Derazantinib

ARQ-087 has anti-proliferative activity in cell lines driven by FGFR dysregulation, including amplifications, fusions, and mutations. Cell cycle studies in cell lines with high levels of FGFR2 protein show a positive relationship between ARQ 087 induced G1 cell cycle arrest and subsequent induction of apoptosis. ARQ 087 inhibits the auto-phosphorylation of FGFR1 and FGFR2 in a dose-dependent manner. In Cos-1 cells overexpressing full-length FGFR1, FGFR2, FGFR3 and FGFR4, ARQ 087 inhibits their phosphorylation with EC50 values of < 0.123 μM, 0.185 μM, 0.463 μM, >10 μM respectively. ARQ 087 inhibits FGFR kinase by an ATP competitive mechanism, and is capable of inhibiting both the inactive and fully active forms of the FGFR kinase. Hence, ARQ 087 delays FGFR activation by inhibiting its autophosphorylation, as well as inhibition of the phosphorylated active kinase^[1].

ARQ 087 attenuates FGFR signaling in SNU-16 human xenograft tumors, leading to a reduction in phospho-FGFR, phospho-FRS2-α, and phospho-ERK, while the total FGFR2 protein is unaffecte. ARQ 087 is effective at inhibiting tumor growth in vivo in FGFR2 altered, SNU-16 and NCI-H716, xenograft tumor models with gene amplifications and fusions. ARQ 087 demonstrated efficacy in multiple in vivo xenograft models, and was well tolerated at doses up to 75 mg/kg^[1].

• Determination of Ki and mode of inhibition

  Kinase inhibitory activity of ARQ 087 was determined for the recombinant FGFR1 or FGFR2 proteins utilizing a biotinylated PYK2 peptide substrate and ATP. ARQ 087 was titrated in DMSO utilizing a 3-fold dilution scheme, and then diluted 10-fold further in deionized water for a final DMSO concentration of 10%. A volume (2.5 μL) of these dilutions or vehicle was added to each well of a reaction plate. FGFR1 or FGFR2 was added to assay buffer (50 mM Tris, pH 8.0, 0.02 mg/mL BSA, 10 mM MgCl2, 1 mM EGTA, 10% glycerol, 0.1 mM Na3PO4, 1 mM DTT) to each well in a volume of 17.5 μL for a final concentration of 0.50 or 0.25 nM, respectively. After a 30-minute pre-incubation period, ATP and substrate were added in assay buffer (5 μL) for final concentrations of 0–1,000 μM ATP and 80 nM biotinylated-PYK2, for a final reaction volume of 25 μL. The plates were incubated for 60 minutes at room temperature, and then stopped in the dark by the addition of 10 μL stop/detection mixture prepared in assay buffer containing EDTA, AlphaScreen™ Streptavidin Donor and P-TYR-100 Acceptor beads for final concentrations of 10 mM EDTA and 500 ng/well of both AlphaScreen™ Donor and Acceptor beads. Assay plates were incubated for 60 minutes at room temperature in the dark, and the plates were read on a Perkin Elmer, Envision Multilabel plate reader. (excitation wavelength: 640 nm, emission wavelength: 570 nm). The effect of enzyme concentration was applied for tight-binding inhibitors, and if necessary, the IC50 values were converted into Ki values if the enzyme concentration was above the IC50 values under the assay conditions utilized.

• Cell lines

  NCI-H716 and SNU-16 cells

• Concentrations

  0.1 μM or 1 μM

• Incubation Time

  24 or 72 hours

• Method

  Cells are plated and incubated at 37°C overnight and subsequently treated with 0.1 μM or 1 μM of ARQ 087 for 24 or 72 hours. The cells were fixed and stained with Cycletest Plus Reagent kit and cell cycle profiles were analyzed using a FACS Calibur flow cytometer.

• Animal Models

  female NCr nu/nu mice (SNU-16) or CB17 SCID mice (NCI-H716)

• Dosages

  0, 25, 50, and 75 mg/kg

• Administration

  oral administration