UNC1999

Protein target name:

EZH1, EZH2

Mechanism of action:

Protein substrate noncompetitive inhibitorSAM competitive inhibitor

Probe Availability

Abcam (1431612-23-5)

MedChem Express (1431612-23-5)

Tocris (1431612-23-5)

MilliporeSigma (1431612-23-5)

Cayman (1431612-23-5)

Probe Information

Target and Probe Information

Target Details

Target class:

Epigenetics

Subclass:

Protein methyltransferase

HUGO symbol:

EZH1 EZH2

Entrez gene ID:

2145 2146

Target alias:

KMT6B, WVS, ENX1, EZH1, KMT6, WVS2, ENX-1, EZH2b, KMT6A

Probe Details

Chemical Probes Portal ID:

CPP48

PubChem CID:

72551585

ChEMBL ID:

CHEMBL3414619

SMILES string:

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

InChi Key:

DPJNKUOXBZSZAI-UHFFFAOYSA-N

Control compound:

UNC2400

Orthogonal probe:

GSK343

PAINs evaluation:

PAINs free

Physico-chemical properties

NMR:

1H NMR (400 MHz, d4-MeOH) δ 8.48 (d, J = 2.4 Hz, 1H), 8.36 (s, 1H), 7.91 (dd, J = 8.9, 2.6 Hz, 1H), 7.87 (s, 1H), 7.75 (d, J = 1.2 Hz, 1H), 6.81 (d, J = 8.9 Hz, 1H), 6.09 (s, 1H), 5.06 (dt, J = 13.3, 6.6 Hz, 1H), 4.58 (s, 2H), 3.61 – 3.46 (m, 4H), 2.70 (ddd, J = 13.1, 8.6, 6.3 Hz, 3H), 2.63 (dd, J = 11.2, 6.0 Hz, 4H), 2.23 (s, 3H), 1.63 (dt, J = 15.3, 7.5 Hz, 2H), 1.55 (d, J = 6.6 Hz, 6H), 1.09 (d, J = 6.5 Hz, 6H), 1.00 (t, J = 7.3 Hz, 3H);

13C NMR (100 MHz, CDCl3) δ 166.44, 165.96, 158.96, 154.78, 146.57, 143.29, 139.87, 136.47, 136.36, 132.35, 128.89, 125.52, 121.47, 120.52, 119.94, 109.10, 108.49, 106.74, 54.65, 50.45, 48.57 (two carbons), 45.59 (two carbons), 36.12, 35.28, 23.70, 22.20 (two carbons), 18.74, 18.62 (two carbons), 14.04.

Validation in Cells and Model Organisms

In vitro validation

On target activity

Potency:

IC50

EZH2 10 nM, EZH1 45 nM

Potency assay:

Transfer of 3H from SAM to peptide substrate in in vitro enzymatic assay. UNC1999's Ki for EZH2/SAM = 4.6 nM.

Off target activity

Potency assay (off target):

UNC1999 is 10,000-fold selective for EZH2 over 15 other lysine, arginine or DNA methyltransferases.

Selectivity against non-family targets:

In a panel of 50 kinases, UNC1999 exhibited 20% max inhibition at 10,000 nM. In a panel of 44 GPCRs, transporters and ion channels (NIMH PDSP), UNC1999 had limited activity at 10,000 nM. Notable activity was detected and quantified for 4 targets: Ki=4700 nM SIGMA1R; Ki=65 nM CXCR2; Ki=300 nM Histamine HRH3; Ki=1500 nM SLC6A2.

In cell validation

On target activity in cells

Potency in cells:

IC50

124 nM

Potency assay (cells):

in-cell western to detect protein substrate methylation in MCF10A cells

Target engagement assay (cells):

Indirect, protein substrate methylation assay in cells.

Toxicity

Cytoxicity assay:

Yes

Notes on cytotoxicity:

EC50 = 19,200 nM in Alamar blue reduction assays.

In vivo validation

Organism:

Swiss albino mice

Dose:

15, 50 or 150 mg/kg

Route of delivery:

Intraperitoneal

Cmax:

9700-1180 nM

Primary Reference

Reference (doi):

10.1021/cb400133j

Reference (PMID):

23614352

This probe is in the process of SAB review. Please continue to check back for new reviews and commentary.

SAB Rating

Rating for use in Cells

2 review(s)

Rating for use in Model Organisms

2 review(s)

SAB Members

SAB Comments

Three days are required for reduction of H3K27me3 in MCF10a cells with IC50 ~135 nM. I recommend using UNC1999 in parallel with its control, UNC2400, and the orthogonal probe, GSK343.

This probe is a dual inhibitor of EZH1 and EZH2 methyltransferase enzymes (in vitro IC50s are 45 nM and 4.6 nM, respectively), which are the components of Polycomb repressive complex 2. This complex converts dimethylated lysines into trimethylated lysines in H3K27. Trimethylation of K27 is a transcriptionally repressive epigenetic mark. The probe inhibits H3K27 trimethylation in MCF10A cells at 5 uM after 3 days treatment, shows toxicity after 7 days of treatment. Potential off-target effects could be due to the GPCR binding with Ki 4,700 nM; 65 nM; 300 nM; and 1,500 nM for Sigma1, Sigma 2, Histamine H3, and Norephedrine transporter, respectively. In vivo studies have shown that the probe has good PK properties; however, its inhibitory effect on EZH1 and EZH2 in different tissues has not been examined, and its organ and tissue distribution profiles were not reported. Therefore, if this probe is used for in vivo studies, careful investigation of its tissue and organ distribution are needed, as well as an analysis of its inhibitory effect of EZH1/2 and off-target GPCR receptors. I recommend using this probe along with UNC2400, which is a negative control molecule.

UNC1999 has utility in cell-based experiments when used in conjunction with UNC2400.

UNC1999

Probe Availability

Probe Information

Target Details

KMT6B, WVS, ENX1, EZH1, KMT6, WVS2, ENX-1, EZH2b, KMT6A

Probe Details

Physico-chemical properties

In vitro validation

In cell validation

Toxicity

In vivo validation

Primary Reference

Supporting Organizations

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UNC1999

Probe Availability

Probe Information

Target Details

KMT6B, WVS, ENX1, EZH1, KMT6, WVS2, ENX-1, EZH2b, KMT6A

Probe Details

Physico-chemical properties

In vitro validation

In cell validation

Toxicity

In vivo validation

Primary Reference

Supporting Organizations

Interested in supporting the portal?