1. Shinkai and Tachibana, Genes&Dev. 2011

3. Activation of Serine-Glycine Synthesis Pathway Repression of Tumor Suppressor gene Tumorigenesis K9 H3K9me1 H3K9me2 H3K9me3 Autophage Tumor suppressor gene Activation & Apoptosis Inhibition of Cancer cell Proliferation & Survival Normal

4. Copeland et al. Nat. Rev. Drug Discovery, 2009 /G9a

5. Wu H, Min J, Lunin VV, Antoshenko T, Dombrovski L, Zeng H, Allali-Hassani A, Campagna-Slater V, Vedadi M, Arrowsmith CH, Plotnikov AN, Schapira M - PLoS ONE (2010)

6. Commercially Available Compounds Leadlike Compound Subset ~ 18 Million 2D library ~ 1.6 Million 3D library Vendor Query with Top 50 hit compounds Standard- ization 3D Structure Generation Tautomer Enumeratio n 1 1 2 2 3 3 4 4 Conformer Enumeratio n 2 nd round similarity search with the compounds having enzyme inhibition activity Functional Screening in Cancer cellsFunctional Screening using ELISA Chemical Modification Anticancer Effect Analyses NCI-60 Human Tumor Cell Line Screen & In Vivo Test SAH for G9a

7. RankingZINC IDMWNAMENCI compounds 1ZINC29786412267.241gamma-L-Glutamyl-4-nitroanilide 2ZINC01635650270.312-amino-3-{[2-(4-nitrophenyl)ethyl]sulfanyl}propanoic acidNSC163413 3ZINC16899833338.385(2S)-2-amino-3-[(3S)-1-(4-ethoxyphenyl)-2,5-dioxo-pyrrolidin-3-yl]sulfanyl-propanoic 4ZINC16899497337.3334-[(3S)-3-[(2S)-2-amino-3-hydroxy-3-oxo-propyl]sulfanyl-2,5-dioxo-pyrrolidin-1-yl]benzoic 5ZINC04188103297.2712-(6-aminopurin-9-yl)-6-(hydroxymethyl)tetrahydropyran-3,4,5-triol 6ZINC58382275323.397(Z)-5-(1,3-benzothiazol-2-yl)-6-(3-pyridyl)hex-5-enoic 7ZINC27525910284.293(2R)-2-amino-3-[2-(4-nitrophenyl)-2-oxo-ethyl]sulfanyl-propanoic 8ZINC01708082335.32acetic-acid-[(2R,3S,5R)-3-acetoxy-5-adenin-9-yl-tetrahydrofuran-2-yl]methyl-esterNSC76760 9ZINC06719206292.362-amino-3-[(8-methoxy-2-methyl-4-quinolyl)sulfanyl]propanoic 10ZINC15657781325.3252'-O-(2-Methoxyethyl)adenosine 11ZINC19720210312.322(2R)-2-amino-3-[(3S)-1-(4-fluorophenyl)-2,5-dioxo-pyrrolidin-3-yl]sulfanyl-propanoic 12ZINC22062133289.289Methimazole Thio-?-D-glucuronide 13ZINC49453260328.4121-{[2-(3,4-dimethylphenyl)-5-methyl-1,3-oxazol-4-yl]methyl}piperidine-3-carboxylic acid 14ZINC49453252334.8031-{[2-(4-chlorophenyl)-5-methyl-1,3-oxazol-4-yl]methyl}piperidine-3-carboxylic acid 15ZINC41689143265.696{[5-(2-Chloro-phenyl)-furan-2-ylmethyl]-amino}-acetic acid 16ZINC02675146287.2734-{[(4-nitrophenyl)sulfonyl]amino}butanoic acid 17ZINC42379800271.3184-[(4-aminophenyl)sulfonyl-methyl-amino]butanoic 18ZINC03999369323.3096-(6-amino-9H-purin-9-yl)-2-ethoxytetrahydrofuro[3,4-d][1,3]dioxol-4-yl]methanol 19ZINC01569549262.2937-adenin-9-ylenanthateNSC88907 20ZINC04514042370.3912-amino-3-[[5-(6-aminopurin-9-yl)-3,4-dihydroxy-tetrahydrofuran-2-yl]methylsulfanyl]propanoic 21ZINC32917705313.311N-[(6-methyl-3-oxo-3,4-dihydro-2H-1,4-benzoxazin-7-yl)sulfonyl]alanine 22ZINC01205282295.255methyl 5-(6-amino-9H-purin-9-yl)-3,4-dihydroxytetrahydro-2-furancarboxylate (non-preferred name) 23ZINC05234530299.2844-[(2-oxo-3H-benzooxazol-6-yl)sulfonylamino]butanoic 24ZINC05549707267.3131-(6-aminopurin-9-yl)-3-(2-hydroxyethyl-methyl-amino)-propan-2-olNSC371362 25ZINC19142033320.299-(3-nitrophenyl)sulfonylpurin-6-amine 26ZINC01732835261.2576-{5,7-dioxo-5H,6H,7H-pyrrolo[3,4-b]pyridin-6-yl}hexanoic acid 27ZINC04627954266.2663-[(6-amino-7H-purin-8-yl)azosulfanyl]propanoicNSC51459 28ZINC19720197342.804(2R)-2-amino-3-[(3S)-1-(3-chloro-4-methyl-phenyl)-2,5-dioxo-pyrrolidin-3-yl]sulfanyl-propanoic 29ZINC09420897280.3242-amino-3-[(6-fluoro-2-methyl-4-quinolyl)sulfanyl]propanoic 30ZINC10474284314.4072-amino-3-[1-(cyclohexylmethyl)-2,5-dioxo-pyrrolidin-3-yl]sulfanyl-propanoic 31ZINC16958662297.271(2R,3R,4R,5S)-2-(6-aminopurin-9-yl)-5-[(1S)-1,2-dihydroxyethyl]tetrahydrofuran-3,4-diolNSC670001 32ZINC40546444312.283(3-Methyl-2,4-dioxo-1,2,3,4-tetrahydro-quinazoline-6-sulfonylamino)-acetic acid 33ZINC02169568280.2164-Nitrobenzoylglycylglycine 34ZINC04513827366.314-[[5-(6-aminopurin-9-yl)-3,4-dihydroxy-tetrahydrofuran-2-yl]methoxy]-4-oxo-butanoic 35ZINC32917690299.284N-[(6-methyl-3-oxo-3,4-dihydro-2H-1,4-benzoxazin-7-yl)sulfonyl]glycine 36ZINC01681686265.229(2S)-2-[(1R)-1-adenin-9-yl-2-keto-ethoxy]-3-hydroxy-propionaldehyde(Adenosine-2′,3′-dialdehyde) 37ZINC58332159264.315-([1,2,4]triazolo[4,3-a]pyridin-3-ylsulfonyl)pentanenitrile 38ZINC13547652309.282[(2R,3R,4R,5R)-5-(6-aminopurin-9-yl)-3,4-dihydroxy-tetrahydrofuran-2-yl]methyl 39ZINC54448642326.36N-[(1R)-1-(2,3-dihydro-1,4-benzodioxin-6-yl)-2-methyl-propyl]tetrazolo[5,1-f]pyridazin-6-amine 40ZINC55284374263.272{[5-(4-fluorophenyl)-1H-pyrazol-4-yl]methyl}(methyl)amino]acetic acid 41ZINC41689200245.2783-[(5-Phenyl-furan-2-ylmethyl)-amino]-propionic acid 42ZINC13548142252.23NebularineNSC 65423 43ZINC01517335330.7474-[2-(4-chlorobenzoyl)anilino]-4-keto-butyrate 44ZINC06691827300.382-amino-3-(1-cyclohexyl-2,5-dioxo-pyrrolidin-3-yl)sulfanyl-propanoic 45ZINC09115078336.359N-[(4-methoxyphenyl)-(1-methylimidazol-2-yl)-methyl]-5,6,7,8,9-pentazabicyclo[4.3.0]nona-2,4,7,9-tet 46ZINC32917656318.7143-[(6-chloro-3-oxo-3,4-dihydro-2H-1,4-benzoxazin-7-yl)sulfonyl]propanoic acid 47ZINC13424933345.208[(2S,3S,4R,5R)-2-(6-aminopurin-9-yl)-4-hydroxy-5-(hydroxymethyl)tetrahydrofuran-3-yl]

8. Green = SAH taken from G9a protein crystal structure (2O8J)

9. G9a Activity / Inhibition Assay Add H3K9 mono-, di-, or trimethy specific Ab G9a SAH Inhibitor candidates Streptavidin coated plate G9a SAM Mono- ( di-, or Tri-) methylated H3K9 1 hr incubation Read chemiluminescence in a luminometer H3K9 mono- ( di-, or tri-) methyl specific Ab Poly His HRP Ab Add Poly His HRP Ab Biotin-H3K9 Me0 ( Me1, or Me2) peptides B B B BBBB BBBB BBBB Add HRP chemiluminescent substrate

10. Figure. Lineweaver-Burk plots showing G9a H3K9 inhibition in a competitive mode with SAM.

11. IC 50 G9a H3K9G9a H3K27GLP H3K9 CompoundMonoDiTriMonoDiMonoDiTri SAH38.7 uM1.253 uM1.704 uM14.08 uM7.655 uM10.7 uM4.921 uM84.01 uM BIX0129416 uM0.131 uM0.871 uM0.583 uM1.01 uM3.424 uM0.075 uM0.104 uM BRD4770 N.C. >200 uM N.C. >429 uM>393 uM>264 uM NuP_01483.384 uM1.1 uM3.596 uM2.49 uM4.217 uM14.54 uM53.52 uM55.97 uM NuP_0179140 uM5.086 uM0.298 uM34.45 uM5.01 uM44.03 uM7.461 uM>221 uM NuP_0260 N.C. 3.095 uM0.348 uM16.45 uM3.281 uM>181 uM>136 uM19.29 uM NuP_0318 N.C. 2.579 uM>732 uM82.14 uM N.C. >762 uM N.C. N. C. indicates Not Calculated NuP_0179NuP_0260NuP_0318NuP_0148 IC50 of Hits BIX01294 UNC0638 BRD4770

12. Gynecological cancer cellsLeukemia cells MOLT4 RS411 HL60K562 THP1 Kasumi1 CCRF-CEM HeLaBJF SK-LMS-1 Caov-3HT-3 SK-UT-1 MES-SA HeLa BJF ME-180 SW626KLE AN3-CA

13. Gynecological cancer cellsLeukemia cells MOLT4 RS411 HL60K562 THP1 Kasumi1 CCRF-CEM HeLaBJF SK-LMS-1 Caov-3HT-3 SK-UT-1 MES-SA HeLa BJF ME-180 SW626KLE AN3-CA

14. Cell proliferation assays in 6 gastric cancer cell lines (G9a inhibitor-treated for 48h)

15. HT-3 gynecological cell line was treated with inhibitors for a week 1 st lane: Untreat (DMSO) 2 nd lane: 100uM of Decitabine 3 rd lane: 100uM of NuP_0148 Amplication with 30 cycles CCNA1CHFRFHITPAX1GAPDH SFRP4CADM1 CEACAM6 CDH1PTEN ESR1SCNN1ALCN2CST6 DNMT3bΒ-ActinRUNX3MLH1DNMT3a GSTP1EPCAMTP53CDKN1ADLC1 RASSF1 G9aGLPAPCBCL2 DNMT1 BRCA1 PCDHGB6 PGRRARA CDKN2A FAS PMNP1PIDDIGF1RBAX TFAP2ARB1

16. IGF2R CDKN2BCDKN3E2F1BRCA2 S100A4 MGMTNF1NF2MEN1 TSC1 WT1WWOXXRCC1VHL ATM CCND2Twist2THRBRARB SMAD4STK11TP73SERPINB5 1 st lane: Untreat (DMSO) 2 nd lane: 100uM of Decitabine 3 rd lane: 100uM of NuP_0148 Amplication with 30 cycles

17. Structure of G9a complexed with SFG 2.4 Å R work /R free = 0.224/0.265