PRMT5 (IC50 = 22 nM)

EPZ015666 (GSK3235025) suppresses SmD3 methylation and cell death in MCL cell lines, showing IC50 values in the nanomolar range [1]. EPZ015666 (GSK3235025) is a strong peptide-competitive and SAM-cooperative inhibitor with over 10,000-fold selectivity against PRMT5 compared to other methyltransferases[2].

EPZ015666 (GSK3235025) is bioavailable when taken orally and suitable for in vivo research. We conducted 21-day efficacy studies in mice with severe combined immunodeficiency (SCID) that were given subcutaneous Z-138 and Maver-1 xenografts. The mice were given oral doses twice a day (BID) at four different dose levels: 25, 50, 100, and 200 mg/kg. The animals are put to sleep after receiving a dose continuously for 21 days, and samples of blood and tissues are examined to ascertain the connection between tumor-growth inhibition (TGI) and methyl-mark pharmacodynamics. In both MCL models, EPZ015666 (GSK3235025) demonstrated dose-dependent exposure and TGI after 21 days. When weighed against vehicle-treated tumors, all EPZ015666 (GSK3235025) dose groups' tumors demonstrated statistically significant differences in weight, volume, and tumor growth on day 21. 200 mg/kg BID dosage resulted in tumor stasis in Z-138 cells, which showed >93% TGI after 21 days, while Maver-1 cells only displayed >70% TGI. Furthermore, the Granta-519 cell line, a rapidly expanding model that reached endpoint on day 18 and demonstrated dose-dependent efficacy with 45% TGI in the 200 mg/kg group, is used to test a third MCL xenograft. With little bodyweight loss in the 200 mg/kg dose group and no additional clinical observations, EPZ015666 (GSK3235025) is well tolerated in all three models[1].

Methyltransferase Assay.[2]
The inhibitory activity of MTA against the catalytic activity of 31 histone methyltransferases (including histone lysine methyltransferases and histone arginine methyltransferases) was assayed using the HotSpotSM radioisotope filter-binding platform as described. Here, MTA was incubated in the presence of a histone methyltransferase, substrate, and tritium-labeled SAM, and detection of the methylated radiolabeled reaction product was performed using a filter-binding method. Briefly, an MTA stock solution was prepared in DMSO at 100 mM. MTA was tested in 10-dose titrations with 3-fold serial dilution starting at 3 mM. The methyltransferase inhibitors SAH (S-(5'-Adenosyl)-L-homocysteine) and chaetocin were used as positive controls; these were tested in 10-dose titrations with 3-fold serial dilution starting at 100 or 200 μM, respectively. All reactions were carried out with 1 μM tritium-labeled SAM and 5 μM peptide or protein substrate. Details about the identity and source of profiled methyltransferases and corresponding substrates are available in Additional Data Table S8. Assays were performed in one of four buffers: Buffer A (50 mM Tris-HCl, pH 8.5, 5 mM MgCl2, 50 mM NaCl, 0.01% Brij35, 1 mM DTT), Buffer B (50 mM Tris-HCl, pH 8.0, 1 mM EDTA, 0.01% Brij35, 1 mM DTT), Buffer C (50 mM Bicine, pH 8.5, 5 mM MgCl2, 50 mM NaCl, 0.01% Brij35, 1 mM DTT), or Buffer D (50 mM Tris-HCl, pH 8.5, 0.01% Brij35, 1 mM DTT). Buffers used for each enzyme assay are listed in Additional Data Table S8. Reactions were performed for 1 hr at 37oC. Curve fits and IC50 determination were performed as described in (31). The dendrogram shown in Fig. 4B was generated using Reaction Biology’s HMT Mapper (http://www.reactionbiology.com/webapps/site/HMTMapper.aspx?map=Methyltransferase).

TA Mechanism of Action Study. [2]
PRMT5 activity was measured using AlphaLISA performed in 384-well AlphaPlates. For MTA IC50 determination, 30 nM PRMT5/MEP50 expressed in HEK293 was incubated for 2 hrs at RT with 1M histone H4 (1-21)-lys(biotin), using varying SAM concentration in 20 L reaction buffer (20 mM sodium phosphate pH 8.5, 1 mM EDTA, 1 mM TCEP, and 0.01% Tween-20) containing compound or DMSO. Reactions were quenched with the addition 20 uL detection solution containing Streptavidin Donor Beads and AlphaLISA® Anti-methyl-Histone H4 Arginine 3 Acceptor Beads diluted to 20 ng/L in 1x Epigenetics Buffer. After 1 hr incubation at RT, luminescence was measured on the Envision 2104 plate reader. Percent activity values were calculated by setting the average background (no enzyme wells) to 0% and the average DMSO wells to 100% activity. Standard deviations were determined from four replicate measurements for compound concentration. Data were analyzed and plotted using GraphPad PRISM v6 and IC50 values were determined using the ‘log(inhibitor) vs normalized reponse –variable slope’ analysis module.

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Cell viability IC50 measurement. [2]
Cell plating densities were determined by evaluating the number of cells required to achieve 70% confluency after 12 days in culture. Cells were seeded in 100 µL of media in 96-well plates (excluding wells in the first or last row or column) using the following densities in cells per well: LU99 at 360, H647 at 100, SF-172 at 35, SU8686 at 250, MIAPACA2 at 135, H838 at 73, H2126 at 1042, HCC44 at 62, KP2 at 292, H661 at 166, and H2030 at 250. Isogenic cell line pairs were seeded at the same densities for all experiments.
MTA and EPZ015666 were dissolved in DMSO at 100 mM and 10 mM, respectively. Drug was administered using the HP D300 digital dispenser. DMSO concentration did not exceed 0.32%. Each drug concentration was plated in six replicates, and the MTA concentration range was titrated logarithmically over 316 µM to 31.6 nM while EPZ015666 concentration range was titrated logarithmically over 31.6 µM to 3.16 nM. Media and drug were changed every 4 days.
Consistent with previously-published inhibitor studies with EPZ015666, total cell viability was assessed after 12-19 days using Cell Titer-Glo luminescent cell viability assay. Isogenic cell line pairs were plated and cultured in parallel for the same time period. Luminescence was measured according to the manufacturer’s instructions using the EnVision Multilabel Reader. Luminescence data from each well was normalized to luminescence from wells containing untreated cells on that same plate. IC50 was calculated by non-linear regression using the ‘log(inhibitor) vs. response (three parameters) analysis in GraphPad Prism 6.

Dissolved in 0.5 % MC; 200 mg/kg; p.o.;
MCL (Z-138, and Maver-1) xenograft models

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Male CD-1 mice (25-40 g; n=6, with 3 per time point) were treated with a single dose of EPZ015666 at 2 mg/kg by intravenous tail-vein injection and 10 mg/kg by oral gavage administration, with both doses formulated in 20% N-N-dimethylacetamide in water. Animals are fasted overnight and weighed before dose administration on the day of dosing. Approximately 30 μL of blood are taken from animals by submandibular or retro-orbital bleeding at pre-specified time intervals (seven time points). For the last time point (24 h), samples are collected via cardiac puncture while the animals are under anesthesia (70% CO2:30% O2). Blood samples are transferred into K2-EDTA tubes and placed on wet ice before centrifugation at 4°C (3,000g, 15 min) to obtain plasma within 30 min after sample collection. Plasma samples are stored at −70±10°C before protein precipitation and LC-MS/MS analysis. We constructed standard calibration curves by analyzing a series of control plasma aliquots containing 100 ng/mL labetalol as an internal standard and 1-3,000 ng/mL EPZ015666. Four levels of quality control are also included in the analysis (3-2,400 ng/mL EPZ015666). Data are analyzed using Phoenix WinNonlin 6.2.1.[1]

[1]. Chan-Penebre E, et al. A selective inhibitor of PRMT5 with in vivo and in vitro potency in MCL models. Nat Chem Biol. 2015 Jun;11(6):432-7.
[2]. Kryukov GV, et al. MTAP deletion confers enhanced dependency on the PRMT5 arginine methyltransferase in cancer cells. Science. 2016 Mar 11;351(6278):1214-8

Protein arginine methyltransferase-5 (PRMT5) is reported to have a role in diverse cellular processes, including tumorigenesis, and its overexpression is observed in cell lines and primary patient samples derived from lymphomas, particularly mantle cell lymphoma (MCL). Here we describe the identification and characterization of a potent and selective inhibitor of PRMT5 with antiproliferative effects in both in vitro and in vivo models of MCL. EPZ015666 (GSK3235025) is an orally available inhibitor of PRMT5 enzymatic activity in biochemical assays with a half-maximal inhibitory concentration (IC50) of 22 nM and broad selectivity against a panel of other histone methyltransferases. Treatment of MCL cell lines with EPZ015666 led to inhibition of SmD3 methylation and cell death, with IC50 values in the nanomolar range. Oral dosing with EPZ015666 demonstrated dose-dependent antitumor activity in multiple MCL xenograft models. EPZ015666 represents a validated chemical probe for further study of PRMT5 biology and arginine methylation in cancer and other diseases.[1]

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The discovery of cancer dependencies has the potential to inform therapeutic strategies and to identify putative drug targets. Integrating data from comprehensive genomic profiling of cancer cell lines and from functional characterization of cancer cell dependencies, we discovered that loss of the enzyme methylthioadenosine phosphorylase (MTAP) confers a selective dependence on protein arginine methyltransferase 5 (PRMT5) and its binding partner WDR77. MTAP is frequently lost due to its proximity to the commonly deleted tumor suppressor gene, CDKN2A. We observed increased intracellular concentrations of methylthioadenosine (MTA, the metabolite cleaved by MTAP) in cells harboring MTAP deletions. Furthermore, MTA specifically inhibited PRMT5 enzymatic activity. Administration of either MTA or a small-molecule PRMT5 inhibitor showed a modest preferential impairment of cell viability for MTAP-null cancer cell lines compared with isogenic MTAP-expressing counterparts. Together, our findings reveal PRMT5 as a potential vulnerability across multiple cancer lineages augmented by a common "passenger" genomic alteration.[2]

C20H25N5O3

383.44

383.2

C, 62.65; H, 6.57; N, 18.26; O, 12.52

1616391-65-1

90241673

White to off-white solid powder

1.3±0.1 g/cm3

686.4±55.0 °C at 760 mmHg

368.9±31.5 °C

0.0±2.2 mmHg at 25°C

1.652

2.14

99.61

O=C(C1=NC=NC(NC2COC2)=C1)NC[C@H](O)CN3CC4=C(C=CC=C4)CC3

ZKXZLIFRWWKZRY-KRWDZBQOSA-N

InChI=1S/C20H25N5O3/c26-17(10-25-6-5-14-3-1-2-4-15(14)9-25)8-21-20(27)18-7-19(23-13-22-18)24-16-11-28-12-16/h1-4,7,13,16-17,26H,5-6,8-12H2,(H,21,27)(H,22,23,24)/t17-/m0/s1

(S)-N-(3-(3,4-dihydroisoquinolin-2(1H)-yl)-2-hydroxypropyl)-6-(oxetan-3-ylamino)pyrimidine-4-carboxamide

GSK-3235025; GSK 3235025; GSK3235025; EPZ-015666; EPZ 015666; EPZ015666.

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)

Solubility in Formulation 1: ≥ 2.5 mg/mL (6.52 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: ≥ 2.5 mg/mL (6.52 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

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Solubility in Formulation 3: ≥ 2.5 mg/mL (6.52 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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Solubility in Formulation 4: 2% DMSO+30% PEG 300+ddH2O:5mg/mL

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 (Please use freshly prepared in vivo formulations for optimal results.)