BI-9564

Reagent Authentication Certificate:

Chemical Probes Portal BI-9564.pdf

Protein target name:

BRD9, BRD7

Mechanism of action:

AntagonistInhibits interaction between BRD9/7 and acetylated histone

Recommended Concentration for use in cells:

300 nM - 1 uM

Probe Availability

MedChem Express (1883429-22-8)

Tocris (1883429-22-8)

Cayman (1883429-22-8)

MilliporeSigma (1883429-22-8)

Probe Information

Target and Probe Information

Target Details

Target class:

Epigenetics

Subclass:

Bromodomain

HUGO ID:

HGNC:25818 HGNC:14310

Entrez gene ID:

65980 29117

UNIPROT ID:

Q9H8M2 Q9NPI1

Target alias:

LAVS3040, PRO9856, BP75, CELTIX1, NAG4

Probe Details

Chemical Probes Portal ID:

CPP552

Probe alias:

None

PubChem CID:

117072549

SMILES string:

CN1C=C(C2=C(C1=O)C=NC=C2)C3=CC(=C(C=C3OC)CN(C)C)OC

InChi Key:

BJFSUDWKXGMUKA-UHFFFAOYSA-N

Control compound:

Not available

Orthogonal probe:

I-BRD9LP-99dBRD9

Physico-chemical properties

Solubility - preferred solvent:

DMSO or aqueous buffer (pH 6.8)

Solubility - concentration:

≥10 mM or >90 µg/mL

Potential reactivities:

Unknown

NMR:

Not available

Storage Recommendations

Storage (dry powder):

Product is stable at room temperature as a solid (at least for 6 months), Recommended storage <0 °C as solid for long term

Storage (solution):

-20 °C DMSO stock solution (long term)

Storage sensitivities:

None

Validation in Cells and Model Organisms

In vitro validation

On target activity

Potency:

BRD9 14 nM

Potency assay:

Isothermal calorimetry (ITC) KD (DiscoveRx)=6 nM

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

5F1H

Structure-activity relationship:

2‐methyl‐1,2‐dihydro‐2,7‐naphthyridin‐1‐one anchor binder makes two H-bonds to Asn100 of BRD9, water-bridged interaction with Tyr57 of BRD9, π-stacking with Tyr106 of BRD9, 4-[(Dimethylamino)methyl]-2,5-dimethoxyphenyl linker: C−H π-interaction with Ile53 of BRD9, T-stacking with Phe44 of BRD9, and induced fit Phe47 of BRD9/C−H π-interaction Selectivity versus BRD4-BD1: Clashes with BRD4-BD1 BD amino acids (Trp81, Gln85, and Leu92)

Off target activity

Potency (off target):

Off target protein and potency:

BRD7 239 nMBRD4 >20 µMCECR2 200 nM

Potency assay (off target):

Thermal shift measured against 48 bromodomains BRD9 +9.2°C BRD7 +6.5°C CECR2 +5.6°C BETs <1°C All other bromodomains ≤2°C

Probe Selectivity in Vitro:

Kinase selectivity: percent control inhibition at 10 µM ACVR1 76%, IC50 (ACVR1) = 5090 mM TGFBR1 74%, IC50 (TGFBR1) = 5140 nM ACVR2B 72%, IC50 (ACVR2B) = 7680 nM All other 321 kinases <40% GPCR selectivity: percent control inhibition at 10µM M1(h) 75% M3(h) 86% All other 53 GPCR <40%

In cell validation

On target activity in cells

Potency in cells:

EC50

BRD9 275 nM

Potency assay (cells):

BRD9 FRAP assay ~90% inhibition at 100 nM Recovery After Photobleaching (FRAP) assay U2OS cells transfected with GFP-BRD9: measurement of recovery half times of wild-type cells treated with DMSO in the absence or presence of 2.5 µM SAHA or treated with BI-9564 at different concentrations

Target engagement assay (cells):

FRAP assay measures target engagement in cell: Recovery After Photobleaching (FRAP) assay U2OS cells transfected with GFP-BRD9.

Off target activity in cells

Potency in cells, off target:

Not done

Potency assay, off target (cells):

BRD7 FRAP: full inhibition at 1 µM CECR2 FRAP: no inhibition at 1 µM

Probe Selectivity in Cell:

Not available

Toxicity

Cytoxicity assay:

Yes

Notes on cytotoxicity:

No compound-related toxicity was observed in U2OS cell lines after 24 h.

In vivo validation

Organism:

Mouse

Dose:

180 mg/kg

Route of delivery:

Oral

Plasma half life:

2.1 h

Systemic clearance:

39 mL/min/kg

Fraction of probe bound to plasma protein:

0.35

Cmax:

35 µM

Tmax:

0.7 h

Organ of Interest

Organ (O):

None

Primary Reference

Reference (doi):

doi: 10.1021/acs.jmedchem.5b01865

Reference (PMID):

26914985

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

This is a pharma probe optimized by SBD from a fragment screen. More potent BRD9 (1-80 nM) vs. BRD7 (200-4000 nM), and selective (>1000-fold vs. BRD2, BRD4, BET). The probe hits one BRD protein outside BRD 9/7, namely, CECR2. However, CECR2 binding did not translate in cells to functional consequence (no CECR2 inhibition at 1 µM in FRAP; also not cytotoxic).

Aug 12 2016 - 5:45pm

BI-9564 was discovered by fragment-based screening and optimized by structure-based design. BI-9564 has a higher affinity for BRD9 (ITC Kd =14) than BRD7 (ITC Kd = 239 nM), and the affinities for BET bromodomains are reported >100 μM (by AlphaScreen). It displays cellular activity in a FRAP assay (1 μM). Although it displays some off-target binding to CECR2 (ITC Kd = 258 nM), no cellular inhibition of CECR2 was observed in the CECR2 FRAP assay at 1 μM, and the off-target binding effect does not translate into other types of cellular experiments.The use of BI-9564 in murine leukemia cells with a BRD4/BRD9 domain-swap has shown that the BRD9 bromodomain is responsible for mediating the antiproliferative effects of BI-9564. However, the compound only displays an IC50 of 800 nM (cellular proliferation assay) in the most sensitive cell line in vitro to BRD9 inhibition (EOL-1). Some off-target effects were observed when used at concentrations >5 uM. A disseminated mouse model of AML was then used to assess the efficacy of the compound, using the human acute myeloid eosinophilic leukemia cell line EOL-1. The compound can be used to evaluate the effects of the in vivo modulation of BRD9 activity.

Aug 17 2016 - 7:18pm

This probe appears better for both in vitro and cell work than the companion probe in the paper (BI-7273). The selectivity in vitro appears quite good for BRD9 even versus the closely related BRD7. In cells, the response to this probe also seems to be better than for the companion probe, though with lower fold selectivity. In mice, the probe was extensively tested only at one dose, though lower doses were used in some of the PK studies.

Sep 6 2016 - 2:01pm

BI-9564

Probe Availability

Probe Information

Target Details

LAVS3040, PRO9856, BP75, CELTIX1, NAG4

Probe Details

Physico-chemical properties

In vitro validation

In cell validation

Toxicity

In vivo validation

Primary Reference

Supporting Organizations

Interested in supporting the portal?

Search form

BI-9564

Probe Availability

Probe Information

Target Details

LAVS3040, PRO9856, BP75, CELTIX1, NAG4

Probe Details

Physico-chemical properties

In vitro validation

In cell validation

Toxicity

In vivo validation

Primary Reference

Supporting Organizations

Interested in supporting the portal?