BLU9931

Reagent Authentication Certificate:

Chemical Probes Portal BLU9931.pdf

Protein target name:

FGFR4

Mechanism of action:

Covalent inhibitor

Recommended Concentration for use in cells:

50 - 500 nM

Probe Availability

MedChem Express (1538604-68-0)

Probe Information

Target and Probe Information

Target Details

Target class:

Protein kinase

Subclass:

HUGO ID:

HGNC:3691

Entrez gene ID:

2264

UNIPROT ID:

P22455

Target alias:

Fibroblast growth factor receptor 4, CD334, JTK2, TKF

Probe Details

Chemical Probes Portal ID:

CPP1201

PubChem CID:

72710839

SMILES string:

CC1=C(C(=CC=C1)NC(=O)C=C)NC2=NC=C3C=C(C=CC3=N2)C4=C(C(=CC(=C4Cl)OC)OC)Cl

InChi Key:

TXEBNKKOLVBTFK-UHFFFAOYSA-N

Orthogonal probe:

Roblitinib

Validation in Cells and Model Organisms

In vitro validation

On target activity

Potency:

Kinact/KI

0.6 X 10^5/(mol/L) s

Potency assay:

Omnia assay technology (Life Technologies); Incubation of recombinant FGFR4 with BLU9931 led to covalent modification of the receptor at Cys552, confirmed by mass shift and proteomics. In enzyme activity assays FGFR4 IC50=3 nM.

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

4XCU

Off target activity

Potency (off target):

IC50

Off target protein and potency:

FGFR1: 591 nM, FGFR2: 493 nM, FGFR3: 150 nM

Potency assay (off target):

In kinome-wide selectivity panel containing 456 kinases (Kinomescan, DiscoveRx), BLU9931 bound significantly to only FGFR4 (KD=6 nM) and CSFR1 (KD=2,716 nM) at 3 uM, yielding a selectivity score S(10)=0.005.

In cell validation

On target activity in cells

Potency assay (cells):

In the FGFR4-dependent cell line MDA-MB-453, BLU9931 potently and dose-dependently inhibited phosphorylation of FGFR4 substrates (FRS2, MAPK, AKT).

Target engagement assay (cells):

Indirect, inhibition of protein substrate phosphorylation

Off target activity in cells

Potency assay, off target (cells):

In FGFR1-driven cells (DMS114), BLU9931 had minimal activity.

In vivo validation

Organism:

Female Balb/c nude mouse

Dose:

10, 30 and 100 mg/kg

Route of delivery:

OralIntravenous

Plasma half life:

2.3 h (oral, 10 mg/kg)

Organ of Interest

Organ (O):

Hep 3B xenograft

Target engagement assay:

Indirect, assessment of CYP7A1 biomarker confirmed modulation of FGFR4.

Primary Reference

Reference (doi):

10.1158/2159-8290.CD-14-1029

Reference (PMID):

25776529

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

Overall, the selectivity (and limits thereof) has been appropriately validated. Direct evidence for cellular or in vivo target engagement is lacking. In addition, it should also be noted than IC50 values for irreversible inhibitors are time-dependent.

Mar 28 2017 - 8:37pm

This molecule represents the most selective FGFR4 inhibitor available. KINOMEscan methodology indicated that the only other kinase outside the FGFR family that is potentially inhibited is CSF1R (KD is significantly weaker at 2,716 nM vs. 6 nM for FGFR4). The compound demonstrates prolonged inhibitory activity in cell lines after removal of compound (~8 hrs), consistent with its covalent mode of action and the rate of resynthesis of EGFR4 protein. Addition of glutathione to enzyme activity assays produces a negligible IC50 shift indicating that the compound does not elicit general thiol reactivity. Impressive efficacy was demonstrated in vivo in xenograft models of HCC after oral administration at the well tolerated 100-300 mg/kg, twice daily doses.

Mar 28 2017 - 10:00pm

BLU9931

Probe Availability

Probe Information

Target Details

Fibroblast growth factor receptor 4, CD334, JTK2, TKF

Probe Details

In vitro validation

In cell validation

In vivo validation

Primary Reference

Supporting Organizations

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BLU9931

Probe Availability

Probe Information

Target Details

Fibroblast growth factor receptor 4, CD334, JTK2, TKF

Probe Details

In vitro validation

In cell validation

In vivo validation

Primary Reference

Supporting Organizations

Interested in supporting the portal?