A-395

Reagent Authentication Certificate:

Chemical Probes Portal A-395.pdf

Protein target name:

EED

Mechanism of action:

Antagonizes protein-protein interaction

Recommended Concentration for use in cells:

100-300 nM

Probe Information

Target and Probe Information

Target Details

Target class:

Epigenetics

Subclass:

Polycomb family

HUGO ID:

HGNC:3188

Entrez gene ID:

8726

UNIPROT ID:

O75530

Target alias:

Embryonic ectoderm development, HEED, WAIT-1, WD protein associating with integrin cytoplasmic tails 1

Probe Details

Chemical Probes Portal ID:

CPP1009

PubChem CID:

123132213

ChEMBL ID:

CHEMBL4104741

SMILES string:

CN(C)[C@H]1CN(C2CCC3=C2C(F)=CC=C3)C[C@@H]1C4=CC=C(N5CCN(CC5)S(=O)(C)=O)C=C4

InChi Key:

REVJNSVNICWODC-KIDMSAQOSA-N

Control compound:

A-395N

Orthogonal probe:

EED226UNC1999GSK126

Physico-chemical properties

Solubility - preferred solvent:

DMSO

Solubility - concentration:

20 mM, 5-10 microL aliquots

Storage Recommendations

Storage (dry powder):

-20 oC for 2 years

Storage (solution):

-80 oC

Storage sensitivities:

Limit DMSO stock solutions to one freeze-thaw cycle

Validation in Cells and Model Organisms

In vitro validation

On target activity

Potency:

0.4 nM

Potency assay:

TR-FRET Compounds were tested in a modified LanthaScreen competition assay in which the in vitro potency of compounds (Ki) was measured through the disruption of a TR-FRET donor-acceptor complex of terbium-labeled anti-GST antibody and a custom synthesized Oregon green (488) labeled probe using a potent, pyrrolidine-based inhibitor. By SPR, the KD=1.5 nM, and A-395 inhibits PRC complex in vitro with a IC50=18 nM.

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

5K0M

Structure-activity relationship:

Some SAR is discussed in the primary publication. Thermal shift assay (TSA) was used.

Off target activity

Potency (off target):

Ki, Tm

Potency assay (off target):

We assessed A-395's activity against 32 epigenetic methyltransferases by Scintillation proximity assay (SPA). A filter-based assay was used for DOT1L, NSD1, NSD2, NSD3, ASH1L, DNMT3A/3L, DNMT3B/3L, and a Thermal shift assay (TSA) for selectivity against eight other proteins containing reader domains. Fluorescence polarization was used for CBX7. Off-target activity was only detected at 1 and 50 uM against PRC2.

In cell validation

On target activity in cells

Potency in cells:

IC50

90 nM (H3K27me3), 390 nM (H3K27me1/2)

Potency assay (cells):

Cell (lymphoma and multiple myeloma) proliferation assays were conducted with the CellTiter-Glo Luminescent Cell Viability Assay (Promega Corporation). Specifically, 1,000 cells were seeded in each well of 96-well cell culture plates and treated with various compounds or DMSO control for 10 d before the cell proliferation assay. A-395 antagonist resulted in data similar to the EZH2 SAM-competitive inhibitors.

Off target activity in cells

Potency assay, off target (cells):

A395 had no detectable impact on histone acetyltransferase activity.

In vivo validation

Organism:

SCID mice

Dose:

300 mg/kg (with extended release vehicle)

Route of delivery:

Subcutaneous

Organ of Interest

Organ (O):

Pfeiffer xenograft

Target engagement assay:

Indirect, decrease in H3K27m3.

Primary Reference

Reference (doi):

10.1038/nchembio.2306

Reference (PMID):

28135237

Endorsed probe

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

SAB Rating

Rating for use in Cells

3 review(s)

Rating for use in Model Organisms

3 review(s)

SAB Members

SAB Comments

A-395 associates tightly to EED in a 1:1 binding model with a KD value of 1.5 nM (shown by SPR). A-395 inhibits the trimeric PRC2 complex (EZH2–EED–SUZ12) with an IC50 of 18 nM using a radioactivity-based assay. The structurally related, negative control, A-395N, was significantly less potent against PRC2 in this assay, with an IC50 >50,000 nM. A-395 has no activity against any of the other protein lysine methyltransferases, arginine methyltransferases, or DNA methyltransferases tested (panel of 32 diverse proteins). In cellular assays, A-395 inhibits H3K27 methylation with IC50=90 nM (rhabdoid tumor cell line RD; 3 days). The negative control, A-395N, exhibits no activity in the cellular assays. In vivo activity of A-395 was tested in a DLBCL Pfeiffer xenograft model. Subcutaneous (s.c.) administration of compound in an extended-release vehicle was the only route that showed a sufficient exposure profile for effectiveness. Pharmacokinetic analysis suggested that s.c. delivery of A-395 at 300 mg/kg can maintain an unbound 90% effective concentration (EC90) for at least 48 h.

Mar 28 2017 - 2:57pm

This compound closely phenocopies EZH2 enzyme inhibitors, which are better-characterized and have greater in vivo utility. Therefore, this would be an alternative choice for EZH2 inhibitor-resistant mutant cells or to specifically probe the role of EED WD40-H3K27me3 interactions.

Jul 13 2017 - 2:00pm

A-395 is a potent and selective inhibitor of EED/PRC2 interaction (SPR Kd=1.5 nM, 1:1 EED/A-395 binding; Ki=0.5 nM). Calculated physicochemical properties: MW=486.65, PSA=55.5, AlogP=3.37, clogD pH 7=1.74, HBDon=0, HBAcc=5, Nrot=5, Ligand Efficiency (LE)=0.371, Lipophilicity Ligand Efficiency (LipE)=7.655. A co-crystal structure is available (PDB code 5K0M, 1.83 Å).

It works well in cells and was reported to inhibit both H3K27me2 and H3K27me3 marks with IC50=390 nM, 90 nM, respectively, in human rhabdoid tumor cell line RD after 72 hours. A-395 inhibited proliferation in multiple myeloma cell lines with EC50s=1-2 uM after 10 days in RPMI 8226, MM.1s and L-363 cell lines. A-395 is highly selective and, thus, can be used to tease out the EED pharmacology in cells and its role in cancer and other diseases. It was selective against a panel of 32 diverse epigenetic methyltransferases including lysine, arginine, and DNA methyltransferases as well as against a survey of other epigenetic ‘reader domains’, including the WD-40-containing protein WDR5 (as measured by TSA) and against the chromodomain of PcG protein CBX7 (as measured by fluorescence polarization).

A-395 was tested in a xenograft model using the DLBCL Pfeiffer cells in SCID mice. A-395 was administered at a very high concentration (300 mg/kg subcutaneously, twice per week for 35 days) and showed significant reduction of the tumor volume up to 150 days as well as the H3K27m3 levels measured after 1 week compared with vehicle. Based on reported pharmacokinetic studies, subcutaneous delivery of A-395 at 300 mg/kg can maintain an unbound 90% effective concentration (EC90) for at least 48 hours. Based on the long lasting exposure after the subcutaneous administration, I suspect that A-395 may also be administered IV or IP with a proper formulation provided that the compound is soluble at the tested concentration. Development of an improved analog with good oral, IV or IP exposure would be beneficial. Selectivity profiling in a broad CEREP panel would help determine if any of the phenotypic or in vivo readout is partially due to some off-target activity. Also there is no evidence that the H3K27m3-mark reduction is responsible for the phenotypic and in vivo response.

Aug 16 2017 - 3:37pm

A-395 is a very potent and selective inhibitor of EED/PRC2 interaction (SPR Kd 1.5 nM, 1:1 EED/A-395 binding; Ki 0.5 nM). Calculated physicochemical properties: MW 486.65, PSA 55.5, AlogP 3.37, clogD pH 7 1.74, HBDon 0, HBAcc 5, Nrot 5, Ligand Efficiency (LE) 0.371, Lipophilicity Ligand Efficiency (LipE) 7.655. Co-crystal structure is available (PDB code 5K0M, 1.83 Å). Other IDs: ZINC584904831, PubChem 123132213, 6PU in 5K0M PD.

It works well in cells and was reported to inhibit both H3K27me2 and H3K27me3 marks with IC50 values of 390 nM and 90 nM, respectively, in human rhabdoid tumor cell line RD after 72 hours. A-395 inhibited proliferation in multiple myeloma cell lines with EC50s of 1-2 uM after 10 days in RPMI 8226, MM.1s and L-363 cell lines.

A-395 is highly very selective and thus can be used to tease out the EED pharmacology in cells its role in cancer and other diseases. It was selective a panel of 32 diverse epigenetic methyltransferases including lysine, arginine, and DNA methyltransferases as well as against a survey of other epigenetic ‘reader domains’, including the WD-40-containing protein WDR5, as measured by TSA and against the chromodomain of PcG protein CBX7 as measured by fluorescence polarization.

The xenograft model was done using the DLBCL Pfeiffer cells in SCID mice. A-395 was administered at a very high concentration 300 mpk s.c. (subcutaneously), twice per week for 35 days and showed significant reduction of the tumor volume up to 150 days as well as the H3K27m3 levels measured after 1 week compared to vehicle. Based on reported pharmacokinetic studies, s.c. delivery of A-395 at 300 mg/kg can maintain an unbound 90% effective concentration (EC90) for at least 48 hours. Based on the long lasting exposure after the subcutaneous administration, I suspect that A-395 may also be administered IV or IP with a proper formulation provided if the compound is soluble at the tested concentration. Development of an improved analog with good oral, IV or IP exposure would be beneficial. Selectivity profiling in a broad CEREP panel would help determine if any of the phenotypic or in vivo readout is partially due to some off-target activity. Also there seems to be no evidence that the H3K 27m3 mark reduction is responsible for the phenotypic and in vivo response.

Jul 9 2020 - 10:23am

A-395

Probe Information

Target Details

Embryonic ectoderm development, HEED, WAIT-1, WD protein associating with integrin cytoplasmic tails 1

Probe Details

Physico-chemical properties

In vitro validation

In cell validation

In vivo validation

Primary Reference

Supporting Organizations

Interested in supporting the portal?

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A-395

Probe Information

Target Details

Embryonic ectoderm development, HEED, WAIT-1, WD protein associating with integrin cytoplasmic tails 1

Probe Details

Physico-chemical properties

In vitro validation

In cell validation

In vivo validation

Primary Reference

Supporting Organizations

Interested in supporting the portal?