EZH2 [1]

In vitro activity: In KARPAS-422 cells, CPI-169 shows a dose-dependent inhibitory effect on cell viability, and produces synergy anti-proliferative activity when used in combination with ABT-199. In 16 out of 25 NHL cell lines, CPI-169 also suppresses cell growth with GI50 of<5 μM.
Kinase Assay: Compound potency is also assessed through incorporation of 3H-SAM into a biotinylated H3 peptide. Specifically, PRC2 containing either EZH1 (160 pM), wt EZH2 (40 pM), or Y641N mutant EZH2 (80 pM, both EZH2 prepared in-house) is pre-incubated with 3H-SAM (0.9 µM), 2 µM H3K27me3 activating peptide (H2N-RKQLATKAAR(Kme3)SAPATGGVKKP-amide) and compounds (as 10 point duplicate dose response titrations) for 120 min in a buffer consisting of 50 mM Tris (pH 8.5), 1 mM DTT, 0.07 mM Brij-35, 0.1% BSA, and 0.8% DMSO in a total volume of 12.5 µl in a black 384 well plate. Reaction is initiated with biotinylated H3 substrate peptides (H3K27me1 for wt EZH2, H3K27me2 for Y641N mutant EZH2; H2N-RKQLATKAAR(Kmen)SAPATGGVKKP-NTPEGBiot) as a 2 µM stock in 12.5 µL and allowed to react at room temperature for 5 h. Quenching is accomplished by addition of 20 µl of STOP solution (50 mM Tris (pH 8.5), 200 mM EDTA, 2 mM SAH). 35 µL of the quenched solution is transferred to Streptavidin Flashplates, incubated overnight, washed, and read in a TopCount Reader. For titrations all compound dilutions are in DMSO, final DMSO concentrations are 0.8% (v/v), and turnover is kept to less than < 5%. IC50s are calculated using non-linear least square four parameter fits (GraphPad 6.0).
Cell Assay: Relative cell numbers are assessed by Cell Titer-Glo (CTG) luminescent cell viability assay using an Envision instrument. GraphPad Prism 6.0 is used for curve fitting, IC50/GI50 and Hill coefficient (H) calculations. The GI90 is calculated using the formula: EC90 = (90 /100-90)1/H EC50. Cell line: 25 NHL cell lines

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In KARPAS-422 cells, CPI-169 reduced global H3K27me3 levels with an EC90 of 0.388 μM after 11 days of treatment (Figure S5C)[1].
CPI-169 combined with ABT-199 showed synergy in suppressing growth of KARPAS-422 cells as determined by the Bliss independence criterion (Figure 6C)[1].
In a panel of 25 NHL cell lines, combination of CPI-169 with ABT-199 showed synergy in cell models that phenotypically responded to both single agents, but synergy did not correlate with EZH2 mutation status or BCL2 genomic aberrations (Figure 7 and Table S3)[1].

In mice bearing KARPAS-422 xenografts, CPI-169 (200 mg/kg, s.c.) effectively suppresses H3K27me3 levels and results in lymphoma tumor regression without affecting body weight or causing any overt adverse effects.

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In KARPAS-422 subcutaneous xenograft model, CPI-169 dosed at 200 mg/kg subcutaneously twice daily (SC BID) resulted in tumor regression (Figures 5E and S5E)[1].
Tumors showed dose-dependent reduction in global H3K27me3 levels and induction of PRC2 target genes (ABAT, APOL3, FBXO39) correlating with dose and impact on tumor growth (Figures 5G and 5H)[1].
The proliferative potential of tumor cells was substantially reduced upon CPI-169 treatment (Figure S5I)[1].
CPI-169 achieved plasma concentrations that covered the target for at least 12 hours (Figure S5F)[1].

For cell viability and long-term growth assays, lymphoma cell lines were plated in compound-containing 96-well plates and passaged every 4 days to fresh plates containing CPI-169. Relative cell numbers were assessed by Cell Titer-Glo luminescent cell viability assay using an Envision instrument. Curve fitting was performed using Prism 6.0 (Figure 6C and 7)[1].
For cell cycle and apoptosis analysis, KARPAS-422 cells were treated with various doses of CPI-169 for up to 14 days, split on days 4, 8, and 11, and collected on days 4,6,8,10,14. Cells were fixed in 70% ice-cold ethanol overnight at -20°C, stained with propidium iodide (20 μg/ml), and DNA content assessed on Guava Easycyte. Apoptosis was assessed by staining with propidium iodide and Annexin-FITC using an Annexin V-FITC Apoptosis Detection Kit, with data acquired on Guava Easycyte and analyzed using Guava Cytosoft program[1].
For measurement of H3K27me3 reduction, KARPAS-422 cells were treated with CPI-169 for 11 days and global H3K27me3 levels were determined by ELISA (Figure S5C)[1].
For combination studies with ABT-199, cells were treated with various dose combinations of CPI-169 and ABT-199, and synergy was calculated using the Bliss independence criterion (Figure 6C and 7)[1].

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

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For the KARPAS-422 xenograft model, each mouse was inoculated subcutaneously in the right flank with 1×10^7 KARPAS-422 tumor cells in 0.2 mL 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. CPI-169 was dosed at 200 mg/kg subcutaneously twice daily (BID). Vehicle was 10% DMSO + 60% polyethylene glycol 400 + 30% ddlH2O. Tumor size was measured three times a week using a caliper, and tumor volume (V) was expressed in mm^3 using the formula V = 0.5 × a × b^2, where a and b were the long and short diameters. Mice were weighed every day. Tumor growth inhibition was calculated as TGI (%) = (1 - (T1 - T0)/(C1 - C0)) × 100 (Figures 5E, S5E, S5I)[1].
For assessment of target engagement and gene expression in tumors, tumors were collected at 14 and 28 days and analyzed for global H3K27me3 and H3K27me1 levels by ELISA, and for expression of PRC2 target genes (ABAT, APOL3, FBXO39) by qPCR (Figures 5G, 5H)[1].

CPI-169 had improved microsomal stability compared to CPI-360 (Figures S5A-S5G)[1].
In KARPAS-422 xenograft model, plasma concentration of CPI-169 at 12 hr post last dose was 2.9 μM (Figure S5F)[1].
The compound achieved plasma concentrations that covered the target for at least 12 hours, as compared to the EC90 of 0.388 μM required to reduce global H3K27me3 levels in KARPAS-422 cells at day 11 of treatment (Figure S5F and S5C)[1].

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

1-[1-(1-ethylsulfonyl-4-piperidinyl)ethyl]-N-[(4-methoxy-6-methyl-2-oxo-1H-pyridin-3-yl)methyl]-2-methyl-3-indolecarboxamide is an indolecarboxamide.

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CPI-169 is a more potent EZH2 inhibitor than CPI-360 with improved microsomal stability[1].
It does not affect global H3K27me1 levels at efficacious doses in vivo (Figure 5G)[1].
It shows synergy with the BCL2 inhibitor ABT-199 in KARPAS-422 cells[1].
The compound is being optimized with the goal to create a therapeutic agent for clinical applications[1].

C27H36N4O5S

528.66

528.24

1450655-76-1

78357814

Typically exists as solid at room temperature

1.3±0.1 g/cm3

1.631

2.67

2

6

8

37

1040

0

O=C(C1=C(C)N(C(C2CCN(S(=O)(CC)=O)CC2)C)C3=C1C=CC=C3)NCC4=C(OC)C=C(C)NC4=O

LHGUZCKPFXXVPV-UHFFFAOYSA-N

InChI=1S/C27H36N4O5S/c1-6-37(34,35)30-13-11-20(12-14-30)18(3)31-19(4)25(21-9-7-8-10-23(21)31)27(33)28-16-22-24(36-5)15-17(2)29-26(22)32/h7-10,15,18,20H,6,11-14,16H2,1-5H3,(H,28,33)(H,29,32)

1-[1-(1-ethylsulfonylpiperidin-4-yl)ethyl]-N-[(4-methoxy-6-methyl-2-oxo-1H-pyridin-3-yl)methyl]-2-methylindole-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)