Home Epigenetics Histone Methyltransferase inhibitor - EZH1/2 inhibitor - Autophagy activator - Apoptosis related activator UNC1999 For research use only.

UNC1999

UNC1999 is a potent, orally bioavailable and selective inhibitor of EZH2 and EZH1 with IC50 of 2 nM and 45 nM in cell-free assays, respectively, showing >1000-fold selectivity over a broad range of epigenetic and non-epigenetic targets. UNC1999 is a potent autophagy inducer. UNC1999 specifically suppresses H3K27me3/2 and induces a range of anti-leukemia effects including anti-proliferation, differentiation, and apoptosis.

UNC1999 Chemical Structure

UNC1999 Chemical Structure

CAS No. 1431612-23-5

Purity & Quality Control

UNC1999 Related Products

Signaling Pathway

Histone Methyltransferase Signaling Pathways

Histone Methyltransferase Signaling Pathway

Cell Data

Cell Lines Assay Type Concentration Incubation Time Formulation Activity Description PMID
human MCF10A cells Function assay 72 h Inhibition of EZH2 in human MCF10A cells assessed as reduction of H3K27me3 level after 72 hrs by Western blot analysis, IC50=0.124 μM 25406853
human MCF10A cells Cytotoxic assay Cytotoxicity against human MCF10A cells assessed as cell viability by Alamar Blue assay, EC50=19.2 μM 25406853
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Biological Activity

Description UNC1999 is a potent, orally bioavailable and selective inhibitor of EZH2 and EZH1 with IC50 of 2 nM and 45 nM in cell-free assays, respectively, showing >1000-fold selectivity over a broad range of epigenetic and non-epigenetic targets. UNC1999 is a potent autophagy inducer. UNC1999 specifically suppresses H3K27me3/2 and induces a range of anti-leukemia effects including anti-proliferation, differentiation, and apoptosis.
Features The first orally bioavailable inhibitor against wild-type and mutant EZH2 as well as EZH1.
Targets
EZH2 [2]
(Cell-free assay)		 EZH1 [2]
(Cell-free assay)
2 nM 45 nM
In vitro
In vitro UNC1999 is highly potent for both EZH2 Y641N and EZH2 Y641F mutants in vitro. UNC1999 causes concentration-dependent reductions of H3K27me3 in MCF10A cells with IC50 of 124 nM , while shows low cellular toxicity. UNC1999 displays potent, concentration-dependent inhibition of cell proliferation with EC50 of 633 nM in a DLBCL cell line harboring the EZH2Y641N mutant. In addition, biotinylated UNC1999 enriches EZH2 from HEK293T cell lysates, and thus may be used in chemoproteomics studies. [1]
Kinase Assay Scintillation Proximity Assay
Methyltransferase activity assays are performed by monitoring the incorporation of tritiumlabeled methyl group from S-adenosylmethionine (3H-SAM) to biotinylated peptide substrates using Scintillation Proximity Assay (SPA) for PRC2-EZH2 trimeric complex (EZH2:EED:SUZ12), PRC2-EZH1 pentameric complex (EZH1:EED:SUZ12:RBBP4:AEBP2), SETD7, G9a, GLP, SETDB1, SETD8, SUV420H1, SUV420H2, SUV39H2, MLL1 tetrameric complex (MLL:WDR5:RbBP5:ASH2L), PRMT1, PRMT3, PRMT5-MEP50 complex and SMYD2. The reaction buffer for SMYD2 and SMYD3 is 50 mM Tris pH 9.0, 5 mM DTT, 0.01% TritonX-100; for G9a, GLP and SUV39H2 is 25 mM potassium phosphate pH 8.0, 1 mM EDTA, 2 mM MgCl2 and 0.01% Triton X-100; and for other HMTs 20 mM Tris pH 8.0, 5 mM DTT, 0.01% TritonX-100. To stop the enzymatic reactions, 10 μL of 7.5 M guanidine hydrochloride is added, followed by 180 μL of buffer, mixed and transferred to a 384-well FlashPlate. After mixing, the reaction mixtures are incubated and the CPM counts are measured using Topcount plate reader. The CPM counts in the absence of compound for each data set are defined as 100% activity. In the absence of the enzyme, the CPM counts in each data set are defined as background (0%). IC50 values are determined using compound concentrations ranging from 100 nM to 100 μM. The IC50 values are determined using SigmaPlot software. EZH2-Y641F assays are performed using 30 nM of enzyme in 20 mM Tris pH 8, 5 mM DTT, 0.01% Triton X-100, 5 μM SAM and 1 μM of H3 (1-24) peptide (same as for the wild-type PRC2-EZH2 complex). For DNMT1, the assay is performed using hemimethylated dsDNA as a substrate. The dsDNA substrate is prepared by annealing two complementary strands (biotintlated forward strand: BGAGCCCGTAAGCCCGTTCAGGTCG and reverse strand: CGACCTGAACGGGCTTACGGGCTC), synthesized by Eurofins MWG Operon. Reaction buffer is 20 mM Tris-HCl, pH 8.0, 5mM DTT, 0.01% Triton X-100. Methyltransferase activity assays for DOT1L is performed using Filter-plates. Reaction mixtures in 20 mM Tris-HCl, pH 8.0, 5 mM DTT, 2 mM MgCl2 and 0.01% Triton X-100 are incubated at room temperature for 1h, 100 μL 10% TCA is added, mixed and transferred to filter-plate. Plates are centrifuged at 2000 rpm for 2 min followed by 2 additional 10% TCA wash and one ethanol wash (180 μL) followed by centrifugation. Plates are dried and 100 μL MicroO is added and centrifuged. 70 μL MicroO is added and CPM are measured using Topcount plate reader.
Cell Research Cell lines DLBCL cell line harboring the EZH2Y641N mutant
Concentrations ~5 μM
Incubation Time 8 days
Method

DB cells, a diffuse-large B-cell lymphoma cell line harboring the EZH2 Y641N mutation, are obtained from ATCC and cultured in RPMI 1640 supplemented with 10% fetal bovine serum, antibiotics, and various concentrations of compounds (DMSO control, UNC1999, or UNC2400). The medium containing the test compound or control is refreshed every 3 days. The numbers of viable cells from at least three independent experiments are measured using TC20 automated cell counter system. Total histones are prepared from cell nuclei using an acidic extraction protocol. About 1 μg of total histones is separated using 15% SDS-PAGE, transferred to PVDF membranes, and probed with histone antibodies. Antibodies used in this study are those against EZH2, general H3, and H3K27me3.
Experimental Result Images Methods Biomarkers Images PMID
Western blot H3K27Me3 / Cleaved PARP / LC3B H3K27me2 / H3K27me1 / H3K27ac / H3K4me3 / H3K9me2 / H3K36me2 27449082
Immunofluorescence EZH2 / H3K27me3 NF-κB 23614352
Growth inhibition assay Cell viability 27449082
In Vivo
In vivo Treatment of UNC1999 (150 and 50 mg/kg, i.p.) results in the plasma concentrations of UNC1999 above its cellular IC50 over 24 hours in vivo. In addition, UNC1999 is also orally bioavailable in mice, which makes chronic animal studies more practical and convenient. [1]
Animal Research Animal Models Male Swiss albino mice
Dosages ~150 mg/kg (i.p.), ~50 mg/kg (oral)
Administration Intraperitoneal administration or oral administration

Chemical Information & Solubility

Molecular Weight 569.74 Formula

C33H43N7O2
CAS No. 1431612-23-5 SDF Download UNC1999 SDF
Smiles CCCC1=C(C(=O)NC(=C1)C)CNC(=O)C2=C3C=NN(C3=CC(=C2)C4=CN=C(C=C4)N5CCN(CC5)C(C)C)C(C)C
Storage (From the date of receipt)

In vitro
Batch:

DMSO : 50 mg/mL ( (87.75 mM) Moisture-absorbing DMSO reduces solubility. Please use fresh DMSO.)

Ethanol : 50 mg/mL

Water : Insoluble


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Tech Support

Answers to questions you may have can be found in the inhibitor handling instructions. Topics include how to prepare stock solutions, how to store inhibitors, and issues that need special attention for cell-based assays and animal experiments.

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