and chromatin state . 
 pmts are divided into two categories : 
 protein lysine methyltransferases ( pkmts ) and protein arginine methyltransferases 
 ( prmts ) . 
 there has been a steadily growing interest in these enzymes 
 as potential therapeutic targets and therefore discovery of pmt inhibitors 
 has also been pursued increasingly over the past decade . here , we 
 present a perspective on selective , small - molecule inhibitors of pmts 
 with an emphasis on their discovery , characterization , and applicability 
 as chemical tools for deciphering the target pmts physiological 
 functions and involvement in human diseases . 
 we highlight the current 
 state of pmt inhibitors and discuss future directions and opportunities 
 for pmt inhibitor discovery .