type I PRMT; PRMT1 (IC50 = 3.1 nM); PRMT3 (IC50 = 48 nM); PRMT4 (IC50 = 1148 nM); PRMT6 (IC50 = 5.7 nM); PRMT1 (IC50 = 1.8 nM)

GSK3368715 (EPZ019997), one of 249 cancer cell lines that represented 12 tumor types, exhibited growth inhibition of at least 50% in comparison to cells treated with DMSO [1].

At all tested doses, GSK3368715 (EPZ019997) significantly inhibited the growth of BxPC3 xenografts, with tumor growth reductions of 78% and 97% in the 150 mg/kg and 300 mg/kg dose groups, respectively [1].

High Throughput Screen[1]
Type I PRMT inhibitors were found through screening Epizyme’s proprietary HMT-biased library (Mitchell et al., 2015). In summary, compound was incubated with PRMT1 for 30 minutes at room temperature (384-well plate) and reactions were initated upon the addition of SAM and peptide. Final assay conditions were 0.75 nM PRMT1 (NP_001527.3, GST-PRMT1 amino acids 1-371), 200 nM 3H-SAM (specific activity 80 Ci/mmol), 1.5 μM SAM , and 20 nM peptide (Biotin-Ahx-RLARRGGVKRISGLI-NH2, 21st Century Biochemicals) in 20 mM bincine (pH 7.6), 1mM TCEP, 0.005% bovine skin gelatin, 0.002% Tween-20 and 2% DMSO. Reactions were quenched by the addition of SAM (400 μM final). Terminated reactions were transferred to a Streptavidin-coated Flashplate, incubated for at least 1 hour and then the plate was washed with 0.1% Tween-20 using a Biotek ELx405 plate washer. The quantity of 3H-peptide bound to the Flashplate was measured using a PerkinElmer TopCount plate reader.

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PRMT Biochemical Assays[1]
All assays were performed with compound or DMSO prestamped (49x, 2% final) in 96 well plates. Assays for PRMT1, PRMT3, PRMT6 and PRMT8 used H4 1-21 peptide and a buffer comprised of 50 mM Tris (pH 8), 0.002% Tween-20, 0.5 mM EDTA and 1 mM DTT. Briefly, Flag-his-tev-PRMT8 (61-394) was expressed in a baculovirus expression system and purified using Ni-NTA agarose affinity chromatography and Superdex 200 gel filtration chromatography. For all assays, final Adenosyl-L-Methionine (SAM) concentration listed contains a mixture of unlabeled SAM and 3H-SAM All reactions were quenched upon the addition of SAH (0.5 mM final).[1]
For competition studies, substrate was added to the compound plate followed by the addition of enzyme. For SAM competition studies, final assay concentrations consisted of 2 nM PRMT1, 40 nM peptide and titrating SAM (50-8000 nM). For peptide competition studies, final assay concentrations consisted of 2 nM PRMT1, 1000 nM and titrating peptide (1.6-1000 nM). Reactions were incubated at room temperature for 18 minutes prior to quench.[1]
For time dependence studies, enzyme/SAM mix was added to the compound plate and incubated for 3-60 minutes prior to addition of the peptide. For no preincubation assay, peptide was added to the compound plate followed by enzyme/SAM mix to initiate the reaction. Final PRMT1 assay concentrations were 0.5 nM PRMT1, 40 nM peptide and 1100 nM SAM. Reactions were incubated at room temperature for 20 minutes prior to quench.

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Methyltransferase Biochemical Assays[1]
In summary, methyltransferase was added to substrate solution and gently mixed. Substrate varied based on methyltransferase tested and was either nucleosome, core histones, histone H3, histone H4 or H3 1-21 peptide. Compound (10 μM final) was added and incubated at room temperature for 10 minutes. Reaction was initiated upon the addition of 3H-SAM (1 μM) and incubated for 1 hour at 30°C. Reaction mixture was delivered to P81 filter-paper and washed with PBS for detection via HotSpot proprietary technology.

In Cell Western[1]
RKO cells were seeded in a clear bottom 384 well plates andtreated with a 20-point two-fold dilution series of GSK3368715 (29,325.5 to 0.03 nM) or 0.15% DMSO. Plates were incubated for 3 days at 37°C in 5% CO2. Cells were fixed with ice-cold methanol for 30 minutes at room temperature, washed with phosphate buffered saline (PBS), then incubated with Odyssey blocking buffer for 1 hour at room temperature. Blocking buffer was removed and cells were incubated overnight at 4°C with rabbit anti-mono-methyl Arginine and mouse anti- α-tubulin diluted in blocking buffer plus 0.1% Tween-20. Following PBS washes, secondary antibodies IRDye 800CW goat anti-Rabbit IgG (H+L) and IRDye 680RD goat anti-mouse IgG (H+L) were applied for 1 hour. Plates were washed thoroughly with PBS, then ddH2O and allowed to dry at room temperature. Plates were scanned and analyzed using the Li-Cor Odyssey imager and software. The relative MMA expression was determined by dividing the integrated intensity of MMA by the integrated intensity of tubulin using Microsoft Excel. The MMA level was then plotted against the log concentration of the compound and plotted using a 4-parameter fit equation using GraphPad Prism 6.0.

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Cell Proliferation Assay[1]
Growth inhibition in response to GSK3368712 and GSK3368715 was evaluated as previously described (McCabe et al., 2012). Data were fit with a four-parameter equation to generate a concentration response curve. The growth IC50 (gIC50) and growth IC100 (gIC100) are the points at which 50% and 100% inhibition of growth are achieved, respectively. Growth Inhibition is the percent maximal inhibition and was calculated as 100-((ymin-100)/(ymax-100)∗100). Ymin-T0 values were calculated by subtracting the T0 value (100%) from the ymin value on the curve, and are a measure of net population cell growth or death. Growth Death Index (GDI) is a composite representation of Ymin-T0 and precent maximal inhibition. If Ymin-T0 values are negative, then GDI equals Ymin-T0; otherwise, GDI represents the fraction of cells remaining relative to DMSO control (ymax) and (ymin): (ymin-100)/(ymax-100)∗100). A minimum of two biological replicates were evaluated for each assay.

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Evaluation of Synergistic Effects on Cell Proliferation[1]
A double titration of GSK3368715 (or GSK3368712) and GSK3203591 was performed for 6 days as described above, except that cells were dosed with a 16-pt, 2-fold dilution matrix of both agents, ranging in concentration from 0.3 to 10,000 nM. Single agent titrations were run in parrallel. Bliss independence analysis was performed using growth inhibition value for each combination and a synergy score determined as previously described (McGrath et al., 2016).

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Cell Cycle Analysis[1]
The Toledo or OCI-Ly1 DLBCL cell lines were treated with a 5-point, 10-fold dilution series GSK3368715 or 0.1% DMSO for 10 days. On days 3, 5, 7, and 10 cell nuclei were isolated and DNA was stained with propidium iodide using CycleTEST PLUS DNA Reagent Kit (Becton Dickinson) per the manufacturer’s instructions. Fluorescence was measured using a Becton Dickinson FACS Calibur flow cytometer. Cell cycle phase distribution was determined by the Watson Pragmatic mathematical model using FlowJo software.

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Caspase 3/7 Assay[1]
The effect of GSK3368715 treatment on cellular caspase-3/7 activity was measured with Caspase-Glo™3/7 assay kit. Assays were performed according to the manufacturer's instructions. Cells were plated and dosed with GSK3368715 or DMSO as described for the cell proliferation assay. At each timepoint, CellTiter-Glo reagent was added to duplicate plates to assess cell viability and Caspase-Glo 3/7 reagent was added to another pair of duplicate platesto assess cell death. The luminescence signal was measured with an EnVision Plate Reader. Caspase 3/7 Glo and CTG values for GSK3368715 and DSMO were background subtracted for each plate. To account for cell number, Caspase 3/7 Glo values for each dose were then normalized to their corresponding CTG value. Normalized Caspase 3/7 Glo values were expressed as a fold increase over the average DMSO Caspase 3/7 Glo value for each dose of GSK3368715. Fold-increases for replicate plates were then averaged for each biological replicate.

In vivo antitumor efficacy study[1]
Efficacy studies of GSK3368712 in a pancreatic patient derived xenograft model (PAXF 2076) were carried out at Charles River Discovery Research Services Germany. Tumor fragments were implanted into Female NMRI nu/nu mice (NMRI-Foxn1nu). Animals and tumor implants were monitored daily until solid tumor growth was detectable in a sufficient number of animals. Following randomization, animals were assigned into study groups and dosed once daily with vehicle or GSK3368712.

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Toxicology Assessment[1]
The toxicological profile of once-daily, oral dosing of GSK3368715 was evaluated in rising and repeat dose toxicity studies. Doses up to the maximal tolerated dose were evaluated in dose range studies. Studies were conducted using pharmacologically relevant rodent (rat; 10-12 week old Wistar:Han; n=10-16 per sex per group) and non-rodent (dog; 10-12 month old beagle; n=3-5 per sex per group) species. Assessments were GLP compliant and consistent with ICH S9 guidance. All studies were conducted in accordance with the GSK Policy on the Care, Welfare and Treatment of Laboratory Animals and were reviewed by the Institutional Animal Care and Use Committee at GSK.

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Pharmacokinetic study[1]
Pharmacokinetic analysis of GSK3368715 and GSK3368712 revealed that both compounds had suitable PK properties for oral administration and in vivo assessment of anti-tumor activity (Table S3). In toxicology studies conducted in rats and dogs, primary on-target toxicity affected the gastrointestinal tract and mild-to-moderate changes to hematopoetic lineages (Table S4), while doses used in mice were well tolerated. The efficacy of type I PRMTi in mice bearing xenografts of cell lines that had cytotoxic responses was examined. The Toledo DLBCL cell line has a cytotoxic response to GSK3368715 with a gIC50 of 59 nM in vitro (Figure 2C). Once-daily administration of GSK3368715 induced dose-dependent inhibition of Toledo tumor growth, with tumor regression in mice treated with >75 mg/kg (Figure 2D). The BxPC3 pancreatic adenocarcinoma cell line has a gIC50 of 2,100 nM, and was cytotoxic at concentrations above 10 μM GSK3368715 (Figure 2E). Once-daily administration of type I PRMTi had significant effects on the growth of BxPC3 xenografts at all doses tested, reducing tumor growth by 78% and 97% in the 150- and 300-mg/kg dose groups, respectively (Figure 2F). Efficacy studies with once-daily administration of 150 mg/kg GSK3368715 in cell line xenograft models of clear cell renal carcinoma (ACHN) and triple-negative breast cancer (MDA-MB-468) revealed tumor growth inhibition of 98% and 85%, respectively (Figures S2F and S2G). In a patient-derived xenograft model of pancreatic adenocarcinoma, type I PRMTi had significant effects on tumor growth, with inhibition >90% in a subset of animals within the 300-mg/kg cohort (Figure 2G).These data demonstrate that GSK3368715 has potent, anti-proliferative activity across cell lines representing a range of solid and hematological malignancies and can completely inhibit tumor growth or cause regressions of tumor models in vivo.

[1]. Anti-tumor Activity of the Type I PRMT Inhibitor, GSK3368715, Synergizes with PRMT5 Inhibition through MTAP Loss. Cancer Cell. 2019 Jul 8;36(1):100-114.e25.

Type I protein arginine methyltransferases (PRMTs) catalyze asymmetric dimethylation of arginines on proteins. Type I PRMTs and their substrates have been implicated in human cancers, suggesting inhibition of type I PRMTs may offer a therapeutic approach for oncology. The current report describes GSK3368715 (EPZ019997), a potent, reversible type I PRMT inhibitor with anti-tumor effects in human cancer models. Inhibition of PRMT5, the predominant type II PRMT, produces synergistic cancer cell growth inhibition when combined with GSK3368715. Interestingly, deletion of the methylthioadenosine phosphorylase gene (MTAP) results in accumulation of the metabolite 2-methylthioadenosine, an endogenous inhibitor of PRMT5, and correlates with sensitivity to GSK3368715 in cell lines. These data provide rationale to explore MTAP status as a biomarker strategy for patient selection.[1]
• GSK3368715 is a potent inhibitor of type I protein arginine methyltransferases
• GSK3368715 alters exon usage and has activity against multiple cancer models
• GSK3368715 synergizes with the PRMT5 inhibitor GSK3326595 to inhibit tumor growth
• MTAP gene deficiency impairs PRMT5 activity, sensitizing cancer cells to GSK3368715

C20H41CL3N4O2

475.9241

366.3

C, 65.54; H, 10.45; N, 15.29; O, 8.73

1629013-22-4

GSK3368715 dihydrochloride;1628925-77-8;GSK3368715 trihydrochloride;2227587-26-8; 1629013-22-4; 2227587-25-7 (HCl); 2227587-26-8 (3HCl)

90462880

Solid powder

1.5

2

5

12

26

365

0

SPEGERVLTUWZPA-UHFFFAOYSA-N

InChI=1S/C20H38N4O2/c1-5-25-15-20(16-26-6-2)9-7-17(8-10-20)19-18(13-22-23-19)14-24(4)12-11-21-3/h13,17,21H,5-12,14-16H2,1-4H3,(H,22,23)

N1-((3-(4,4-bis(ethoxymethyl)cyclohexyl)-1H-pyrazol-4-yl)methyl)-N1,N2-dimethylethane-1,2-diamine

EPZ019997;SK3368715; GSK-3368715; GSK 3368715; EPZ019997; EPZ-019997; GSK 3368715; EPZ019997; EPZ-019997;GSK-3368715; EPZ 019997

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)

May dissolve in DMSO (in most cases), if not, try other solvents such as H2O, Ethanol, or DMF with a minute amount of products to avoid loss of samples

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.

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Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution.
Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)
Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil)
Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals).

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Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)]
*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.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

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Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium)
Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose
Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals).

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Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

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Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

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