EZH2 (Ki = 2.5 nM); EZH2 (IC50s = 11 nM, 16 nM)

In SCID mice bearing s.c. G401 xenografts, EPZ-6438 induces tumor stasis during the administration period and produces a significant tumor growth delay with minimal effect on body weight.

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Tazemetostat (EPZ6438) Leads to Complete and Sustained Regression of SMARCB1 Mutant MRT Xenografts.[1]
A study in SCID mice bearing s.c. G401 xenografts was performed where animals were dosed orally for 21 d with EPZ-6438. One-half of the mice per group were euthanized on day 21 to collect blood and tissues, while the remaining animals were treated for an additional 7 d and then left without dosing for another 32 d. EPZ-6438 was well tolerated at all doses with minimal effect on body weight (Fig. S4A). Oral dosing at 250 or 500 mg/kg twice daily (BID) for 21–28 d practically eliminated the fast-growing G401 tumors (Fig. S4 B and C and Fig. 4A). Regrowth was not observed for 32 d after dose cessation. EPZ-6438 dosed at 125 mg/kg induced tumor stasis during the administration period and produced a significant tumor growth delay compared with vehicle after the dosing period. Measuring EPZ-6438 plasma levels either 5 min before or 3 h after dosing on day 21 revealed a clear dose-dependent increase in systemic exposure (Fig. S4D). Tumors that were harvested from subsets of mice from each group on day 21 showed strong inhibition of H3K27Me3, correlating with the antitumor activity (maximum effect achieved at 250 mg/kg; Fig. 4B). In addition, dose-dependent changes in the expression of CD133, PTPRK, DOCK4, and GLI1 were detected in the G401 xenograft tumors.

Biochemical Enzyme Assays [2]
Preparation of recombinant purified human PRC2 complex containing WT EZH2, Y641 EZH2 mutants, A677G EZH2 or WT EZH1 was performed as previously described 1,2. Chicken erythrocyte oligonucleosomes were purified as previously described 3. Biotinylated histone peptides were synthesized by 21st Century Biochemicals and HPLC-­‐purified to > 95% purity. 384-­‐well Flashplates and Microscint 0 scintillation fluid were purchased commercially and 96-­‐well Multiscreen HTS filter-­‐binding plates were from Millipore. 3H-­‐labeled S-­‐ adenosylmethionine (3H-­‐SAM) was obtained commercially with a specific activity of 80 Ci/mmol. Unlabeled SAM and S-­‐adenosylhomocysteine (SAH) were obtained commercially. Flashplates were washed in a Biotek ELx-­‐ 405 with 0.1% Tween. 384-­‐well Flashplates and 96-­‐well filter binding plates were read on a TopCount microplate reader . Compound serial dilutions were performed on a Freedom EVO and spotted into assay plates using a Thermo Scientific Matrix PlateMate.

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Determination of Enzyme Inhibition IC50 Values [2]
10-­‐point curves of EPZ005687 were made using serial 3-­‐fold dilutions in DMSO, beginning at 2.5 mM (final top concentration of compound was 50 μM and the DMSO was 2%). A 1 μL aliquot the inhibitor dilution series was spotted in a 384-­‐well microtiter plate. The 100% inhibition control consisted of 1 mM final concentration of the product inhibitor S-­‐adenosylhomocysteine, (SAH). Compound was incubated for 30 min with 40 μL per well of 5 nM PRC2 (final assay concentration in 50 μL was 4 nM) in 1X assay buffer (20 mM Bicine [pH 7.6], 0.002% Tween 20, 0.005% Bovine Skin Gelatin and 0.5 mM DTT). 10 μL per well of substrate mix comprising assay buffer 3H-­‐SAM, unlabeled SAM, and peptide representing histone H3 residues 21 – 44 containing C-­‐terminal biotin (appended to a C-­‐terminal amide-­‐capped lysine) were added to initiate the reaction (both substrates were present in the final reaction mixture at their respective Km values, an assay format referred to as ‘‘balanced conditions’’4. The final concentrations of substrates and methylation state of the substrate peptide are indicated for each enzyme in Supplementary Table 7. Reactions were incubated for 90 min at room temperature and quenched with 10 μL per well of 600 μM unlabeled SAM, then transferred to a 384-­‐well Flashplate and washed after 30 min.

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In Vitro EPZ005687-­Substrate Competitions [2]
Experimental conditions for the SAM competitions in 50 μL total volume in 384-­‐well format were similar to the IC50 experiments with the following exceptions.EPZ005687 was serially diluted 2-­‐fold in DMSO (final concentration 2%) to make 10-­‐point curves. SAM was titrated over a range between 0.012 to 15 μM. To monitor the reaction, a radioactive tracer 3H-­‐SAM was used in each reaction, equivalent to up to 250 nM (which was accounted for in the total SAM concentration). At SAM concentrations less than 250 nM, the reaction contained only 3H-­‐SAM. Each SAM concentration had equivalent wells with no enzyme present in the reaction as negative controls to subtract the contribution to background signal of the 3H-­‐SAM. Reactions were incubated for 90 min and quenched with 10 μL per well of 600 μM SAM. Oligonucleosome competitions were performed in 96-­‐well format in the same assay buffer as described in the IC50 determination, except that it was supplemented with 100 mM KCl. EPZ005687 was serially diluted 2-­‐fold in DMSO (final concentration 2%) to make 10-­‐point curves. The oligonucleosome was titrated over 8 points using a 2-­‐fold dilution scheme with top concentration of 500 nM in the final enzyme reaction volume of 50 μL. Reactions were initiated by adding a cocktail of SAM and 3H-­‐SAM, with final respective concentrations of 450 and 150 nM. Reactions were quenched with the addition of 10 μL SAM (600 μM) and 30 μL of the reactions were added to 96-­‐well filter binding plates. The membranes were washed 3 times with 200 μL of 10% tricarboxylic acid followed by washing once with 95% ethanol. The membranes were air dried and 30 μL Microscint 0 was added before reading in a TopCount.

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Magnetic pull-­down of EZH2 to determine effect of EPZ005687 on protein-­ protein interactions within the PRC2 complex [2]
50% suspension of Anti-­‐FLAG M2 magnetic beads was obtained commercially and washed 3 times in 1X assay buffer, consisting of 20 mM bicine (pH = 7.6) and 0.002% Tween20. PRC2 complex (500 nM) containing FLAG-­‐tagged EED was incubated with 10 μM EPZ005687 in 1X assay buffer. The DMSO contribution from addition of the compounds was 1%, so a vehicle control of 1% DMSO was also included. The incubations were then added to 50 μL of washed beads that were resuspended in 50 μL of 1X assay buffer in the wells of a 96-­‐well polypropylene microplate and incubated for 1 h at room temperature. The beads were pulled down using a microplate magnet, and 50 μL of supernatant was removed. The beads were washed 3 times with 100 μL of 1X assay buffer and resuspended in 50 μL of 1X assay buffer. The supernatant and beads were then mixed with an equal volume of 2X SDS-­‐PAGE gel loading buffer and boiled for 10 minutes. The samples were run on an 8% Tris-­‐glycine SDS-­‐PAGE gel for 90 min at 125V, then visualized with GelCode Blue staining.

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Determination of Mutually Exclusive Binding of SAH and EPZ005687 by Yonetani-­Theorell Analysis [2]
SAH and EPZ005687 were serially diluted and spotted into a 384-­‐well microplate in a grid pattern such that all combinations of concentrations of SAH and EPZ005687 were obtained. To this, 40 μL of a mixture containing PRC2 (4 nM) and biotin in 1X assay buffer (20 mM Bicine [pH 7.6], 0.002% Tween 20, 0.005% Bovine Skin Gelatin and 0.5 mM DTT). 10 μL per well of substrate mix comprising assay buffer 3H-­‐SAM (150 nM), unlabeled SAM (1800 nM), and peptide representing histone H3 residues 21 – 44 containing C-­‐terminal biotin (appended to a C-­‐terminal amide-­‐capped lysine) (185 nM) were added to initiate the reaction (both substrates were present in the final reaction mixture at their respective Km values, an assay format referred to as ‘‘balanced conditions’’4. Reactions were incubated for 90 min at room temperature and quenched with 10 μL per well of 600 μM unlabeled SAM, then transferred to a 384-­‐well Flashplate and washed after 30 min. The inverse of the reaction velocity was plotted as a function of SAH concentration at several different concentrations of EPZ005687 to yield a Yonetani-­‐Theorell plot.

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Selectivity of EPZ005687 Against a Panel of 77 Human Ion Channels and GPCRs [2]
EPZ005687 was tested for the displacement of radiolabeled ligands known to bind to 77 human ion channels and GPCRs in a standard panel offered by Cerep. EPZ005687 was tested in duplicate at a concentration of 10 μM and the specific ligand binding to the receptor is defined as the difference between the total binding and the non-­‐specific binding determined in the presence of an excess of unlabeled ligand. The results in Supplementary Table 1 are expressed as percent of control of specific binding ((measured specific control/specific control binding) X 100). Also shown in Supplementary Table 1 are the identities of the reference radioligands and their binding affinities.

In Vitro Cell Assays.[1]
For the adherent cell line proliferation assays [all cell lines except KYM-1, which was analyzed as previously described for suspension cell lines, plating densities for each cell line were determined based on growth curves (measured by ATP content) and density over a 7-d time course. On the day before compound treatment, cells were plated in either 96-well plates in triplicate (for the day 0–7 time course) or 6-well plates (for replating on day 7 for the remainder of the time course). On day 0, cells were either untreated, DMSO-treated, or treated with Tazemetostat (EPZ6438) starting at 10 µM and decreasing in either threefold or fourfold dilutions. Plates were read on day 0, day 4, and day 7 using Cell Titer Glo, with compound/media being replenished on day 4. On day 7, the six-well plates were trypsinized, centrifuged, and resuspended in fresh media for counting by Vi-Cell. Cells from each treatment were replated at the original density in 96-well plates in triplicate. Cells were allowed to adhere to the plate overnight, and cells were treated as on day 0. On days 7, 11, and 14, plates were read using Cell Titer Glo, with compound/media being replenished on day 11. Averages of triplicates were used to plot proliferation over the time course, and calculate IC50 values. For cell cycle and apoptosis, G401 and RD cells were plated in 15-cm dishes in duplicate at a density of 1 × 106 cells per plate. Cells were incubated with Tazemetostat (EPZ6438) at 1 µM, in a total of 25 mL, over a course of 14 d, with cells being split back to original plating density on day 4, 7, and 11. Cell cycle analysis and TUNEL assay were performed using a Guava flow cytometer, following the manufacturer’s protocol.

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Gene Expression Analysis.[1]
G401 and RD cells were plated in T-75 flasks at 175,000 cells per flask and 117,000 cells per flask, respectively, and allowed to adhere overnight. On day 0, cells were treated in duplicates with DMSO or 1 µM Tazemetostat (EPZ6438). Cells were harvested and pelleted on days 2, 4, and 7 with media and compound being replenished on day 4. Tumor tissue from the G401 xenograft animals dosed for 21 d [vehicle, 125 mg/kg, and 250 mg/kg (six animals each) and 500 mg/kg (four animals) Tazemetostat (EPZ6438) dose groups] were used for gene expression analysis. Total mRNA was extracted from cell pellets and tumor tissue using the RNeasy Mini Kit and reverse transcribed by the High Capacity cDNA Reverse Transcription Kit. RT-PCR was performed by ViiA 7 Real-Time PCR Systems (AB) using TaqMan Fast Advanced Master Mix (AB; 4444964) and TaqMan primer/probe sets. Gene expression was normalized to 18S (AB; Hs99999901_s1), and fold change was calculated using the ΔΔCt method. For the in vivo samples, the average Ct value ± SD was determined for each dose group and fold change compared with vehicle dose group was calculated using the ΔΔCt method.

For the in vivo study, mice were inoculated s.c. at the right flank with G401 tumor cells (5 × 106 cells per mouse) in 0.2-mL mixture of base media and Matrigel (McCoy’s 5A/Matrigel, 1:1) for tumor development. The treatments were started when the tumor size reached ∼157 mm3 for the tumor efficacy study (n = 16 mice per group). Tazemetostat (EPZ6438) or vehicle (0.5% NaCMC plus 0.1% Tween 80 in water) was administered orally BID at a dose volume of 10 µL/g for either 21 or 28 d. Animal body weights were measured every day during the first week, and then twice weekly for the remainder of the study. Tumor size was measured twice weekly in two dimensions using a caliper, and the volume was expressed in cubic millimeters. For pharmacokinetic–pharmacodynamic analysis, eight mice with the largest tumor burden were euthanized for tumor and blood collection after 21 d of dosing. The remaining mice continued dosing for 1 more week, and from day 29, treatment was stopped and the mice were enrolled in a tumor growth delay study. Mice were observed as individuals until they reached the tumor weight endpoint (2,000 mm3) or until day 60 (whichever came first). [1]

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Dissolved in 0.5% NaCMC plus 0.1% Tween 80 in water; 500 mg/kg; Oral administration

SCID mice bearing s.c. G401 xenografts.

Absorption, Distribution and Excretion
Tazemetostat 800mg twice daily leads to a Cmax of 829ng/mL, with a Tmax of 1-2 hours , and an AUC of 3340ng\*h/mL. Absorption is not significantly affected by a high fat, high calorie meal. Tazemetostat is 33% bioavailable.
Tazemetostat is 15% eliminated in urine and 79% eliminated in feces.
Tazemetostat has a volume of distribution of 1230L.
Tazemetostat has an apparent total clearance of 274L/h.

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Metabolism / Metabolites
Tazemetostat is metabolized by CYP3A4 to an inactive desethyl metabolite and one other inactive metabolite not described.

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Biological Half-Life
Tazemetostat has a terminal elimination half life of 3.1h.

Hepatotoxicity
In clinical trials, serum ALT elevations occurred in 14% and AST elevations in 18% of patients on tazemetostat therapy and rose to more than 5 times ULN in 3.5%. Nevertheless, there were no instances of clinically apparent liver injury with symptoms or jaundice in several multicenter open-label trials of tazemetostat. Despite the frequency of adverse events during tazemetostat therapy, discontinuations due to adverse events are uncommon. Clinical experience with tazemetostat, however, is limited and the frequency of de novo serum enzyme elevations during treatment raises the issue of its potential for causing hepatotoxicity.
Likelihood score: E* (unproven but suspected rare cause of clinically apparent liver injury).

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Protein Binding
Tazemetostat is 88% protein bound in plasma.

[1]. Durable tumor regression in genetically altered malignant rhabdoid tumors by inhibition of methyltransferaseEZH2. Proc Natl Acad Sci U S A. 2013 May 7;110(19):7922-7.

[2]. A selective inhibitor of EZH2 blocks H3K27 methylation and kills mutant lymphoma cells. Nat Chem Biol . 2012 Nov;8(11):890-6.

Tazemetostat is a methyltransferase inhibitor used to treat metastatic or locally advanced epithelioid sarcoma not eligible for complete resection. Tazemetostat was first named in literature as EPZ-6438. Tazemetaostat was granted FDA approval on 23 January 2020.
Tazemetostat is a Methyltransferase Inhibitor. The mechanism of action of tazemetostat is as a Methyltransferase Inhibitor, and Multidrug and Toxin Extrusion Transporter 1 Inhibitor, and Multidrug and Toxin Extrusion Transporter 2 K Inhibitor.
Tazemetostat is a methyltransferase inhibitor and antineoplastic agent used in the therapy of advanced epithelioid sarcoma. Tazemetostat is associated with a moderate rate of transient serum enzyme elevations during therapy, but has not been implicated in cases of clinically apparent acute liver injury with jaundice.
Tazemetostat is an orally available, small molecule selective and S-adenosyl methionine (SAM) competitive inhibitor of histone methyl transferase EZH2, with potential antineoplastic activity. Upon oral administration, tazemetostat selectively inhibits the activity of both wild-type and mutated forms of EZH2. Inhibition of EZH2 specifically prevents the methylation of histone H3 lysine 27 (H3K27). This decrease in histone methylation alters gene expression patterns associated with cancer pathways and results in decreased tumor cell proliferation in EZH2 mutated cancer cells. EZH2, which belongs to the class of histone methyltransferases (HMTs), is overexpressed or mutated in a variety of cancer cells and plays a key role in tumor cell proliferation.
See also: Tazemetostat Hydrobromide (active moiety of); Tazemetostat hydrochloride (is active moiety of); Tazemetostat dihydrobromide (is active moiety of) ... View More ...

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Drug Indication
Tazemetostat is indicated to treat adult and pediatric patients 16 years and older with metastatic or locally advanced epithelioid sarcoma that is not eligible for complete resection. It is also indicated to treat adult patients with relapsed or refractory follicular lymphoma whose tumors are positive for an IZH2 mutation and who have received at least 2 prior systemic therapies. Additionally, it is indicated in adult patients with relapsed or refractory follicular lymphoma who have no satisfactory alternative treatment options.

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Mechanism of Action
EZH2 is a methyltransferase subunit of the polycomb repressive complex 2 (PRC2) which catalyzes multiple methylations of lysine 27 on histone H3 (H3K27). Trimethylation of this lysine inhibits the transcription of genes associated with cell cycle arrest. PRC2 is antagonized by the switch/sucrose non-fermentable (SWI/SNF) multiprotein complex. Abnormal activation of EZH2 or loss of function mutations in SWI/SNF lead to hyper-trimethylation of H3K27. Hyper-trimethylation of H3K27 leads to cancer cell de-differentiation, a gain of cancer stem cell-like properties. De-differentiation can allow for cancer cell proliferation. Tazemetostat inhibits EZH2, preventing hyper-trimethylation of H3K27 and an uncontrollable cell cycle.

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Pharmacodynamics
Tazemetostat is a methyltransferase inhibitor that prevents hyper-trimethylation of histones and inhibits cancer cell de-differentiation. The duration of action is long as it is given twice daily. Patients should be counselled regarding the risk of secondary malignancies and embryo-fetal toxicity.

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Inactivation of the switch/sucrose nonfermentable complex component SMARCB1 is extremely prevalent in pediatric malignant rhabdoid tumors (MRTs) or atypical teratoid rhabdoid tumors. This alteration is hypothesized to confer oncogenic dependency on EZH2 in these cancers. We report the discovery of a potent, selective, and orally bioavailable small-molecule inhibitor of EZH2 enzymatic activity, (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). The compound induces apoptosis and differentiation specifically in SMARCB1-deleted MRT cells. Treatment of xenograft-bearing mice with (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) leads to dose-dependent regression of MRTs with correlative diminution of intratumoral trimethylation levels of lysine 27 on histone H3, and prevention of tumor regrowth after dosing cessation. These data demonstrate the dependency of SMARCB1 mutant MRTs on EZH2 enzymatic activity and portend the utility of EZH2-targeted drugs for the treatment of these genetically defined cancers.[1]

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EZH2 catalyzes trimethylation of histone H3 lysine 27 (H3K27). Point mutations of EZH2 at Tyr641 and Ala677 occur in subpopulations of non-Hodgkin's lymphoma, where they drive H3K27 hypertrimethylation. Here we report the discovery of EPZ005687, a potent inhibitor of EZH2 (K(i) of 24 nM). EPZ005687 has greater than 500-fold selectivity against 15 other protein methyltransferases and has 50-fold selectivity against the closely related enzyme EZH1. The compound reduces H3K27 methylation in various lymphoma cells; this translates into apoptotic cell killing in heterozygous Tyr641 or Ala677 mutant cells, with minimal effects on the proliferation of wild-type cells. These data suggest that genetic alteration of EZH2 (for example, mutations at Tyr641 or Ala677) results in a critical dependency on enzymatic activity for proliferation (that is, the equivalent of oncogene addiction), thus portending the clinical use of EZH2 inhibitors for cancers in which EZH2 is genetically altered.[2]

C34H44N4O4

572.74

572.336

C, 71.30; H, 7.74; N, 9.78; O, 11.17

1403254-99-8

1467052-84-1 (HCl);1467052-75-0 (HBr);1403254-99-8;

66558664

Off-white to light yellow solid powder

1.2±0.1 g/cm3

750.8±60.0 °C at 760 mmHg

407.9±32.9 °C

0.0±2.5 mmHg at 25°C

1.589

0.98

2

6

9

42

992

0

NSQSAUGJQHDYNO-UHFFFAOYSA-N

InChI=1S/C34H44N4O4/c1-5-38(29-10-14-41-15-11-29)32-20-28(27-8-6-26(7-9-27)22-37-12-16-42-17-13-37)19-30(25(32)4)33(39)35-21-31-23(2)18-24(3)36-34(31)40/h6-9,18-20,29H,5,10-17,21-22H2,1-4H3,(H,35,39)(H,36,40)

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

Tazemetostat; E7-438; EPZ 6438; Tazemetostat; 1403254-99-8; 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; Tazemetostat [INN]; E 7438; EPZ6438; tazerik; E7438; EPZ-6438;

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)