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Purity: ≥98%
EPZ015866 (also known as EPZ-015866; GSK-3203591; GSK-591) is an orally bioavailable and selective inhibitor of PRMT5 (protein arginine methyltransferase 5) with potential anticancer activity. EPZ015866 was discovered based on the precursor and analog EPZ015666 (GSK3235025).
| Targets |
PRMT5 (protein methyltransferase 5; IC50 = 4 nM)
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| ln Vitro |
Treatment with GSK591 (5 μM; MCF7, T47D, and MCF10A cells) inhibits the proliferation and self-renewal of breast cancer stem cells (BCSCs). In vitro, GSK591 lowers the quantity of BCSCs[2].
Pharmacological Inhibition of PRMT5 Reduces BCSC Numbers In Vitro [2] Because we demonstrated that the catalytic activity of PRMT5 was required for driving BCSC function and FOXP1 expression (Figures 1G and 6C), we wanted to determine whether a small-molecule inhibitor targeting PRMT5 could affect BCSC function. We therefore treated MCF7 cells with the validated PRMT5 inhibitor GSK3203591 (EPZ015866; herein referred to as GSK591) (Figure 7A; Duncan et al., 2015). Similar to PRMT5 depletion, inhibition of PRMT5 significantly suppressed MCF7-derived BCSC proliferation and self-renewal and the number of ALDEFLUOR+ T47D cells (Figures 7B and S7I), while overexpression of FOXP1 was able to rescue the BCSC proliferative defect induced by GSK591 (Figure 7C). Our data thus imply that drug targeting PRMT5 reduces the activity of BCSCs, but given that depletion of FOXP1 and GSK591 was not epistatic in reducing primary and secondary mammospheres (Figure 7B), FOXP1 is important but not sufficient for all PRMT5-dependent events in BCSCs. PRMT5 has been reported as a critical component of normal stem cell function (Chittka et al., 2012, Liu et al., 2015), hence one potential limitation of PRMT5-directed therapies for breast cancer is the suppression of normal mammary stem cell function. Interestingly, although knockdown and inhibition of PRMT5 reduces the number of primary mammospheres of MCF10A cells, self-renewal was unaffected (Figures 7B, S7I, and S7J). In support, stem cell numbers, as determined by ALDEFLUOR staining was unaffected by GSK591 (Figure S7I). Moreover, this lack of effect of PRMT5 deletion or inhibition on MCF10A self-renewal was not due to receptor status, as treatment of the triple-negative breast cancer cells SUM159 with GSK591 phenocopies that of MCF7 cells, reducing both BCSC proliferation and self-renewal (Figure 7B). Hence, it appears that PRMT5 activity specifically affects self-renewal of cancer rather than normal mammary stem cells. |
| ln Vivo |
EPZ015666 (an analog of EPZ01586) or vehicle (0.5% methylcellulose in water) was administered orally BID at a dose volume of 10 mL/kg for 21 d (Granta-519 was dosed for 18 d).
EPZ015666 (GSK3235025, an analog of EPZ015866) 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 [Nat Chem Biol. 2015 Jun;11(6):432-7.]. |
| Enzyme Assay |
PRMT Biochemical Assays[3]
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). 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. 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. Methyltransferase Biochemical Assays[3] 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. |
| Cell Assay |
Cell Proliferation Assay[2]
Cell Types: MCF7, T47D, and MCF10A cells Tested Concentrations: 5 μM Incubation Duration: Experimental Results: Suppressed BCSCs proliferation and self-renewal. Mammosphere Assay[2] MCF7, T47D, and MCF10A cells were plated onto pHEMA-coated six-well plates and cultured at 37°C for 5 days. Mammospheres >50 μm were scored using a graticle. For serial replating, mammospheres were disaggregated and cultured for a further 5 days prior to scoring. For drug treatment, cells were incubated with 5 μM GSK591 or 2.5 μM 4-OHT or vehicle controls. For further details, see Supplemental Experimental Procedures. |
| Animal Protocol |
Dissolved in 0.5% methylcellulose in water; 10 and 100 mg/kg; oral
Male CD-1 Mouse Xenograft Studies and In Vivo Imaging[2] Animal experiments were conducted in accordance with United Kingdom Home Office regulations. For the limiting dilution assay, 5- to 7-week-old female NSG mice were injected with the appropriate number of cells and a slow release estrogen pellet was subcutaneously implanted at the base of the tail. Two hours prior to harvest at the experimental endpoint, mice were injected with 100 mg/kg BrdU. For in vivo depletion of PRMT5, female NSG mice were injected with 5 × 106 cells and once tumors were palpable, maintained on a diet of dox chow. For imaging, mice were injected intraperitoneally (i.p.) with 150 mg/kg of luciferin, and images were captured on an IVIS Spectrum. See Supplemental Experimental Procedures for more detail. From these combined data, EPZ015666 emerged as the compound with the best combination of low plasma clearance and high oral bioavailability maintaining potent antiproliferative activity against a known mantle cell lymphoma cell line (Z-138 cells). Plasma protein binding in mouse plasma was determined to be approximately 30%. Further PK studies revealed that at 100 mg/kg in mouse, the unbound plasma concentration was equivalent to or above, the methyl mark IC90 for a period of 12 h, thus suggesting that BID dosing of EPZ015666 could effectively inhibit PRMT5 in vivo (Figure Figure44). Additional species PK and ADME characterization data on EPZ015666 were reported by Rioux et al.[1] |
| References |
[1]. Kenneth W. Duncan et al. Structure and Property Guided Design in the Identification of PRMT5 Tool Compound EPZ015666. ACS Med. Chem. Lett., 2016, 7 (2), pp 162-166.
[2]. Chiang K, et al. PRMT5 Is a Critical Regulator of Breast Cancer Stem Cell Function via Histone Methylation and FOXP1 Expression. Cell Rep. 2017 Dec 19;21(12):3498-3513. [3]. Fedoriw A, et al. 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. |
| Additional Infomation |
GSK591 (EPZ015866) is a potent and selective inhibitor of protein methyltransferase 5 (PRMT5).
The recent publication of a potent and selective inhibitor of protein methyltransferase 5 (PRMT5) provides the scientific community with in vivo-active tool compound EPZ015666 (GSK3235025) to probe the underlying pharmacology of this key enzyme. Herein, we report the design and optimization strategies employed on an initial hit compound with poor in vitro clearance to yield in vivo tool compound EPZ015666 and an additional potent in vitro tool molecule EPZ015866 (GSK3203591).[1] Breast cancer progression, treatment resistance, and relapse are thought to originate from a small population of tumor cells, breast cancer stem cells (BCSCs). Identification of factors critical for BCSC function is therefore vital for the development of therapies. Here, we identify the arginine methyltransferase PRMT5 as a key in vitro and in vivo regulator of BCSC proliferation and self-renewal and establish FOXP1, a winged helix/forkhead transcription factor, as a critical effector of PRMT5-induced BCSC function. Mechanistically, PRMT5 recruitment to the FOXP1 promoter facilitates H3R2me2s, SET1 recruitment, H3K4me3, and gene expression. Our findings are clinically significant, as PRMT5 depletion within established tumor xenografts or treatment of patient-derived BCSCs with a pre-clinical PRMT5 inhibitor substantially reduces BCSC numbers. Together, our findings highlight the importance of PRMT5 in BCSC maintenance and suggest that small-molecule inhibitors of PRMT5 or downstream targets could be an effective strategy eliminating this cancer-causing population.[2] |
| Molecular Formula |
C22H28N4O2
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|---|---|---|
| Molecular Weight |
380.48
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| Exact Mass |
380.221
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| Elemental Analysis |
C, 69.45; H, 7.42; N, 14.73; O, 8.41
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| CAS # |
1616391-87-7
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| Related CAS # |
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| PubChem CID |
117072552
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| Appearance |
White to off-white solid powder
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| Density |
1.3±0.1 g/cm3
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| Boiling Point |
648.4±55.0 °C at 760 mmHg
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| Flash Point |
345.9±31.5 °C
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| Vapour Pressure |
0.0±2.0 mmHg at 25°C
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| Index of Refraction |
1.645
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| LogP |
3.66
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| Hydrogen Bond Donor Count |
3
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| Hydrogen Bond Acceptor Count |
5
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| Rotatable Bond Count |
7
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| Heavy Atom Count |
28
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| Complexity |
513
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| Defined Atom Stereocenter Count |
1
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| SMILES |
O[C@@H](CNC(C1C=CN=C(C=1)NC1CCC1)=O)CN1CC2=CC=CC=C2CC1
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| InChi Key |
TWKYXZSXXXKKJU-FQEVSTJZSA-N
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| InChi Code |
InChI=1S/C22H28N4O2/c27-20(15-26-11-9-16-4-1-2-5-18(16)14-26)13-24-22(28)17-8-10-23-21(12-17)25-19-6-3-7-19/h1-2,4-5,8,10,12,19-20,27H,3,6-7,9,11,13-15H2,(H,23,25)(H,24,28)/t20-/m0/s1
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| Chemical Name |
(S)-2-(cyclobutylamino)-N-(3-(3,4-dihydroisoquinolin-2(1H)-yl)-2-hydroxypropyl)isonicotinamide
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| Synonyms |
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| HS Tariff Code |
2934.99.9001
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| Storage |
Powder -20°C 3 years 4°C 2 years In solvent -80°C 6 months -20°C 1 month |
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| Shipping Condition |
Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)
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| Solubility (In Vitro) |
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| Solubility (In Vivo) |
Solubility in Formulation 1: ≥ 2.25 mg/mL (5.91 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 22.5 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. Solubility in Formulation 2: ≥ 2.25 mg/mL (5.91 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 22.5 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. View More
Solubility in Formulation 3: ≥ 2.25 mg/mL (5.91 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution. |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 2.6283 mL | 13.1413 mL | 26.2826 mL | |
| 5 mM | 0.5257 mL | 2.6283 mL | 5.2565 mL | |
| 10 mM | 0.2628 mL | 1.3141 mL | 2.6283 mL |
*Note: Please select an appropriate solvent for the preparation of stock solution based on your experiment needs. For most products, DMSO can be used for preparing stock solutions (e.g. 5 mM, 10 mM, or 20 mM concentration); some products with high aqueous solubility may be dissolved in water directly. Solubility information is available at the above Solubility Data section. Once the stock solution is prepared, aliquot it to routine usage volumes and store at -20°C or -80°C. Avoid repeated freeze and thaw cycles.
Calculation results
Working concentration： mg/mL；
Method for preparing DMSO stock solution： mg drug pre-dissolved in μL DMSO (stock solution concentration mg/mL). Please contact us first if the concentration exceeds the DMSO solubility of the batch of drug.
Method for preparing in vivo formulation:：Take μL DMSO stock solution, next add μL PEG300, mix and clarify, next addμL Tween 80, mix and clarify, next add μL ddH2O，mix and clarify.
(1) Please be sure that the solution is clear before the addition of next solvent. Dissolution methods like vortex, ultrasound or warming and heat may be used to aid dissolving.
(2) Be sure to add the solvent(s) in order.
Co-crystal structures of PRMT5:MEP50 (green) with compound8(A, magenta), compound9(B, orange), compound10(C, yellow), and compound15(D, cyan).ACS Med Chem Lett.2015 Dec 2;7(2):162-6. |
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Plot of cLogD vs scaled clearance (CL) from human microsomes (A) and mouse liver microsomes (B).Plot of proliferation IC50in Z-138 cells vs observed CL in mouse PK (C) and percentage oral bioavailability %F from mouse PK (D) dosed at 2 mg/kg IV and 10 mg/kg PO.ACS Med Chem Lett.2015 Dec 2;7(2):162-6. |
Plasma PK profile ofEPZ015666in male CD-1 mouse following single dose administration at 10 and 100 mg/kg p.o. Methyl mark IC90corrected for PPB shown as dotted line. ACS Med Chem Lett.2015 Dec 2;7(2):162-6. |
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