Pazopanib HCl (GW786034 HCl) 635702-64-6

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Molecular Weight:

473.98 Pazopanib (GW786034) is a novel Multi-Target Inhibitor of VEGFR1, VEGFR2, VEGFR3, PDGFR, FGFR, c-Kit and c-Fms with IC50 of 10 nM, 30 nM, 47 nM, 84 nM, 74 nM, 140 nM and 146 nM, respectively.


Biological Activity


Pazopanib potently inhibits VEGF-induced phosphorylation of VEGFR2 in HUVEC cells with IC50 of 8 nM. Pazopanib shows dose-dependent growth inhibition in all synovial
sarcoma cell lines including SYO-1 and HS-SY-II cells. Proliferation of
SYO-1 and HS-SY-II cells is inhibited even at 1 µg/mL of Pazopanib and
is completely abolished at 5 µg/mL. Pazopanib induces G1 arrest, and
thereby suppresses the growth of synovial sarcoma cells.


Phosphorylation
of Akts, GSK-3β, JNKs, p70 S6 Kinase, and mTOR is suppressed in
Pazopanib-treated SYO-1 cells compared with that in the vehicle-treated
cells. Pazopanib between 20 m g/mL and 22.5 m g/mL shows an increasing reduction of RPE cell viability.


The mice treated with 30 mg/kg or 100 mg/kg Pazopanib reveals a
significant decrease in tumor burden compared with the mice treated with
vehicle or 10 mg/kg Pazopanib. Treatment with Pazopanib is
well-tolerated and there is no significant difference in the body weight
among the mice in each group.






Kinase enzyme assays


VEGFR enzyme assays for VEGGR1, VEGFR2, and VEGFR3 are run in
homogeneous time-resolved fluorescence (HTRF) format in 384-well
microtiter plates using a purified, baculovirus-expressed
glutathione-S-transferase (GST) fusion protein encoding the catalytic
c-terminus of human VEGFR receptor kinases 1, 2, or 3. Reactions are
initiated by the addition of 10 μL of activated VEGFR2 kinase solution
[final concentration, 1 nM enzyme in 0.1 M HEPES, pH 7.5, containing 0.1
mg/mL bovine serum albumin (BSA), 300 μM dithiothreitol (DTT)] to 10 μL
substrate solution [final concentration, 360 nM peptide,
(biotin-aminohexyl-EEEEYFELVAKKKK-NH2), 75 μM ATP, 10 μM MgCl2],
and 1 μL of titrated Pazopanib in DMSO.


Plates are incubated at room
temperature for 60 min, and then the reaction is quenched by the
addition of 20 μL of 100 mM ethylene diamine tetraacetic acid (EDTA).
After quenching, 20 μL HTRF reagents (final concentration, 15 nM
Streptavidin-linked allophycocyanin, 1 nM Europium-labeled
antiphosphotyrosine antibody diluted in 0.1 mg/mL BSA, 0.1 M HEPES, pH
7.5) is added and the plates incubated for a minimum of 10 min. The
fluorescence at 665 nM is measured with a Wallac Victor plate reader
using a time delay of 50 μs.






Method






Phosphorylation of VEGFR2 is assessed in HUVEC stimulated with VEGF.
HUVEC are plated in type-I collagen-coated 10 cm plates in Clonetics
EGM-MV medium at 1.0-1.5 × 106 cells/plate. After 24 hours,
the confluent cells are serum starved overnight by replacing the growth
medium with Clonetics EBM medium containing 0.1% BSA, 500 μg/mL
hydrocortisone. Cells are treated with Pazopanib at various
concentrations for 1 hour, followed by addition of 10 ng/mL VEGF or
vehicle for 10 min. Cells are solubilized in lysis buffer.


VEGFR2 is
immunoprecipitated using antiflk-1 antibody and analyzed by sodium
dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) followed
by Western blotting and detection with antiflk-1 or with
antiphosphotyrosine (anti-P-tyr-biotin) antibody. The VEGFR2
phosphorylation level is quantified by densitometry and normalized to
the total VEGFR2 level.

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Product Categories : Protein Tyrosine Kinase > VEGFR Inhibitor