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HMT inhibitors 2018-02-04T09:02:59+00:00

To cite this section of the Cancer Epigenetics Drug Database

ReferenceI. Hübscher, M. Wachtel, and I.M. Bennani-Baiti. Histone methyltransferase inhibitors. Cancer Epigenetics Drug Database (CEDD) – Experimental dataset, Cancer Epigenetics Society (https://ces.b2sg.org/cedd/experimental_hmti/), 2016.

Chemical structures of cancer epigenetic drugs

Brief description of cancer epigenetic drugs

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A-366 is a highly selective G9a/GLP HMTi (IC50 = 3.3 nM) that exhibits little activity against other methyltransferases (IC50 > 3 µM). Despite a good cellular activity on H3K9me2, A-366 shows little to no cytotoxic effects on tumor cell lines, but induces cellular differentiation of leukemia cell lines.

Molecular Weight: 329.44 g/mole

A-366 is soluble at 100 mM in DMSO or in ethanol. It is insoluble in water.

Storage Condition: Dry, dark and -20 C for the long term

A-366 chemical structure is 5′-methoxy-6′-[3-(pyrrolidin-1-yl)propoxy]spiro[cyclobutane-1,3′-indole]-2′-amine .

BIX01294 is a selective G9a HMTi (IC50 = 2.7 µM in a cell-free assay which does not inhibit SUV39H1 and PRMT1. BIX01294 reduces G9a-mediated H3K9 dimethylation (IC50 =  1.7 μM) in a SAM- independent manner and GLP-mediated H3K9 trimethylation (IC50 = 38 μM).

Molecular Weight: 490.64 g/mole

BIX-01294 is soluble at 100 mM in DMSO or in water. It is soluble at < 10 mM in ethanol.

Storage Condition: Dry, dark and -20 C for the long term

BIX-01294 chemical structure is N-(1-benzylpiperidin-4-yl)-6,7-dimethoxy-2-(4-methyl-1,4-diazepan-1-yl)quinazolin-4-amine.

BIX-01338 is a SAM-dependent inhibitor of several HMTs including G9a, GLP, PRMT1, SET7/9, and SUV39H1.

Molecular Weight: 603.55 g/mole

BIX-01338 is soluble at 10 mg/ml in DMSO

Storage Condition: Dry, dark and -20 C for the long term

BIX-01294 chemical structure is 1-[2-[4-[(4-methoxyphenyl)-oxomethoxy]phenyl]ethyl]-2-[[oxo-[4-(trifluoromethyl)phenyl]methyl]amino]-5-benzimidazolecarboxylic acid.

BRD-4770, a selective HMTi of G9a (IC50 = 6.3 µM), also induces cell senescence. BRD-4770 reduces G9a –mediated H3K9me2 and H3K9me3 and inhibits anchorage-dependent and -independent proliferation in PANC-1 cells.

Molecular Weight: 413.47 g/mole

BRD-4770 is soluble at 10 mM in DMSO. It is insoluble in ethanol or water

Storage Condition: Dry, dark and -20 C for the long term

BRD-4770 chemical structure is Methyl 2-benzamido-1-(3-phenylpropyl)-1H-benzo[d]imidazole-5-carboxylate.

Chaetocin, extracted from Chaetomium species, is a HMTi (IC50 = 0.8 μM for Su(var)3-9, IC50 = 2.5 μM for mouse G9a and IC50 = 3 μM for Neurospora crassa DIM5). Chaetocin is a potent inhibitor of several cancer cell lines proliferation and colony formatiom (IC50 = 2-10 nM).

Molecular Weight: 696.84 g/mole

Chaetocin is soluble at 100 mM in DMSO. It is insoluble in ethanol or water

Storage Condition: Dry, dark and -20 C for the long term

Chaetocin chemical structure is (3S,3’S,5aR,5aR,10bR,10’bR,11aS,11’aS)-2,2′,3,3′,5a,5’a,6,6′-octahydro-3,3′-bis(hydroxymethyl)-2,2′-dimethyl-[10b,10’b(11H,11’H)-bi3,11a-epidithio-11aH-pyrazino[1′,2′:1,5]pyrrolo[2,3-b]indole]-1,1′,4,4′-tetrone.

3-Deazaneplanocin A or DZNep (a carbocyclic analog of adenosine) is a potent and competitive S-adenosylhomocysteine hydrolase inhibitor (Ki = 50 pM in cell-free assays). 3-Deazaneplanocin A (DZNep) is also an EZH2 inhibitor.

Molecular Weight: 262.265 g/mole

DZNep is soluble at 50 mM in DMSO or in water. It is insoluble in ethanol

Storage Condition: Dry, dark and -20 C for the long term

DZNep chemical structure is (1S,2R,5R)-5-(4-Amino-1H-imidazo[4,5-c]pyridin-1-yl)-3-(hydroxymethyl)-3-cyclopenten-1,2-diol and is also known as 3-Deazaneplanocin A.

E72, a quinazoline analog, is a selective G9a/GLP HMTi (IC50 = 100 nM). E72 displays an IC50 > 5 µM against Suv39H2.

Molecular Weight: 544.78 g/mole

UNC1999 chemical structure is 7-[(5-Aminopentyl)oxy]-N-[1-(5-aminopentyl)-4-piperidinyl]-N-[3-(dimethylamino)propyl]-6-methoxy-2,4-quinazolinediamin.

EI1 is a highly selective EZH2 HMTi with and IC50 = 15 nM. In DLBCL cells, EI1 reduces H3K27me3, activates EZH2 target gene p16 expression, and causes growth cell cycle arrest and apoptosis.

Molecular Weight: 544.78 g/mole

EI1 is soluble at 50 mM in DMSO. It is insoluble in ethanol or in water.

Storage Condition: Dry, dark and -20 C for the long term

EI1 chemical structure is 6-Cyano-N-((4,6-dimethyl-2-oxo-1,2-dihydropyridin-3-yl)methyl)-1-(pentan-3-yl)-1H-indole-4-carboxamide.

EPZ004777 is a highly selective DOT1L HMTi (IC50 = 0.4 nM in a cell-free assay). EPZ004777 inhibits methylation of H3K79 and expression of MLL fusion target genes. EPZ004777 induces differentiation, apoptosis and inhibists proliferation in MLL-rearranged cells.

Molecular Weight:  539.67 g/mole.

EPZ004777 is soluble up to 100 mM in DMSO or ethanol . It is insoluble in water.

Storage Condition: Dry, dark and -20 C for the long term

EPZ004777 chemical structure is 1-(3-((((2R,3S,4R,5R)-5-(4-Amino-7H-pyrrolo[2,3-d]pyrimidin-7-yl)-3,4-dihydroxytetrahydrofuran-2-yl)methyl)(isopropyl)amino)propyl)-3-(4-(tert-butyl)phenyl)urea.

EPZ005687 is a highly selective SAM-dependent EZH2 HMTi (Ki = 24 nM in a cell-free assay, Ki = 1.2 µM for EZH1 and Ki > 10 µM against 15 other HMTs). EPZ005687 inhibits methylation of H3K27 in various lymphoma cells.

Molecular Weight:  539.67 g/mole

EPZ005687 is soluble at 100 mM in DMSO .It is insoluble in water or in ethanol.

Storage Condition: Dry, dark and -20 C for long the long term

EPZ005687 chemical structure is 1-cyclopentyl-N-((4,6-dimethyl-2-oxo-1,2-dihydropyridin-3-yl)methyl)-6-(4-(morpholinomethyl)phenyl)-1H-indazole-4-carboxamide.

EPZ011989 is a highly selective wt and mt EZH2 HMTi (Ki < 3 nM). EPZ011989 also inhibits EZH1 (Ki < 50 nM), but not other HMTs (Ki > 9 µM). EPZ011989 reduces H3K27me3 levels in the Y641F, mutant-bearing human lymphoma cell line: WSU-DLCL2 (IC50 < 100 nM). EPZ011989 also reduces H3K27 trimethylation in wild-type EZH2 AML cell lines Kasumi-1, MOLM-13, and MV4-11 with an IC50 < 1 μM.

Molecular Weight: 605.82 g/mole.

EPZ011989 is soluble at 10 mM in DMSO and at 100 mM in ethanol. It is insoluble in water.

Storage Condition: Dry, dark and -20 C for the long term.

EPZ011989 chemical structure is N-((4,6-Dimethyl-2-oxo-1,2-dihydropyridin-3-yl)methyl)-3-(ethyl((trans)-4-((2-methoxyethyl)(methyl)amino)cyclohexyl)amino)-2-methyl-5-(3-morpholinoprop-1-yN-1-yl)benzamide .

EPZ015666 is a highly selective PRMT5 HMTi (Ki = 5 nM versus Ki = 100 µM for other PMTs). EPZ015666 inhibits proliferation of multiple mantle cell lymphoma cell lines (Z-138, Granta-519, Maver-1, Mino, and Jeko-1) at submicromolar concentrations  (IC50 = 0.1 -1 µM)

Molecular Weight: 383.44 g/mole.

EPZ015666 is soluble at 100 mM in DMSO or in ethanol. It is insoluble in water.

Storage Condition: Dry, dark and -20 C for the long term.

EPZ015666 chemical structure is (S)-N-(3-(3,4-Dihydroisoquinolin-2(1H)-yl)-2-hydroxypropyl)-6-(oxetan-3-ylamino)pyrimidine-4-carboxamide.

EPZ-5676 is a highly selective SAM-dependent DOT1L HMTi (Ki = 80 pM in a cell-free assay versus Ki ≥ 3 µM for other PMTs). EPZ-5676 reduces H3K79me2 levels in MLL-AF4 rearranged MV4-11 cells (IC50 = 2.6 nM). EPZ-5676 also inhibits proliferation of MV4-11 cells (IC50 = 9 nM).

Molecular Weight: 562.71 g/mole.

EPZ-5676 is soluble at 100 mM in DMSO or in ethanol. It is insoluble in water.

Storage Condition: Dry, dark and -20 C for the long term.

EPZ-5676 chemical structure is (2R,3R,4S,5R)-2-(6-amino-9H-purin-9-yl)-5-((((1r,3S)-3-(2-(5-(tert-butyl)-1H-benzo[d]imidazol-2-yl)ethyl)cyclobut and is also known as Pinometostat.

EPZ-6438is a highly selective EZH2 HMTi (Ki = 2.5 nM and IC50 = 11nM in a cell-free assay versus > 11µM for other HMTs). EPZ-6438 also inhibits EZH1 (Ki = 87.5 nM) and reduces H3K27 trimethylation levels in wild-type or SMARCB1 mutant cells. EPZ-6438 also inhibits proliferation of SMARCB1-deleted MRT cell lines (32 nM < IC50  < 1 µM). EPZ-6438 cytostatic activity is enhanced by prednisolone or dexamethasone in several EZH2 mutant lymphoma cell lines.

Molecular Weight: 572.74 g/mole.

EPZ-6438 is soluble at 10 mM in DMSO. It is insoluble in water or ethanol.

Storage Condition: Dry, dark and -20 C for the long term.

EPZ-6438 chemical structure is N-((4,6-dimethyl-2-oxo-1,2-dihydropyridin-3-yl)methyl)-5-(ethyl(tetrahydro-2H-pyran-4-yl)amino)-4-methyl-4′-(morpholinomethyl)-[1,1′-biphenyl]-3-carboxamide. EPZ-6438 is also known as Tazemetostat.

GSK126 is a highly selective EZH2 HMTi (IC50 = 10 nM versus ≥ 10 µM for other HMTs). In vitro, GSK126 effectively reduces H3K27 trimethylation levels followed by H3K27 dimethylation levels in both EZH2 wild-type and mutant Diffuse large B-cell lymphoma (DLBCL) cell lines. GSK126 also inhibits EZH2 mutant DLBCL cell lines.

Molecular Weight: 526.67 g/mole.

GSK126 is soluble in warmed DMSO (25 C). It is insoluble in water or ethanol.

Storage Condition: Dry, dark and -20 C for the long term.

GSK126 chemical structure is (S)-1-(sec-Butyl)-N-((4,6-dimethyl-2-oxo-1,2-dihydropyridin-3-yl)methyl)-3-methyl-6-(6-(piperazin-1-yl)pyridin-3-yl)-1H-indole-4-carboxamide.

GSK343 is a highly selective EZH2 HMTi (IC50 = 4 nM in a cell-free assay, IC50 = 240 nM for EZH1 and IC50 > 4µM against other histone methyltransferases). GSK343 reduces H3K27 trimethylation levels in HCC1806 breast cancer cells (IC50 = 174 nM) and also inhibits proliferation of several cancer cell lines.

Molecular Weight: 541.69 g/mole.

GSK343 is soluble at 20 mM in DMSO (warm).

Storage Condition: Dry, dark and -20 C for the long term.

GSK343 chemical structure is 1-(1-Methylethyl)-N-[(6-methyl-2-oxo-4-propyl-1,2-dihydro-3-pyridinyl)methyl]-6-[2-(4-methyl-1-piperazinyl)-4-pyridinyl]-1H-indazole-4-carboxamide.

SAH-EZH2 is a cell-permeable a-helical 27-mer peptide (FSSNRXKILXRTQILNQEWKQRRIQPV) that accumulates in the nucleus. SAH-EZH2 directly binds to EED with a Kd of 320 nM, leading to the disruption of EED-EZH2 interaction and thus of the PRC2 complex. SAH-EZH2 is an HMTi that reduces cellular H3K27me3 at micromolar concentrations.

SAH-EZH2 is soluble at 10 mM in water.

Storage Condition: Dry, dark and -20 C for the long term.

SGC 0946 is a highly selective DOT1L HMTi (IC50 = 0.3 nM in a cell-free assay versus IC50 > 30 nM for other HMTs), and is ineffective against DNMT1. SGC 0946 inhibits H3K79 dimethylation (IC50 in the range of 2.5-10 nM in cell-based assays) and selectively induces apoptosis in cells containing an MLL translocation. SGC 0946 is also more active than its EPZ004777 analog.

Molecular Weight: 618.57 g/mole.

SGC 0946 is soluble at 100 mM in DMSO or in ethanol. It is insoluble in water.

Storage Condition: Dry, dark and -20 C for the long term.

SGC 0946 chemical structure is 1-(3-((((2R,3S,4R,5R)-5-(4-Amino-5-bromo-7H-pyrrolo[2,3-d]pyrimidin-7-yl)-3,4-dihydroxytetrahydrofuran-2-yl)methyl)(isopropyl)amino)propyl)-3-(4-(tert-butyl)phenyl)urea

UNC0224 is a highly selective G9a/GLP HMTi (IC50 = 15 – 20 nM), but shows only little activity against SET7, SET8 and SET9 (IC50 > 15 µM).

Molecular Weight: 485.67 g/mole.

UNC0224 is soluble at 100 mM in DMSO or in 1eq. HCl. It is insoluble in water.

Storage Condition: Dry, dark and -20 C for the long term.

UNC0224 chemical structure is 7-[3-(Dimethylamino)propoxy]-6-Methoxy-2-(4-Methyl-1,4-Diazepan-1-Yl)-N-(1-Methylpiperidin-4-Yl)quinazolin-4-Amine.

UNC0321, a quinazoline analog, is a highly selective for G9a HMTi (IC50 = 6 – 9 nM) and GLP (IC50 = 15 nM), and shows little activity against other HMTs such as JMJD2E, SET7/9, SET8/PreSET7, and PRMT3 (IC50 > 10 µM). UNC0321 inhibits H3K9 dimethylation in MDA-MB-231 cells at an IC50 of 40 µM.

Molecular Weight: 515.69 g/mole.

UNC0321 is soluble at 10 mM in DMSO. It is insoluble in water.

Storage Condition: Dry, dark and -20 C for the long term.

UNC0321 chemical structure is 7-(2-(2-(Dimethylamino)ethoxy)ethoxy)-6-methoxy-2-(4-methyl-1,4-diazepan-1-yl)-N-(1-methylpiperidin-4-yl)quinazolin-4-amin.

UNC0638 is a highly selective G9/GLP HMTi (IC50 < 20 nM), and shows little activity against several other HMTs such as SET7/9 , SET8 , PRMT3, and SUV39H2. UNC0638 inhibits H3K9 dimethylation in MDA-MB-231 cells (IC50 = 81 nM).

Molecular Weight: 509.73 g/mole

UNC0638 is soluble at 100 mM in DMSO or in ethanol. UNC0638 is also soluble in 1eq. HCl.

Storage Condition: Dry, dark and -20 C for the long term.

UNC0638 chemical structure is 2-Cyclohexyl-N-(1-isopropylpiperidin-4-yl)-6-methoxy-7-(3-(pyrrolidin-1-yl)propoxy)quinazolin-4-amine.

UNC0642 is a highly selective HMTi for G9a and GLP (IC50 < 2.5 nM), and for example has little activity against EZH2 (IC50 > 5 µM). UNC0642 was shown to selectively inhibit G9a-catalyzed H3K9 dimethylation in an S-adenosyl-methionine (SAM)-independent manner. UNC0642 inhibits the H3K9me2 mark in the MDA-MB-231 breast cancer line at an IC50 of 110 nM.

Molecular Weight: 546.70 g/mol

UNC0642 is soluble at 100 mM in DMSO and at 50 mM in 1eq. HCl.

Storage Condition: Dry, dark and -20 C for long term

UNC0642 chemical structure is 2-(4,4-Difluoro-1-piperidinyl)-6-methoxy-N-[1-(1-methylethyl)-4-piperidinyl]-7-[3-(1-pyrrolidinyl)propoxy]-4-quinazolinamine.

UNC0965 is the biotinylated version of UNC0638. UNC0965 is a highly selective G9a/GLP HMTi (IC50 < 2.5 nM) which reduces H3K9me levels in MDA-MB-231 cells (IC50 > 3 µM).

UNC0965 is soluble in DMSO.

UNC1999, a highly selective EZH2 (IC50 = 2 nM) and EZH1 (IC50 = 45 nM) HMTi in cell-free assays. UNC1999 displays an IC50 of  > 2 µM for other HMTs. UNC1999 reduces H3K27 trimethylation levels in MCF10A cells (IC50= 124nM) and inhibits proliferation of EZH2Y641N mutant bearing DLBCL cells (EC50 = 633 nM).

Molecular Weight: 569.74 g/mole

UNC1999 is soluble at 100 mM in DMSO or at 50 mM in 1eq. HCl.

Storage Condition: Dry, dark and -20 C for the long term.

UNC1999 chemical structure is N-[(6-methyl-2-oxo-4-propyl-1H-pyridin-3-yl)methyl]-1-propan-2-yl-6-[6-(4-propan-2-ylpiperazin-1-yl)pyridin-3-yl]indazole-4-carboxamide.

Functional assays used to assess the activity of cancer epigenetic drugs

Annexin V Assay is a cell viability assay that relies on the premise that Annexin V, a cell-impermeable protein, binds with high affinity membrane phosphatidylserine phospholipids when exposed at the cell surface. Though phosphatidylserine phospholipids localize to the inner, cytoplasmic side of the plasma membrane in healthy cells, they translocate from the inner to the outer side of the plasma membrane early in apoptosis, and become accessible to Annexin V. Following staining with Annexin V and propidium iodide (see PIA), the assay allows to quantify: i. (Annexin V / PI) viable cells; ii. Annexin V+ / PI cells undergoing early apoptosis; iii. (Annexin V / PI)+ dead cells and those in late apoptosis. Annexin V is usually labeled with a fluorochrome such as FITC as to analyze staining by flow cytometry.

Automated Cell Counting regroups a number of automated procedures including for example electrical impedance-based technologies (e.g Bio-Rad’s Coulter), or microscopy imaging-based methods (e.g. Beckman’s Vi-CELL™ Cell Counter) to assess cell viability and proliferation. These techniques are mostly automated versions of the trypan blue exclusion assay (see TBE).

BrdU Cell Incorporation Assay is a cell proliferation assay used to quantify 5-bromo-2′-deoxyuridine or BrdU incorporation into DNA during DNA replication that occurs in the course of cellular proliferation. Cells in culture are incubated in the presence of large amounts of BrdU, which is incorporated into DNA instead of thymidine. Following cell fixation and DNA denaturation, a labelled monoclonal anti-BrdU antibody is used to quantify the amount of incorporated BrdU, which in turn reflects the extent of de novo replicated DNA / cell proliferation.

Caspase 3/7 Apoptosis Assay: the Caspase 3 and 7 cysteine protease family members are integral components of mammalian apoptosis. Several variants of this assay are available, including for example those that utilize caspase 3 and 7 substrates that emit light or fluorescence once cleaved by the caspases. The substrate being in excess, the amount of light/fluorescence is proportional to the amount of caspases, and by extension, to the number of apoptotic cells.

Calcein AM allows to assess cell metabolism, and consequently, cell viability. Calcein AM is a non-fluorescent, cell permeable hydrophobic compound that is hydrolyzed by intracellular esterases to produce Calcein, a hydrophilic, cytoplasm-contained fluorescent compound in metabolically active (live) cells. Spectrophotometry of Calcein AM-labeled cells with an excitation at 490 nm and emission at 520 nm permits the quantification of metabolically active cells.

Cell Cycle Analysis is carried out by flow cytometry analysis of cellular DNA content after staining of nuclei with a DNA binding dye (usually propidium iodide). Cells distributes into the G0/G1 phase (cells with about 1n DNA content), S phase (~1.5n), and G2/M phase (~2n). This assay allows to assess (and quantify) if a drug induces a cell cycle block at a given cell cycle phase/checkpoint.

Colony Formation Assay or Colony Forming Units is an assay that is usually carried out by visualizing colonies with the crystal violet dye (see CVA) and allows to assess a cell’s capacity to overcome contact inhibition by growing into a colony, usually in soft agar. This assay is considered to be an in vitro measure of cellular tumorigenicity. The assay is based on counting the number of colonies, their size, or both.

Crystal Violet Assay is a cell viability and proliferation assay based on crystal violet, a cell permeable tri-(dimethylaniline)-methane dye that binds to both DNA and proteins, thus allowing for the visualization of cells (e.g. see CFA), or their quantification by use of spectrophotometry at 570 nm. Since crystal violet stains equally well living and dead cells, this assay is appropriate only for adherent cells that detach after cell death. After the medium is discarded and the dead cells washed away, only the adherent cells remain and are stained.

CyQUANT is a cell proliferation assay that relies on a cell permeable green fluorescent nucleic acid dye (CyQUANT GR dye) and a fluorescence suppression dye that quenches background cellular fluorescence. This is a one-step assay whereby reagents are added directly to cells in culture, incubated 30-60min, and fluorescence is measured with an excitation at 485 ± 10 nm and emission at 530 ± 12.5 nm. The assay essentially measures the amount of nucleic acids in living cells in culture, and is linear over a range of 50 – 250,000 cells in a 200 µl volume.

4′,6-diamidino-2-phenylindole or DAPI is a fluorescent dye that stains the nucleus by binding to AT-rich DNA. Apoptosis can be assessed in fluorescent microscopy by counting the number of DAPI-stained cell nuclei that show condensed and fragmented chromatin, two early signs of apoptosis.

Erythrosine B Exclusion assay is an alternative to trypan blue (see TBE) in quantifying cell viability and proliferation. Similarly to trypan blue, live cells are impermeable to Erythrosine B, whereas dead cells readily incorporate it.

CellTiter-Glo Luminescent Cell Viability Assay assesses the levels of cellular ATP as a gauge of cellular metabolism. The assay is based on luciferase, which requires ATP (and Mg2+) to convert luciferin into oxiluciferin, and which in the process emits luminescence. The intensity of luminescence signal is proportional to the amount of available cellular ATP, and is thus indicative of metabolic activity.

Guava Viability Assay allows to assess both total cell count and cell viability. The assay is based on quantifying two dyes, one that binds DNA in all cells, and a second dye that binds DNA in dead cells only.

Hemocytometer Cell Counting is based on counting the number of cells in visible light microscopy in multiple fields of a hemocytometer, and then calculating the average number of cells per field.

Lactate Dehydrogenase assay measures the amount of LDH, a non-secreted cytosolic enzyme, in cell culture media as an indicator of cytotoxicity. In cytotoxic conditions, the plasma membrane is damaged and LDH is released into the cell culture medium. The amount of LDH in the medium can be indirectly assessed using MTT type of assays that quantify the amount of NADH in the cell culture medium produced during the conversion of lactate into pyruvate by LDH.

Mitochondrial Membrane Potential assay: intact cells present with a mitochondrial membrane potential due to the polarity differential between the overall electronegatively charged mitochondrial matrix and electropositively charged cytosol. In this assay, cells are incubated in the presence of cell permeable potential-sensitive fluorescent dyes such as JC-1, which accumulate into mitochondria and shift fluorescence emission from green to red (from about 529 to 590 nm). In cytotoxic conditions or apoptosis, mitochondrial membranes are damaged and mitochondrial membrane polarity is compromised, leading to a shift in the fluorescence emission ratio, which can be quantified and reflects cell viability.

A variation of the MTT assay (see below) that uses the MTS reagent (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium) that also relies on formazan quantification to assess cellular metabolism, which may under certain conditions reflect cell number. Contrary to the multistep MTT assay, MTS involves a 1-step procedure.

Mosmann’s Thiazolyl blue Tetrazolium salt assay is a colorimetric assay to quantify cellular metabolic activity. MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) is a yellow tetrazole that is metabolized into a non-hydrosoluble purple formazan by NADH/NADPH-dependent mitochondrial dehydrogenases. After solubilization in DMSO or SDS/HCl, formazan is spectrophotometrically quantified at 500-600 nm to assess the extent of tetrazole reduction, and by inference the activity of cellular dehydrogenases. Cellular metabolic activity may under certain conditions reflect cell number.

Neutral Red Method uses a vital dye, neutral red (a.k.a. Basic Red 5, Toluylene Red) that is actively uptaken by viable cells, whereby the dye accumulates in lysosomes, whereas non-viable cells remain impermeable to the dye. After washing out the excess of dye, the incorporated dye is released from the cells and is ionized following incubation in an acidified ethanol solution, which yields its characteristic red color. The amount of neutral red incorporated by the cells can then be quantified spectrophotometrically and is proportional to the number of viable cells.

Propidium Iodide (PI) Assay is a cell viability assay based on the fact that healthy cells are impermeable to PI. Under certain cytotoxic conditions and during apoptosis, cell membranes are compromised and show increased permeability to PI, which accumulates in the cell nuclei, wherein it intercalates into DNA. PI is fluorescent and can therefore be quantified by fluorescence microscopy or spectrophotometry. Since PI also binds RNA, cleaner results may be obtained by pretreating cells with RNAses.

Resazurin Conversion Assay is based on the reduction of the non-fluorescent resazurin reagent into fluorescent resorufin by cellular dehydrogenases, and thus serves to measure the level of cellular metabolism (or as some interpret it, to assess the number of metabolically active -and thus viable- cells).

Syto 60 Apoptosis Assay: Syto probes are cell permeable fluorescent compounds that bind to DNA and RNA. As cells undergo apoptosis, there is a caspase-dependent loss of fluorescence. The amount of fluorescence emitted by Syto is, therefore, proportional to the number of viable cells and inversely proportional to the number of apoptotic cells.

Spheroid Formation Assay is an assay that allows to assess a cancer cell’s capacity to grow in 3D, and thus to overcome contact inhibition. Cells are embedded in soft agar in the presence or absence of the drug, and spheroids are allowed to grow some 10-14 days depending on cell proliferation rates. The number of spheroids are counted at the end of the experiment for each test sample.

is a cell viability and proliferation assay that utilizes Sulforhodamine B, an anionic aminoxanthene dye that engages into electrostatic interactions with basic amino acids under moderately acidic conditions. The binding of Sulforhodamine B to cellular proteins is linearly dependent on the amount of proteins (and therefore cells), as measured spectrophotometrically at 560-580nm (after washing out the excess of non-bound dye and solubilization in SDS). Sulforhodamine B, however, permeates both living and dead cells, thus limiting the scope of the assay to adherent cells that detach after cell death.

Trypan Blue Exclusion Assay is based on counting in visible light microscopy the number of cells dyed in blue (trypan blue-permeable cells are cells whose membrane is no longer intact, which is indicative of cell death) and trypan blue-impermeable cells (viable cells). Trypan blue is also referred to as diamine blue or Niagara blue.

3H-Thymidine Incorporation assay allows to measure the amount of thymidine incorporated into de novo replicated DNA during cell proliferation. 3H-thymidine is added directly to the cell culture and permitted to incubate some 4-12 hours depending on cell division rates. Labelled DNA is next harvested onto glass fiber filter paper, which can be processed in a scintillation beta-counter. The amount of beta counts/amount of 3H-thymidine incorporated is proportional to the amount of replicated DNA and cell proliferation.

Terminal deoxynucleotidyl transferase (TdT) dUTP Nick End Labeling (TUNEL) allows the detection of DNA fragmentation that accompanies apoptosis. The assay takes advantage of the capacity of TdT to add labeled dUTPs to nicks in the fragmented DNA of cells undergoing apoptosis. The incorporation of dUTPs labeled with either a fluorochrome (e.g. fluorescein-dUTP) or a hapten (e.g. biotin-dUTP, BrdUTP) can be readily quantified and is proportional to the extent of apoptosis.

Transwell Assay, also referred to as the Boyden chamber transwell assay, can be used to quantify cellular motility, migration, chemotaxis, or invasion. The assay is based on the capacity of some cells to traverse a (usually 8 µm) porous membrane (motility / migration) that may in addition be coated with a basement membrane extract, Collagen I or IV, or Laminin I (invasion). The compartment to which the cells would migrate may contain a chemoattractant (e.g. SDF1α/CXCL12), thus allowing to assess chemotaxis. At the end of the experiments, cells that crossed the membrane are stained and counted.

The Wound Healing Assay, a.k.a Scratch Assay, is a technique to assess cell migration propensity in vitro. The assay relies on tracking cells movements on a slide onto which a gap (wound, scratch) is applied. While this method is widely accepted as a cell migration assay, we at the Cancer Epigenetics Society have concerns about its validity as its results may be compromised by the proliferation, or lack thereof, of the cells under investigation.

Water-soluble Tetrazolium salts assays utilize water-soluble dyes that yield formazans with different absorption spectra as an alternative to the water-insoluble and single-spectrum reagents of the MTT assay. Assay based on WST-1 (2-(4-Iodophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium) and perhaps more so on WST-8 (2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium) offer several advantages as they are reduced outside the cells, include 5-methyl-phenazinium methyl sulfate that serves as an electron transfer mediator, and yield a water-soluble formazan. Unlike MTT, WST assays are accompanied with lower cell toxicity, do not require a solubilization step, and emit stronger signals. WST assays allow to quantify cellular metabolism extracellularly. Cellular metabolic activity may under certain conditions reflect cell number.

A variant of the MTT, MTS and WST assays that relies on 2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide as the tetrazole, and akin to WST, is reduced to a water-soluble formazan, thus eliminating the last solubilization step. XTT displays higher sensitivity and dynamic range than MTT, and as the latter, allows to quantify cellular metabolism. Cellular metabolic activity may under certain conditions reflect cell number.

A word about the Cancer Epigenetics Drug Database – HMTi section

Welcome to the Cancer Epigenetic Drug Database – Experimental dataset for histone methyltransferase inhibitors (HMTi). The epigenetics drug database gives an overview of cancer cell lines sensitivity to HMTi in various functional assays (see assays abbreviations below). The page starts off with the chemical structures of the epigenetic drugs, a succinct description of the epigenetic drugs and functional assays used to test them in cancer cell lines, followed by Tables summarizing the data. Some of the epigenetic drugs also have clinical activities which are summarized in this page. Please note that the epigenetic drug solubility values provided here are only to be used as a guide, and that solubility depends on several variables such as the purity of the solute and solvent used, as well as the temperature used. Similarly, the IC50 values provided herein can be highly dependent on the type of functional assay used and length of incubation of cells with the epigenetic drugs.

The Cancer Epigenetics Drug Database is a work in progress and we rely on our members and readers to bring to our attention data that ought to be included here. We, therefore, welcome any information that you may provide that would help us make this database as useful a tool for your research as possible. You may email us your suggestions at info@ces.b2sg.org, Subject line: CEDD – HMTi, Experimental dataset. The Cancer Epigenetics Drug Database is periodically updated, so please check it again for new data that may be relevant to your research.

Notes:

1Inc: Incubation of the cells in the presence of the drug in days.
2Assay: Please see details on functional assays above.
3Gene vs protein expression: Symbols in plain text denote proteins (e.g. EZH2), those in italics the mRNA (e.g. EZH2), and those in bold (e.g. EZH2) imply that both the mRNA and the protein are affected. ↑ indicates an increase, whereas ↓ indicates a decrease in mRNA or protein expression or in the levels of an epigenetic mark. Example of epigenetic marks: H3K27me3 is the abbreviation for histone H3 trimethylated at lysine 27, and H3K9me2/3 stands for histone H3 di- or tri-methylated at lysine 9.

Histone methyltransferase inhibitor (HMTi) activities in cancer cells

Cell line
IC50 (µM) Inc1 Assay2 Additional information3 ID#
22Rv1 > 10
5 GLO
Calu-1 > 10
5 GLO
Calu-6 > 10
5 GLO
Daoy > 10
7 GLO
DU145 > 10 5 GLO
G-401 > 10 7 GLO
HL-60 > 10 5 GLO
HT-1080 > 10 5 GLO
IMR-32 > 10 12 GLO
K-562 > 10 5 GLO
Kasumi-1 > 10 5 GLO
KMS11 > 10 5 GLO
KMS12 > 10 5 GLO
L-363 > 10 5 GLO
LN-229 > 10 5 GLO
LNCap > 10 5 GLO
MOLT-16 > 10 5 GLO ↓ global H3K9me2, ↑ G9a
MV4-11 > 10 5 GLO G0/G1 arrest
NCI-H1975 > 10 5 GLO
NCI-H2009 > 10 5 GLO
NCI-H358 > 10 5 GLO
NCI-H441 > 10 5 GLO
NCI-H522 > 10 5 GLO
NCI-H661 > 10 5 GLO
NCI-H929 > 10 5 GLO
OPM2 > 10 5 GLO
PA-1 > 10 7 GLO
RPMI 8226 > 10 5 GLO
RS4;11 > 10 5 GLO
SK-BR-3 > 10 5 GLO
SK-N-MC > 10 7 GLO
TC-71 > 10 7 GLO
THP-1 > 10 5 GLO
U-2 OS > 10 7 GLO

© CEDD, Cancer Epigenetics Society & B² Scientific Group, Ltd. All rights reserved, 2016. Data may be included in academic publications contingent on the citation of the reference on top of this page.

Cell line
IC50 (µM) Inc1 Assay2 Additional information3 ID#
ACC-LC-319 4 – 6 2 WST-8
C6 1 – 5 3 MTT
HeLa 4 – 16 2 MTT G9a IC50 = 1.7 µM,
GLP IC50 = 38 µM
HeLa < 5 2 TBE
MCF7 > 2 3 HCC Sox2
MCF7 > 2 4 HCC Sox2
NCI-H2170 > 6 2 WST-8
SBC-5 < 2 2 WST-8
SHEP-1 < 5 2 TBE
SW780 2 – 4 2 WST-8
U-2 OS < 5
2 TBE
UM-UC-3 2 – 4 2 WST-8

© CEDD, Cancer Epigenetics Society & B² Scientific Group, Ltd. All rights reserved, 2016. Data may be included in academic publications contingent on the citation of the reference on top of this page.

Cell line
IC50 (µM) Inc1 Assay2 Additional information ID#
HeLa > 13.3
2 MTT

© CEDD, Cancer Epigenetics Society & B² Scientific Group, Ltd. All rights reserved, 2016. Data may be included in academic publications contingent on the citation of the reference on top of this page.

Cell line
IC50 (µM) Inc1 Assay2 Additional information ID#
HeLa ~ 10 3 GLO
HeLa > 10 3 CAA
HPAC ~ 1
3 GLO
HPAC > 10 3 CAA
MCF7 2.5 3 GLO
MCF7 5
3 CAA
PANC-1 ~ 10 3 GLO
PANC-1 > 10 3 CAA
PC-3 ~ 10 3 GLO
PC-3 > 10 3 CAA

© CEDD, Cancer Epigenetics Society & B² Scientific Group, Ltd. All rights reserved, 2016. Data may be included in academic publications contingent on the citation of the reference on top of this page.

Cell line
IC50 (µM) Inc1 Assay2 Additional information3 ID#
A-549 0.14 1 SRB ↓ SUV39H1, ↑ ATF3, CHOP
AML-193 0.05 – 0.1
3 MTT Sub-G1 arrest (same by SUV39H1 shRNA); ↓ H3K9me2/3 on p15, CDH1
C6 0.01 – 0.05
3 MTT
Calu-1 0.15
1 SRB
CML-LSC ~ 0.3
2 TBE
HL-60 ~ 0.1 1 A5A ↓ SUV39H1 & H3K9me3 on p15, CDH1, FZD9
K-562 ~ 0.1 1 A5A ↓ SUV39H1 & H3K9me3 on CDH1, FZD9
Kasumi-1 < 0.1 1 A5A ↓ SUV39H1 & H3K9me3 on p15, CDH1, FZD9
KG-1 ~ 0.1 1 A5A ↓ SUV39H1 & H3K9me3 on p15, CDH1, FZD9
NCI-H157 0.1 1 SRB ↓ SUV39H1, ↑ ATF3, CHOP
NCI-H1650 0.19 1 SRB
NCI-H1792 0.22 1 SRB ↓ SUV39H1, ↑ ATF3, CHOP
NCI-H460 0.15 1 SRB
THP-1 < 0.1 1 A5A ↓ SUV39H1 & H3K9me3 on CDH1, FZD9

© CEDD, Cancer Epigenetics Society & B² Scientific Group, Ltd. All rights reserved, 2016. Data may be included in academic publications contingent on the citation of the reference on top of this page.

Cell line
IC50 (µM) Inc1 Assay2 Additional information3 ID#
A-549 ~ 20
3 MTT
DMS53 < 5
5 WST-8
HepG2 > 20
3 MTT
HL-60 ~ 1 2 TBE ↓ EZH2, SUZ12, Cyclin E
HL-60 0.2 – 0.5
3 A5A ↓ EZH2, SUZ12, Cyclin E
Jurkat 12
2 MTT Cell cycle S-phase arrest
Lu130 ~ 5
6 WST-8
MCF10A > 100 6 MTS
MCF7 9.84 2 MTT EZH2 & DNMT1, RASSF2A
MCF7 0.2 6 MTS G2/M arrest
MDA-MB-231 > 20
3 MTT
MDA-MB-231 0.24 6 MTS G2/M arrest
MPNST-724 ~ 10 3 MTT
NCI-H209 < 5
6 WST-8
OCI-AML3 ~ 0.5
2 TBE ↓ EZH2, SUZ12, Cyclin E
OCI-AML3 0.2 – 0.5
3 A5A ↓ EZH2, SUZ12, Cyclin E
S462 1 – 2 3 MTT G2 arrest
SAS < 10 2 WST-8
SK-BR-3 ~ 0.8 6 MTS

© CEDD, Cancer Epigenetics Society & B² Scientific Group, Ltd. All rights reserved, 2016. Data may be included in academic publications contingent on the citation of the reference on top of this page.

Cell line
IC50 (µM) Inc1 Assay2 Additional information3 ID#
DB 0.37 – 1.1
15 ACC
G-401 ~ 1.1
15 ACC ↓ global H3K27me2/3
KARPAS 422
0.37 – 1.1 15 ACC
OCI-Ly19 > 10
10 CFA ↓ global H3K27me2/3
SU-DHL-4 3.3 – 10
15 ACC
Toledo 3.3 – 10 15 ACC
WSU-DLCL2 < 1
10 CFA

© CEDD, Cancer Epigenetics Society & B² Scientific Group, Ltd. All rights reserved, 2016. Data may be included in academic publications contingent on the citation of the reference on top of this page.

Cell line
IC50 (µM) Inc1 Assay2 Additional information ID#
697 36.57 14 GVA
HL-60 > 50 14 GVA
Jurkat > 50 14 GVA
Kasumi-1 33
14 GVA
KOPN-8 0.62
14 GVA
MOLM-13 0.72
14 GVA
MV4-11 0.17
14 GVA
Reh 13.9
14 GVA
RS4;11 6.47
14 GVA
SEM 1.72 14 GVA
THP-1 3.36 18 GVA
U937 > 50 14
GVA

© CEDD, Cancer Epigenetics Society & B² Scientific Group, Ltd. All rights reserved, 2016. Data may be included in academic publications contingent on the citation of the reference on top of this page.

Cell line
IC50 (µM) Inc1 Assay2 Additional information ID#
Aska-SS 0.72 14 MTT
DoHH2 3.8 11 GVA
Fuji 1.5 14 MTT
HT 3
11 GVA
KARPAS 422 0.45
11 GVA
OCI-Ly19 1.7
11 GVA
Pfeiffer 0.005
11 GVA
SU-DHL-6 0.32
11 GVA
SYO-1 2.1
14 MTT
Toledo 13.4 11 GVA
WSU-DLCL2 0.3 11 GVA
Yamato-SS 3.5
14
MTT

© CEDD, Cancer Epigenetics Society & B² Scientific Group, Ltd. All rights reserved, 2016. Data may be included in academic publications contingent on the citation of the reference on top of this page.

Cell line
IC50 (µM) Inc1 Assay2 Additional information ID#
WSU-DLCL2 0.002-0.01
11 GVA Erradicates tumor in mice at 250 mg/kg

© CEDD, Cancer Epigenetics Society & B² Scientific Group, Ltd. All rights reserved, 2016. Data may be included in academic publications contingent on the citation of the reference on top of this page.

Cell line
IC50 (µM) Inc1 Assay2 Additional information ID#
Granta-519 0.06
12 GVA
Jeko-1 0.9
12 GVA
Maver-1
0.45 12 GVA  
Mino 0.1 12 GVA
Z-138 0.1
12 GVA

© CEDD, Cancer Epigenetics Society & B² Scientific Group, Ltd. All rights reserved, 2016. Data may be included in academic publications contingent on the citation of the reference on top of this page.

Cell line
IC50 (µM) Inc1 Assay2 Additional information3 ID#
697 32.7 14 GVA
HL-60 > 50 14 GVA
Jurkat > 50 14 GVA
Kasumi-1 12.2
14 GVA
KOPN-8 0.17
14 GVA
MOLM-13 0.06 14 GVA
MOLM-13 > 1
7 A5A HOXA9, MEIS1
MOLM-13 ~ 0.16
7 A5A HOXA9, MEIS1
MV4-11 0.004
14 GVA Sub-G1 arrest
NOMO-1 0.03
14 GVA
Reh 1.2 14 GVA
RS4;11 1.3 14 GVA
SEM 0.2
14
GVA
SKM-1 > 4 7 GLO

© CEDD, Cancer Epigenetics Society & B² Scientific Group, Ltd. All rights reserved, 2016. Data may be included in academic publications contingent on the citation of the reference on top of this page.

Cell line
IC50 (µM) Inc1 Assay2 Additional information3 ID#
Fuji 0.15 14 GLO ATF3
Fuji 0.15 11 CAA ATF3
G-401 0.16
14 GLO
HS-SY-2 0.52
14 GLO CDKN2A
HS-SY-2 0.63
11 GLO CDKN2A
SW982 > 10
14 GLO

© CEDD, Cancer Epigenetics Society & B² Scientific Group, Ltd. All rights reserved, 2016. Data may be included in academic publications contingent on the citation of the reference on top of this page.

Cell line
IC50 (µM) Inc1 Assay2 Additional information ID#
697 ~ 7.5 6 GLO
BC-1 8.29
6 GLO
BC-2 4.76
6 GLO
BC-3 2.22
6 GLO
CA46 6.59
6 GLO
Ci-1 4.28 6 GLO
COLO 205
~ 5
3 MTT
CRO-AP2 1.64 6 GLO
Daudi 1.27 6 GLO
DB 0.86 6 GLO
DB 0.25 9 ACC
DG-75 3.25 6 GLO
DMS53 ~ 8
8 WST-8
Farage 1.72 6 GLO
HCT 116
~ 5
3 MTT
HD-MY-Z 10.72 6 GLO
Hs 445 3.53 6 GLO
HS-Sultan 2.28 6 GLO
HT 0.52 6 GLO
HT-29 2 – 5 3 MTT
Jiyoye 0.23 6 GLO
KARPAS 231 ~ 3 6 GLO
KARPAS 422 0.23 6 GLO
KARPAS 422 > 3 7 GLO
KARPAS 422 ~ 3 8 GLO
KARPAS-1106P 4.54 6 GLO
L-428 4.7 6 GLO
LNCaP > 10 2