CPI-360 targets Enhancer of Zeste Homolog 2 (EZH2) (wild-type and Y641N mutant) and to a lesser extent EZH1. In biochemical assays using reconstituted PRC2, CPI-360 is a subnanomolar inhibitor of EZH2 methyltransferase activity (exact IC50 values not specified in text; approximately 5-fold more potent on wild-type EZH2 compared to Y641N mutant). It inhibits EZH1 about 100-fold less potently. The compound is SAM-competitive as demonstrated by an ~5-fold shift in IC50 at higher SAM concentrations. CPI-360 shows exquisite selectivity across a panel of 30 histone lysine and arginine methyltransferases and DNA methyltransferases, with no inhibition observed at concentrations up to 10 µM except for EZH1 and EZH2. [1]

In vitro activity: In KARPAS-422 cells, CPI-360 potently reduces global H3K27me3 and H3K27me2 levels with EC50 of 56 nM and 65 nM, respectively. CPI-360 also causes time-dependent transcriptional changes, and affects the viability of Y641N mutant EZH2-containing KARPAS-422 cells. In addition, CPI-360 gradually arrests KARPAS-422 cells in the G1 cell cycle stage followed by the induction of apoptosis.
Kinase Assay: CPI-360 is a potent, selectiveEZH2inhibitor with IC50 of 0.5 nM and 2.5 nM nM for wt EZH2 and Y641N EZH2, respectively.
Cell Assay: CPI-360 functions on the basis of S-adenosyl-Lmethionine (SAM)-competition, inhibits EZH1 about 100-fold less and shows exquisite selectivity across a large panel of histone lysine and arginine, and DNA methyltransferases. CPI-360 potently reduced global H3K27me3 and H3K27me2 levels in a dosedependent manner. CPI-360 effectively suppressed heavy H3K27me3 incorporation in KARPAS-422 cells without affecting total histone turnover. CPI-360 treatment causes time-dependent transcriptional changes in germinal center B cell-like diffuse large B cell lymphoma.

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CPI-360 potently reduces global H3K27me3 and H3K27me2 levels in a dose-dependent manner in HeLa cells (ELISA assay) and KARPAS-422 cells, without affecting H3K27me1, other tri/dimethylation marks, or protein levels of PRC2 subunits (EZH2, SUZ12, EED). Western blot analysis confirmed dose-dependent reduction of H3K27me3 after 4 days of treatment. Cellular thermal shift assay (CETSA) showed that CPI-360 induces thermal stabilization of EZH2 in KARPAS-422 and HT cells at 52°C in a dose-dependent manner, with EC50 values lower after 24h compared to 4h treatment; the effect is reversible upon compound removal. NanoBRET assays demonstrated that CPI-360 (10 µM) does not disrupt EZH2-EED or EZH2-SUZ12 protein-protein interactions, nor does it globally affect EZH2 binding to chromatin. SILAC LC-MS/MS analysis revealed that CPI-360 treatment decreases H3K27me3- and H3K27me2-containing peptide species, increases unmodified H3K27 peptides, and interestingly increases H3K27me1 peptide abundance (except when combined with H3K36me3). Isotopic labeling turnover experiments showed that CPI-360 (3 µM) suppresses H3K27me3 turnover (t1/2 ~37-60 hr) but not H3K27me1 turnover (t1/2 ~6-25 hr) in KARPAS-422 cells; very high concentration (30 µM) partially affects H3K27me1 turnover. RNA-seq analysis after 4 days treatment with 1.5 µM CPI-360 (along with GSK-126 and EPZ-6438) showed significant alteration of 1,248 genes, with induced genes marked by H3K27me3. Time-dependent gene expression induction (ABAT, APOL3, FBXO39) was observed only after prolonged treatment (6-10 days), correlating with loss of promoter-proximal H3K27me3. CPI-360 treatment (1.5 µM) for 10 days caused gradual G1 cell cycle arrest in KARPAS-422 cells, and apoptosis was observed starting after 13 days of treatment. In long-term growth assays, CPI-360 affected viability of Y641N mutant EZH2-containing KARPAS-422 cells (time-dependent, not by day 4) but not wild-type EZH2-containing OCI-LY19 cells (at 4,7,10 days). Across a panel of 43 NHL cell lines, CPI-360 showed selective cell killing, with GCB-DLBCL being the most prevalent affected subtype, though responses were also seen in ABC-DLBCL, BL, and MCL lines. Combination of CPI-360 (or CPI-169) with ABT-199 showed synergy in KARPAS-422 cells (Bliss independence method) and in some NHL cell lines that respond to both single agents. [1]

In mice bearing KARPAS-422 xenografts, CPI-360 (200 mg/kg, s.c.) reduces tumor growth by 44%.

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CPI-360 demonstrated in vivo efficacy in a KARPAS-422 subcutaneous xenograft model (EZH2 Y641N mutant). Twice daily subcutaneous administration of 200 mg/kg CPI-360 for 28 days reduced tumor growth with tumor growth inhibition (TGI) of 44% without affecting body weight or causing overt adverse effects. However, inadequate pharmacological properties of CPI-360 did not allow for complete target coverage. In the same model, a more potent analog CPI-169 achieved tumor regression. CPI-360 treatment resulted in dose-dependent reduction of global H3K27me3 levels in tumors (ELISA assay, p<0.0001) while H3K27me1 levels remained unchanged. Induction of PRC2 target genes (ABAT, APOL3, FBXO39) was observed in compound-treated tumors compared to vehicle. [1]

Biochemical assays: PRC2 and oligonucleosome reconstitution and radiometric biochemical assays using oligonucleosomal substrates were carried out as described previously. Methyltransferase selectivity panel profiling with CPI-360 was carried out using services of Reaction Biology Corp. Detailed methods: Enzymatic assays were carried out with either 20 or 200 µM PRC2 (standard assay and 10X enzyme, respectively) and with 10X K50app of H3K27me1 (10X peptide) or SAM (10X SAM). The ~5 fold shift of IC50 values at higher SAM is consistent with CPI-360 being SAM-competitive. Data are represented as the mean of triplicate experiments ± SD. Selectivity testing across a panel of recombinant DNA and histone methyltransferases (30 different enzymes) was performed under balanced conditions, with IC50 values obtained from a 10-point dose response curve with 3-fold serial dilutions starting from a top concentration of 10 µM. [1]
Cellular Thermal Shift Assay (CETSA): Experiments were carried out as described previously (Martinez Molina et al., 2013). KARPAS-422 or HT cells were incubated with DMSO or various concentrations of CPI-360 (0.01-40 µM) for indicated times (2, 4, or 24 hr), then exposed to temperature shifts (room temperature, 37°C, 52°C) for 3 min. Cells were lysed, soluble and insoluble proteins separated by centrifugation, and soluble protein pool examined for EZH2 protein levels by western blotting. Vinculin served as control. Densitometry-based quantification of western blot signals (EZH2 intensities normalized to vinculin) was performed. [1]
NanoBRET assays: Human embryonic kidney (HEK)293 cells were transfected with NanoLuc-EZH2 and Halo-EED (or Halo-SUZ12). 48 hr post transfection, BRET signal was detected indicating direct interaction. CPI-360 (10 µM) was added over the 48 hr period. For chromatin binding, KARPAS-422 cells were treated with DMSO or CPI-360 (10 µM) for 2 days, and BRET signal was measured using Halo-tagged H3.1/H3.3 and NanoLuc-tagged EZH2. [1]
SILAC LC-MS/MS: KARPAS-422 cells were grown in DMSO and heavy medium or in CPI-360 (0.63, 2.5, and 10 µM) and light medium for 4 days. Cell populations mixed, histones isolated using EpiQuik kit, propionylated (Sigma) to block free amines, trypsin digested, and another round of propionylation. Peptides desalted via Stage Tip and analyzed by Orbitrap MS. Peptides resuspended in 0.1% formic acid, injected into LTQ-Orbitrap XL ETD mass spectrometer with Agilent 1260 HPLC. Chromatographic separation over 0-55% acetonitrile with 0.1% formic acid over 55 min on a 100 µm x 15 cm column packed with HALO C18, flow rate 300 nl/min. Survey scans acquired in Orbitrap from m/z 300-2000 at 30,000 resolution. [1]
Isotopic labeling turnover assay: KARPAS-422 cells shifted to RPMI medium containing methyl-13C, d3-L-methionine in presence of DMSO or CPI-360 (3 µM or 30 µM) at time 0. Cells collected at 11 time-points from 6 hr to 168 hr. Histone extraction, derivatization, desalting LC-MS/MS procedure as described. Abundance of isotopically labeled (heavy) and unlabeled histone peptide species harboring specific H3K27 methylation states determined. [1]

Cell culture and viability assays: Lymphoma cell lines obtained from ATCC or DSMZ, grown in recommended media with 10% fetal bovine serum and 1% penicillin/streptomycin. For long-term assays, cells plated onto compound-containing 96-well plates, passaged every 4 days to plates with fresh EZH2 inhibitors. Relative cell numbers assessed by Cell Titer-Glo (CTG) luminescent cell viability assay using an Envision instrument. Curve fitting with Prism 6.0. [1]
Western blot and ELISA: Whole cell extracts analyzed by western blotting with antibodies against H3K27me3, H3K27me2, H3, EZH2, SUZ12, EED, GAPDH, etc. Meso Scale Discovery ELISA assay performed as described previously (Garapaty-Rao et al., 2013) to quantify global H3K27me3 and H3K27me1 levels. [1]
Cell cycle analysis: KARPAS-422 cells treated with various doses of CPI-360 for up to 14 days, split on days 4,8,11. Cells collected on days 4,6,8,10,14, fixed in 70% ice-cold ethanol overnight at -20°C, stained with propidium iodide (20 µg/ml), DNA content assessed on Guava Easycyte, analysis with Guava Cytosoft program. [1]
Apoptosis assay: KARPAS-422 cells treated with CPI-360, stained with propidium iodide and Annexin-FITC using Annexin V-FITC Apoptosis Detection Kit I, data acquired on Guava Easycyte, percentage of apoptotic cells calculated. [1]
RNA-seq and ChIP-seq: KARPAS-422 cells treated with DMSO, CPI-360 (1.5 µM), GSK-126, or EPZ-6438 for 4 days, samples subjected to RNA-seq. ChIP-seq performed to determine H3K27me3 sites across genome. Data available under GEO accession GSE62058. [1]
Quantitative PCR: Cells pelleted, lysed in buffer RLT, RNA purified using RNeasy columns. 200 ng RNA converted to cDNA using SuperScript III Reverse Transcriptase and random primers. qPCR performed using cDNA, Roche FastStart Universal Probe Master Mix, and TaqMan probes on Stratagene MX3005P instrument. Ct and delta Ct values analyzed. [1]
CETSA: As described in Enzyme Assay section. [1]

Dissolved in 10% DMSO + 60% polytheylene glycol 400 + 30% ddH2O; 200 mg/kg; s.c.injection, Mice bearing KARPAS-422 tumors

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Animal protocol for xenograft studies: Each mouse inoculated subcutaneously in the right flank with KARPAS-422 tumor cells (1x10^7) in 0.2 ml of PBS with matrigel (1:1). Treatments commenced when average tumor size reached approximately 100-200 mm^3. Each group consisted of ten randomly assigned tumor-bearing mice. Mice were dosed with vehicle (10% DMSO + 60% polyethylene glycol 400 + 30% ddlH2O) or CPI-360 (200 mg/kg, subcutaneous, twice daily (BID)) for 28 days. Tumor size measured three times a week using a caliper, tumor volume (V) expressed in mm^3 using formula V = 0.5 a x b^2 (a and b long and short diameters). Mice weighed every day. Tumor growth inhibition (TGI %) = (1 - (T1-T0)/(C1-C0)) x 100. [1]

The paper states that CPI-360 has inadequate pharmacological properties that did not allow for complete target coverage, but no specific ADME parameters (e.g., half-life, bioavailability, clearance) are provided for CPI-360. [1]

Chem Biol.2014 Nov 20;21(11):1463-75.

CPI-360 is an EZH2 small molecule inhibitor identified from a hybrid compound series derived from CPI-905. It is SAM-competitive and shows subnanomolar potency against EZH2. The compound selectively affects turnover of trimethylated H3K27 but not monomethylated H3K27 at pharmacologically relevant doses, suggesting that EZH2 inhibition alone (without EZH1 inhibition) is sufficient for efficacy in NHL. CPI-360 treatment causes time-dependent transcriptional changes, G1 arrest, and apoptosis in GCB-DLBCL cells. Surprisingly, CPI-360 shows efficacy also in NHL models with wild-type EZH2, indicating broader potential application. The compound was used as a tool to explore EZH2 biology and its mechanism of action in non-Hodgkin's lymphoma. [1]

C25H31N3O4

437.53

437.231

C, 68.63; H, 7.14; N, 9.60; O, 14.63

1802175-06-9

73442743

Solid powder

1.3±0.1 g/cm3

712.2±60.0 °C at 760 mmHg

384.5±32.9 °C

0.0±2.3 mmHg at 25°C

1.621

2.2

2

4

6

32

786

1

O1CCC(CC1)[C@@H](C)N1C2C=CC=CC=2C(C(NCC2C(NC(C)=CC=2OC)=O)=O)=C1C

PFPSFENQCNMITC-MRXNPFEDSA-N

InChI=1S/C25H31N3O4/c1-15-13-22(31-4)20(24(29)27-15)14-26-25(30)23-17(3)28(21-8-6-5-7-19(21)23)16(2)18-9-11-32-12-10-18/h5-8,13,16,18H,9-12,14H2,1-4H3,(H,26,30)(H,27,29)/t16-/m1/s1

N-[(4-methoxy-6-methyl-2-oxo-1H-pyridin-3-yl)methyl]-2-methyl-1-[(1R)-1-(oxan-4-yl)ethyl]indole-3-carboxamide

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)