EPZ015666

Reagent Authentication Certificate:

Chemical Probes Portal EPZ015666.pdf

Protein target name:

PRMT5

Mechanism of action:

Protein substrate competitive inhibitorSAM uncompetitive inhibitor

Recommended Concentration for use in cells:

300 nM - 2.5 uM

Probe Availability

MedChem Express (1616391-65-1)

Cayman (1616391-65-1)

MilliporeSigma (1616391-65-1)

Probe Information

Target and Probe Information

Target Details

Target class:

Epigenetics

Subclass:

Protein methyltransferase

HUGO ID:

HGNC:10894

Entrez gene ID:

10419

UNIPROT ID:

O14744

Target alias:

JBP1, SKB1, IBP72, SKB1Hs, HRMT1L5

Probe Details

Chemical Probes Portal ID:

CPP154

Probe alias:

1616391-65-1, 3XV, GTPL8287

PubChem CID:

90241673

SMILES string:

C1CN(CC2=CC=CC=C21)C[C@H](CNC(=O)C3=CC(=NC=N3)NC4COC4)O

InChi Key:

ZKXZLIFRWWKZRY-KRWDZBQOSA-N

Control compound:

EPZ019896

Orthogonal probe:

GSK591DS-437

PAINs evaluation:

PAINs free

Physico-chemical properties

NMR:

1H NMR (400MHz, MeOD-d4) δ 8.24 (s, 1H), 7.15 - 7.05 (m, 4H), 7.02 - 6.98 (m, 1H), 5.09 (s, 1H), 4.95 (t, J=6.8 Hz, 2H), 4.59 (t, J=6.3 Hz, 2H), 4.10 - 4.03 (m, 1H), 3.72 (s, 2H), 3.56 - 3.46 (m, 2H), 2.96 - 2.91 (m, 2H), 2.87 - 2.80 (m, 2H), 2.65 (d, J=6.3 Hz, 2H);

13C NMR (101 MHz, DMSO-d6) δ ppm 29.7, 45.7, 47.4, 52.8, 57.6, 63.7, 68.1, 79.4, 104.9, 126.8, 127.4, 127.6, 129.6, 135.2, 135.5, 155. 8, 158.8, 164.1, 165.9

Storage Recommendations

Storage (solution):

-20 oC for 2 years

Validation in Cells and Model Organisms

In vitro validation

On target activity

Potency:

5 nM

Potency assay:

Homogeneous time-resolved fluorescence (HTRF) assay under balanced conditions to monitor the monomethylation of H4R3 on a histone H4 peptide by PRMT5:MEP50

PDB ID for probe-target interaction (3D structure):

4GQB, 4X60, 4X63

Structure-activity relationship:

Yes,

Off target activity

Potency assay (off target):

No inhibition of 20 Protein methyltransferases at 50 uM (PRMT8, PRMT1, PRMT3, PRMT6, PRMT7, CARM1, DOT1L, SMYD2, SMYD3, SETD7, EZH1, EZH2, SUV39H1, EHMT1, EHMT2, SETDB1, SETD2, NSD1, WHSC1L1, WHSC1)

In cell validation

On target activity in cells

Potency in cells:

IC50

9 nM

Potency assay (cells):

In-cell western (ICW) assay following inhibition of symmetric dimethylation of arginine-containing substrates in Z-138 cells over 4 days.

Target engagement assay (cells):

Yes, indirect: see potency assay; direct: cellular thermal shift assay (CETSA) in A375 cells.

In vivo validation

Organism:

CD-1 mice

Dose:

10 mg/kg2 mg/kg (IV)

Route of delivery:

Oral

Plasma half life:

1.62 hours (IV)

Systemic clearance:

30.0 mL/min/kg (IV)

Cmax:

3500 ng/mL (oral)

Tmax:

0.333 hours (oral)

Organ of Interest

Organ (O):

xenograft

Target engagement assay:

In the SDMA ELISA, Z-138 xenograft tumors collected on day 21 showed dose-dependent changes of >40% and >95% inhibition (>48% and >87% for Maver-1 tumors at day 21; >66% and >95% for Granta-519 tumors at day 18) of the methyl mark achieved at the lowest dose and highest dose, respectively.

Primary Reference

Reference (doi):

10.1021/acsmedchemlett.5b00380 10.1038/nchembio.1810

Reference (PMID):

25915199

Endorsed probe

The Chemical Probes Portal endorses this compound as chemical probe for use in cells and in model organisms.

SAB Rating

Rating for use in Cells

3 review(s)

Rating for use in Model Organisms

3 review(s)

SAB Members

SAB Comments

The reported data for this compound shows potent biochemical and cellular inhibition of the target, and preliminary single dose pharmokinetic (PK) data suggests good coverage above IC90 for 12 hr @ 100 mg/kg. Physicochemical properties are acceptable. Note that no selectivity data against related family targets are reported and, as such, there remains a possibility that off-target pharmacology may be observed. However, on-target pharmacology is demonstrated with reduction of downstream products.

Jun 12 2016 - 4:02am

EPZ015666 is an excellent potent and selective PRMT5 probe that can be used in cellular systems.

Jun 12 2016 - 4:03am

There is clear evidence showing EPZ015666 inhibits PRMT5 directly in cell-free experiments, including several orthogonal biochemical assays, X-ray crystallography, surface plasmon resonance, SAR, appropriate counter-screens for assay artifacts and binding kinetics. There is also solid evidence showing target engagement in cells, notably through the use of CETSA and a functional readout (i.e., reduction in SmD3me2s signal). Available evidence suggests this is an sound chemical probe with oral bioavailability and demonstrated efficacy in mouse xenograft models, both with reduction in tumor volume (Z-138 and Maver-1 cells) and PRMT5 target inhibition (SDMA levels). Additional evidence that would strengthen confidence in the use of EPZ015666 includes additional selectivity data (e.g., other epigenetic enzyme classes and unrelated classes as well), cytotoxicity data versus other standard cell lines, additional PK data (e.g., under conditions where the dosage and formulation more closely approximates the xenograft studies), and demonstration of in vivo target engagement (e.g., CETSA). This probe should be used in conjunction with its inactive analog, EPZ019896, to control for off-target effects.

Jun 30 2016 - 3:59am

EPZ015666

Probe Availability

Probe Information

Target Details

JBP1, SKB1, IBP72, SKB1Hs, HRMT1L5

Probe Details

Physico-chemical properties

In vitro validation

In cell validation

In vivo validation

Primary Reference

Supporting Organizations

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EPZ015666

Probe Availability

Probe Information

Target Details

JBP1, SKB1, IBP72, SKB1Hs, HRMT1L5

Probe Details

Physico-chemical properties

In vitro validation

In cell validation

In vivo validation

Primary Reference

Supporting Organizations

Interested in supporting the portal?