FGFR1-4 biochemical assays

FGFR kinase inhibition assays were performed at KM for ATP. Picomolar to low nanomolar concentrations of FGFR proteins were incubated in 1 × Kinase Reaction Buffer with 1 μM of CSKtide and 50 to 250 of μM ATP at 25 ℃ for 90 mins in the presence or absence of a dosed concentration series of inhibitor. All reactions were terminated by the addition of Stop Buffer, and plates were read on a Caliper EZReader2. IC50 values were fit with a four-parameter log[Inhibitor] versus response model with floating Hill Slope.

Cell lines

MDA-MB-453, DMS114 and Hep 3B cells

Preparation method

This compound is soluble in DMSO. General tips for obtaining a higher concentration: Please warm the tube at 37 ℃ for 10 minutes and/or shake it in the ultrasonic bath for a while. Stock solution can be stored below - 20 ℃ for several months.

Reacting condition

0.3 ~ 300 nM; 1 hr

Applications

In MDA-MB-453 cells, BLU9931 dose-dependently and effectively reduced phosphorylation of FGFR4 signaling pathway components, including FRS2, MAPK and AKT. In DMS114 cells, BLU9931 exhibited minimal reduction of phosphorylation in all FGFR1 signaling pathway components. In Hep 3B cells, BLU9931 also potently inhibited phosphorylation of the FGFR4 pathway components (except pAKT).

Animal models

Hep 3B tumor-bearing mice

Dosage form

10, 30 and 100 mg/kg; p.o.; b.i.d., for 21 days

Applications

BLU9931 dose-dependently inhibited the growth of Hep 3B tumors. At the dose of 100 mg/kg, BLU9931 resulted in tumor regression. Moreover, 2 of the 9 mice showed no signs of tumor 30 days after cessation of treatment.

Other notes

Please test the solubility of all compounds indoor, and the actual solubility may slightly differ with the theoretical value. This is caused by an experimental system error and it is normal.

BLU9931 is a potent and irreversible inhibitor of FGFR4. BLU9931 potently inhibited FGFR4 activity (IC50 = 3 nmol/L), but weakly inhibited FGFR1 (IC50 = 591 nmol/L), FGFR2 (IC50 = 493 nmol/L), and FGFR3 (IC50 = 150 nmol/L) activity.
Fibroblast growth factor receptor 4 (FGFR4) is the receptor of fibroblast growth factor 19 (FGF19), which is a tightly controlled hormone that regulates bile acid synthesis and hepatocyte proliferation in the normal liver. FGFR4 acts as a targeted therapy to treat patients with HCC whose tumors have an activated FGFR4 signaling pathway. BLU9931 is highly selective for FGFR4 versus other FGFR family members and all other kinases. BLU9931 binds within the ATP-binding pocket of FGFR4, forming a covalent bond with Cys552. The anilino-quinazoline core of BLU9931 makes a bidentate hydrogen-bonding interaction with the hinge residue (Ala553) of FGFR4, whereas the dichlorodimethoxyphenyl group occupies the hydrophobic pocket, providing FGFR-family selectivity. BLU9931 displayed significant binding to only two of the 398 wild-type kinases, FGFR4 (99.7% inhibition relative to DMSO control; Kd = 6 nmol/L) and CSF1R (90.1% inhibition relative to DMSO control; Kd = 2716 nmol/L) by KINOMEscan method. [2]
BLU9931 shows remarkable antitumor activity in mice bearing an HCC tumor xenograft that overexpresses FGF19 due to amplification as well as a liver tumor xenograft that overexpresses FGF19 mRNA but lacks FGF19 amplification.
References:
[1]. Hagel M, Miduturu C, Sheets M et al.First Selective Small Molecule Inhibitor of FGFR4 for the Treatment of Hepatocellular Carcinomas with an Activated FGFR4 Signaling Pathway. Cancer Discov. 2015 Mar 16. [Epub ahead of print]
[2]. Davis MI, Hunt JP, Herrgard S, Ciceri P, Wodicka LM, Pallares G, Hocker M, Treiber DK, Zarrinkar PP. Comprehensive analysis of kinase inhibitor selectivity. Nat Biotechnol. 2011 Oct 30;29(11):1046-51.