CPI-1205

CPI-1205, a highly potent and selective EZH2 inhibitor (biochemical IC50=2 nM, cellular EC50=32 nM).

Signaling Pathways >> Epigenetics >> Histone Methyltransferase

Research Areas >> Cancer

IC50: 2 nM (EZH2)[1]

When tested within an in vitro hERG binding assay at concentration ranges of 45 nM to 100 μM, CPI-1205 shows an IC50 of 21.3 μM. CPI-1205 demonstrates modest selectivity (EZH1 IC50 of 52±11 nM) when tested against enhancer of zeste homologue 1 (EZH1), a methyltransferase highly related to EZH2. CPI-1205 achieves unbound exposures well above their respective cellular potencies; however, only the unbound exposure for CPI-1205 remains well above the cellular EC50 up to 4 h[1].

CPI-1205 is dosed at 160 mg/kg orally twice daily (po BID) for 25 days in tumor bearing female CB-17 SCID mice. Upon treatment of tumor-bearing CB-17 SCID mice with CPI-1205, tumor regression is observed within 2 weeks. By the end of day 25, significant tumor growth inhibition is recorded (>97% TGI relative to vehicle). CPI-1205 demonstrates robust antitumor effects in a Karpas-422 xenograft model when dosed at 160 mg/kg BID. CPI-1205 shows excellent oral bioavailability. CPI-1205 exhibits moderate clearance of 2.16 L/h/kg (40% liver blood flow), a half-life of ~1.6 h, similar volume of distribution (1.4 L/kg), and excellent bioavailability (100% F)[1].

PRC2 (wt or Y641N mutant), biotinylated nucleosome, H3K27me3 activator peptide and CPI-1205 (in DMSO) are incubated in 50 mM Tris, pH 8.5, 5 mM MgCl2, 1 mM DTT, 70 uM Brij-35, and 0.1 mg/mL BSA for 30 minutes. Reaction is initiated by addition of [3H]-SAM to final conditions of 5 nM PRC2, 200 nM nucleosome (concentration expressed as H3), activator peptide (3.6 μM) and 200 nM [3H]-SAM in a total volume of 25 μL in 384 well Greiner plates. For CPI-1205 analysis assays are either single point or ten point dose-responses with final total DMSO of 0.8 or 1.6% (v/v). Typically assays are run for 60 minutes with <35% substrate turnover. After reaction assays are quenched by addition of 20 μL of 2 mM SAH and 200 mM EDTA in 50 mM Tris, pH 8.5. Reactions are transferred to streptavidin-coated FlashPlates, incubated for 2 h, aspirated, washed, and read on a TopCount[1].

Ten different doses of each test compound (in a series of 3-fold dilutions) are plated in duplicate 384-well tissue culture treated plates. HeLa cells grown in culture are trypsinized and counted. Cell are diluted to 67,000 cells per mL in10% DMEM and 15 μL (1,000 cells) are plated into each well using the Biotek MicroFloTM Select Dispenser. Plates are incubated at 37°C/5% CO2 for 72 hrs. One of the duplicate plates is processed for AlphaLISA and the other for viability. Cell viability is assayed by adding 15 μL of Cell Titer Glo to each well with cells with media. The plates are incubated at RT for 15-20 minutes on a plate shaker at low speed. The plates are then read using an EnVision-Alpha Reader[1].

Rats[1] CPI-1205 is orally administered in a GLP compliant toxicity study for 4 weeks to both Sprague-Dawley rats and beagle dogs followed by a 4-week recovery period. CPI-1205 is administered by oral gavage at single daily doses (QD) of 100, 300, and 600 mg/kg to rats for 28 days and at twice daily doses (BID) of 50, 150, and 500 mg/kg for 28 days to dogs. In general, CPI-1205 is well-tolerated in the 28-day GLP toxicology studies, and any findings are reversible over the recovery period.

[1]. Vaswani RG, et al. Identification of (R)-N-((4-Methoxy-6-methyl-2-oxo-1,2-dihydropyridin-3-yl)methyl)-2-methyl-1-(1-(1-(2,2,2-trifluoroethyl)piperidin-4-yl)ethyl)-1H-indole-3-carboxamide (CPI-1205), a Potent and Selective Inhibitor of Histone Methyltransferase EZH2, Suitable for Phase I Clinical Trials for B-Cell Lymphomas. J Med Chem. 2016 Nov 10;59(21):9928-9941.

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